T Cell Receptor structure and cytokine

responses in lung targeted inflammatory

models

A thesis submitted for the degree of Doctor of Philosophy

University of London

Catherine Jane Reynolds

Lung Immunology Group

National Heart and Lung Institute

Sir Alexander Fleming Building

Imperial College

London

SW7 2AZ

Supervised by: Dr Rosemary Boyton Professor Daniel Altmann

2008

Acknowledgements

Over the course of my PhD there have been many people without whom I would never

have been able to get to this point. Whether it was the kind donation of reagents,

training and advice in lab techniques, scientific input or simply morale support, all

help has been invaluable and very much appreciated.

I would particularly like to thank my supervisors, Rosemary Boyton and Danny

Altmann. There is definitely no way that this thesis would exist without your

unrelenting support, vision and enthusiasm. Your unwavering belief that I would get

there in the end, even through the darker times of transgenic founder screening, and

the enormous commitment, both in terms of your time and your energy, made my PhD

an extremely rewarding and enjoyable experience.

Many people contributed reagents, training and advice to me throughout my PhD and

I've tried to list all of these people, to whom I am extremely grateful, below. There

have also been lots of people who have contributed less specifically, but no less

importantly, particularly in terms of moral support and maintaining my mental well-

being, during the course of this thesis. I'd particularly like to thank all members past

and present of the Boyton, Altmann and Dallman labs, especially Tracey and

Xiaoming who were important extra pairs of hands in many big experiments!

Finally I'd like to thank my partner, Matt. A massive thank you for your never ending

support and patience including all the meals cooked, washing done and pep talks

delivered! I couldn't have done it without you.

2

Reagents, help and support

Transgenesis

Helen Bodmer — HEL expression cassette

Brian Sauer — floxed stop cassette

Jeffrey Whitsett — CC10 promoter

Diane Mathis and Christophe Benoist — TCR cassette vectors

Maggie Dallman f3m70 Cre transgenic line

Laurence Bugeon

Colin Hetherington

Robert Sumner

Karla Watson

Pronuclear injections at Nuffield Department of Medicine (University of Oxford)

Zoe Webster — pronuclear injections at MRC Clinical Sciences Centre (Imperial

College)

Keith Murray

Daniel Wells

Darren Bilton

Amy Wathen

South Kensington campus animal facility technicians

Technical help and support

Kate Choy Lung function studies

Sara Mathie

Lorraine Lawrence — Lung histology

Julia Llewellyn-Hughes — DNA sequencing

Katlin Takacs — DNA preparation for pronuclear injection

Yogesh Singh — Spectratyping

Alan Ahern Molecular biology techniques

Jennifer Rowland

Gurman Kaur — Real time PCR

3

Abstract

Thl or Th2 type immune responses are an important feature of many human diseases,

including several lung pathologies. Multiple factors are involved in determining

whether a naïve CD4+ T cell differentiates into a Thl or Th2 effector cell and several

lines of evidence point towards an important role for the interaction between the T cell

receptor (TCR) and its peptide ligand. A previous study showed that highly polarised

Th2 CD4+ T cells selected under Th2 polarising conditions favour a structurally

distinct TCR with an elongated TCR a chain complimentarity determining region 3

(CDR3), while those selected under Thl polarising conditions favour a shorter TCR

CDR3 a chain. Molecular modelling predicted that this elongated TCR a chain

results in a less optimal (lower avidity) interaction between the TCR and peptide/MHC

complex. In this thesis, clonotypic analysis was used to identify dominant TCR

chains from established Thl and Th2 polarised T cell lines. Dominant TCR a chains

were cloned into TCR transgenic expression vectors and lines of mice with transgenic

expression of these chains were identified. In vitro and in vivo studies were used to

investigate T cell responses in these transgenic lines and the data suggests that

expression of the Th2 derived TCR a chain can cause an inherent bias in CD4+ T cell

phenotype. A long term aim of this work is to use the Thl and Th2 TCR transgenic

animals to develop novel mouse models of lung disease and this thesis also describes

the generation of transgenic mice that express the Thl and Th2 TCR cognate epitope,

PLP 56-70, inducibly and specifically in the lung. Also implicated in the pathogenesis

of many lung diseases is the CXC chemokine, interleukin 8. The final chapter of this

thesis describes the generation of transgenic mice overexpressing this human protein

constitutively in the lung and reports initial characterisation of protein expression and

consequent lung inflammation in these animals.

4

Table of contents page number

Title page 1

Acknowledgements 2

Abstract 4

Table of contents 5

List of figures 10

List of tables 15

List of appendices 16

List of abbreviations 18

Chapter 1. Introduction

1.1 The ar3 T cell receptor 24

1.2 T cell receptor gene rearrangement 25

1.3 T cell receptor sequence diversity 28

1.4 T cell receptor assembly 29

1.5 Thymic selection 34

1.6 Dual TCR expression 36

1.7 TCR editing and revision 37

1.8 Structure of the TCR/CD3 signalling complex 38

1.9 pMHC binding by TCR 44

1.10 TCR binding affinity and conformational change 46

1.11 TCR triggering 49

1.12 CD4+ T cell phenotype 53

1.13 Thl and Th2 cell differentiation 56

1.14 The TCR and CD4+ T cell differentiation 61

1.15 Thl and Th2 responses in human lung disease 64

1.16 Mouse models of human lung disease 70

1.17 Approaches to transgenesis 74

1.18 Interleukin 8 80

1.19 Neutrophil function 83

5

1.20 Interleukin 8 and human lung disease 86

1.21 Aims of this thesis 90

Chapter 2. Materials and methods

2.1 Genomic DNA extraction 94

2.2 RNA extraction 94

2.3 DNase treatment of RNA samples 95

2.4 cDNA synthesis 95

2.5 Polymerase Chain Reaction (PCR) 96

2.6 Restriction enzyme digest 96

2.7 Preparation of XL-Gold competent cells 96

2.8 E-coli transformation 97

2.9 Standard cloning and ligation protocol 97

2.10 Blunt end generation 98

2.11 Oligonucleotide annealing 99

2.12 TA cloning of PCR products 99

2.13 Preparation and screening of plasmid DNA 99

2.14 T cell receptor PCR analysis 100

2.15 Sequencing and T cell receptor sequence analysis 100

2.16 TCR CDR3 spectratyping 101

2.17 Real-time PCR 101

2.18 Preparation of DNA for oocytes pronuclear injection 102

2.19 Genotyping 102

2.20 Southern blotting 103

2.21 Southern blot Digoxigenin (DIG) probe labelling, hybridisation 103

and detection

2.22 Tamoxifen induction 105

2.23 Footpad/flank peptide immunisation 105

2.24 Immediately ex vivo T cell proliferation assay 105

2.25 Culture of T cell lines 106

2.26 Measurement of bronchial hyperreactivity (BHR) 106

6

2.27 Measurement of Resistance (R) and Compliance (Cdyn) 107

2.28 Collection of Bronchoalveolar lavage (BAL) and serum 107

2.29 Cell extraction from whole lung lobe 108

2.30 Differential cell counting 108

2.31 Lung homogenate 108

2.32 Lung histology — tissue preparation 109

2.33 Haematoxylin and Eosin (H+E) staining 109

2.34 Periodic Acid Schiff (PAS) staining and scoring 109

2.35 Immunohistochemistry — tissue fixation 110

2.36 Peroxidase based immunohistochemistry 110

2.37 Immunofluorescence tissue staining 111

2.38 Inflammatory cell scoring 112

2.39 Induction of allergic airway disease using ovalbumin (OVA) 112

2.40 Enzyme Linked ImmunoSorbant Assay (ELISA) 112

2.41 Flow cytometric analysis 113

Chapter 3. Generation of Thl and Th2 TCR a chain transgenic lines

Results

3.1 Determination of a and p TCR gene usage in Thl and Th2 lines 114

specific for PLP 56-70

3.2 Construction of Thl and Th2 TCR a chain transgenes 124

3.3 Identification of Thl and Th2 TCR a chain transgenic founder 132

animals

3.4 Determination of TCR a chain transgene expression 136

Discussion 146

Chapter 4. TCR 13 chain selection and cytokine phenotype in Thl and

Th2 a chain transgenics

Results

4.1 Analysis of TCR p chain usage in unprimed Th2 TCR a chain 153

transgenics

7

4.2 Analysis of TCR 13 chain usage in Th2 TCR a chain transgenic 157

in the context of antigen specificity

4.3 Analysis of PLP (56-70) specific Th2 TCR a chain transgenic 160

T cell lines

4.4 Construction of a Th2 TCR (3 chain transgene 171

4.5 Identification of a Th2 TCR p chain transgenic founder animal 174

4.6 Analysis of a PLP (56-70) specific Thl TCR a chain transgenic 176

T cell line

4.7 Altered cytokine phenotype of an in vivo memory response to antigen 178

in Th2 TCR a chain transgenic animals

Discussion 182

Chapter 5. Generation of transgenic lines for inducible, lung targeted,

antigen expression

Results

5.1 Design and construction of transgenes for inducible, lung targeted, 192

expression of the antigen PLP 56-70

5.2 Identification of MIPLP transgenic founder animal 203

5.3 Determination of stop sequence excision and MIPLP transgene 205

expression

5.4 Re-design of the SIPLP transgene 209

5.5 Identification of SIPLP transgenic founder animals 212

Discussion 216

Chapter 6. Generation and initial characterisation of transgenic lines

with lung targeted expression of human interleukin 8

Results

6.1 Design and construction of a transgene for lung targeted expression 222

of human interleukin 8

6.2 Identification of lung targeted hIL-8 transgenic founder animals 226

6.3 Determination of lung specific hIL-8 transgene expression 228

8

6.4 Lung function and lung cell infiltrate in lung targeted hIL-8 233

transgenics

6.5 Investigation of the impact of IL-8 expression in the murine lung 239

during ovalbumin induced lung inflammation

Discussion 246

Overview 256

Bibliography 260

Appendices 307

9

List of Figures

Page number

Chapter 1. Introduction

1.1 TCR a and p loci gene arrangement and recombination 26

1.2 Timing of TCR gene rearrangement and T cell selection during 30

normal thymocyte development

1.3 Signalling pathways downstream of the T cell receptor 40

1.4 Cytokine environment and CD4+ T cell differentiation 58

1.5 The mechanistic basis of reverse tetracycline-controlled 77

transcriptional activator (rtTA) and Cre/Lox systems of inducible

gene targeting

1.6 Transgenic strategies of this thesis 93

Chapter 3. Generation of Thl and Th2 TCR a chain transgenic lines

3.1 The PCR strategy used to determine a and (3 TCR gene usage in Thl 115

and Th2 line cDNA

3.2 A comparison of TCR a and 13 gene usage in Thl and Th2 T cell lines 122

in order to select chains for transgenic expression

3.3 Presence or absence of the dominant Thl and Th2 CDR3 a motifs in 123

Thl and Th2 lines

3.4 The different states of arrangement of TCR a genes 125

3.5 A schematic diagram showing the cloning strategy used to create the 127

Thl a chain construct

3.6 A schematic diagram showing the cloning strategy used to create the 128

Th2 a chain construct

3.7 Assembly of Thl and Th2 TCR a chain constructs 130

3.8 Complete Thl and Th2 TCR a chain constructs 131

3.9 Identification of Th2 TCR a chain transgenic founder animals 134

3.10 Identification of the Thl TCR a chain transgenic founder animal 135

10

3.11 Demonstration of correct Th2 TCR a chain transgene processing 139

and expression by PCR

3.12 Demonstration of correct Thl TCR a chain transgene processing and 140

expression by PCR

3.13 An increased percentage of double negative thymocytes and a 143

decreased CD4:CD8 ratio suggests expression of the TCR a chain

transgene at the cell surface early in thymocyte development

3.14 An increased percentage of double negative thymocytes suggests 144

expression of the Thl a chain transgene at the cell surface early in

thymocyte development

3.15 Transgenic expression of Thl or Th2 T cell receptor a chains does 145

not affect the levels of TCR at the cell surface

Chapter 4. TCR 13 chain selection and cytokine phenotype in Thl and

Th2 a chain transgenics

4.1 TCR 13 chain sequences from splenocytes of unprimed Th2 TCR a 155

chain transgenic and littermate control animals

4.2 Spectratype analysis of TCR 13 chain gene usage in splenocytes of 156

unprimed Th2 TCR a chain transgenic and littermate control animals

4.3 TCR p chain sequences from primed draining lymph node of Th2 158

TCR a chain transgenic and littermate control animals

4.4 Spectratype analysis of TCR 13 chain gene usage in primed draining 159

lymph node T cells of Th2 TCR a chain transgenic and littermate

control animals

4.5 Rapid clonal expansion of TCR over successive restimulations of a 161

Th2 TCR a chain transgenic T cell line

4.6 Spectratype analysis of Th2 a chain transgenic and littermate derived 162

T cell lines through successive restimulations

4.7 Cytokine phenotype of Th2 TCR a chain transgenic and littermate T 165

11

cell lines over successive restimulations

4.8 Real time PCR analysis of GATA3 and Tbet transcripts in Th2 TCR a 166

chain transgenic and littermate T cell lines over successive

restimulations

4.9 Cytokine phenotype of Th2 TCR a chain transgenic and littermate T 167

cell lines after 15 restimulations

4.10 Clonal expansion of a TCR over successive restimulations of a Th2 169

TCR a chain transgenic T cell line

4.11 Cytokine phenotype of a line 34 Th2 TCR a chain transgenic T cell 170

line

4.12 A schematic diagram showing the cloning strategy used to create the 172

Th2 (3 chain construct

4.13 Cloning of the rearranged Th2 TCR p chain into the TCR (3 expression 173

cassette

4.14 Identification of the Th2 TCR p chain transgenic founder animal 175

4.15 Analysis of a Thl TCR a transgenic derived T cell line 177

4.16 An in vivo memory response to peptide demonstrates a difference in 180

cytokine profile between Th2 TCR a chain transgenic and littermate

animals

4.17 TCR analysis of splenocytes from Th2 TCR a transgenic and 181

littermate animals at day 32 after priming

Chapter 5. Generation of transgenic lines for inducible, lung targeted,

antigen expression

5.1 The strategy for inducible, lung specific, expression of antigen 195

PLP (56-70)

5.2 The localisation of Cre and CC10 proteins in uninduced and tamoxifen 197

induced lung sections from (3m70 MerCreMer mice

5.3 Co-localisation of Cre and CC10 in the lung of a tamoxifen induced 198

r3m70 MerCreMer mouse

12

5.4 A schematic diagram of the cloning strategy used to create the 200

inducible, lung specific PLP (56-70) transgenes

5.5 Assembly of the inducible, lung specific, membrane bound and secreted 201

PLP (56-70) transgenes

5.6 Complete inducible, lung specific, PLP constructs (membrane [MIPLP] 202

and secreted [SIPLP])

5.7 Identification of an MIPLP transgenic founder animal 204

5.8 Removal of the floxed stop sequence and expression of MIPLP in lung 207

tissue following induction with tamoxifen

5.9 Complete removal of the floxed stop sequence occurs over many days 208

and persists for many weeks following induction with tamoxifen

5.10 A schematic diagram to demonstrate the potential importance of loxP 210

site orientation

5.11 A schematic diagram of the cloning strategy used to invert the floxed 211

stop sequence of the SIPLP construct

5.12 Identification of SIPLP transgenic founder animals 214

5.13 Removal of the floxed stop sequence and expression of SIPLP in lung 215

tissue following induction with tamoxifen

Chapter 6. Generation and initial characterisation of transgenic lines

with lung targeted expression of human interleukin 8

6.1 A schematic diagram of the cloning strategy used to create the lung 224

targeted hIL-8 transgene

6.2 Cloning of human IL-8 cDNA into a lung targeted expression 225

construct

6.3 Identification of lung targeted hIL-8 transgenic founder animals 227

6.4 Tissue specific expression of hIL-8 in both transgenic lines 230

6.5 Levels of hIL-8 cDNA transcripts and hIL-8 protein in the lung of both 231

lines of hIL-8 transgenic mice

6.6 hIL-8 expresion is confined to bronchial epithelial cells of the lung in 232

both lines of transgenic mice

13

6.7 Baseline cell infiltrates in the lungs of IL-8 transgenics and littermate 235

animals

6.8 Neutrophil staining by immunohistochemistry of lung sections from 236

IL-8 transgenic and littermate animals

6.9 Mild inflammation in line 19 transgenic compared to line 33 and 237

littermate animals

6.10 Lung function of IL-8 transgenic and littermate animals 238

6.11 Lung function measurements in IL-8 transgenic and littermate animals 242

following ovalbumin sensitisation and challenge

6.12 Differential BAL and lung cell counts from IL-8 transgenic and 243

littermate animals following ovalbumin sensitisation and challenge

6.13 Lung inflammation in OVA sensitised and challenged IL-8 transgenic 244

and littermate animals

6.14 Prevalence of mucus secreting goblet cells in inflated lung sections 245

from OVA sensitised and challenged IL-8 transgenic and littermate animals

14

List of Tables

Page number

Chapter 3. Generation of Thl and Th2 TCR a chain transgenic lines

3.1 TCR a sequences from a Thl polarised NOD.E T cell line against 118

PLP (56-70)

3.2 TCR a sequences from a Th2 polarised NOD.E T cell line against 119

PLP (56-70)

3.3 TCR p sequences from a Thl polarised NOD.E T cell line against 120

PLP (56-70)

3.4 TCR l sequences from a Th2 polarised NOD.E T cell line against 121

PLP (56-70)

3.5 TCR a chain sequences from splenocytes of Th2 TCR a chain 141

transgenic and littermate control animals

3.6 TCR a chain sequences from 2 Thl a chain transgenic animals 142

15

List of Appendices

Page number

1. Stock solutions and buffers 307

2. Antibodies and recombinant proteins 310

3. Restriction enzymes and buffers 311

4. PCR primers for analysis of TCR a and p gene usage 312

5. TCR CDR3r3 spectratyping primers 313

6. Thl and Th2 TCR a chain primers 314

7. MIPLP and SIPLP transgene primers and oligonucleotides 315

8. PCR primers 316

9. Wright-Giemsa stain — cell morphology 317

10. Thl TCR a chain construct - plasmid map 318

11. Th2 TCR a chain construct — plasmid map 319

12. Th2 TCR I chain construct — plasmid map 320

13. MIPLP construct — plasmid map 321

14. SIPLP construct — plasmic! map 322

15. Inverted stop sequence SIPLP construct — plasmid map 323

16. Lung targeted hIL-8 construct — plasmid map 324

17. Thl TCR a complete V-J insert nucleotide sequence 325

18. Thl TCR a rearranged V-J insert nucleotide/protein sequence 326

19. Th2 TCR a complete V-J insert nucleotide sequence 327

20. Th2 TCR a rearranged V-J insert nucleotide/protein sequence 328

21. Th2 TCR 13 complete V-J insert nucleotide sequence 329

22. Th2 TCR 3 rearranged V-J insert nucleotide/protein sequence 330

23. MIPLP construct — complete nucleotide sequence 331

24. Transmembrane HEL/PLP (56-70) nucleotide/protein sequence 337

25. SIPLP construct — complete nucleotide sequence 339

26. Secreted HEL/PLP (56-70) nucleotide/protein sequence 345

27. Inverted stop sequence SIPLP construct — complete nucleotide 346

16

sequence

28. Lung targeted hIL-8 construct — complete nucleotide sequence 352

29. hIL-8 nucleotide/protein sequence 356

30. Inflammatory cell scoring system 357

17

List of abbreviations

AHR

AIRE

AP

APC

APECED

APL

ARDS

ATP

BAL

Bax

BCG

Bcl

BHR

Blys

bp

BSA

C

°C

CC10

CD

cDNA

CDR

Cdyn

CF

CFA

CFTR

Airway hyperresponsiveness

Autoimmune regulator

Activating protein

Antigen presenting cell

Autoimmune polyendocrinopathy candidiasis ectodermal dystrophy

Altered peptide ligand

Acute respiratory distress syndrome

Adenosine triphosphate

Bronchoalveolar lavage

Bc12 associated X protein

Bacille Calmette-Guerin (strain of Mycobacterium bovis)

B cell leukaemia protein

Bronchial hyperreactivity

B lymphocyte stimulator

base pair

Bovine serum albumin

Constant

Centigrade

Clara cell protein 10

Cluster of differentiation

Complimentary DNA

Complimentarity determining region

Compliance

Cystic fibrosis

Complete Freund's adjuvant

Cystic fibrosis transmembrane conductance regulator

18

CIAP Calf intestinal alkaline phosphatase

CMA Cytomegalovirus

COPD Chronic obstructive pulmonary disease

cTEC Cortical thymic epithelial cells

CTLA Cytotoxic T lymphocyte associated antigen

CTP Cytidine triphosphate

D Diversity

DAG Diacylglycerol

DC Dendritic cell

DC-SIGN Dendritic cell-specific ICAM3-grabbing nonintegrin

DIG Digoxigenin

DLN Draining lymph node

DN Double negative

DNA Deoxyribonucleic acid

DP Double positive

DPB Diffuse panbronchiolitis

DTT Dithiothreitol

E Enhancer

EAE Experimental autoimmune encephalomyelitis

EBV Epstein barr virus

ELISA Enzyme linked immunosorbant assay

ER Endoplasmic reticulum

ERK Extracellular signal-regulated kinase

F Forward

FCS Foetal calf serum

FITC Fluorescein Isothiocyanate

g gram

g gravitational acceleration

19

GATA Gene activator of TCR a

GTP Guanosine triphosphate

H+E Haematoxylin and Eosin

HCMV Human cytomegalovirus

HEL Hen egg lysozyme

hIL-8 human Interleukin 8

HPRT Hypoxanthine Phosphoribosyltransferase

Hsp Heat shock protein

i.m intra-muscular

i.p intra-peritoneal

ICAM Intracellular adhesion molecule

IFA Incomplete Freund's adjuvant

IFN Interferon

Ig Immunoglobulin

IL Interleukin

IMGT International immunogenetics

IP3 Inositol- 1 ,4,5-triphosphate

IPF Idiopathic pulmonary fibrosis

IPTG Isopropyl-P-d-thiogalactopyranoside

ITAM Immunoreceptor tyrosine based activation motif

IU International unit

J Joining

KB Kilobase

kD Kilodalton

1 litre

LAT Linker for activation of T cells

20

LB

Luria-Bertani

Lck

Lymphocyte-specific-protein-tyrosine kinase

LFA

Lymphocyte function associated antigen

M Molar

m meter

m milli

mAb monoclonal antibody

MAPK Mitogen activated protein kinase

MBP Myelin basic protein

MDNCF Monocyte derived neutrophil chemotactic factor

Mer Mutated oestrogen receptor

MHC Major histocompatibility complex

MIPLP Membrane bound, inducible, proteolipoprotein

MONAP Monocyte derived neutrophil activating peptide

MPO Myeloperoxidase

mRNA messenger ribonucleic acid

MS Multiple sclerosis

mTEC medullary thymic epithelial cell

n nano

N random nucleotide

NAF Neutrophil activating factor

NFAT Nuclear factor of activated T cells

NF-KB Nuclear factor K B

NK Natural killer

NOD Non-obese diabetic

NTP Nucleotide triphosphate

OVA Ovalbumin

21

P Promoter

PAS Periodic acid Schiff

PBMC Peripheral blood mononuclear cell

PBS Phosphate buffered saline

PCC Pigeon cytochrome c

PCD Pulmonary ciliary dyskinesia

PCR Polymerase chain reaction

PE Phycoerythrin

phox phagocyte oxidase

PI3K Phosphatidylinositol 3 kinase

PIP2 Phosphatidylinositol 3,4-bisphosphate

PLC Phospholipase C

PLP Proteolipoprotein

pMHC peptide/MHC complex

pTa pre-TCR a chain

R Resistance

R Reverse

r recombinant

RAG Recombination activating gene

reg regulatory

RNA Ribonucleic acid

RNAi RNA interference

RS S Recombination signal sequence

rTS reverse transcriptional silencer

rtTA reverse tetracycline transcriptional activator

s.c sub-cutaneous

SCID Severe combined immunodeficiency disease

SD Standard deviation

SE Standard error

22

SH Src homology

SIPLP Secreted, inducible, proteolipoprotein

SLP SH2 domain containing leukocyte protein

SLPI Secretory leukocyte protease inhibitor

SOCS Suppressor of cytokine signalling

SP Single positive

SP Surfactant protein

STAT Signal transducer and activator of transcription

tetO Tetracycline operator

TGF Transforming growth factor

Th T helper cell

TLR Toll like receptor

TNF Tumor necrosis factor

TRITC Tetramethyl rhodamine iso-thiocyanate

TTP Thymidine triphosphate

V

Variable

V

Volt

ZAP z-chain associated protein kinase

23

Chapter 1

Introduction

1.1 The afi T cell receptor

The af3 T cell receptor (TCR) is a transmembrane heterodimer expressed on the

surface of CD4+ and CD8 + T lymphocytes. TCRs on CD4+ and CD8+ T cells

recognise peptides bound to class I or class II major histocompatibility complex

(MHC) molecules respectively and engagement of the TCR with peptide and MHC

can result in activation of the T cell and initiation of an adaptive immune response.

The TCR protein was first identified in the early 1980's using antibodies raised against

lymphomas or T cell clones (Allison et al., 1982 and Haskins et al., 1983). It was

noted that these antibodies, specific for a component of the T cell, could block antigen

recognition and that this blocking or binding did not occur when the antibodies were

used against T cells of different antigen specificities. Precipitation of the proteins

bound by these antibodies revealed a dimer of approximately 80 kilodaltons (kD),

which, on reduction of disulphide bonds, could be separated into two monomers of 39

and 41 kD. Tryptic peptide analysis of the two TCR monomers, now known as the

alpha chain and the beta chain, began to reveal the basis of TCR clonality and

peptide/MHC specificity (Kappler et al., 1983), with comparisons of peptides derived

from TCRs of different specificities suggesting the presence of constant and variable

regions in both chains. Understanding of the genetic basis for these constant and

variable regions of the TCR became possible following the isolation of alpha and beta

chain cDNA (Chien et al., 1984 and Hedrick et al., 1984) which showed the TCR

chains to be composed of rearranged variable (V), diversity (D), joining (J) and

constant (C) region sequences, a concept previously encountered in studies of the B

cell antigen receptor immunoglobulin genes (Hozumi and Tonegawa, 1976).

24

Following the discovery of the a13 TCR, another TCR molecule, known as the y8

TCR, was also described (Brenner et al., 1986).

1.2 T cell receptor gene rearrangement

There are three distinct TCR loci in the genome of both humans and mice, Tcrb,

Tcra/Tcrd and Tcrg, containing the V, D, J and C genes necessary for assembly of af3

and y8 T cell receptors. An outline of the murine germline configuration of genes

within the Tcrb and Tcra/Tcrd loci is shown in figure 1.1. The process of gene

rearrangement and the joining of V, D, J and C genes to form a functional TCR chain

occurs in the thymus during lymphocyte development and utilises highly conserved

recombination signal sequences (RSS) that flank each gene segment (Akira et al.,

1987). The recombination signals consist of conserved heptamer and nonamer

sequences that flank relatively non-conserved 12 bp (12RSS) or 23 bp (23RSS)

spacers. Point mutations in the conserved sequences, or changes in the length of the

spacers, drastically reduce recombination efficiency (Akira et al., 1987) and, although

non-conserved, changes in the sequence of the spacer regions have also been shown to

have an impact (Montalbano et al., 2003). Recombination between gene segments

occurs via a mechanism of double strand breaks (Roth et al., 1992) and is mediated by

the recombination activating genes, RAG1 and RAG2 (McBlane et al., 1995). Mice

deficient in either RAG1 or RAG2 fail to develop mature T cells, due to an inability to

initiate V(D)J recombination (Mombaerts et al., 1992a and Shinkai et al., 1992), and

as such, transgenic expression of rearranged TCR alpha and beta chain genes can

restore T cell development in these animals (Shinkai et al., 1993). The RAG proteins

control the specificity of cleavage events during recombination by binding to the

conserved RSS motifs, but also control the configuration in which gene segments can

be joined together, by dictating that a gene with a 5' 23RSS can only be joined to a

gene with a 3' 12RSS and vice versa (van Gent et al., 1996). This is known as the

12/23 rule of recombination (Akira et al., 1987). However, this seemingly simple

mechanism of control over the configuration of TCR genes cannot account for the

25

Va and VS (a) genes

DS Jo ES CS VS Ja Ca Ea

__ I

Vf3 genes PD(31 DP1 Jf31

ti

Cf31 Dp2 J132

CP2 E(3 V(3 r) C) I

[

(b)

RAG mediated cleavage

Random nucleotide (N) addition by TdT

(c) NN

—11111111E11— Va Ja

N N 4

Vf3 Df3 Jr3 Vf3 DP JP

Figure 1.1 TCR a and 13 loci gene arrangement and recombination. (a) Schematic diagrams of the arrangement of V, D, J and C genes and enhancer elements at the murine Tcra/d and Tcrb loci are shown. (b) Gene segments are combined by a mechanism of double strand breaks, mediated by the RAG recombinase protein complex. Junctional diversity during the joining together of gene segments is brought about by random nucleotide (N) addition by the Terminal deoxynucleotidyl transferase (TdT) enzyme. (c) The order in which gene segments are combined is strictly controlled with DP-J(3 recombination occurring before VP-DP joining. Points at which N nucleotide addition creates junctional diversity in the a and [3 TCR chains are marked (N).

26

strict control imposed on the order in which different genes and indeed different

chains are rearranged. Figure 1.1 outlines the basic mechanism of the V(D)J

recombination reaction and also shows the order in which gene segments for each

TCR chain are recombined. According to the 12/23 rule direct VP-J13 rearrangement

should be possible. However, DP-JP rearrangements are found to occur preferentially,

followed by VP - D.43 (Born et al., 1985). A greater level of control than simply

12RSS or 23RSS is now known to reside with the recombination signal sequence

itself. A study using a modified TCR (3 locus containing just one DP gene segment

and one JP gene cluster demonstrated that the 12RSS of the JP was unable to substitute

for the 12RSS of the DP in allowing recombination with VP gene segments (Bassing

et al., 2000). However, although this finding sheds more light onto how the

configuration of gene segments in TCR chains may be controlled, the complex

temporal regulation of gene rearrangement is still unexplained. Temporal control of

TCR gene rearrangement is important not only in determining the order in which

genes of a single chain are rearranged (Born et al., 1985), but also in controlling

rearrangement of (3 or y chains prior to rearrangement of a or 8 chains (Raulet et al.,

1985) and in controlling allelic exclusion of the TCRP locus, where productive

rearrangement of one TCR (3 allele impedes further gene rearrangement on another

(Uematsu et al., 1988). To explain these processes, the concept of locus accessibility

has emerged whereby multiple mechanisms are employed to alter the accessibility of

genes within a TCR locus to the RAG V(D)J recombinase. Experiments have shown

the presence of nucleosomes (the simplest form of chromatin packaging) around RSSs

to be inhibitory to cleavage by the V(D)J recombinase (Golding et al., 1999) and

changes in the chromatin accessibility of VP genes, as measured by sensitivity to

DNase I digestion and levels of histone acetylation have been observed upon transition

of developing T cells from double negative (DN) to double positive (DP) (Tripathi et

al., 2002). As well as acetylation, chromatin accessibility is also affected by

methylation. Increased levels of methylation as a consequence of deletion of the

promoter PD(31 (figure 1.1) from the TCR (3 locus results in decreased D131 gene

rearrangement, although D(32 rearrangement is unaffected (Whitehurst et al., 2000).

This suggests that chromatin remodelling may be one mechanism by which promoters

27

within TCR loci exert control over local rearrangements. Longer range control over

chromatin structure has been observed for the enhancer sequence (EP) of the TCR P

locus (figure 1.1), deletion of which significantly reduces chromatin accessibility

throughout the DpJP region (Oestreich et al., 2006). This study also reported direct

physical contact between EP and PD131, implying that different control elements

within a TCR locus can interact to exert control over TCR gene rearrangement during

T cell development.

1.3 T cell receptor sequence diversity

Mathematical estimates of the number of different TCR sequences that can potentially

exist are in the range of 1012-1015 (Davis and Chien, 1999). However, through the

need for self tolerance, and the processes of negative and positive selection in the

thymus, estimates of the number of different aP T cell receptor sequences present in

the periphery range from 2 x 106 for a naive mouse (Casrouge et al., 2000) to 2 x 107

in human peripheral blood (Arstila et al., 1999). Diversity of TCRs is achieved in one

of three ways. As already discussed, the genomic locus of each TCR chain contains

many different V, D and J gene segments which recombine to generate a functional

TCR chain. There are therefore many possible combinations of gene sequences that

can exist following recombination. Secondly, each TCR is composed of an a and a P

chain. Diversity is therefore amplified by receptor heterodimerisation where, for

example, a particular p chain sequence is able to form a functional receptor with

multiple TCR a chain partners (Listman et al., 1996). The third mechanism by which

TCR sequence diversity is achieved is by imprecise joining of gene segments during

the recombination process itself. Following cleavage of the TCR locus, coding ends

of gene segments, brought into close proximity by the RAG V(D)J recombinase

complex, must be joined together to create a functional TCR chain. During the joining

phase of rearrangement reactions, random nucleotides (N) are added in a template

independent fashion onto the 3' OH ends of the combining gene segments by the

terminal deoxynucloetidyl transferase (TdT) enzyme. Regions of N nucleotides at the

junctions of gene segments are absent in the TCRs of T lymphocytes that lack this

28

enzyme (Komori T et al., 1993) and the size of the a(3 TCR repertoire in TdT null

mice has been estimated to be just 10% of the size of a normal wild-type animal

(Cabaniols et al., 2001). However, immune responses appear relatively unimpaired in

these animals, which remain able to mount robust responses against a range of

different antigens (Gilfillan et al., 1995). Regions of each TCR chain sequence that

are subject to N region addition are marked on figure 1.1 and lie in a region termed the

CDR3a or CDR313 of the TCR a and 13 chains respectively. The CDR3, along with

the germline encoded CDR1 and CDR2 regions of the TCR, plays an important role in

peptide specificity and in binding of the TCR to peptide/MHC and will be discussed in

more detail in a later section.

1.4 T cell receptor assembly

The assembly of a functional a(3 TCR on the surface of a mature T cell is an ordered

process that is subject to strict control measures at multiple stages during T cell

development. A schematic diagram of the different stages of T cell development in

the thymus and the timing of al3 TCR assembly is shown in figure 1.2. The main

requirements of a TCR on the surface of a T cell, exiting the thymus to join the

peripheral immune system, are that it should be able to recognise peptides bound to

self-MHC molecules but that it should not be reactive to peptides derived from self-

proteins. The nature of TCR gene rearrangement in promoting extensive TCR

sequence diversity, as discussed in the previous section, means that there is estimated

to be potential for the generation of 1012 — 1015 different TCRs. However many of

these will not satisfy the self MHC / self peptide requirements mentioned above and so

processes of positive and negative selection are employed in the thymus to reduce and

restrict the peripheral TCR repertoire to those molecules which will be functional, but

not self reactive.

During T cell development, the TCR (3 chain is rearranged before the a chain (Raulet

et al., 1985) and indeed use of a TCR p chain transgene in scid mice (Bosma et al.,

1983), which carry a mutation in a DNA repair enzyme and so cannot carry out VDJ

29

DN4

DP a chain

SP SP

f

TCR a gene rearrangement

13 chain selection

Positive and negative selection

DN1

DN2

DN3 }

TCR p gene rearrangement

+

Figure 1.2 Timing of TCR gene rearrangement and T cell selection during normal thymocyte development. The main stages of normal thymocyte development are shown, along with cell surface markers used to distinguish each immature cell population. TCR (3 gene rearrangement and cell surface expression occurs before that of the TCR a chain and the TCR (3 chain is stabilised at the cell surface by binding to a surrogate a chain (pTa). Formation of a pTa complex at the cell surface mediates transition of thymocytes from the DN stage to the DP stage and following allelic exclusion at the TCR (3 locus, TCR a gene rearrangement and expression is permitted. On formation of a stable a13 heterodimer, positive and negative selection events ensure that mature SP T cells exiting the thymus are able to recognise foreign peptides bound to self MHC, but are not reactive to peptides derived from self proteins.

30

recombination, demonstrated expression of the TCR (3 chain on the cell surface of DN

thymocytes in the absence of the a chain (Hiroyki et al., 1991). Stable cell surface

expression of the 13 chain is permitted by the formation of a heterodimer with a 33 kDa

glycoprotein known as the pre-TCR a chain (Groettrup et al., 1993). af3 T cell

development is found to be severely impaired in animals that lack the pre-TCR a gene

(Fehling et al., 1995), with T cell development blocked at the transition between DN

and DP thymocytes (figure 1.2), although the block is incomplete compared to that

seen in the absence of the 13 chain (Mombaerts et al., 1992b). This finding implies that

proteins other than the pre-TCR a (pTa) are able to facilitate transition of thymocytes

from DN to DP and indeed in pTa deficient mice, a13 TCR complexes can rescue T

cell development and promote maturation and expansion of T cells expressing a

productively rearranged (3 chain (Buer et al., 1997). However, the a chain cannot act

as a direct substitute for pTa, as T cells manipulated to express a TCR a chain at the

point of normal pTa gene expression, but lacking endogenous pTa, demonstrate

impaired development, proliferation and differentiation (Borowski et al., 2004). In

this study, inefficiencies in T cell development and survival were partly overcome by

expression of a hybrid TCR a chain, containing the cytoplasmic domain of pTa, and

multiple lines of evidence now point to an important role for pTa in mediating

signalling events that promote T cell development and allelic exclusion at the TCR p

locus. pTa is a type 1 transmembrane protein (Saint-Ruf et al., 1994) which pairs

with the TCR p chain via a disulphide bond. The extracellular portion of the protein is

an immunoglobulin (Ig) like domain and the intracellular cytoplasmic tail contains

several potential phosphorylation sites and a Src homology 3 (SH3) domain binding

sequence. Mutation studies of this cytoplasmic tail have demonstrated an important

role in T cell development (Aifantis et al., 2002) and in intracellular Ca2+ mobilisation,

an important mediator of intracellular signalling events which has been shown to

increase, alongside activation of the transcription factors NF-KB and NFAT, during

constitutive signalling through the pre-TCR complex (Aifantis et al., 2001). The

process by which a developing lymphocyte expressing a pre-TCR (TCR (3 / pTa)

complex is permitted to move from the DN to DP compartment of the thymus, thereby

31

undergoing proliferation and escape from apoptosis, is known as p selection and is

accompanied by allelic exclusion at the TCR (3 locus, ensuring that each developing T

cell expresses a single TCR (3 chain. Signalling through the pre-TCR is known to be

important in this allelic exclusion as thymocytes from mice lacking pTa show an

increased number of cells with two rearranged TCR (3 alleles (Aifantis et al., 1997), a

finding also observed in mice lacking the signalling pathway adapter, SH2 Domain-

containing Leukocyte Protein (SLP)-76 (Aifantis et al., 1999). These SLP-76

knockout mice also show a block at the DN stage of thymocyte development, which

cannot be overcome by expression of TCR a and p chain transgenes. However,

signalling through the pre-TCR during (3 selection does not occur in isolation and other

signalling pathways, such as those initiated by the Notch receptor and its ligands have

also been shown to play a role (Ciofani et aL, 2004).

One of the functional outcomes of pre-TCR signalling, leading to allelic exclusion at

the TCR (3 locus, is the down regulation of the RAG genes responsible for gene

rearrangement (Wilson et al., 1994). Expression levels of RAG-1 and RAG-2 are high

at two stages during lymphocyte development. The first is early in DN thymocytes,

prior to (3 chain selection, and the second later in DP thymocytes, permitting

rearrangement at the TCR a locus (figure 1.2). Unlike the TCR (3 locus, both alleles

of the TCR a locus are permitted to undergo rearrangement simultaneously and this

rearrangement is not inhibited by expression of an aP TCR at the cell surface

(Borgulya et al., 1992). As such, rearrangement at the TCR a locus continues until an

a(3 pair on the surface of the T cell successfully engages with a major

histocompatibility complex molecule and results in positive selection and

downregulation of RAG gene expression (Brandle et al., 1992). A single TCR a allele

is able to make multiple rearrangements, so that if the first rearrangement does not

result in a functional TCR when paired with the TCR (3 chain expressed by that T cell,

the TCR a locus can excise the first rearrangement and recombine different V and J

genes upstream and downstream of the original rearrangement respectively in a

process termed TCR a chain revision (Marolleau et al., 1988). Mice engineered to

32

express a modified TCR a locus containing a very limited number of V and J genes,

which cannot undergo effective TCR cc chain revision, show a reduced number of a(3

T cells in the periphery (Huang et al., 2005), highlighting the importance of this

process in normal T cell development. At the point of TCR a chain rearrangement

and expression, the pre-TCR complex is downregulated from the cell surface via a

mechanism of competitive displacement (Trop et al., 2000). Immature double positive

thymocytes (TCRI') often express two cell surface TCR a chains, derived from each

TCR a, allele, both forming pairs with the same TCR 13 chain (Alam et al., 1995).

However, most mature thymocytes and peripheral T cells express just one cc(3 TCR

and this process of selection of one TCR cc chain over another is known as allelic

exclusion, although the functional rearrangement of both TCR cc chain alleles that can

precede exclusion makes it a functionally different process from that previously

described for allelic exclusion at the TCR (3 locus. Several different hypotheses have

been proposed to explain allelic exclusion of TCR a chains. The first proposes that

competition between the chains for pairing with a limited number of TCR (3 chain

molecules dictates which pair becomes dominant. This is supported by experiments

demonstrating good expression of both chains intracellularly, despite the presence of

just one on the cell surface, arguing against a mechanism of downregulation of one

chain (Alam and Gascoigne, 1998). However, T cells engineered to express two

independent TCRs, i.e. with different cc and (3 chains, still preferentially express just

one receptor at the cell surface, although both TCR proteins can be detected

intracellularly and the receptor which dominates is not fixed between T cell clones

(Sant'Angelo et al., 2001). This finding makes the competition hypothesis harder to

rationalise, but does point to a post-translational mechanism of control. A second

hypothesis proposes an active mechanism of exclusion whereby signalling events are

required to select one a(3 pair over another. In a study using immature thymocytes

expressing two TCR cc chains with different peptide specificities, allelic exclusion was

achieved upon ligand recognition with subsequent loss of the non-ligated receptor in

mature T cells (Boyd et al., 1998). This receptor specific signal for allelic exclusion

was also found to be CD45 dependent, implicating a mechanism of signalling through

33

the TCR, a finding subsequently supported by studies involving other mediators of the

TCR signalling pathway (Niederberger et al., 2003).

1.5 Thymic selection

The process of positive selection, whereby an ocr3 TCR demonstrates binding and

recognition of self-MHC, occurs in the thymic cortex. Interactions between T cells

and cortical thymic epithelial cells (cTECs) have been shown to be dependent upon

MHC (Bousso et al., 2002) and lead to the migration of thymocytes towards the

thymic medulla, a process mediated by the upregulation of chemokine receptors, such

as CCR7, and their ligands (Kwan and Killeen, 2004). In the thymic medulla T cells

interact with other cell types, such as dendritic cells (DCs) and medullary thymic

epithelial cells (mTECs), which present a diverse array of peptide/MHC complexes

with peptides derived from ubiquitously expressed, but also tissue specific proteins

(Sospedra et al., 1998). A high affinity interaction between an a(3 TCR and a self

peptide/MHC complex results in negative selection and deletion of the thymocyte

bearing the autoreactive TCR (Kappler et al., 1987) thus establishing tolerance to self

proteins. A recent study highlighting the importance of negative selection in

establishing tolerance used mice able to express an MHC molecule (IA") bound to just

a single peptide, permitting positive selection of thymocytes but severely impairing the

process of negative selection. The authors demonstrated that mature T cells specific

for this MHC molecule were highly cross reactive, not only recognising other peptides

bound to IA", but also binding other MHC alleles (Huseby et al., 2005).

Expression of peripheral antigens in the thymus was found to be a function of mTECs

(Derbrinksi et al., 2001) and in this study, expression of a peripheral protein in these

cells was found to be sufficient for the generation of peripheral tolerance. However,

although expression of the tissue specific antigen is a function of mTECs, they are not

necessarily the cell type that ultimately presents the antigen to the developing

lymphocyte. A study of antigen presentation in the thymus, using mice in which

mTECs could express ovalbumin (OVA) but not MHC class I, demonstrated that DCs

34

in the thymus could generate tolerance to this protein via a mechanism of indirect

antigen presentation (Gallegos and Bevan, 2004). Expression of tissue specific

antigens in mTECs is now known to be primarily mediated by the transcription factor

AIRE (Anderson et al., 2002), although some genes can still be upregulated in the

absence of this protein (Derbinski et al., 2005). Humans with mutations in the AIRE

gene suffer from systemic autoimmunity (Finnish-German APECED Consortium,

1997), as do AIRE deficient mice (Anderson et al., 2002). More recently, variations

between individuals in thymic expression levels of autoantigens controlled by the

AIRE protein, such as insulin, have been correlated with susceptibility to disease

(Taubert et al., 2007). However, thymic expression of a protein does not necessarily

result in efficient negative selection and tolerance. A study investigating tolerance

versus autoimmunity to the membrane protein H+/K+ ATPase, the main autoantigen of

autoimmune gastritis, found that although one subunit of the protein (H/Ka) is

expressed in the thymus, T cells specific for this molecule are not deleted (Allen et al.,

2005). The reason for this failure of negative selection was found to be the absence of

the (3 subunit (H/K(3), which is required to stabilise the H/Ka subunit in the

membrane. Many proteins are also expressed differentially between tissues as

different splice variants of the same gene. This can mean that, despite induction of

gene expression in the thymus during T cell development, the epitope associated with

disease is not encountered by autoreactive T cells until they reach the periphery (Klein

et al., 2000). Therefore, although the expression of tissue specific antigens in the

thymus is critical for the generation of self-tolerance, tolerance may be harder to

achieve for more complicated proteins with multiple subunits or complex genetic

expression mechanisms.

The main mechanism of central tolerance induction in the thymus is negative selection

by apoptosis. T cells deficient in two key pro-apoptotic proteins, Bak and Bax

demonstrate impaired thymocyte development, including an impaired ability to

undergo negative selection (Rathmell et al., 2002), and NOD mice, which display a

general susceptibility to autoimmunity, have been shown to have reduced expression

of another mediator of apoptosis, the bim protein (Liston et al., 2004). However, an

35

alternative mechanism of central tolerance, where T cells bearing autoreactive TCRs

are induced to acquire a regulatory T cell (CD4+CD25±) phenotype has also been

described (Jordan et al., 2001). T cells proceeding along this pathway of selection

have been shown to require a high affinity interaction with their peptide/MHC

complex. That a high affinity interaction between a TCR and self/peptide MHC in the

thymus can result in development of a regulatory T cell phenotype in some cases, but

apoptosis and negative selection in other cases, and that a low affinity interaction

spares the T cell from deletion resulting in positive selection (Alam et al., 1996),

raises important, but yet broadly unanswered questions about how the T cell interprets

a signal through the TCR to generate different functional outcomes. This issue will be

explored further in later sections discussing TCR structure, TCR signalling and T cell

differentiation.

1.6 Dual TCR expression

Although most mature CD4+ and CD8+ T cells express just a single type of al3 TCR at

the cell surface, allelic exclusion, particularly at the TCR a locus, is not always

complete. This results in the expression of two TCR a chains, each pairing with the

same TCR p chain to form two independent receptors (Padovan et al., 1993). It is

estimated that as many as 30% of peripheral human T cells (Padovan et al., 1993) and

10-30% of peripheral mouse T cells (Elliott and Altmann, 1995 and Sarukhan et al.,

1998) may carry dual a chains and although dual TCRs are most commonly associated

with incomplete allelic exclusion at the a locus, very low frequencies of T cells

expressing two p chains have also been reported (Padovan et al., 1995). Since the

demonstration of dual TCR expression on the surface of a single T cell in the

periphery, much attention has focussed on the potential implication of this finding in

terms of antigen specificity and autoimmunity. It has been postulated that autoreactive

TCRs may escape negative selection in the thymus by being expressed at low levels on

the surface of a T cell which is then subsequently positively selected based on low

affinity self peptide/MHC recognition by a second, more highly expressed, TCR. This

idea was supported by a study using dual TCR transgenic mice where one receptor,

36

specific for the natural self-antigen C5, was rescued from negative selection in the

thymi of C5+ mice by expression of a second unrelated TCR. Both TCRs were

present on the surface of mature T cells in the periphery, but levels of the autoreactive

TCR were very low and these CD8+ T cells were only able to mediate killing of a C5

carrying target cell when activated via the second TCR (Zal et al., 1996). That this

phenomenon may then lead to increased susceptibility to autoimmunity also has some

experimental support (Sarukhan et al., 1998). In this study a TCR transgenic model of

autoimmune diabetes was used to demonstrate that, in the context of an active central

tolerance mechanism, T cells expressing low levels of autoantigen specific TCR could

still be found in the periphery and that these cells also expressed a second TCR

molecule and were capable of causing disease. However, other studies in different

disease systems using mice hemizygous for the TCR a locus and therefore unable to

express dual TCRs have not always supported this link (Elliott and Altmann, 1996 and

Corthay et al., 2001). Another proposed implication of dual TCR expressing cells is

that they may serve to increase the repertoire of TCRs in the periphery which are

capable of binding to foreign peptides by rescuing from deletion those TCRs which do

not bind self peptide/MHC with high enough affinity to be positively selected in the

thymus (He et al., 2002).

1. 7 TCR editing and revision

Although discussions of negative selection in the thymus usually refer to deletion of a

T cell bearing an autoreactive TCR, there is some evidence that T cell apoptosis is not

always the default pathway for removing these receptors from the repertoire. As an

alternative, a mechanism of receptor editing has been proposed to occur whereby

autoreactive TCRs are internalised and further rearrangement at the TCR a locus is

permitted (McGargill et al., 2000). This was demonstrated using a transgenic TCR

specific for a peptide of OVA in a mouse expressing this peptide specifically in the

thymus. Why the binding of some autoreactive TCRs may activate this receptor

editing pathway of negative selection compared to the apoptotic pathway initiated by

others is not currently understood, but does seem to be an outcome intrinsic to the

37

TCR and antigen being recognised. When a different TCR (2C) and ligand (HY) were

used in the system described above, deletion was the main outcome (Mayerova and

Hogquist, 2004) and the choice between receptor editing and deletion was found to be

unaffected by the cell type involved in presentation of the antigen within the thymus.

When a T cell exits the thymus and enters the peripheral immune system, the TCR(s)

it bears on the cell surface are generally considered to be fixed, i.e. no further changes

in TCR sequence occur. However, a study using a TCR Vp5 transgenic mouse

demonstrated that the RAG genes could be re-expressed in peripheral CD4+ T

lymphocytes, and that active V(D)J recombination was detectable in these cells

(McMahan and Fink, 1998). In this study, this process of receptor revision was seen

to occur over time, with numbers of V135+ cells in the periphery decreasing as mice

aged, but it has also been reported to occur in response to stimulation with

superantigen (Huang et al., 2002) and the notion that receptor revision is restricted to

recent thymic emigrants still undergoing secondary rearrangement events has also

been challenged (Cooper et al., 2003). Any changes in the TCR of a T cell once in the

periphery and out of reach of thymic selection could be potentially dangerous to self

as, although receptor revision may serve as a mechanism of peripheral tolerance and

deletion, the generation of autoreactive TCR is also a possibility. In one study using

non-obese diabetic (NOD) mice, which are susceptible to autoimmune diabetes, a

population of peripheral, autoagressive, CD40+ T cells were seen to express RAG

genes and alter their surface expression of Va (Vaitaitis et al., 2003). However, a

good understanding of the importance of TCR revision in relation to disease

mechanisms, peripheral tolerance or TCR repertoire expansion is still lacking.

1.8 Structure of the TCR/CD3 signalling complex

As previously discussed, the ar3 and y8 TCRs are both transmembrane heterodimers

whose individual subunits are composed of rearranged V, D, J and C region

sequences. Many crystal structures for both al3 TCRs and yo TCRs have now been

solved (e.g. Garcia et al., 1996 and Allison et al., 2001). Each TCR chain is revealed

38

to form 2 immunoglobulin (Ig) like domains on the extracellular side of the cell

membrane, one composed of rearranged V, D and J sequences and the other formed

from part of the C region, although the Ig fold formed by the Ca region is atypical due

to the absence of a top (3 sheet. A connecting peptide, the transmembrane domain and

a short cytoplasmic tail are also encoded by the C regions and the two chains of each

TCR are joined together by disulphide bonds. Cell surface expression of the TCR

heterodimer is stabilised by, and is indeed dependent upon, association with another 3

protein dimers (CD3ye, CD38E and CD3t) and a TCR molecule at the cell surface in

association with these additional polypeptides is known as the TCR/CD3complex

(Samelson et al., 1985, Sussman et al., 1988 and Geisler, 1992). The extracellular

domains of the y,E, and 8 chains also form Ig folds (Arnett et al., 2004 and Kjer-

Nielsen et al., 2004) while the extracellular domains of the chains are only 9 amino

acids in length and are of unknown structure. A schematic diagram of the 4 protein

dimers that make up the TCR/CD3 complex is shown in figure 1.3. However,

although the identity of the components of the complex are now fairly well

established, the lack of a crystal structure for the complex in its entirety means that our

understanding of the three dimensional organization of the complex is still incomplete.

Although precise interactions between subunits, particularly the extracellular portions

are ill defined, acidic and basic charged residues in the transmembrane helices of TCR

and CD3 subunits, which are highly conserved, are known to play an important role in

complex assembly (Call et al., 2002). In this study, isolation of radiolabelled

assembly intermediates from the cell endoplasmic reticulum (ER), and a mutagenesis

approach, allowed the exact transmembrane residue requirements of each intermediate

to be determined. It was found that the intermediates of (TCRa - CD38E), (TCR(i -

CD3yE) and (TCRa - CD3) all required one basic and two acid residues for

assembly with the basic residues provided by transmembrane regions of the TCR

chains and acidic residues by transmembrane regions of the CD3 chains. This study

also provided evidence that each TCR/CD3 complex contained just a single TCR aP

heterodimer, a concept that has been controversial, with other groups reporting

evidence of multiple aP molecules per complex (Fernandez-Miguel et al., 1999). To

date, the exact stoichiometry of the TCR/CD3 complex is still debated, but more

39

H

(Raf--1)

CSIlfjneurin

Transcriptional activation

TCR

8 6

CD4

Figure 1.3 Signalling pathways downstream of the T cell receptor. This schematic diagram shows the major components of the intracellular signalling pathways of T cell activation but does not cover all known proteins and interactions. Phosphorylation of ITAM motifs in the intracellular domains of the CD3 proteins by Lck results in recruitment and activation of ZAP-70. ZAP-70 phosphorylates the adaptor molecule LAT, which is then able to recruit a multitude of other adapter proteins and enzymes ultimately leading to activation of the transcriptional activation by the transcription factors AP-1, NF-xl3 and NFAT

40

recent evidence points towards the existence of both monovalent and multivalent

complexes on the cell surface (Schamel et al., 2005) a finding that may have important

implications for current models of TCR signalling.

Much is now known about the nature of the intracellular signalling events that occur

following ligation of the TCR with a peptide/MHC complex (pMHC). A review of all

of the different kinases, phosphatases and adapter molecules currently known to be

involved in the propagation of a productive TCR signal will not be attempted here, but

the main components of the signalling cascade are shown in figure 1.3. The earliest

signalling events following TCR ligation are phosphorylation of immunoreceptor-

tyrosine-based activation motif (ITAM) tyrosines in the intracellular domains of CD3

chains by the Src family kinases Lck and Fyn, although the contribution of Fyn under

normal physiological conditions is thought to be minimal as it is largely unable to

compensate for the loss of a functional Lck molecule (Straus and Weiss, 1992). The

CD3 y, 8 and 6 chains each contain 1 ITAM while the CD3 chain contains 3 and

phosphorylation of these motifs creates docking sites for the Src homology 2 (SH2)

domains of the Syk family kinase ZAP-70 (Chan et al., 1991). Expression of the

ITAM containing region of the CD3 chain as a chimeric transmembrane protein that

is oligomerized by an extracellular stimulus such as a monoclonal antibody (Irving and

Weiss, 1991) or the aggregation of chimeric proteins bearing ZAP-20 and Fyn as their

intracellular components (Kolanus et al., 1993) are both able to transduce normal

activation signals by themselves, independently of the rest of the TCR complex.

Recruitment of ZAP-70 to the TCR/CD3 signalling complex results in

phosphorylation of ZAP-70 by Lck at position Tyr-493 (Chan et al., 1995) and the

critical importance of this molecule in normal T cell function is clear from the severe

immunodeficiencies in both CD8÷ and CD4+ responses in patients which lack a

functional ZAP-70 protein (Elder et al., 1994). Activated ZAP-70 then itself

phosphorylates the adapter protein LAT (linker for activation of T cells) (Zhang et al.,

1998), the multiple phosphotyrosine residues of which act to recruit the many other

enzymes and linker proteins involved in the main Ras and Ca2+ mediated pathways of

cell signalling (Zhang et al., 1999). Activation of these signalling pathways ultimately

41

lead to the activation of transcription factors such as AP-1 and NFAT (figure 1.3)

which then act, often cooperatively (Jain et al., 1992) to direct the expression of the

many different genes known to be upregulated during T cell activation. Binding sites

for these transcription factors were originally identified in regulatory regions upstream

of the interleukin-2 (IL-2) gene (Serfling et al., 1989 and Shaw et al., 1988) but a role

in regulation of expression of many other proteins, particularly cytokine genes, is now

known. Mice with T cells that lack the main lymphocyte NFAT proteins, NFAT1 and

NFAT2, show deficiencies in many of the main effector outcomes of T cell activation

(Peng et al., 2001) such as cytotoxicity, due to failure to upregulate effector molecules

such as CD40 ligand and Fas ligand, and cytokine production, with production of

cytokines such as IL-2, interferon-y (IFN-y), IL-4, IL-10, IL-5, and tumor necrosis

factor-a (TNF-a) all severely impaired in these animals. The question of whether

these generic T cell activation transcription factors can influence the effector

phenotype of an activated T cell, for example in determining whether a T cell

differentiates to become a Thl cell, producing predominantly IL-2 and IFN-y, or a Th2

cell, producing cytokines such as IL-4, IL-5 and IL-13, has been much debated. The

processes governing Thl and Th2 T cell differentiation will be discussed in more

detail in a later section, but important studies addressing this issue include those

utilising mice deficient in one or other of the NFAT1 and NFAT2 proteins. Mice

lacking NFAT1 show impaired IFN-y production by activated T cells (Kiani et al.,

2001), are more susceptible to infections that rely on the development of a Thl

effector cell response (Kiani et al., 1997) and show exacerbated Th2 type responses

(Ranger et al., 1998a). Conversely, NFAT2 deficient T cells show impaired IL-4

production (Yoshida et al., 1998) and reduced titres of the IL-4 dependent antibody

isotypes IgG1 and IgE (Ranger et al., 1998b). However, from more recent data, the

simplistic explanation of these knockout studies, that NFAT1 positively regulates Thl

cell differentiation but negatively regulates Th2 cell differentiation and that NFAT2 is

required for Th2 cell differentiation, has not held true. Expression of constitutively

active forms of NFAT1 and NFAT2 are both able to induce transcription of Thl

(Porter and Clipstone, 2002) and Th2 cytokines (Monticelli and Rao, 2002) and

cooperative binding of NFAT1 with Thl and Th2 lineage specific transcription factors

42

has been demonstrated in vivo at the promoter regions of both the IFN-y and IL-4

genes (Avni et al., 2002).

Although the TCR signalling cascade discussed in brief above was described as being

mediated by a series of phosphorylation events, effective control over TCR signalling

also requires the coordinated action of tyrosine phosphatases. Upon TCR engagement,

increased protein tyrosine phosphorylation within the cell is detectable within a matter

of just a few seconds (June et al., 1990). However, to be a well controlled signalling

mechanism, the tyrosine residues in question must be kept in an unphosphorylated

state prior to TCR ligation. In keeping with this, treatment of T cells with

pervanadate, a protein tyrosine phosphatase inhibitor, results in activation of some of

the tyrosine kinase mediated signalling events normally associated with ligation of the

TCR, such as activation of Lck, and the induction of some effector outcomes of TCR

signalling, such as production of IL-2 (Secrist et al., 1993). In relation to T cell

signalling, the most well studied tyrosine phosphatase protein to date is CD45 (Tonks

et al., 1988). CD45 is a membrane bound protein expressed on the surface of all

nucleated hematopoietic cells, a deficiency in which results in profound defects in T

cell development and function (Mee et al., 1999 and Weaver et al., 1991). Perhaps

counter intuitively to the findings of the pervanadate experiment described above,

CD45-/- T cells show an increase in the strength of the TCR stimulus that is required

for TCR signalling (Mee et al., 1999). This can be explained by the action of CD45

phosphatase activity on the protein tyrosine kinase, Lck. CD45 activity was found to

keep Lck in an easily activated form by removal of a tyrosine phosphate that

negatively regulates the kinase activity of this protein (Mustelin et al., 1989). The

balance between phosphorylation and dephosphorylation of proteins that make up and

surround the TCR/CD3 signalling complex is therefore of paramount importance in

dictating whether or not a TCR mediated signalling event is propagated into the T cell.

How ligation of the TCR leads to the triggering of these signalling events and how the

nature of ligand binding to TCR is interpreted by the signalling machinery to lead to

different T cell effector outcomes is still very poorly understood and multiple different

models of TCR triggering have been proposed.

43

1.9 pMHC binding by TCR

Although the way in which the TCR activation signal is propagated from TCR ligation

to initiation of intracellular signalling events is poorly defined, better understood is the

nature of the physical interaction between the TCR and its pMHC, studies of which

have helped to shed some light on possible mechanisms of TCR triggering. Despite

the technical difficulties involved in crystallising complexes of multiple, multimeric,

transmembrane proteins, the crystal structures of at least 24 TCRs bound to either

class I or class II pMHC complexes have been solved (Garcia et al., 1996 and

Reinherz et al., 1999). In terms of the pMHC structure being recognised by the TCR,

the general architecture of class I and class II MHC molecule peptide binding grooves

are quite similar, with the floor of the groove formed by a seven stranded (3 sheet and

sides of the groove by two long a helices. However, differences in the pMHC surface

which must be bound by TCR are brought about by the different lengths of peptide

bound preferentially by each class of MHC, with class I molecules tending to bind

shorter peptides than those bound by class II molecules (Stern and Wiley, 1994).

Class I MHC molecules generally bind peptides of 8-10 residues in length, the termini

of which are anchored into the peptide binding groove by conserved, MHC allele

specific, pockets (Colbert et al., 1993). These conserved pockets mean that allele

specific motifs in peptides binding to a particular MHC molecule have also been

identified (Falk et al., 1991). The anchoring of peptide termini in MHC class I

complexes results in longer peptides being forced to bulge out of the binding groove

(Speir et al., 2001) and, along with generally shallower binding of peptide in the

groove, means that peptide residues in MHC class I complexes are generally more

accessible to the TCR. However, as the termini of peptides bound to MHC class II

molecules are not fixed, and class II molecules are able to bind peptides of

significantly longer length, peptide termini extending from the ends of MHC class II

peptide binding grooves can also play a major role in interacting with the TCR

(Carson et al., 1997). Despite recognition of different MHC molecules bound to

peptides of different lengths and conformations, the general orientation of the TCR,

with respect to pMHC, upon binding is seen to be relatively similar between

44

complexes. Generally the TCR molecule binds such that its orientation is diagonal

with respect to the MHC peptide-binding groove. The Va and VP domains of the

TCR lie over the N and C terminal portions of the peptide respectively and the

majority of peptide contacts are made via the hypervariable CDR3a and CDR.3(3

loops. Contacts with the MHC a helices that form the walls of the peptide binding

groove are made predominantly by the less variable and germline encoded CDR1 and

CDR2 regions, although peptide contacts with these loops have also been noted

(Garcia et al., 1996). Although, from the structural data obtained to date for different

TCR/pMHC complexes, this diagonal mode of binding is by far the most commonly

observed, there are notable exceptions. For example, recently solved structures of two

autoimmune TCRs binding to their pMHC ligands revealed that compared to other

conventional TCR binding positions, these MBP specific autoimmune TCRs were not

centred over the pMHC surface, but were shifted towards the N terminus of the

peptide (Hahn et al., 2005 and Li et al., 2005). Determining whether an unusual

binding pattern is a general feature of autoreactive TCRs requires the resolution of

many more structures, not least because the features of a TCR or pMHC that define

conventional binding are still not clear. Although at the level of each individual

TCR/pMHC whose structure has been solved, the precise residues involved in binding

of the TCR in this orientation can be identified, there are as yet no general rules to

describe the location and identity of residues involved in this docking and resultant

MHC restriction of TCR. Indeed, different TCRs recognising the same pMHC

complex have been shown to use assume the same orientation, yet use completely

different TCR amino acids for binding (Ding et al., 1998) and in a recent study, the

same TCR was shown to utilise different binding residues and adopt an altered

orientation when binding to a self versus a foreign MHC molecule (Colf et al., 2007).

This data argues against the presence of conserved residues in TCR molecules that

dictate binding orientation, although some amino acid positions are suggested to make

more frequent contacts than others and analysis of TCR reactivity to MHC from T

cells which had not undergone negative or positive selection suggested that MHC

reactivity is indeed an inherent feature of TCR germline sequences (Zerrahn et al.,

1997). More convincing data to explain MHC restriction comes from study of

45

residues in the MHC molecule itself. When all known structures are compared,

several MHC residues are consistently observed to make important contacts, for

example residues a65 and a155 of MHC class I molecules. These residues were also

recently shown to be 2 of only 3 residues involved in binding to TCR in a complex

with a highly bulged peptide which severely restricted possible TCR/MHC

interactions (Tynan et al., 2005). The authors of this study proposed binding via these

residues to comprise the minimal "generic footprint" of MHC restriction, but in the

absence of many more conserved TCR and MHC contacts other mechanisms that drive

the docking of the TCR with a pMHC complex have been proposed. To reconcile the

need for a TCR to scan many different pMHC complexes on the surface of an APC but

then to initiate signalling and T cell activation only when specific binding is achieved,

a two step mechanism of TCR binding to pMHC has been put forward (Wu et al.,

2002). This study, using a mutagenesis approach and measurement of TCR/pMHC

binding interactions using surface plasmon resonance, demonstrated that mutation of

MHC contact residues had a profound effect on initial association of the TCR with

pMHC but that these residues contributed very little to the overall stability of the

interaction. Conversely, mutations of peptide contacts profoundly influenced the

stability of the complex, affecting off rates of binding, but made only a minor

contribution to complex association. The model therefore proposes that initial

interactions between the TCR and MHC guide and position the receptor over the

peptide ligand and that the TCR, most likely using the CDR3 loops, then forms a more

stable interaction with its ligand which, if of sufficiently high affinity, will allow

transmission of a positive signal and activation of the T cell.

1.10 TCR binding affinity and conformational change

Interactions between TCRs and pMHC are usually characterised as being of relatively

low affinity with slow association rates and faster dissociation rates (Matsui et al.,

1994). That the low affinity of this interaction may be due to insufficient contacts

between the TCR and the pMHC is not supported by extensive interactions revealed

by structural studies. Instead, the demonstration that the kinetics of binding becomes

46

faster with increased temperature (Boniface et al., 1999) led to an induced fit model of

TCR binding where productive binding required the TCR to overcome a large energy

barrier. Calculation of binding energies showed that binding was associated with

unfavourable changes in entropy (Willcox et al., 1999), which suggests an increase in

molecule ordering. This could be brought about by conformational changes, and as a

consequence reduced flexibility, of a part of the TCR/pMHC complex itself, or by the

incorporation of ordered water molecules at the binding interface. As water molecules

are excluded from the TCR/pMHC interface upon binding this does not seem a likely

explanation for unfavourable entropy. Instead, structural studies support a model in

which parts of the TCR, particularly the CDR3 loops, undergo a conformational

change on binding, such that a greater number of contacts are made with the peptide

and stability of the complex is increased (Garcia et al., 1998 and Reiser et al., 2002).

However, a more recent analysis of the well studied LC13 TCR, specific for an

Epstein-Barr virus (EBV) peptide, showed that not all TCR/pMHC interactions

conform to this thermodynamic model of binding (Ely et al., 2006). In this study,

CDR loops were found to be well ordered in the unliganded state suggesting that in

this case, although a conformational change did occur on binding, binding was

entropically favoured rather than disfavoured. Another study with the same TCR

molecule used mutagenesis and surface plasmon resonance to examine the

contribution of binding energy from the CDR1, CDR2 and CDR3 loops of this

receptor. Contrary to the two-step mechanism of binding described above, the CDR1

and CDR2 loops were found to contribute only minimally to ligand binding with

stabilization of the complex mediated mainly by the CDR3 loops (Borg et al., 2005).

Despite increasingly apparent differences in the binding mechanisms employed by

different TCR/pMHC complexes, the important contribution made by the CDR3 loops

to binding affinity and specificity, but also degeneracy, is clear. That the flexibility of

the CDR3 loops, and therefore their ability to undergo conformational change on

ligand binding, could explain how a single receptor is able cross react with multiple

peptides, has been demonstrated using structural studies where the structure of a single

TCR was solved in complex with different peptides bound to the same MHC molecule

(Reiser et al., 2003). CDR3 loops were found to adopt very different conformations

47

depending on the ligand bound, although changes in other parts of the molecule were

also observed. Indeed, specificity of binding has recently been shown to be influenced

by residues of the pMHC molecule that do not make any direct contacts with the TCR

molecule at all (Huseby et al., 2006).

Despite the ability of many TCRs to bind multiple ligands, the affinity of this binding

is invariably not the same. That this is because the TCR molecule is flexible only up

to a point was addressed in a recent study using a single TCR recognising 3

structurally distinct peptides (Mazza et al., 2007). Although overall binding

orientations in the 3 structures were comparable, different peptide structures forced

some adaptation of the TCR molecule which, when combined with the retention of

many TCR/MHCa helix contacts, resulted in suboptimal and lower affinity binding

interactions with some peptides compared to others. Suboptimal binding was

associated with impaired effector outcomes of T cell activation, such as reduced

proliferation and IL-2 production. The observation that TCR/pMHC interactions of

different affinity can result in different functional outcomes for the T cell is a well

documented one (Sykulev et al., 1994, Matsui et al., 1994). Studies linking TCR

binding affinity to effector outcome have been used to investigate processes such as

thymocyte positive selection (Alam et al., 1996) and many different groups have

reported the ability to generate receptors with increased affinity for pMHC by

mutating residues of the TCR, most commonly in the CDR1, CDR2 or CDR3 regions

(Chlewicki et al., 2005 and Manning et al., 1999). However, as mentioned previously,

the way in which information about the affinity of the TCR binding interaction is

transmitted into the cell to initiate the TCR signalling machinery, and how different

biological outcomes might arise from the transmission of this information is still

poorly understood. Some of the different mechanisms that have been proposed to

explain the process of TCR triggering are discussed below.

48

1.11 TCR triggering

The two earliest models of TCR triggering were based on the concepts of

multimerization and conformational change. The multimerization model proposed

that specific pMHC binding by TCRs would result in clustering of pMHC/TCR

complexes at the cell surface, bringing intracellular signalling molecules into close

proximity with one another and allowing signalling to be initiated. Evidence was

provided by the demonstration that, in solution, TCR/pMHC complexes could

oligomerise upon ligand binding (Reich et al., 1997). However, these results were

subsequently disputed (Baker and Wiley, 2001) and, as this model requires the

simultaneous ligation of multiple TCR molecules, the observation that a T cell could

be activated by just a single pMHC could not be explained (Sykulev et al., 1996 and

Irvine et al., 2002). To resolve this problem variants of the multimerization model

have been proposed (Trautrnann and Randriamampita, 2003), such as the

heterodimerization model, where triggering is initiated by simultaneous pMHC

binding of the TCR and its CD4 or CD8 co-receptor (Delon et al., 1998), and the

pseudo-dimerisation model, where TCR oligomers form by engagement of both

specific peptide and self peptide MHC (Irvine et al., 2002). As for the

multimerization model however, some data remains unexplained by these

mechanisms. Mice that lack co-receptors are still able to mount effector T cell

responses (Schilham et al., 1993) and the same is true for T cells with greatly reduced

expression of self-peptide MHC (Sporri and e Sousa, 2002). Conformational change

has been another mechanism proposed to explain TCR triggering. Small scale

conformational changes that occur in the TCR molecule, particularly the CDR loops,

upon ligand binding have now been noted from comparisons of non-ligated and ligated

structures of several TCRs (Garcia et al., 1998 and Reiser et al., 2002). However,

with the exception of one study, where upon ligand binding a conformational change

in the TCRa constant domain was observed (Kjer-Nielsen et al., 2003), large

conformational changes in the extracellular domains or constant regions of the TCR

have not been seen and so argue against a mechanism of signal transduction by

conformational changes through the TCRaf3 heterodimer itself. In support of a

49

conformational change mechanism though, thermodynamic data of TCR binding does

find a relationship between flexibility at the TCR/pMHC interface and T cell

activation (Krogsgaard et al., 2003) and more recently attention has focused on the

idea that binding induces changes in the structural relationship between the TCRar3

heterodimer and CD3 components of the signalling complex. A study using a yeast

two-hybrid approach, to identify molecules that bind to the CD3 subunits of the

signalling complex during T cell activation, showed that an adaptor protein, Nck,

binds to a proline-rich sequence present in the cytoplasmic tail of CDR. This

sequence was only available for Nck binding following ligation of the TCR and was

proposed to be exposed as a consequence of a conformational change induced by

ligand binding (Gil et al., 2002). Studies subsequently showed that this

conformational change occurred in thymocytes upon presentation by MHC of both

positively and negatively selecting peptides (Gil et al., 2005) although more recently

conformational change was found to be more associated with thymocytes undergoing

negative selection than positive selection (Risueno et al., 2006) suggesting that rather

than being an all or nothing event, conformational change could be a mechanism by

which the T cell could transmit information about the strength of TCR/pMHC

interactions. It remains to be seen whether the cytoplasmic tails of other CD3 proteins

may show similar rearrangements upon TCR ligation, and the normal T cell

development observed in mice that lack this proline rich sequence of CDR has called

into question the real importance of this conformational change in the mechanism of

TCR triggering (Szymczak et al., 2005).

A third model of TCR triggering also gaining experimental support is the kinetic

segregation model (van der Merwe et al., 2000). This model hypothesises that, in a

resting cell, phosphorylation of signalling molecules is maintained at a low level by

tyrosine phosphatases such as CD45 and CD148 and signalling is therefore not

initiated. Upon ligation of the TCR, proteins at the cell surface passively segregate

according to size, with larger molecules such as CD45, CD148 and LFA-1 becoming

separated from the smaller proteins that comprise the TCR signalling complex. The

exclusion of bulky phosphatases from around TCR signalling molecules means that,

50

once phosphorylated, they are more likely to remain that way and, as TCR/pMHC

binding would be likely to prohibit the diffusion of signalling complexes away from

these small contact zones, a TCR signal can be potentiated. The advantage of this

model over those already discussed is that it is able to explain observations of ligand-

independent triggering, for example where CTLA-4 is able to inhibit T cell activation

in the absence of its ligand, B7 (Chikuma et al., 2005), as the presence or absence of

phosphatases around the signalling machinery is mediated by passive diffusion rather

than a specific ligation signal. In support of this kinetic segregation model both CD45

and CD148 are found to be excluded from sites of TCR triggering (Bunnell et al.,

2002 and Lin and Weiss, 2003). In the study involving CD148 (Lin and Weiss, 2003),

chimeric proteins were used to demonstrate that exclusion was mediated, in large part,

by presence of the bulky extracellular domain of the protein and that if the intracellular

phosphatase domain was targeted to the immune synapse by creating a fusion protein

with the adapter molecule LAT, signalling through the TCR could be inhibited.

Further evidence that the segregation of molecules is dependent upon the sizes of their

extracellular domains and that this process is important in TCR triggering was also

provided by a study in which the size of an MHC class I extracellular domain was

increased using immunoglobulin spacers. A larger MHC molecule was associated

with reduced TCR triggering with very little depletion of CD45 at the site of cell

contact, but no effect on ligation of the TCR with pMHC was observed (Choudhuri et

al., 2005). That protein segregation alone is sufficient to mediate TCR triggering has

yet to be demonstrated and it is possible that several of the proposed mechanisms

discussed here may act together in controlling this process. Indeed a recent study

examining the requirement of the CDR conformational change for activation has

reported that the conformational change induced in this subunit must be accompanied

by TCR/pMHC clustering in order to be productive (Minguet et al., 2007).

Whatever the answer to the question of how TCR triggering is initiated, a slightly

separate issue is one of how the TCR signalling complex interprets the variable

binding properties of different pMHC ligands in order to generate different effector

outcomes, whether that be positive versus negative thymic selection (Alam et al.,

51

1996) or other effector decisions such as CD4+ T cell differentiation (Lezzi et al.,

1999). Differences in phosphorylation states of TCR signalling molecules in response

to different ligands have been well documented (Sloan-Lancaster et al., 1994 and

Madrenas et al., 1995) but several hypotheses exist for how these different signalling

patterns may be achieved. The idea that conformational changes may occur in the

signalling complex to different extents depending on the nature of the TCR/pMHC

binding interaction has been mentioned above, but currently there is very little data to

support this idea. More widely accepted is the view that information about binding is

transmitted into the cell via serial triggering events (Lanzavecchia et al., 1999) and

that signalling is therefore dependent upon the half-life or "dwell time" of the

TCR/pMHC complex. This model proposes that a threshold number of TCRs must be

engaged for T cell activation and that each pMHC can serially engage multiple TCRs.

The shorter the half-life of a pMHC, the less efficient the triggering will be and the

longer it will take to reach the required threshold. Equally however, if the half-life of

a complex exceeds the time required to trigger the TCR, efficiency of signalling will

also be decreased as less serial engagements will occur within a given time period.

Consistent with this theory were the findings of a mutagenesis study where mutations

in the antigen binding site of a TCR were used to increase or decrease the half-life of

the TCR/pMHC complex (Kalergis et al., 2001). Mutations that resulted in decreased

and increased half-lives of TCR/pMHC complexes, as measured by binding to pMHC

tetramers, both resulted in impaired T cell activation. Additional evidence that

TCR/pMHC complexes with a short half-life have been associated with incomplete

phosphorylation of the CD3 chain also supports this hypothesis (Kersh et al., 1998)

highlighting the importance of binding kinetics and serial phosphorylation events in

TCR signalling. Some data however remains unexplained by this model, such as the

observation that the binding of TCR/pMHC complexes with very similar half-lives can

result in very different functional outcomes (Baker et al., 2000).

Although the preceding discussion has addressed the central role of the TCR and its

associated signalling molecules in T cell activation, in a normal biological setting, the

complex does not act alone and many other molecules and mechanisms interact to

52

elicit the final effector outcome for the T cell. T cell contact with an antigen

presenting cell (APC) can last for many hours (Stoll et al., 2002). During this time

TCR/pMHC complexes, but also other receptor/ligand pairs including co-stimulatory

molecules (e.g. CD28-B7) and adhesion molecules (e.g. LFA-1-ICAM-1) accumulate

and adopt a defined arrangement at the T cell-APC interface which is now referred to

as the "immune synapse" (Monks et al., 1998). Although formation of the immune

synapse is dependent upon signalling through the TCR, the myriad of other molecules

present at this interface serves to highlight the many other mechanisms that must be

considered when deciphering the pathway to a functional outcome of T cell activation.

This thesis will attempt to define the role that cq3 TCR structure has in influencing the

outcome of CD4+ T cell differentiation into T cells of different effector phenotypes.

The nature of the signal that a T cell receives through its TCR is known to have some

influence over this process, but many other factors and signalling pathways have also

been implicated. The process of CD4÷ T cell differentiation and the mechanisms that

may be involved in its control are discussed below.

1.12 CD4+ T cell phenotype

The two major lineages of cq3 TCR bearing T cells that emerge from the thymus are

classified according to their expression of co-receptors. Cells bearing the co-receptor

CD8 are termed CD8+, but when activated are also referred to as cytotoxic T cells due

to their primary function in killing infected target cells (Blanchard et al., 1987). CD8+

T cells will not be discussed further here, but instead attention focussed on cells

bearing the co-receptor CD4, termed CD4+ T cells. CD4+ T cells are also known as T

helper (Th) cells and have been shown to play a role in many different types of

immune response, mainly through their ability to co-ordinate the actions of other cell

types into a response appropriate to the initial insult or infection. This functional

diversity of CD4+ T cells was partly explained by the findings of Mosmann et al. that

mouse CD4+ T cell clones could be classified into distinct populations (named Thl

and Th2) on the basis of their patterns of cytokine production. It was subsequently

53

found that these different cytokine patterns could be linked to the different functional

properties of these cells (Mosmann and Coffman, 1989). Thl cells produce mainly

IL-2 and IFN-y, are involved in cell mediated inflammatory reactions such as those in

response to bacterial infections (Andersen et al., 1995) and have been implicated in a

variety of autoimmune diseases (Trembleau et al., 1995). Th2 cells can produce IL-4,

IL-5, IL-9, IL-10 and IL-13 and are involved in expulsion of extracellular parasites

(Bancroft et al., 1994) as well as strong antibody reactions and allergy (Robinson et

al., 1992). This fundamental role of Thl and Th2 cells in adaptive immunity, and the

finding that populations of these highly polarised cells can be identified in a variety of

disease states means that there is much interest in understanding the mechanisms

involved in their generation. The many factors now known to be involved in Thl

versus Th2 differentiation, including the hypothesis that the structure of the ap TCR

may be important, are discussed in the section below. However, since the original

classification of CD4+ T cells into one or other of these subsets, other populations of

CD4+ T cells that are distinct from Thl or Th2 have also been identified.

Aside from Thl and Th2 effector cells, the CD4+ T cell subset on which the most

attention has been focused in recent years is the regulatory CD4+ T cell (Treg). Most

commonly, Tregs are now defined by the expression of CD4 and CD25 at the cell

surface and by their ability to downregulate the immune responses mediated by other

T cell subsets. Depletion of lymph node cells and splenocytes of CD4+CD25+ T cells,

and then transfer of the remaining cells into nude BALB/c mice, results in the

development of spontaneous autoimmune pathologies (Sakaguichi et al., 1995).

However, the mode of action of T„g cells remains ill defined. Cytokines that have

been implicated in the suppressive mechanisms of CD4+CD25+ T cells include IL-10

and transforming growth factor p (TGF(3). CD4+CD25+ T cells have been shown to

produce IL-10 in vivo (Annacker et al., 2001) and IL-10 production by these cells

mediates suppression of some forms of autoimmunity, although others are IL-10

independent (Surf-Payer and Cantor, 2001). For TGFP there is little evidence to

suggest that CD4+CD25+ T cells mediate suppression of CD4+ T cells via this

cytokine, but TGFP mediated suppression of CD8+ T cells has been documented

54

(Chen M et al., 2005). Other proposed modes of action are based on mechanisms of

direct cell-cell contact as T cells deficient in the co-stimulatory molecules CD80 and

CD86 are resistant to suppression (Paust et al., 2004). That regulatory T cells may

develop during the process of thymic selection has already been mentioned and central

to this process is TCR signalling. Mice deficient in two downstream molecules of

TCR signalling, Bc1-10 and PKC-O have reduced numbers of Treg cells (Schmidt-

Supprian et al., 2004) and understanding the signalling mechanisms and genetics of

Treg lineage commitment was greatly advanced by the identification of the supposed

CD4+CD25+ T cell specific transcription factor, Foxp3 (Hori et al., 2003 and Fontenot

et al., 2005). A recent study in which the Cre recombinase system of gene targeting

(discussed later) was used to ablate Foxp3 expression specifically in Treg cells resulted

in an altered cytokine expression profile towards Thl cytokines such as IL-2 and IFN-

y, an altered pattern of gene expression where genes known to be controlled by Foxp3

such as Socs2 and Ctla-4 were downregulated, and a loss of suppressor function

(Williams and Rudensky, 2007). Many gene targets of the Foxp3 transcription factor

are now being identified and the recent identification of other transcription factors that

cooperate with Foxp3 (Wu et al., 2006 and Ono et al., 2007) should enable rapid

identification of the patterns and sequences of gene expression that are required to

bestow regulatory cell phenotype and function.

Another CD4+ T cell lineage specific transcription factor recently identified is RORyt,

reported to be expressed by the newly described Th17 subset of CD4+ T cells (Ivanov

et al., 2006) that have been associated with inflammatory responses such as those seen

in autoimmunity (Langrish et al., 2005). Th17 cells were identified as a subset of T

cells whose differentiation could be induced by the cytokine IL-23 (Aggarwal et al.,

2003) and it is known that mice deficient in IL-23 are resistant to several autoimmune

diseases, such as experimental autoimmune encephalomyelitis (EAE) (Cua et al.,

2003). This link with IL-17 producing cells and the demonstration that

proteolipoprotein (PLP) specific, IL-17 producing T cells could induce EAE when

transferred into naive mice while classical Thl cells could not (Langrish et al., 2005)

helped to explain why previous data regarding the pathogenicity of Thl responses was

55

confusing. Data showing that ablation of Thl responses can in fact worsen

autoimmune disease (Ferber et al., 1996) can now be reconciled with data showing a

reciprocal relationship between Thl, Th2, Th17 and indeed Treg cells where cytokines

promoting the development of one subset act to prevent the development of others

(Park et al., 2005 and Bettelli et al., 2006). This concept of antagonistic development

of one subset by another will be discussed further in the section below in relation to

Thl and Th2 cell differentiation, but it is becoming ever clearer that the original

classification of CD4+ T cells into either Thl or Th2 is no longer by any means the

whole picture and it is likely that in the future, important roles of other subsets of

CD4+ T cells, in addition to the ones discussed here, will be elucidated.

1.13 Thl and Th2 cell differentiation

One of the most important and well described factors influencing CD4+ T cell

differentiation is local cytokine environment. Among the most well characterised Thl

promoting cytokines is IL-12. IL-12 is a heterodimer composed of p40 and p35

subunits, and is produced primarily by antigen presenting cells, including

macrophages, monocytes and dendritic cells (Macatonia et al., 1995). Mice deficient

in IL-12 are impaired, although not completely lacking, in their ability to produce

IFN-y and mount an effective Thl response (Magram et al., 1996) and exogenous

addition of this cytokine is now used extensively in vitro as a means to generate a Thl

phenotype in primary T cells. IL-12 acts via the signal transducer and activator of

transcription 4 (STAT4), following binding to the IL-12 receptor on the surface of the

T cell and, primarily, activation of this signalling pathway leads to the upregulation of

IFN-y expression (Thierfelder et al., 1996). The residual IFN-y production in IL-12

deficient mice also points towards the importance of other cytokines in the

development of Thl responses and IFN-y itself, as well as other cytokines such as IL-

18, IL-27 and IFN-a have all been implicated (Schmitt et al., 1994, Kohno et al.,

1997 and Hibbert et al., 2003). Important in the understanding of Thl phenotype

development was the discovery of the Thl lineage specific transcription factor, Tbet.

T-bet is a member of the T-box family of transcription factors and the antagonistic

56

nature of Thl versus Th2 differentiation is exemplified by the demonstration that IFN-

y production can be induced, and IL-4 and IL-5 production repressed, by the

introduction of Tbet into T cells that were defined as having a Th2 phenotype (Szabo

et al., 2000). In addition, mice lacking Tbet spontaneously develop a phenotype

similar to that seen in human asthma, a predominantly Th2 mediated disease (Finotto

et al., 2002). Lineage specific transcription factors identified in Th2 cells include

GATA-3 and c-maf (Zheng and Flavell, 1997 and Ho et al., 1996) and as observed for

T-bet, ectopic expression of GATA-3 can induce Th2 cytokine production in

committed Thl cells (Lee et al., 2000). GATA-3 expression is induced in Th2

differentiating cells by the transcription factor STAT6, which is activated by T cell

binding of the main Th2 promoting cytokine, IL-4. Additionally very recent data

suggests that GATA-3 expression is additionally regulated by Notch signals (Fang et

al., 2007). T cells from IL-4, STAT6 and GATA-3 knockout mice are all greatly

impaired in their ability to undergo differentiation into Th2 cells (Kopf et al., 1993,

Shimoda et al., 1996 and Pai et al., 2004). Less is known about the regulation of the

c-maf transcription factor, but more recently signalling via IL-6 and the TCR were

both implicated (Yang et al., 2005). A schematic diagram of the main cytokines and

transcription factors involved in the differentiation of Thl and Th2 cells is shown in

figure 1.4. As well as the action of Thl and Th2 associated transcription factors in

directly promoting the expression of some cytokines and repression of others,

epigenetic mechanisms involving chromatin remodelling and locus accessibility have

been proposed to explain the fixed phenotype of fully differentiated T cells and the

heritability of phenotype as cells proliferate and clonally expand during the course of

an immune response. In support of this, additional DNase I hypersensitivity sites

(HS), indicators of increased locus accessibility, appear at the 114 locus and Ifng locus

in Th2 and Thl differentiating cells respectively (Agarwal and Rao, 1998) and

changes in acetylation and methylation, other markers of chromatin remodelling, have

also been observed (Avni et al., 2002 and Lee et al., 2002). There is evidence that

lineage specific transcription factors are involved in mediating these epigenetic

changes, with ectopic expression of GATA-3 able to mediate chromatin remodelling at

the 114 locus, even in Thl cells (Lee et al., 2000). However, a direct role for T-bet in

57

Naïve CD4f T cell

Thl CD4+ T cell Th2 CD4+ T cell

Figure 1.4 Cytokine environment and CD4+ T cell differentiation. Cytokines IL-12 and IL-4 promote Thl and Th2 cell differentiation respectively. Intracellular signalling pathways activated by TCR and cytokine receptor ligation result in activation of the Stat protein and lineage specific transcription factors Tbet, GATA3 and cMaf. These transcription factors drive the expression of genes for Thl and Th2 CD4+ T cell differentiation, including the cytokines IFN-y and IL-4 which act to potentiate the development of Thl and Th2 cell lineages respectively.

58

modifications at the Ifng locus has recently been questioned with evidence suggesting

that previously observed remodelling events at this locus maybe an indirect

consequence of GATA-3 downregulation by the Thl transcription factor (Usui et al.,

2006).

With the clear importance of cytokines in the process of CD4+ T cell differentiation, it

follows that APCs, as the primary source of these cytokines during the initiation phase

of an immune response, should also have an important role to play. The types of

cytokines that an APC produces are greatly dependent upon the type of pathogen or

stimulus that it meets. For example, dendritic cells mature into Th2 promoting

effectors (DC2s) in the presence of protein extract from the parasite Schistosoma

mansoni, but Thl promoting effectors (DC1s) result from exposure to toxins from

intracellular bacteria (de Jong et al., 2002). Differential activation of Toll like

receptors (TLRs) is proposed as one mechanism by which these different APC

phenotypes arise. In one study, using an OVA based disease induction protocol, TLR2

ligation was associated with promotion of a Th2 type immune response, but TLR9

ligation with a more Thl type response (Redecke et al., 2004) and dendritic cells

isolated from BALB/c mice infected with Chlarnydia show increased expression of

TLR9 and can inhibit in vivo allergic responses to OVA when adoptively transferred

(Han et al., 2004). However, understanding the possible effects of TLR ligation on

differentiation are complicated by the presence of TLRs on T cells as well as APCs

and a recent study has linked direct ligation of TLR2 on T cells to activation of Thl

effector functions in already differentiated T cells (Imanishi et al., 2007). Also to be

considered is the timing, cytokine environment and location of APC activation.

Highlighting the temporal and historic importance of APC activation, one study

showed that mice infected with Influenza virus 30 days before exposure to allergen

developed more severe allergic disease than control animals (Dahl et al., 2004). The

cytokine environment of the APC itself at the time of activation was also shown to

have an influence on T cell differentiation in a murine model of Leishmania infection.

BALB/c mice are normally susceptible to this pathogen as a result of a dominant Th2

immune response. When IL-4 was present in vivo during initial activation of DCs,

59

rather than during T cell priming, mice developed a Thl response and were resistant to

disease (Biedermann et al., 2001). Other evidence suggests that the site at which the

APC encounters antigen is also important and several studies have shown that antigen

encountered by APC at mucosal sites, such as the lung and the intestine, preferentially

induce Th2 cell differentiation, even in mouse strains that are strongly biased towards

the development of Thl CD4 T cells (Constant et al., 2000 and Iwasaki and Kelsall,

1999). That some mouse strains, such as C57BL/6 and BALB/c are biased towards

developing Thl and Th2 responses respectively suggests an important genetic

component. Studies in this area have mainly focussed on MHC genotype (Murray et

al., 1992), although a locus on murine chromosome 11 has also been implicated

(Gorham et al., 1996).

As previously mentioned, the immune synapse that develops at the site of T cell-APC

contact during activation contains many more proteins than just those of the

TCR/pMHC complex. Co-stimulation is an important component of T cell activation

and acts not only to deliver a second signal to the T cell on top of the signal received

through the TCR, but also to increase the avidity (strength) of the T cell-APC

interaction. The two main co-stimulatory molecules upregulated on APCs following

encounter with antigen are B7-1 (CD80) and B7-2 (CD86) (Reiser et al., 1992 and

Freeman et al., 1993). CD86 has been shown to preferentially induce IL-4 production

(Freeman et al., 1995) and distinct regions in the cytoplasmic domain of CD28, the

receptor for CD80 and CD86 on the surface of the T cell, that are required for Th2

differentiation have now been identified (Andres et al., 2004). There is also evidence

that the co-stimulatory molecule OX40 as well as the CD4 molecule itself can play a

supportive role in Th2 development (Ohshima et al., 1998 and Fowell et al., 1997).

With the large array of other proteins on the surface of, and secreted by, both T cells

and APCs, other molecules proposed to influence CD4+ T cell differentiation are

continually being identified (Oosterwijk et al., 2007 and Lund et al., 2007 and Fang et

al., 2007), but the challenge will be how to fit these into an already complex picture of

decision making at the time of T cell priming.

60

1.14 The TCR and CD4+ T cell differentiation

Despite the importance of co-stimulation, key to the overall avidity of a T cell-APC

interaction is the binding of TCR to its pMHC ligand, and there are now multiple lines

of evidence pointing towards a role for both avidity and affinity of TCR/pMHC

interactions in influencing CD4+ T cell differentiation. Indeed, some recent data

tentatively suggests that for some TCR/pMHC interactions, TCR mediated signals

alone, in the absence of co-stimulation, or Thl promoting cytokines and even T-bet,

are sufficient to drive development of Thl cells (Ariga et al., 2007). That the strength

of the signal received through the TCR can influence T cell effector outcome was

supported by data on the duration of TCR signalling, where prolonged stimulation of

naïve CD4+ T cells in vitro with pMHC and an anti-CD28 antibody was required to

induce a Th2 phenotype (Lezzi et al., 1999). It can be imagined that the most simple

way in which TCR/pMHC interactions could influence avidity of T cell-APC binding

would be the number of binding interactions occurring at any given time, with a

greater number of binding events increasing binding avidity. In support of this, both

peptide dose and the density of MHC molecules on the surface of an APC have been

shown to be important (DiMolfetto et al., 1998). Data on antigen dose are slightly

conflicting and depend upon the antigen used in the study but, in general, very high

and low doses of antigen elicit Th2 responses, while moderate and high antigen doses

favour Thl responses (Constant et al., 1995 and Hosken et al., 1995). Alternatively,

strength of TCR signalling could be influenced by the kinetics of binding and affinity

for pMHC of each individual TCR molecule, i.e. at the level of structural interactions

between the TCR and its pMHC ligand. Early evidence that this may be true came

from studies using altered (mutated) peptide ligands (APLs). It was observed that

amino acid substitutions in peptide ligands, usually by as little as just a single residue,

could shift the effector cell phenotype of CD4+ T cells responding to this antigen from

Thl to Th2 (Pfeiffer et al., 1995 and Tao et al., 1997). That these switches in T cell

phenotype can alter disease outcome has also been seen in several studies involving

the Thl mediated murine autoimmune disease, EAE (Brocke et al., 1996 and

Nicholson et al., 1995). In the study by Nicholson et al. SJL mice, normally

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susceptible to EAE induction using a peptide of PLP (PLP 139-151), were prevented

from developing, or displayed greatly reduced severity of disease when immunised

with an altered peptide ligand (Q144). Disease could be prevented by immunising

with APL alone or in combination with the wild-type peptide and the adoptive transfer

of T cell lines specific for APL Q144 could also confer protection. T cells isolated

from mice immunised with Q144 were found to secrete the Th2 type cytokines IL-4

and IL-10 and were able to proliferate in response to both Q144 and the native peptide.

This suggested that T cells were able generate productive TCR signals in response to

the mutated peptide and that Q144 was not simply acting as a competitive MHC

blocker inhibiting productive TCR/pMHC interactions. The mutated residue of this

APL was known to be the primary contact residue in TCR binding and a subsequent

study revealed that the primary contact points of APL Q144 had shifted to residues

141 and 142, towards the N terminal end of the peptide (Das et al., 1997). Others had

demonstrated APLs, described as antagonistic in these studies, to have reduced affinity

for TCR (Lyons et al., 1996) and the generation of altered affinity TCR/pMHC

interactions by mutating residues in the TCR itself has already been discussed. A link

between mutations in TCR structure and CD4+ T cell differentiation came with the

demonstration that a change in a single amino acid in the CDR2 region of a TCR could

shift the immune response from Thl to Th2 (Blander et al., 2000). Taken together, all

of this data leads to a hypothesis which suggests that the structure of a TCR and, as a

consequence, the binding interaction between TCR and pMHC can bias CD4+ T cell

differentiation, with high affinity interactions promoting Thl development and lower

affinity interactions favouring a Th2 response.

However, despite this data, all of which involves manipulation of TCR interactions

artificially in vitro, what is the relevance in vivo to any role that TCR structure may

have in determining the CD4+ T cell phenotype of populations of T cells with a normal

and diverse repertoire of TCRs? The finding that TCRs with relatively high affinity

can be selected and come to dominate the repertoire during the course of an immune

response has been observed both in vivo and in vitro. In an in vitro study, PCR

mutagenesis was used to mutate the CDR3a regions of TCRs and through successive

62

rounds of in vitro selection using a yeast expression system, high affinity mutants were

found to become dominant (Holler et al., 2000). In vivo, pMHC multimers have been

used to demonstrate expansion of both MHC class I and MHC class II restricted, high

affinity TCRs, during development of an immune response to pathogen in mouse

models of infection (Busch and Pamer, 1999 and Malherbe et al., 2000) and high

affinity TCRs, sequences of which are conserved between patients, have also been

seen in studies assessing T cell clonal dominance to human cytomegalovirus (HCMV)

(Trautmann et al., 2005). In the study by Malherbe et al. looking at expansion of

MHC class II restricted CD4+ T cells in mice during the course of infection with the

Leishmania major parasite, T cell populations expanding in a resistant strain of mice

(D10.D2), which develop a Thl type immune response to the pathogen, were found to

bind pMHC with higher affinity and have a different pattern of Va and CDR3a usage

to T cell populations expanding in the susceptible BALB/c strain of mouse, which

develop a predominantly Th2 type response. Other studies have also observed

selection of preferred CDR3 motifs during immune responses (McHeyzer-Williams et

al., 1999). In this study, CDR3 sequences from T cells present at time points

throughout the course of an immune response to whole pigeon cytochrome c (PCC)

protein were analysed. By day 3, 40% of cells analysed were using CDR3 sequences

containing at least 6 of 8 conserved features identified, for example a CDR3a length

of 8 amino acids or a serine residue at position a95, and this rose to more than 80%

after 2 weeks. In a subsequent memory response to PCC, 96% of T cells were using a

restricted TCR. As, unlike B cells, T cells do not undergo affinity maturation

(mutation of CDR amino acids to generate higher affinity ligand binding), the

dominant sequences must have been selected from the original starting repertoire of

pMHC specific TCRs. Combined, this data led to the idea that not only could

preferred structural motifs be selected for during an immune response, but that the

structural features selected may bear some relation to the phenotype of the cells

responding, i.e. structural features of TCR selected during a Thl immune response

may be different from those selected during a Th2 immune response. This question

was addressed by a study in our laboratory, published in 2002, which determined the

TCR gene usage, CDR3a and CDR313 sequences from T cell clones and lines selected

63

under Thl versus Th2 polarising conditions in vitro (Boyton et al., 2002). This study

found that CD4+ T cell clones selected under Th2 polarising conditions favoured an

elongated TCR CDR3a chain and from modelling studies predicted that these

elongated TCR a chains may result in less optimal (lower affinity) interactions

between the pMHC complex, which in this case was a PLP peptide (PLP 56-70)

complexed to the MHC class II molecule I-Ag7, and CDR loops of the TCR. No clear

V gene bias between Thl and Th2 cell derived TCRs was observed and there were no

differences in the lengths of CDR3(3 regions. In short term Thl and Th2 polarised T

cell lines, dominant Thl and Th2 TCR CDR3a sequences could be identified, and

when oligonucleotides of these CDR3a sequences were radiolabelled and used to

probe cDNA libraries of CDR3a sequences, it was found that the longer Th2 derived

CDR3a motif was present only in Th2 lines while the shorter Thl CDR3a motif

seemed to be permissible in both, although was more dominant in the Thl lines.

The hypothesis that emerged from this study was that, if structurally distinct TCRs

arising from Thl and Th2 polarised T cells, i.e. a TCR bearing an elongated CDR3a

region from a Th2 cell and a shorter CDR3a region from a Thl cell, are expressed

transgenically, an inherent bias towards Thl or Th2 will be observed in CD4+ T cell

responses from these animals. Chapters 3 and 4 of this thesis describe the generation

and characterisation of such transgenic animals. Further to this hypothesis is a second

question of whether these Thl and Th2 transgenic animals could be used to create new

models of Thl and Th2 mediated disease. The lung is one organ in which

understanding Thl and Th2 type responses is of much interest. The roles played by

Thl and Th2 CD4+ T cells in human lung diseases are discussed below, and chapter 5

of this thesis describes the generation of a new transgenic model that could be of use

in the modelling of these diseases in the mouse.

1.15 Thl and Th2 responses in human lung disease

CD4+ T cells in the lung are an area of major interest, as a number of lung diseases are

believed to be associated with a bias for either Thl or Th2 type responses. Pulmonary

64

sarcoidosis is an example of a chronic lung disease that has been associated with a

predominantly Thl type immune response (Moller et al., 1996) while asthma,

bronchopulmonary aspergillosis and cryptogenic fibrosing alveolitis are among other

diseases thought to be predominantly Th2 mediated (Robinson et al., 1992, Kurup et

al., 1994 and Wallace et al., 1995). In most cases the underlying cause, or nature of

the initiating insult to the lung, is not well understood and other major challenges still

remaining include a good understanding of the regulation of lung inflammation, and

the long term processes of repair and lung remodelling that occur in the context of

prolonged, chronic, inflammatory responses.

Allergic asthma is probably the best studied lung disease thought to be associated with

Th2 type responses. Many different allergens have been associated with asthma, but

among the most common and well studied include proteins from the house dust mite,

grass pollen and cat dander. Allergic asthma is characterised by airway

hyperreactivity, which for the most part is reversible, high levels of IgE antibodies and

airway inflammation. The main cell types found in the asthmatic lung are CD4+ T

cells, eosinophils, mast cells, basophils and neutrophils, and cytokines found to be

elevated in the lungs of asthmatics are predominantly of the Th2 type (Robinson et al.,

1992). Since the discovery of a role for CD4+ T cells and Th2 cytokine production in

allergy, much effort has gone into understanding the mechanisms by which they act to

cause disease. A major function of IL-4 and IL-13 is to mediate IgE antibody class

switching by B cells (Lebman and Coffman, 1988 and Punnonen et al., 1993). These

cytokines function though activation of the transcription factor STAT6, which can

synergise with the transcription factor NF-KB to promote germline transcription of

IgE. The process of IgE transcription is also mediated by signalling through CD40

(Kawabe et al., 1994), the ligand for which is transiently expressed on T cells

following antigen stimulation, and polymorphisms in the IL-4, IL-13 and CD40 genes

have all been associated with high IgE levels in patients with asthma (Suzuki et al.,

2000, Hunninghake et al., 2007, Park et al., 2007). IgE class switching is a process

mediated by a germline recombination event, similar to those described above for

TCR gene rearrangement, although it is RAG independent (Lansford et al., 1998). It

65

is proposed to occur predominantly in lymphoid tissue (Muramatsu et al., 1999)

although active class switching in the bronchial mucosa of asthmatics has also been

demonstrated (Takhar et al., 2007). IgE acts by binding to high affinity receptors

(FCERI) on the surface of mast cells and basophils, and crosslinking of this IgE by

specific antigens leads to the release of inflammatory mediators such as histamine,

leukotrienes, enzymes and cytokines (Segal et al., 1977). Mast cell and basophil

degranulation, and the release of these inflammatory mediators by specific IgE

crosslinking in the lungs of asthmatics upon exposure to allergen, is proposed to be the

initiating sequence of events in an asthmatic response. Consequences include mucus

secretion, smooth muscle contraction and the recruitment of other effector cells to the

lung, some of which contribute to the observed increase in airway

hyperresponsiveness (AHR) that is synonymous with asthma. In keeping with an

essential role for IgE in the early phase of an asthmatic response, administration of an

anti-IgE antibody to asthmatic patients has been shown to inhibit allergen induced

AHR (Fahy et al., 1997), although mouse models of asthma have demonstrated

development of AHR and eosinophilia in the absence of IgE (Mehlhop et al., 1997).

As such, the exact nature of the mechanisms behind the development of AHR are not

yet fully understood and the roles played by inflammatory cells, such as the

eosinophil, are still controversial. Recruitment of eosinophils to the lung is mediated

by chemokines such as eotaxin (Ganzalo et al., 1996), and cytokines such as IL-5.

Mice deficient in IL-5 fail to develop lung eosinophilia and AHR (Foster et al., 1996),

and although this may be interpreted as evidence for a role for IL-5 and eosinophils in

AHR development this has been complicated by the subsequent demonstration that

this effect was dependent upon the genetic background of the IL-5-/- mice (Hogan et

al., 1998). The key roles of the prototypic Th2 cytokines, IL-4, IL-5, and IL-13

discussed so far in the context of asthma serves to highlight the important role of Th2

type, allergen specific, CD4+ T cells in the pathogenesis of this disease, and with

evidence that Thl cytokines are antagonistic towards the development of T cells with

a Th2 phenotype (Wynn et al., 1995), the prospect that promotion of a Thl type

environment may ameliorate Th2 mediated disease in the lung has provoked much

interest. However, results have been conflicting and while some groups have

66

demonstrated reduced airway inflammation and AHR upon transfer of antigen specific

Thl T cells in established mouse models of asthma (Huang et al., 2001), others have

not, but have instead seen enhanced inflammatory cell recruitment to the lung (Hansen

et al., 1999). In a recent study where IFN-y was overexpressed in the mouse lung,

levels of IL-5 and IL-13 were found to be increased upon induction of allergic airway

inflammation (Koch et al., 2006). Some studies have reported increased number of T

cells expressing IFN-y in the lungs of patients with asthma (Krug et al., 1996) and

viral infections, immune responses to which are predominantly Thl mediated, have

been associated with an increased risk of developing this disease (Sigurs et al., 2005).

In the Thl associated lung disease of pulmonary sarcoidosis, although the pathology

of the disease is well characterised in terms of the presence of granulomas,

accumulation of activated CD4+ T lymphocytes and macrophages and production of

Thl type cytokines (Moller et al., 1996), the antigen(s) to which the immune system is

responding is not yet known. The presence of activated CD4+ T cells at the sites of

disease has led to much research to try to identify the antigens for which these T cells

are specific but, although some studies have reported some V gene bias in the TCRs of

these lymphocytes, with bias being linked to MHC class II genotype (Grunewald et

al., 2002), the activating peptides remain elusive. Some studies have pointed towards

mycobacterial antigens as possible targets of T cell responses in sarcoidosis (Song et

al., 2005 and Hajizadeh et al., 2007), although attempts to identify mycobacterium

tuberculosis in sarcoid biopsies have been unsuccessful (Marcoval et al., 2005) and the

possibility that Thl T cells in sarcoidosis may be responding to endogenous rather

than bacterial antigens still remains. Additionally, the finding that patients with

sarcoidosis are deficient in, or have greatly reduced numbers of, a recently described

subset of CD 1 d-restricted NKT cells, which have immunoregulatory functions, has

also led to the suggestion that defects in immune regulation may play a role in the

development of this disease (Ho et al., 2005).

Despite the Thl type nature of immune responses during development of sarcoidosis,

severe fibrosis is a common end point (Moller, 2003). Fibrosis is more commonly

67

associated with Th2 type responses, with cytokines IL-4, IL-5 and IL-13 all implicated

in pathogenesis and in a common mouse model of fibrosis, promotion of Thl type

immunity using IL-12 was seen to markedly reduce fibrotic disease development

(Wynn et al., 1995). How the Thl environment of sarcoidosis may then lead to

fibrosis is therefore unclear, although other pro-fibrotic mediators such as TGF-p are

found to be expressed in the sarcoidosis lung (Zissel et al., 1996) and more recently

polymorphisms in the gene for TGF-133 have been implicated in progression to fibrosis

in sarcoid patients (Kruit et al., 2006). Fibrosis is a mechanism of tissue repair and

occurs when connective tissue grows in place of normal parenchymal tissue. If limited

to small areas of an organ, fibrosis is beneficial, as it can reverse tissue damage.

However, if the process of fibrosis becomes continuous and progressive, organ

function, particularly of those such as the lung, where tissue architecture is delicate,

can become severely compromised and can ultimately cause organ failure.

A common fibrotic lung disease in humans is cryptogenic fibrosing alveolitis, one of a

group of interstitial lung diseases that are characterised by lung inflammation and

fibrosis. Lung infiltrates include activated T cells, macrophages, B cells and, in

keeping with the Th2 nature of the disease, eosinophils and a Th2 like pattern of

cytokines (Haslam et al., 1980 and Wallace et al., 1995). Similar to sarcoidosis, the

exact nature of the lung antigens that initiate the disease process is unknown, however,

the inflammatory pathways that lead to fibrosis are becoming better defined. Key

roles for the cytokines IL-13 and TGF-P have been proposed. Overexpression of both

of these cytokines in transgenic animals leads to severe fibrosis (Sime et al., 1997 and

Zhu et al., 1999), while neutralisation can attenuate disease (Belperio et al., 2002).

That this is not just a feature of all Th2 type cytokines was demonstrated by the lack of

airway fibrosis seen in transgenic mice that overexpressed IL-4 (Rankin et al., 1996).

Signalling pathways that mediate fibrosis and are initiated by these cytokines are still

being elucidated, but IL-13 has been proposed to act in both a TGF-p dependent (Lee

et al., 2001) and independent manner (Kaviratne et al., 2004). Signalling molecules

shown recently to be upregulated by IL-13 include the chemokine receptor CCR5 and

IL-11Ra and blocking the functions of both of these receptors ameliorated IL-13

68

induced inflammation and fibrosis (Ma et al., 2006 and Chen Q et al., 2005). The

Smad3 signalling pathway has been implicated in TGF-13 mediated pulmonary fibrosis,

with Smad3 mice shown to be resistant to the severe fibrosis induced by

overexpression of TGF-13 in the lung (Bonniaud et al., 2004) and more recently,

importance of the mitochondrial mediated pathway of apoptosis, involving the

apoptosis proteins Bax and Bid has also been suggested (Kang et al., 2007). This

demonstration of a link between TGF-I3 signalling and pathways of apoptosis provide

an explanation for the dependence on epithelial cell apoptosis previously observed for

TGF-13 induced fibrosis (Lee et al., 2004). Although fibrosis is the main pathology

observed in lung diseases such as cryptogenic fibrosing alveolitis, that it is observed as

a chronic component of other lung diseases, such as sarcoidosis, COPD and asthma,

serves to highlight the interrelated nature of the many inflammatory and repair

processes that can be initiated in the lung in response to a wide range of insults.

There is therefore still much to be learnt about the consequences of Thl and Th2 type

immune responses in the lung and it is becoming increasingly clear that, far from

being exclusively one type or the other, the pathogenesis of many lung diseases,

particularly those that are chronic in nature, probably involves interplay between many

different Thl and Th2 cytokine mediated mechanisms. A further level of complexity

now arises from the description of Treg cells and more recently Th17 cells, a subset of

CD4÷ T cells whose development can be antagonised by both Thl and Th2 type

cytokines, and there will undoubtedly be many examples in the coming months of how

these other T cell subsets may play a role in lung inflammation. IL-17 mRNA has

been reported to be elevated in the sputum of asthmatic patients (Bullens et al., 2006)

and in a study using Tbet /- mice discussed previously as having a spontaneous asthma

like phenotype, increased neutrophilia in the airways of these mice has now been

linked to a greater propensity of T cells from these mice to develop into IL-17

producing cells (Fujiwara et al., 2007). As a result, effective treatments for many of

these diseases are unlikely to be achieved by blocking a single pathway. However,

much of what is currently known about lung inflammation, and the apparent

dependence of some pathologies on single mediators, has come from mouse models

69

using manipulation of single entities using transgenesis or monoclonal antibodies, and

the induction of disease through conditions that may be far from physiologically

normal. Some of the mouse models commonly used in the study of human lung

disease are discussed below.

1.16 Mouse models of human lung disease

Many different mouse models now exist for the study of human lung disease. Some

mimic specific disease pathologies such as granuloma formation, while others have

achieved modelling of multiple disease outcomes, such as the AHR, lung

inflammation and the high IgE levels that result from antigen challenge in several

mouse models of asthma. The disease outcomes associated with the chronic Thl type

and IFN-y mediated lung inflammation of diseases such as sarcoidosis and

bronchiectasis have proved the hardest to model. Bronchiectasis is characterised by

irreversible airway dilation and is found in association with chronic bronchial

infection and inflammation. There are currently no good animal models of

bronchiectasis, and while bacterial lung infection can be easily induced in the mouse

(Cole et al., 2005), the irreversible changes in lung structure that occur in humans over

long periods of time have proved harder to mimic. No spontaneous models have been

described and our lack of understanding surrounding the processes involved in chronic

airway structural change makes it difficult to arrive at a starting point in trying to

establish an in vivo model. Likewise, there are currently no good models of

sarcoidosis, although some protocols to induce granuloma formation in the mouse

have been described. Early models investigated the use of human sarcoid tissue itself

as an inducing agent, with granuloma formation in mice observed following

inoculation with sarcoid tissue homogenate (Mitchell et al., 1976). However, results

were not consistent, with others reporting similar granulomas when control tissue

homogenates were used (Belcher and Reid, 1975) and this model as since fallen out of

favour. Other approaches include the intravenous injection of complete Freund' s

adjuvant in rats (Bergeron et al., 2001) and Schistosome mansoni or

Propionibacterium acnes infection in mice (Wynn et al., 1993 and Nishiwaki et al.,

70

2004). However, the mechanisms of granuloma formation in these models remain

poorly defined and indeed elucidation of mechanisms that are occurring in response to

antigens irrelevant to the human disease of sarcoidosis may be misleading. This may

be particularly true of Schistosome mansoni infection, where formation of granulomas

is seen to be accompanied by a Th2 type pattern of cytokine expression (Wynn et al.,

1993). However, in keeping with the Th2 nature of this disease model, S. mansoni

infection is also used as a model of fibrosis (Wynn et al., 1995). In the S.mansoni

model, fibrosis and granuloma formation occur in the liver. To model fibrosis in the

lung, the most common protocols involve the infliction of lung injury using toxins or

drugs. The antibiotic bleomycin is used most commonly (Adamson and Bowden,

1974) but the acute nature of the fibrotic disease caused by these agents raises doubts

over whether they are faithful mimics of the slower but progressive tissue remodelling

observed in diseases such as idiopathic pulmonary fibrosis. Another infection model

that has been used to cause fibrosis in the lung uses Aspergillus fumigatus (Blease et

al., 2002). Models using this pathogen are of particular interest as this fungus is

known to be the cause of allergic bronchopulmonary aspergillosis and is associated

with asthma. As such, many features of human asthma, such as AHR, eosinophilia

and high levels of IgE, can be modelled in mice by exposure to this pathogen (Kurup

et al., 1992).

Aside from the aspergillosis model, several different and well characterised murine

systems now exist for the study of the more acute phases of allergic lung immune

responses. The most commonly cited of these is the ovalbumin (OVA) model of

murine asthma (Kung et al., 1994). Protocols for disease induction using the OVA

model vary slightly, but the overall scheme of sensitisation to OVA by intraperitoneal

(i.p) injection followed by exposure of the airways to the allergen, either as an aerosol

or by intranasal administration, 2 or 3 weeks after the initial priming, is similar across

most studies. Strains of mouse commonly used are BALB/c and C57BL/6 and mice

sensitised and challenged with antigen demonstrate airway hyperresponsiveness, lung

infiltrates dominated by allergen specific CD4+ T cells and eosinophils, Th2 type

cytokines in the BAL and lung such as IL-4, IL-5 and IL-13, and high levels of

71

allergen specific serum IgE. The existence of transgenic mice that express a TCR

(D011.10), specific for a peptide of OVA (OVA323-339) (Murphy et al., 1990) has

made the use of this model even more widespread. These TCR transgenic mice have

facilitated the development of adoptive transfer models of lung inflammation where

lymphocytes from immunised TCR transgenic mice are cultured in vitro, often in Thi

or Th2 polarising conditions, before being transferred by intravenous injection to naïve

mice that are subsequently challenged with OVA (Randolph et al., 1999). In this way,

allergen specific T cells can be tracked in vivo and this approach has allowed for a

better understanding of the role of both Thl and Th2 CD4+ T cells in the development

of allergy. In the context of human asthma, and indeed murine allergy, as no natural

mouse allergens have been described, OVA is an irrelevant protein. The success of

the model in generating a Th2 type disease profile is mediated in part by the inclusion

of the Th2 promoting adjuvant, alum, in the priming stages of disease induction (Grun

and Maurer, 1989). In this way, the OVA model of asthma is non-ideal as it

predisposes the immune response towards a Th2 phenotype in a way that is

independent of allergen, which in the case of OVA is also physiologically irrelevant to

the human disease. The non-physiological nature of the immune response is also

highlighted by the large doses of protein, administered over a period of many days,

that are required to induce significant disease, compared to the relatively small doses

of antigen that human asthmatics are proposed to be exposed to (Arshad et al., 1998).

In order to more closely mimic human disease, other models that use human allergens

have been developed, for example, the house dust mite allergen, DerP1 (Lee et al.,

1999). However, disease induction protocols with DerP1 also require priming with

alum and high antigen doses. More complicated attempts to create humanised mouse

models of asthma have also used house dust mite allergy as their basis, with SCID

mice reconstituted with peripheral blood mononuclear cells (PBMCs) from house dust

mite allergic patients showing evidence of human cell inflammatory infiltrates and

specific human IgE responses following challenge with antigen (Duez et al., 1996).

Another drawback of original OVA and DerP1 disease induction protocols is that they

fail to mimic the progressive fibrotic and remodelling processes that are seen to occur

over the long term in the lungs of patients with more severe asthma. To try to model

72

these changes, existing protocols have been modified to include extended periods of

allergen challenge. Signs of airway remodelling, such as goblet cell hyperplasia and

collagen deposition, have been observed (McMillan and Lloyd, 2004) although similar

to criticisms of acute airway inflammation, in these models, the doses of antigen

required to elicit these pathologies in the murine lung are extremely high and unlikely

to be physiologically similar to the human disease.

In addition to the use of exogenous agents, such as pathogens, drugs or proteins,

transgenic mice have been used extensively, both in combination with the models so

far described, and alone, to decipher more specifically the roles of individual

molecules or pathways in the mechanisms and potential treatments of disease. For

example, most of the cytokines associated with CD4+ T cell immune responses have

now been overexpressed in the lung. Overexpression of IL-4, IL-5, IL-13, and IL-9 all

result in spontaneous recruitment of inflammatory cells, particularly eosinophils and

lymphocytes, to the airways and are also associated with other pathologies such as

mucus hypersecretion, airway hyperresponsiveness, collagen deposition and fibrosis to

varying degrees (Rankin et al., 1996, Lee et al., 1997, Zhu et al., 1999, and Temann et

al., 1998). Overexpression of IFNI leads to infiltrates of macrophages and

neutrophils, and alveolar enlargement, characteristic of emphysema, is also observed

(Wang et al., 2000). The opposite approach has been to knockout proteins of interest.

The spontaneous asthma like phenotype of Tbefi" mice has already been discussed

(Finotto et al., 2002), as has the lack of eosinophilia and AHR in IL-5-/- animals

(Foster et al., 1996), and a great many molecules have now been manipulated in these

ways. However, results should be interpreted with caution, bearing in mind potential

genetic background effects and also the presence or absence of these proteins from

birth, which could result in abnormal lung or lymphoid tissue development. To

circumvent the problems of abnormal lung development and enable genetic

intervention purely at the point of disease induction, inducible transgenic animals are

now being increasingly employed (Cheng et al., 2007). However, having stated this

caveat to the timing of gene overexpression, the phenotypes observed when IL-13 is

73

inducibly targeted to the lung are found to be very similar to those that result in mice

that express this cytokine constitutively (Zheng et al., 2000).

Chapter 5 of this thesis describes the generation of an inducible transgenic model of

lung disease, where induction results not in the overexpression or ablation of

endogenous protein but in the expression of a peptide antigen for CD4+ T cells. The

peptide in question is the cognate antigen for the T cell receptors used in the

generation of the TCR transgenic animals described in chapters 3 and 4 of this thesis.

Commonly employed strategies in the generation of transgenic animals, along with the

transgenic approaches that have been used in the generation of the mouse models in

this thesis are discussed in the section below.

1.17 Approaches to transgenesis

Transfer of foreign DNA into the mouse genome by injection of a plasmid into the

pronuclei of fertilised mouse oocytes, a technology now known as transgenesis, was

first achieved in 1980 (Gordon et al., 1980). That this transfer of genetic material

could lead to an altered phenotype in genetically modified animals was demonstrated a

couple of years later (Palmiter et al., 1982) and since this time, the use of transgenic

mice in research has become widespread and routine. Improvements in transgene

design have led to better and more predictable expression levels of the proteins of

interest, but pronuclear injection of oocytes remains the most common technique by

which genetic modification of the mouse is achieved, and it has now been employed in

many other species of animal including rats, rabbits, sheep, pigs and cows. Other

approaches to DNA integration that have been tried and which are becoming more

widely used include the use of transposons (DNA sequences that trigger integration)

(Dupuy et al., 2002) and the use of retroviral vectors (Lois et al., 2002). Of these

approaches, retroviral vectors probably hold the most promise, and very high

efficiencies from low titre preparations of virus are now being reported (Ritchie et al.,

2007). Although random recombination events are the most straightforward way to

introduce new genetic material into the genome, modification of endogenous gene

74

function by gene knockout or mutation cannot be achieved in this way. This approach

requires a homologous recombination event at the endogenous loci of the gene of

interest and is achieved using embryonic stem (ES) cells as, in these cells, for

unknown reasons, homologous recombination is the most efficient. Although more

efficient in ES cells, homologous recombination is still a rare event and cells in which

this has occurred must be selected and further used to generate a living embryo. Only

two mouse lines have proven useful in the development of chimeric embryos, which

then transmit the recombined gene to their progeny, and most chimeric mice have been

generated using 129/SV ES cells. The difficulty of this technique is additionally

highlighted by the failure to date to achieve transgenesis by homologous

recombination in any species other than the mouse.

One of the benefits of homologous recombination over random integration of a

transgene is that factors influencing transgene expression are better understood. If the

position of the transgene in the genome is known and controlled, then so are the

corresponding promoter and enhancer elements surrounding the integrated DNA.

When a transgene is integrated randomly into the genome, control over expression

may be mediated not only by the promoter that has been engineered into the transgene

itself, but also by promoter, enhancer and silencer elements that now surround it

(Cranston et al., 2001). Approaches to minimise the influence of surrounding genetic

elements have included the use of so called insulator sequences (Taboit-Dameron et

al., 1999) and the inclusion of long genomic DNA fragments in the transgene itself

(Giraldo and Montoliu, 2001), but removing bacterial sequences prior to pro-nuclear

injection and the inclusion of intronic sequences immediately before coding sequences

are among some straight forward rules now employed in the design of transgenes to

aid correct expression. Aside from the coding sequence of the gene of interest, crucial

to the function of the transgene is the choice of promoter. The promoter used will

determine the timing and tissue specificity of transgene expression and the use of a

wide variety of different promoters have been reported in transgenic mice, from

ubiquitous and constitutive, such as the chicken (3 actin promoter (Wiekowski et al.,

2001a) to the tissue specific and developmentally controlled, such as the CD4

75

promoter (Salmon et aL, 1996). There is also a growing interest in inducible gene

expression technology, where tissue specific or ubiquitous gene expression/ablation

can be exogenously controlled at the most appropriate time point for the scientific

question being addressed. For gene knockout studies this approach was demanded by

the embryo lethal phenotype of many constitutive knockouts and in overexpression

studies it has become important to separate abnormal developmental processes from

those really involved in the biological pathway of interest. Several systems for

inducible gene targeting have now been described with the tetracycline and Cre

systems being the most commonly employed and schematic diagrams that outline the

mechanistic basis behind these two approaches are shown in figure 1.5

In the tetracycline system of inducible gene targeting a ubiquitous or tissue specific

promoter directs expression of a reverse tetracycline transcriptional activator (rtTA).

Temporal control is achieved because this molecule can only initiate transcription of

the target gene by binding an upstream tetracycline operator (tetO) sequence in the

presence of doxycycline (Zhu Z et al., 2002). The main drawback to this approach has

been the low baseline levels of target gene expression found in the absence of

induction, as rtTA can exhibit a degree of residual affinity to tetO in the absence of the

drug. To minimise this problem, some groups have used another transgene coding for

a transcriptional silencer (rTS). rTS binds to tetO only in the absence of doxycyline

and so transcriptional repression is lifted on administration of the drug (Zhu et al.,

2001). An additional problem with this system was highlighted more recently in a

study of rtTA transgene use in the lung (Sisson et al., 2006). This study found that

expression of simply the rtTA protein alone in the lung, in the absence of any target

gene, could cause pulmonary pathology consistent with an emphysema like phenotype

in these mice. This study highlights one of the many dangers of interpreting data

derived from transgenic animals and makes a strong case for the need to control for all

aspects of a particular genetic manipulation.

The Cre/lox system involves a mechanism of site-specific recombination that derives

from bacteriophage P 1. Cre recombinase recognises, and catalyses recombination

76

Promoter rtTA

(et° Promoter

No expression of target gene

• • Addition of • Doxycycline No Doxycycline

Filo- target gene Q•

DO

target gene

Complex retained in the cytoplasm

Hsp90 Addition of Tamoxifen

Nuclear translocation

target gene Promoter STOP p • • .. E. • • • •

U

(a) Tetracycline system

Expression of target gene

(b) Cre/Lox system

I*Mer Cre Mer V A Promoter

)c* target gene Promoter p STOP 1........"..."••••••1

loxP loxP

No expression of target gene Recombination between lox? sites

target gene Promoter Ip

Expression of target gene 4'e El

Figure 1.5 The mechanistic basis of reverse tetracycline-controlled transcriptional activator (rtTA) and Cre/Lox systems of inducible gene targeting. In both systems, spacial control is achieved with the use of one or more tissue specific promoters. (a) In the tetracycline system a transcriptional activator (rtTA) is only active in the presence of the tetracycline derivative, doxycycline. Upon binding to doxycycline, rtTA can bind an operator sequence (tet0) and activate target gene expression. (b) In the Cre/Lox system, the Cre recombinase mutated oestrogen receptor complex must bind tamoxifen and translocate to the cell nucleus in order to activate target gene expression. Gene expression is permitted by the excision of a translational stop sequence. Alternatively, engineering of loxP sites to flank the gene of interest itself can result in temporal control over gene ablation. Cre mediated recombination is prevented in the absence of tamoxifen by Hsp90 retention of the MerCreMer complex in the cytoplasm.

77

between, 34 base pair sequences known as loxP sites. Excision of a stretch of DNA

located between two loxP sites can either knockout a gene or permit expression of an

adjacent gene (figure 1.5) and the system becomes inducible when two mutated

hormone-binding domains of the murine oestrogen receptor are engineered to flank the

Cre protein (MerCreMer) (Metzger et al., 1995). These hormone binding domains

complex with Hsp90 to retain the Cre recombinase in the cell cytoplasm until

administration of the drug tamoxifen. On binding tamoxifen the MerCreMer complex

is able to move to the cell nucleus and mediate recombination. Spatial control over

this system can be achieved by tissue specific expression of Cre and/or the gene of

interest and it has been suggested that further control over specificity may be possible

by expressing the Cre protein as two halves, under the control of two different

promoters (Casanova et al., 2003). Cre toxicity is the most widely reported problem

of this system (Loonstra et al., 2001). It occurs because mammalian genomes contain

pseudo loxP sites whose sequence can deviate considerably from the consensus loxP

sites present in the transgene, but which can still act as functional recognition sites for

the Cre protein. The inducible MerCreMer system does minimise this problem by

reducing the amount of time for which cells are exposed to the Cre protein, but the

possibility of phenotypes arising from cell toxicity means that proper experimental

controls, where animals carry the Cre transgene but not the target gene, should always

be included.

An emerging area in the field of gene targeting is the use of RNA interference (RNAi)

and a recent study has combined RNAi technology with the inducible tetracycline

system of gene expression to achieve inducible, tissue specific and reversible gene

knockdown in transgenic mice (Dickins et al., 2007). In this study, a short hairpin

RNA (shRNA) targeting the mouse Trp53 gene was expressed transgenically under

the control of a tetracycline responsive CMV promoter with rtTA under the control of

a liver specific promoter. Efficient, tissue specific and reversible knockdown of Trp53

was observed upon administration of doxycycline that could be demonstrated to have

functional consequences in an established model of tumoregenesis, where knockdown

of Trp53 led to more rapid disease progression. The Cre system of gene targeting does

not led itself so well to this approach as induction is not reversible, but the use of the

78

tetracycline system and RNAi in this way represents a promising new approach to

controlling endogenous gene expression in vivo.

Chapter 5 of this thesis describes the use of the MerCreMer system of inducible gene

targeting to create a transgenic system in which a T cell epitope (PLP 56-70) is

expressed inducibly and specifically in the lung. There are currently no reports in the

literature of other peptides being targeted for expression in the lung in this way, but

the inducible targeting of proteins to cardiac tissue (Xiong et al., 2007) and tumor cells

(Spiotto et al., 2003) are examples of other studies that have successfully employed

the MerCreMer system of gene expression.

The peptide (PLP56-70), used in the inducible mouse model described in chapter 5, is

the cognate epitope for the T cell receptors used for the generation of the TCR

transgenic animals described in chapters 3 and 4. TCR transgenic animals were first

described in 1988, when mice bearing transgenes encoding the a and (3 chains of a

TCR specific for the male H-Y antigen were generated (Kisielow et al., 1988).

Studies with these animals served to answer important questions about T cell tolerance

and thymic selection. The authors observed that, while in female mice peripheral

CD8+ T cells bearing the transgenic TCR were capable of lysing male target cells,

fewer transgenic TCR bearing cells were present in the periphery of male mice, and

those that were present were unable to react to antigen. Since the publication of these

first TCR transgenic animals, a great many more TCR transgenic models have been

described, serving to answer many more questions about T cell development and

function as well as providing valuable new disease models. The integral role that TCR

transgenic animals have played in the understanding of T cell development has been

discussed earlier in this thesis. Expression of a and (3 chain transgenes, both

independently and together, was pivotal to understanding the way in which TCR

molecules are assembled (Uematsu et al., 1988) and these models have also played an

important role in our understanding the role of the TCR in, and the timing of, negative

selection in the thymus (Douek et al., 1996). In this study by Douek et al., the timing

of negative selection in several different TCR transgenic lines were compared. The

79

site at which negative selection occurred in the thymus, i.e. thymic cortex or thymic

medulla, was found to be influenced by the nature of the antigen recognised by the

TCR. Negative selection as a consequence of superantigen recognition was found to

occur in the medulla, while presentation of cognate self-peptide could induce deletion

in either compartment. TCR transgenics have also been used to answer important

questions about T cell function in the periphery and there are now numerous examples

in the literature of the use of TCR transgenics to investigate T cell function in the

context of a disease. The use of mice bearing a transgenic TCR (D011.10) specific for

an epitope of the ovalbumin protein, in combination with the commonly used

ovalbumin model of allergic airways disease, has already been described in the context

of mouse models of lung disease. Other examples of the use of TCR transgenesis in

disease models include mouse models of multiple sclerosis (MS) (Ellmerich et al.,

2005) and autoimmune gastritis (Candon et al., 2004). In the study by Ellmerich et

al., the TCR in question was a human sequence derived from myelin basic protein

specific T cells of MS patients and was used in conjunction with a human HLA class

II transgenic mouse. Mice expressing both the human TCR and human HLA class II

were shown to develop spontaneous MS like pathologies. The TCR transgenic mice

described in chapters 3 and 4 of this thesis have been used to try to answer basic

biological questions about the role of the TCR in determining CD4+ T cell phenotype,

but are also linked to the inducible transgenic model described in chapter 5, which

uses the MerCreMer inducible system of gene expression. A long-term aim of the

work in this thesis is to combine the TCR transgenic models (chapters 3 and 4) with

the inducible lung gene expression system (chapter 5) to create a new mouse model of

lung inflammation.

1.18 Interleukin 8

Previous discussion surrounding lung diseases and attempts to understand the

processes of lung inflammation has included the mention of many proteins that have

been both ablated and overexpressed in the lung. One cytokine not normally directly

associated with Thl or Th2 type immune responses per se, but nevertheless implicated

80

in the pathogenesis of several human lung diseases, is IL-8. To date there are no

reports in the literature of IL-8 having been overexpressed in the mouse lung and

chapter 6 of this thesis, along with discussion in the following sections, describes

current understanding of this cytokine in the context of lung inflammation and the

generation of transgenic mouse lines with lung targeted, but constitutive, expression of

IL-8.

Interleukin-8 (IL-8) was first identified in 1987 through its role as a neutrophil

activating cytokine. Identification of this protein by several different groups at around

the same time meant that it was assigned different names, being referred to as

monocyte derived neutrophil chemotactic factor (MDNCF) (Yoshimura et al., 1987),

monocyte derived neutrophil activating peptide (MONAP) (Schroder et al., 1987) and

neutrophil activating factor (NAP) (Walz et al., 1987). The name IL-8 was

subsequently proposed and adopted but since that time a whole family of structurally

related cytokines, known also as chemokines, have been identified, and in much of the

current literature IL-8 is referred to as CXCL8. Chemokines are so called because of

the leukocyte chemotactic properties displayed by most. Members of this family are

low molecular weight proteins with cysteine residues at well conserved positions and

it is these cysteine residues that define their nomenclature (Zlotnik and Yoshie, 2000).

IL-8 belongs to the CXC subgroup of chemokines, which are so called because the

first two cysteine residues at the N terminus of the functional protein are separated by

a single amino acid. This is compared to 3 amino acids or adjacent cysteine residues

in the CX3C and CC subgroups respectively. CXC chemokines can be further

classified into ELR+ or ELR- based on the presence or absence of a tripeptide motif

(Glu-Leu-Arg) directly before the first cysteine residue at the N terminus of the

protein. IL-8 is an ELR+ chemokine (appendix 29) that correlates with a potent

chemotactic effect, particularly on neutrophils, (Yoshimura et al., 1987), angiogenic

activity (Strieter et al., 1995) and high affinity binding to the chemokine receptors

CXCR1 and CXCR2 (Holmes et al., 1991 and Murphy and Tiffany, 1991).

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The cDNA of IL-8 encodes a 99 amino acid precursor protein (appendix 29), the N

terminal signal sequence of which is cleaved extracellularly to yield predominantly 77

or 72 amino acid versions of the protein (Matsushima et al., 1988). Processing is

carried out by extracellular proteases and the form that dominates depends upon the

cellular environment. In vitro cultures of fibroblasts and endothelial cells produce

mainly the 77 amino acid form, while the 72 amino acid form is produced by

leukocytes (Padrines et al., 1994, Hebert et al., 1990 and Nakagawa et al., 1991).

Different forms of the protein exhibit different neutrophil chemoattraction activities

with the 72 amino acid form more potent than the 77 amino acid protein and it is the

72 amino acid protein that is identified at sites of inflammation in vivo (Ko et al.,

1993). Crystallographic studies revealed that at high concentrations IL-8 exists as a

homodimer (Baldwin et al., 1991), however at nanomolar concentrations, which most

likely reflects conditions in vivo, this cytokine is predominantly monomeric (Burrows

et al., 1994) and indeed mutation of the protein to prevent dimerization does not

prevent receptor binding or neutrophil activation (Rajarathnam et al., 1994).

IL-8 can be produced by leukocytes such as T cells, monocytes, neutrophils and

natural killer (NK) cells but also by somatic cells such as endothelial cells, fibroblasts

and epithelial cells. However, expression is not generally constitutive and production

is induced by inflammatory cytokines such as IL-1 and TNF-a (Matsushima et al.,

1988). Bacterial proteins and viruses can cause upregulation of IL-8 expression

(Martich et al., 1991 and Johnston et al., 1997), as well as environmental conditions

such as hypoxia (Xu et al., 1999), and the transcription factors NF-K13 and AP-1 have

been shown to bind the IL-8 gene, implicating these proteins in the control of IL-8

gene transcription (Murayama et al., 1997 and Thompson et al., 2006). The biological

activities of IL-8 are mediated through binding to the G protein coupled receptors,

CXCR1 and CXCR2. These receptors are more than 80% homologous at the amino

acid sequence level and differ only in their NH2 terminal portions, giving rise to

slightly different binding specifies of other chemokines. Binding of IL-8 induces

receptor internalisation, although receptors are subsequently recycled to the cell

surface (Samanta et al., 1990) and signalling is mediated by the receptor coupled G

82

proteins which activate pathways such as those mediated by PI3K-y and MAPK

(Hirsch et al., 2000 and Km11 et al., 1996). CXCR1 and CXCR2 receptors have been

identified on the surface of several different cell types such as neutrophils, monocytes

and NK cells (Morohashi et al., 1995), and while the role of IL-8 in NK cell migration

remains slightly controversial, with some studies reporting IL-8 to be chemotactic to

NK cells and others not (Campbell et al., 2001 and Loetscher et al., 1996), neutrophil

chemotaxis in response to IL-8 is better defined. In order for IL-8 produced by a

tissue, for example in response to a bacterial infection, to attract neutrophils, the

cytokine is first internalised by vascular endothelial cells, transcytosed and then

presented to neutrophils at the luminal surface of the vessel (Middleton et al., 1997).

This transcytosis and presentation on the luminal surface is mediated by the C terminal

end of the protein, and cleavage of this C terminal region by a Streptococcus pyogenes

bacterial proteinase has been proposed as a mechanism of immune evasion by this

pathogen (Edwards et al., 2005). On binding to the CXCR1 and CXCR2 receptors on

the surface of passing neutrophils, IL-8 induces the shedding of adhesion molecule L-

selectin and alters the expression levels and avidity of integrins such as CD11 b and

CD1 1 c, stimulating migration across endothelial and epithelial cell layers (Detmers et

al., 1990, Huber et al., 1991 and Mul et al., 2000). Additionally, IL-8 can activate

some of the effector mechanisms employed by neutrophils, such as degranulation and

the respiratory burst (Walz et al., 1987) and migration of other cell types such as

basophils, eosinophils and T cells, both directly and indirectly, has also been reported

(Geiser et al., 1993, Bruijnzeel et al., 1993, Larsen et al., 1989 and Chertov et al.,

1996).

1.19 Neutrophil function

High levels of IL-8 protein are found to be associated with several different diseases

(discussed later), often in conjunction with large neutrophil infiltrates. With the potent

chemotactic and neutrophil activating properties of IL-8, and the important role that

neutrophils are known to play in innate defence mechanisms and in direction of the

adaptive immune response, there is much interest in the relevance of the high levels of

83

IL-8 protein and neutrophils in these disease pathologies. Neutrophils are most

commonly thought of as phagocytes that accumulate at, and are rapidly recruited to,

sites of bacterial infection where they ingest and kill invading microbes. Neutrophils

produce a wide variety of antimicrobial compounds which are stored within the cell to

kill ingested pathogens, but which are also secreted during the processes of

degranulation and the respiratory burst, which occur as a result of neutrophil

activation. Neutrophil granules contain many antimicrobial molecules, such as

lactoferrin, lysozyme and matrix metalloproteinases (Faurschou and Borregaard, 2003)

and products of the respiratory burst, such as hydrogen peroxide, hypochlorous acid

and reactive oxygen intermediates are also toxic to invading pathogens. First to be

discharged upon activation of the neutrophil are the peroxidase-negative secondary

and tertiary granules. As well as the matrix metalloproteinases MMP8, MMP9 and

MMP25, which are thought to have an important role in facilitating neutrophil

recruitment and tissue breakdown (Faurschou and Borregaard, 2003), these granules

contain the antimicrobial proteins lactoferrin, lipocalin, lysozyme and LL37, which act

to kill microorganisms. Lactoferrin, for example, is an iron binding protein that acts to

sequester iron thereby removing an essential microbial growth factor from the

extracellular environment, and transgenic mice that overexpress the human form of

lactoferrin are found to have enhanced immune responses to bacterial infection

(Guillen et al., 2002). Following the release of secondary and tertiary granules, next

to be released by the neutrophil are the peroxidase-positive primary granules. These

contain additional antibiotic proteins, such as cathepsin G, neutrophil elastase,

protease 3, and Azurocidin (Campanelli et al., 1990) as well as myeloperoxidase.

Myeloperoxidase is an iron containing enzyme that converts hydrogen peroxide into

the much more powerful antiseptics, hypochlorous acid, hypobromous acid and

hypoiodous acid. The hydrogen peroxide that serves as a substrate for

myeloperoxidase derives from production of reactive oxygen intermediates by the

neutrophil, the primary mediator of which is a phagocyte oxidase (phox). A

deficiency of this enzyme is fatal as patients are unable to control bacterial infection

(Good et al., 1968). Neutrophils are therefore an essential and powerful first line of

defence against invading pathogens, but the chemicals that they release are also

84

damaging to host cells and if uncontrolled can cause enormous damage to the local

tissue. A small level of tissue damage around a site of infection is probably beneficial

to the host as it may help to isolate microbes and prevent spread of infection, however,

in the longer term neutrophils also generate signals to suppress their own activation,

promote apoptosis and initiate repair processes. Anti-inflammatory compounds

secreted by neutrophils include lipoxins, and both neutrophils and macrophages

secrete the anti-inflammatory factor, secretory leukocyte protease inhibitor (SLPI) (Jin

et al., 1997) that acts to neutralise some of the antimicrobial proteases secreted during

the process of neutrophil degranulation. This protease inhibitor also acts to protect the

epithelial-cell-growth-promoting cytokine, proepithelin, from neutrophil elastase

thereby enabling repair processes to be initiated. If proepithelin is instead degraded by

neutrophil elastase, products of this degradation act to promote IL-8 release by

epithelial cells and the recruitment of activated neutrophils to the site of injury

continues (Zhu J et al., 2002).

Neutrophils are produced continuously by the bone marrow and traffic though the

peripheral blood stream until recruited to sites of infection by chemokines such as IL-

8. As such, they are one of the first cell types to be found at the site of a wound and

there is growing interest in the role that neutrophils may have in directing later stages

of the immune response. That activated neutrophils themselves can be a source of IL-8

has already been mentioned, but other examples include TNF-a, B lymphocyte

stimulator (BLys) and IFN-y. TNF-a drives DC and macrophage differentiation and

activation (Bennouna et al., 2003) and neutrophils can additionally mediate DC

activation by cell-cell contact through engagement of the CD11 b integrin on

neutrophils by the DC specific adhesion molecule, DC-SIGN (van Gisbergen et al.,

2005). BLys secretion by neutrophils promotes the activation of B cells and in a

recent study, levels of BLys secreted by neutrophils from patients with rheumatoid

arthritis were found to be greater than those from healthy control subjects (Scapini et

al., 2005). Incidentally, rheumatoid arthritis is an example of a human disease that has

been associated with elevated levels of IL-8 at the site of disease (Troughton et al.,

1996). A further link between neutrophils and the adaptive immune system comes

85

from the demonstration that in the presence of IL-12, neutrophils can produce IFN-y

(Ethuin et al., 2004) and as well as through the secretion of inflammatory mediators,

another way in which neutrophils may act to initiate adaptive immune responses was

reported in a recent study using a Mycobacterium bovis strain Bacille Calmette-Guerin

(BCG) model of infection (Abadie et al., 2005). In this study by Abadie et al. they

used transgenic BCG expressing enhanced green fluorescent protein to show that

neutrophils rather than dendritic cells carried BCG from a site of infection in the skin

into the draining lymph node. However, there are also examples in the literature of

neutrophils functioning to suppress rather than activate the adaptive immune response

(Schmielau and Finn, 2001) and so a picture is emerging of a role for neutrophils in

both the positive and negative regulation of many aspects of the immune response to a

pathogen, some aspects of which are likely to be intimately linked to the presence or

absence of IL-8 at the site of inflammation.

1.20 Interleukin-8 and human lung disease

High levels of IL-8 are found in association with several human lung diseases. One

well studied example is bronchiectasis (Richman-Eisenstat et al., 1993).

Bronchiectasis is characterised by irreversible airway dilation and is found in

association with chronic bronchial infection and inflammation. As the respiratory

organ of the body, the lung is constantly exposed to airborne microbes and as such has

had to develop a multitude of innate and adaptive defence mechanisms to deal with

these pathogens. To allow for sufficient and efficient gas exchange, the epithelial

surface of the lung is vast and, in humans, much of this epithelial surface is covered by

a layer of mucus. Mucus acts to trap incoming bacteria, and these can then be cleared

from the lung by the action of motile cilia which line the surface of airway epithelial

cells. It therefore follows that an impairment in mucus production, or the function of

cilia, will result in susceptibility to infection, and in the diseases of cystic fibrosis (CF)

and primary ciliary dyskinesia (PCD), which are both known causes of bronchiectasis,

this is indeed the case (Matsui et al., 2006 and Bush et al., 1998). In this recent study

by Matsui et al. the concentrated airway mucus that results from the deficiencies in ion

86

transport seen in CF was found to not only impede clearance of mucus from the

airways, but also provide a favourable environment for bacterial growth and biofilm

formation. In some cases of bronchiectasis, such as those caused by CF and PCD, the

cause of persistent infection is understood. In many cases however, the disease is

idiopathic (of unknown cause). Failure to clear bacteria from the airways in the first

instance by ciliary movement results in bacterial contact with, and activation of, the

lungs innate defence system of which neutrophils are an important component. The

main mechanisms by which neutrophils clear invading microbes has been discussed in

the previous section and the importance of the multitude of antimicrobial peptides

secreted by these cells during a bacterial infection are highlighted by various disease

models such as the disease protection conferred on mice manipulated to express

lysozyme constitutively in the lung (Akinbi et al., 2000), and more recently by the

impaired bacterial clearance observed in mice deficient in this protein (Cole et al.,

2005). It is the persistent nature of infection that lies behind the pathophysiology of

bronchiectasis, as sustained inflammatory responses inevitably lead to tissue damage,

impairing the ability of the lung to fight infection, and creating a vicious cycle of IFN-

y mediated immunity.

As previously mentioned, CF patients suffer from chronic bacterial infections and

elevated levels of IL-8 protein have been found in BAL, sputum and in the bronchial

epithelial cells of these patients (Muhlebach et al., 1999, Sloane et al., 2005 and

Muhlebach et al., 2004), along with pronounced neutrophilic infiltrates (Konstan et

al., 1994). Whether the observed high levels of IL-8 are purely a function of chronic

inflammatory reactions occurring in the lung, increasing IL-8 expression as a

consequence of high levels of inflammatory cytokines and bacterial proteins, or

whether the reason for high IL-8 expression is intrinsic to CF itself is not quite clear.

However, two recent studies have suggested that the mutation in CF transmembrane

conductance regulator (CFTR) gene alone, proposed to be the genetic basis for

disease, is sufficient to cause elevated IL-8 gene expression in the airways (Perez et

al., 2007 and Chan et al., 2006).

87

Another lung disease characterised by large neutrophil infiltrates and high levels of IL-

8 is acute respiratory distress syndrome (ARDS). Caused by a variety of direct or

indirect insults to the lung such as pneumonia, smoke inhalation or surgery, several

groups have reported a correlation between neutrophilia and levels of IL-8 in patients

with ARDS (Aggarwal et al., 2000) and indeed an association between high levels of

IL-8 and an increased risk of subsequently developing this condition has also been

reported (Donnelly et al., 1993). A role for IL-8 in mediating the pathogenesis of this

acute disease, aside from simply recruiting and activating the neutrophils which

amplify lung inflammation and cause tissue damage, has been suggested to be

mediated by anti-interleukin-8 autoantibodies (Kurdowska et al., 2001). This group

reported a significant correlation between concentrations of IL-8:anti-IL-8 antibody

complexes in the lung and the onset of ARDS. More recently, they have also

demonstrated that these IL-8:anti-IL-8 antibody complexes are indeed attached to

FcyRIIa receptors and are therefore likely to be mediating pro-inflammatory signals in

the lung (Allen et al., 2007).

Other diseases for which IL-8 has been found to be a marker of disease severity

include idiopathic pulmonary fibrosis (IPF), COPD and diffuse panbronchiolitis

(DPB) (Cane et al., 1991, Yamamoto et al., 1997 and Koga, 1993). Indeed, anti-IL-8

therapy has been suggested as a possible therapeutic approach for the treatment of

COPD (Mahler et al., 2004). Aside from neutrophil recruitment, additional roles for

IL-8 in the pathogenesis of each of these diseases is currently unclear, but

polymorphisms in the IL-8 gene have been reported in association with DPB (Emi et

al., 1999) and in the COPD study by Yamamoto et al. IL-8 levels were closely

correlated with lung function measurements. A more recent study has attempted to

elucidate a role for IL-8 in directly affecting airway structure and function

(Govindaraju et al., 2006). In this study, smooth muscle cells from human airways

were analysed for expression of the IL-8 receptors, CXCR1 and CXCR2. Both

receptors were found to be present on the surface of these cells and blocking of the

receptors using neutralising antibodies could prevent changes in intracellular Ca2+

observed upon IL-8 stimulation. IL-8 was also found to cause contraction of the

88

muscle cells as well as stimulating cell migration. This data suggests that the lung

function changes observed in many of the lung diseases discussed here could, in part,

be mediated by the high levels of IL-8 found within the lung.

Although asthma is normally considered to be associated with eosinophilic lung

infiltrates, neutrophil influx is found to be a common feature of persistent asthma and

this neutrophilia has also been associated with elevated levels of IL-8 (Gibson et al.,

2001). Increased levels of IL-8 have also been found in sputum samples directly

before late phase exacerbations in acute asthma (Kurashima et al., 1996) and in

keeping with the more recent results of the airway smooth muscle study by

Govindaraju et al., IL-8 inhalation has been shown to directly provoke

bronchoconstriction in guinea pigs, which was observed in the absence of any

leukocyte recruitment into the airways (Fujimura et al., 1999). A positive role for IL-

8 in the regulation of asthma has been proposed following the finding that IL-8 can

selectively inhibit IL-4 induced IgE production (Kimata et al., 1992) and the treatment

of human lungs with IgE results in the release of IL-8 (Erger and Casale, 1998).

However, there must then be a trade off between IgE downregulation and the

neutrophil and eosinophil chemoattractant properties of this cytokine. Aside from IgE,

the reason for elevated IL-8 levels in the airways of asthmatic is not yet clear, but that

the initial source of this cytokine maybe the airway epithelium in response to allergen

is supported by the finding that pulmonary epithelial cells can produce high levels of

IL-8 in the presence of diesel exhaust particles (Takizawa et al., 1999). However the

wide range of toxic chemicals present in diesel exhaust fumes means that this finding

is not necessarily applicable to other more classical allergens.

The neutrophilia observed in many human lung diseases, the central role of IL-8 in

neutrophil recruitment and also emerging roles for IL-8 in disease pathogenesis that

are separate from neutrophil recruitment and activation, all make IL-8 an attractive

target for manipulation in animal models of lung disease. There are currently no

reports in the literature of IL-8 having been overexpressed in the airways of mice,

although this may not be an intuitive experiment as in the mouse genome, the IL-8

89

gene and the gene for its receptor, CXCR1, have been deleted. However, the CXCR2

receptor is expressed, and this receptor is able to mediate neutrophil chemotaxis in

response to the two proposed murine homologues of IL-8, KC and MIP-2 as well as in

response to human IL-8 itself (Bozic et al., 1994). The chemoattractant properties of

human IL-8 have been demonstrated using neutrophils for a variety of different

species, including monkeys, rabbits, dogs and mice (Rot, 1991). Although the potency

of human IL-8 varied between species and was lower than that observed for human

neutrophils, these findings nevertheless suggest that interleukin-8, if expressed in the

murine lung, should be biologically active. Various studies have manipulated the

mouse CXCR2 receptor and its ligands in vivo and in keeping with the role of IL-8 and

its receptors in human studies, mice overexpressing KC in a variety of tissues,

including the lung, do show evidence of neutrophil infiltrates (Lira, 1996). Mice

overexpressing KC in the lung also demonstrate increased resistance to infection (Tsai

et al., 1998), while neutralising the CXCR2 receptor results in increased disease

susceptibility (Tsai et al., 2000). These studies suggest that, as in humans, ELR+ CXC

chemokines are critical mediators of neutrophil mediated host defence mechanisms.

However, as has been discussed in this section, there is increasing evidence that IL-8

mediates lung pathology in ways other than those involving neutrophil recruitment and

additionally, to study potential therapeutic approaches to IL-8 mediated lung

pathology in the mouse, using the human IL-8 protein rather than the mouse

homologues, which are similar but not equivalent, would be preferable. There is

therefore merit in the approach to overexpress human IL-8 in the murine lung and the

generation and initial characterisation of such transgenic animals is described in

chapter 6 of this thesis.

1.21 Aims of this thesis

Much of the discussion in this chapter has centred around the T cell receptor molecule

and around the Thl and Th2 type immune responses that are an important feature of

many human diseases, including several lung pathologies. Multiple factors are

involved in determining whether a naive CD4+ T cell differentiates into a Thl or Th2

90

effector cell and several lines of evidence point towards an important role for the

interaction between the T cell receptor (TCR) and its peptide ligand. A previous study

by Boyton et al. (Boyton et al., 2002) showed that highly polarised Th2 CD4+ T cells

selected under Th2 polarising conditions favour a structurally distinct TCR with an

elongated TCR a chain complimentarity determining region 3 (CDR3), while those

selected under Thl polarising conditions favour a shorter TCR CDR3 a chain.

Molecular modelling predicted that this elongated TCR a chain results in a less

optimal (lower avidity) interaction between the TCR and peptide/MHC complex and

gave rise to the hypothesis that, if structurally distinct TCRs arising from Thl and Th2

polarised T cells, i.e. a TCR bearing an elongated CDR3a region from a Th2 cell and

a shorter CDR3a region from a Thl cell, are expressed transgenically, an inherent bias

towards Thl or Th2 will be observed in CD4+ T cell responses from these animals.

Much of the data in this study by Boyton et al. was derived from T cell responses of

H2-E transgenic NOD (NOD.E) mice to the peptide epitope PLP 56-70. PLP 56-70 is

an epitope used to induce EAE in NOD and NOD.E mice. A deletion in the promoter

region of the I-E a chain gene means that NOD mice only express the MHC class II

molecule I-A and are known to spontaneously develop type 1 diabetes. However,

expression of an I-E a chain transgene protects NOD.E mice from diabetes (Lund et

al., 1990), but makes them more susceptible to EAE (Tackas et al., 1995). In this

study by Tackas et al. I-E expression in NOD.E animals was associated with a shift

away from IL-4 production and towards greater IFN-y production in T cell responses

to PLP 56-70, suggesting an association between Thl responses and disease in this

model, whilst describing Th2 responses in association with protection. In the study by

Boyton et al., multiple NOD.E T cell lines and clones specific for the peptide PLP 56-

70 were grown and polarised in vitro towards a Thl or Th2 phenotype. In all cases,

the peptide response was found to be I-Ag7 rather than I-E restricted. It is Thl and Th2

T cell lines grown during the course of this study, which are specific for the peptide

epitope PLP 56-70, that serve as the starting point for the work described in chapters 3

and 4 of this thesis, where the aim is to express dominant TCRs from Thl and Th2

polarised T cell lines transgenically to determine whether a structurally distinct TCR

can cause an innate bias in CD4+ T cell phenotype (figure 1.6)

91

Further to this hypothesis is a second question of whether these Thl and Th2 TCR

transgenic animals could be used to create new models of Thl and Th2 mediated

disease. The lung is one organ in which understanding Thl and Th2 type responses is

of much interest and a second aim of this thesis is to establish a mouse model whereby

the peptide epitope for these Thl and Th2 TCRs (PLP 56-70) can be expressed

inducibly and specifically in the lung. Chapter 5 of this thesis describes the generation

of this model using a lung specific promoter and the MerCreMer system of inducible

gene expression and a schematic diagram detailing the way in which these mice would

be used to generate novel lung disease models is shown in figure 1.6.

The final aim of this thesis is to generate another mouse model of lung disease, where

lung pathology is as a consequence of overexpression of the human cytokine IL-8

(figure 1.6). Elevated levels of IL-8 protein are found associated with several different

human lung diseases, but the precise role that IL-8 plays in the pathologies of these

diseases has yet to be elucidated. Chapter 6 of this thesis describes the generation of

mice overexpressing the human IL-8 protein in the lung and initial characterisation of

lung pathology in these animals.

92

(b) Lung targeted

Antigen

• Inducible and lung targeted

Antigen

1

(c) (a)

Antigen specific CD4+ T cell lines

Lung targeted

IL-8 f

Lung pathology? CD4+ T cell phenotype?

Thi or Th2 derived TCR

Thl or Th2 type lung inflammation?

Figure 1.6 Transgenic strategies of this thesis. A schematic diagram of the transgenic strategies proposed for the three main aims of this thesis. (a) The use of TCR transgenics to answer questions about a possible role for the TCR in determining CD4+ T cell phenotype. (b) The targeting of antigen inducibly and specifically to the lung to generate, in combination with the TCR transgenics, models of Thi and th2 type lung inflammation. (c) Targeting of human interleukin 8 to the lung to elucidate the role of this cytokine in lung inflammation and pathology.

Chapter 2

Materials and methods

2.1 Genomic DNA extraction

400 µ1 of digestion buffer (appendix 1) and 10 [,t1 of 20 mg/ml proteinase K (Sigma-P-

6556) were added to each tissue sample and samples incubated at 56°C overnight.

After complete digestion, 200 µ1 of 6M (saturated) NaC1 were added and the sample

mixed well by vigorous shaking before centrifuging at 16000 x g for 15 minutes. The

supernatant was removed to a fresh tube and DNA precipitated by the addition of 600

µI of isopropanol. The sample was mixed well by inversion and spun at 16000 x g for

10 minutes to pellet the DNA. The DNA pellet washed once in 70 % EtOH before

being briefly air-dried and dissolved in 50-80 1_11 of TE buffer (appendix 1). Samples

were incubated briefly at 56°C for 15 minutes to ensure the DNA pellet was fully in

solution and samples were then stored at 4°C for up to one week or -20°C for more

long term storage.

2.2 RNA extraction

For tissue samples or samples of greater than 5 x 106 cells, RNA was extracted using

Trizol (Invitrogen-15596-018) a phenol and guanidine thiocyanate based extraction

reagent. Prior to addition of Trizol reagent, tissue samples were homogenized using a

manual glass-glass homogenizer. Samples were lysed by repeated pipetting with 1 ml

of Trizol reagent. If not processed to RNA immediately, samples were stored at 4°C

overnight or at -20°C for up to 1 month. Following cell lysis, 2000 of chloroform

was added, the sample mixed by vigorous shaking for 30 seconds and then incubated

on ice for 5 minutes. Samples were centrifuged at 12,000 x g for 15 minutes at 4°C

and the upper aqueous layer removed to a new tube. RNA was precipitated by

addition of 500 Ill of isopropanol. Samples were mixed by inversion and incubated at

room temperature for 10 minutes before centrifugation at 12,000 x g to pellet the

RNA. RNA pellets were washed once with 75% EtOH, air-dried briefly and dissolved

94

in 20-30 t1,1 of RNase free water (Sigma-W4502). RNA samples were stored at -20°C

in the presence of 20 U RNase OUT (Invitrogen-10777-019).

RNA from cell line samples of less than 5 x 106 cells was extracted using an RNA

binding spin column (Stratagene-400805).

2.3 DNase treatment of RNA samples

To remove contaminating DNA from some RNA preparations prior to cDNA

synthesis, samples were treated with an RNase free DNase (Promega-M610A). 3-51.1g

of RNA in a reaction volume of 20 tx1 containing 2 Ill 10X reaction buffer (400 mM

Tris-HC1 pH 8.0, 100 mM MgSO4 and 10 mM CaC12) and 1 U of DNase per Rg of

RNA were incubated at 37°C for 30 minutes. The DNase was then removed from the

sample by phenol:chloroform (Sigma-P1944) extraction. All traces of phenol were

removed by an additional chloroform:isoamylalcohol (24:1) extraction and RNA

precipitated using 2.5 volumes of 100% EtOH. RNA pellets were washed once with

70% EtOH before being briefly air-dried and dissolved in 10 µ1 of RNase free water.

cDNA synthesis reactions were then set up both with and without the reverse

transcriptase enzyme to ensure that subsequent PCR results obtained were not due to

contaminating DNA.

2.4 cDNA synthesis

RNA was quantitated by spectrophotometer and 10Ong - ltA,g used per cDNA reaction.

The RNA in a volume of 13 1A.1 of sterile water containing 50 ng random hexamers

(Invitrogen-48190-011) and 1 IA of 10 mM dNTPs (Invitrogen-18427-013) was heated

to 65°C for 5 minutes and then placed on ice. 4 iil of 5X cDNA synthesis buffer

(Invitrogen-18080-044), 1 !Al of 0.1M DTT (Invitrogen-18080-044), 40 U of RNase

OUT and 200 U of Superscript III (Invitrogen-18080-044) were added. The mixture

was heated at 25°C for 5 minutes, followed by 50°C for 60 minutes and 70°C for 15

minutes. 1 [11 of completed cDNA reaction mixture was used as a template for each

subsequent PCR reaction. cDNA was stored at -20°C.

95

2.5 Polymerase Chain Reaction (PCR)

A general PCR protocol is described below. All oligonucleotide primer sequences,

reaction product sizes and any reaction conditions that varied from those described in

this section are listed in appendices 6, 7 and 8.

PCR reactions were carried out in a reaction volume of 25 µI containing 50-100 ng of

template DNA, 16 mM (NH4)2SO4, 67 mM Tris-HC1 (pH 8.8), 0.01% Tween-20, 1.5

mM MgC12, 200 µM dATP, dCTP, dGTP, and dTTP (Bioline-BIO-39025), 0.5 ILIM of

each oligonucleotide primer (Sigma Genosys) and 0.6 units of Taq polymerase. The

reaction mixture was incubated at 95°C for 5 minutes, 30 cycles of 95°C for 1 minute,

55°C for 1 minute, and 72°C for 1 minute, and a final cycle at 72°C for 8 minutes. The

result of each PCR was then determined by running the reaction on a 1% agarose TAE

(appendix 1) gel stained with EtBr (Sigma-E1510).

2.6 Restriction enzyme digest Restriction enzyme digests were carried out in a volume of 20 µI containing 0.5-2 µg

of plasmid DNA, 1 U of appropriate restriction enzyme(s), and 2µl of 10X buffer

(appendix 3). Reactions were incubated at 37°C or 50°C (appendix 3) for at least 1

hour. Digests were run alongside a DNA ladder with markers of known molecular

weight (Promega-G5711) on 1% agarose TAE gels at 90V.

2.7 Preparation ofXL-Gold competent cells

XL-Gold cells from a previous preparation were streaked onto a Luria-Bertani (LB)

agar (Sigma-L2897) plate containing 50 µg/m1 tetracycline (Sigma-T8032) and

incubated at 37°C overnight. The following day a single XL-Gold colony was

inoculated into 10 ml of LB broth (Sigma-L3022) containing 50 lag/m1 tetracycline

and incubated at 37°C overnight on a shaker (225 rpm). The 10 ml overnight culture

was inoculated into 200 ml of LB broth containing 0.015M MgC12 and grown until the

optical density Q = 600 nm) of the culture reached 0.6. The culture flask was then

placed on ice for 10 minutes before spinning 100 ml of the culture in a pre-cooled

96

centrifuge (4°C) at 1600 x g for 10 minutes. The supernatant was discarded and the

remaining culture volume spun down in the same tubes. The cell pellets were

resuspended by swirling and gentle pipetting in a total volume of 60 ml of ice cold

solution A (appendix 1) and incubated on ice for 20 minutes. Samples were then

centrifuged again, the supernatant discarded, and the pellets resuspended in a total

volume of 12 ml of ice cold solution B (appendix 1). Competent cells were stored at -

80°C in 200 µ1 aliquots.

2.8 E-coli transformation

All transformations were performed using XL-Gold (see above) or INVaF' competent

cells (Invitrogen-C2020-03). The same transformation protocol was used for both

strains. 2111 of a ligation reaction or 1µl of a plasmid preparation were added to 50 [al

of competent cells. The mixture was incubated on ice for 30 minutes before heat

shock at 42°C for 30 seconds. 250 pi of SOC medium (supplied with INVaF'

competent cells) or LB broth warmed to room temperature were added and the cells

were then incubated with shaking (225 rpm) at 37°C for 1 hour. Bacteria were spread

onto LB agar plates containing 50 µg/ml of ampicillin (Sigma-A9518). Plates were

inverted and incubated at 37°C overnight. For TA cloning ligations, plates were also

spread with 40 ial of a 40 mg/ml stock solution of 5-bromo-4-chloro-3-indolyl-b-D-

galactopyranoside (X-Gal) (Bioline-Bio37035) and 8R1 of a 100 mM stock solution of

Isopropyl 13-D-1-thiogalactopyranoside (IPTG) (Sigma-16758) 30 minutes before

plating of the bacteria. This allowed successfully ligated plasmids to be identified by

blue/white screening of the bacterial colonies. Colonies containing a plasmid with a

PCR product correctly ligated appear white.

2.9 Standard cloning and ligation protocol

With the exception of one step, which used a synthetic oligonucleotide insert, the

fragments for all other cloning steps were obtained by single or double restriction

enzyme digests of the appropriate plasmids. These restriction digests were carried out

overnight in a volume of 40 111. For single enzyme digests it was necessary to

dephosphorylate the ends of the vector fragment after digestion to prevent re-ligation.

97

This was achieved by adding 0.01 U of Calf Intestinal Alkaline Phosphatase (CIAP)

(Promega-M1821) per pmol of DNA ends in the reaction (where 1 !is of 1000 bp

DNA = 3.03 pmol of DNA ends) and incubating at 37°C for 1 hour. All DNA

fragments used for cloning were purified by separation on a 1% agarose (Sigma-

A9539) TAE gel followed by gel extraction using silica beads (Qbiogene-1001-600).

The amount of each purified fragment recovered was quantitated by running 1 [xl of

each sample on an agarose gel alongside a quantitative DNA ladder (Bioline-

BI033025). As optimal ligation conditions for each cloning step were unknown, a

number of reactions using different ratios of vector to insert were set up each time.

The amount of each fragment required to achieve each ratio was calculated using the

equation below.

amount of vector (ng) x size of insert (bp) x molar ratio of insert = amount of insert (ng) size of vector (bp) vector

Standard ratios of vector to insert used were 1:1, 1:3, 1:6 and 1:12. Control reactions

of 1:0 and 1:0 with no ligase added were also set up to assess levels of re-ligated and

uncut vector respectively. Ligation reactions were set up in a volume of 10 [AI

containing 5 U of T4 DNA ligase (Promega-M1801) and 2 [11 of 5 x reaction buffer

(250 mM Tris-HC1 pH 7.6, 50 mM MgC12, 5 mM ATP, 5 mM DTT and 25% w/v

polyethylene glycol-8000). Reactions were incubated at 16°C overnight before

transformation into XL-Gold E-coli (see above).

2.10 Blunt end generation

A restriction digest (see above) of the appropriate plasmid and restriction enzyme was

inactivated by heating to 65°C for 15 minutes. Blunt ends were generated using T4

DNA polymerase (Promega-M4211). The reaction volume of the original restriction

digest was increased to 50 1,t1 using sterile water, and contained 6 U of T4 DNA

polymerase, 100 mM of each dNTP and 3 ill of promega 10X multi-core buffer (250

mM Tris acetate, pH 7.8, 1 M potassium acetate, 100 mM Magnesium acetate and 10

mM DTT). Reactions were incubated at 37°C for 5 minutes before inactivation at

98

75°C for 10 minutes. The plasmid was then purified by agarose gel extraction and

blunt ends ligated using T4 DNA ligase.

2.11 Oligonucleotide annealing

Each oligonucleotide was made up to a concentration of 1 mg/ml in sterile water. 1 121

of each oligonucleotide was then added to 18 itil of TE buffer and the mixture heated at

70°C for 5 minutes. The tube was placed into a beaker of water at 65°C and left to

cool to room temperature before placing on ice. No further purification of this

annealed product was required before ligation.

2.12 TA cloning of PCR products

PCR products amplified using Taq polymerase (Bioline-BIO-21040) were cloned into

the plasmid pCle2.1 by TA cloning (Invitrogen-K2000-01). Vector DNA did not

require any purification before ligation, but PCR products were separated by agarose

gel electrophoresis and gel purified using a silica matrix protocol to preserve the A

overhangs. 10-20 ng of PCR product were ligated into 25 ng of pCle2.1 vector in a

reaction volume of 10 pi containing 4 U of T4 DNA ligase (Invitrogen-15224-041)

and 1 1,t1 of 10 x reaction buffer (60 mM Tris-HC1 pH 7.5, 60 mM MgC12, 50 mM

NaCl, 1 mg/ml BSA, 70mM (3-mercaptoethanol, 1 mM ATP, 10 mM spermidine and

20 mM DTT). Ligation reactions were incubated at 16°C overnight.

2.13 Preparation and screening of plasmid DNA

Successfully transformed bacterial colonies were picked from agar plates using sterile

pipette tips and grown up in 8 ml of LB broth containing ampicillin (50 p.g/m1) at

37°C overnight. Bacteria were pelleted by spinning at 1500 x g for 4 minutes and

plasmid DNA extracted and purified by alkaline lysis (Qiagen-27106 or Sigma

PLN350). Successfully ligated plasmids were identified by restriction enzyme digest

and PCR.

99

2.14 T cell receptor PCR analysis T cell receptor (TCR) a and (3 chain cDNA transcripts were amplified using sets of

primers previously described by Candeias et al. (Candeias et al., 1991). Three rounds

of PCR are required to obtain a chain products while two rounds are required for (3.

The 5' primers for both a and 13 are degenerate and designed to bind all TCR families.

5' primers for a and (3 chain amplification are NW36, NW37, NW38 and NK121,

NK122, NK123 respectively. They were used as an equimolar mixture for all rounds

of amplification. 3' primers are derived from a and (3 constant regions. For the a

chain, 3' primer NJ108 was used in the first round of amplification, followed by

NJ109 and NJ110. For the (3 chain, MQ284 was the first 3' primer used, followed by

MS175. Primer sequences are listed in appendix 4. All PCR rounds were set up as

described in the section above using an annealing temperature of 53°C for 2 minutes

and amplifying for 28 cycles. Products obtained were 300-400 bp in size and were TA

cloned as described. Successfully ligated PCR products were identified by EcoR1

digestion of plasmid DNA and sequenced using the M13 primer (appendix 4).

2.15 Sequencing and T cell receptor sequence analysis

All sequencing reactions were performed at the Natural History Museum sequencing

facility. Plasmid DNA containing the sequence of interest was sequenced using a

sequence specific primer and the Big Dye Terminator v1.1 Cycle Sequencing Kit

(Applied Biosystems). Sequencing reaction products were analysed on an Applied

Biosystems 3130x1 DNA analyser and sequence data obtained was manipulated using

GeneJockeyll software. Raw sequence data files were read by EditView (Perkin

Elmer).

T cell receptor sequences and CDR3 region lengths were identified based on the

classifications of the International Immunogenetics (IMGT) information system

database (http://imgt.cines.fr) (Ruiz et al., 2000). CDR3 length was taken as the

number of amino acids between, and not including, the conserved cysteine residue (C)

at the 3' end of the V gene segment and the conserved phenylalanine residue (F) at the

5' end of the J segment.

100

2.16 TCR CDR3 Spectratyping

The sequences of the specific Vf3 gene primers and the constant Cr3 primer conjugated

to the fluorescent dye 6-carboxyflurorescein-amino-hexy (6-FAM) are shown in

appendix 5. PCR reactions were set up as described above with a final MgCl2

concentration of 3.5 mM. For a given cDNA template, each V13 primer was set up as a

separate PCR reaction with the constant C13-6FAM primer. PCR reaction mixtures

were incubated at 95°C for 10 minutes followed by 36 cycles of 94°C for 20 seconds,

55°C for 40 seconds, 72°C for 40 seconds and a final extension step of 72°C for 5

minutes. 10 til of each PCR reaction was run on a 1% agarose TAE gel to confirm

that the reaction had been successful before proceeding. PCR products were then run

on an ABI Prism 3100 Genetic Analyzer in ABI Prism 96 well optical reaction plates

(Applied Biosystems-403012) where each well of the plate contained 5 IA of PCR

product, 1011,1 of Hi-Di Formamide (Applied Biosystems-4311320) and 0.50 of a

Genescan 500 ROX standard (Applied Biosystems-401734). Data was collected and

analysed by Gene Mapper ID Software version 3.2 (Applied Biosystems).

2.17 Real- time PCR RNA and cDNA were prepared as described. 500 ng of DNase treated RNA was used

as a template in each cDNA reaction. PCR reactions were carried out in a volume of

20 Ill containing 1 !al of cDNA, 8 [Al of RNase free water, 1µ1 of 20x gene specific

primer mix (Applied Biosystems — Assays-on-DemandTM Gene Expression Assay)

(appendix 8) and 10 ttl of 2x TaqMan® Universal PCR Master Mix (Applied

Biosystems - 4304437). Reactions were incubated at 50°C for 2 minutes, 95°C for 10

minutes and then 40 cycles of 95°C for 15 seconds and 60°C for 1 minute. PCR

reactions were set up in triplicate for each set of primers used, and CT values (the PCR

cycle number at which the fluorescence passes a fixed threshold) were obtained using

a Stratagene MX3000P real-time PCR machine and software. Variability in the

amount of RNA used for each cDNA reaction was controlled for by running a PCR for

18s RNA for each sample. PCR of a dilution series of a single sample showed the 18s

and all other primer sets to have different amplification efficiencies. It was therefore

101

not possible to use the 2-mc` mathematical expression for fold change. CT values for

PCR reactions were instead converted to numerical values, using the equation for the

appropriate efficiency curve before normalising each gene of interest value with

respect to the 18s value for the same sample. The sample with the lowest level of gene

expression was assigned a value of 1. Levels of gene expression in all other samples

were expressed as a value relative to 1.

2.18 Preparation of DNA for oocyte pronuclear injection

Plasmid DNA purified using a commercial alkaline lysis based purification kit

(Qiagen-12162) was digested overnight with the appropriate restriction enzyme.

Restriction digests were performed in a volume of 60 Ill containing 15 [t.g of plasmid

DNA and at least 30 U of restriction enzyme. The required band was separated by

running the sample on a 0.8% low melting point agarose (Promega-V2831) TAE gel at

70 V for 6 hours. The fragment for injection was excised and gel purified using a

DNA binding column (Qiagen-28704). An additional wash step of 5 minutes was

added to the recommended protocol. DNA was eluted from the column in embryo

grade water (Sigma—W1503) and the DNA solution sterilised by passing though a

0.22um Costar spin filter (Fisher Scientific —MPA150010C). Purified DNA fragments

were quantitated using a spectrophotometer (Nanodrop® ND-1000) and by running

of the preparation on a 1% agarose TAE gel alongside a quantitative DNA ladder.

Purified DNA fragments were stored at 4°C until the day of injection when they were

diluted to a concentration of 2-4 ng/p,1 in embryo grade water in a final volume of 50

pl. Oocyte pronuclear injections were performed at the Nuffield Department of

Medicine (University of Oxford) or the MRC Clinical Sciences Centre (Hammersmith

campus of Imperial College London)

2.19 Genotyping

Genomic DNA from a small sample of tail tissue was extracted using a high salt DNA

extraction method (see above). Mice were assigned a number for identification based

on cage number and ear marking at the time of tail biopsy. Mice carrying the

transgene of interest were identified by PCR. The genotypes of transgenic founder

102

animals and of animals used for subsequent breeding were also confirmed by Southern

blot (see below).

2.20 Southern blotting

Restriction digests of genomic DNA were carried out in a reaction volume of 50 0

containing 10-15 [tg of genomic DNA, 50 U of appropriate restriction enzyme, 5 !al of

10X buffer supplied with enzyme (appendix 3), 1 mM Spermidine (Sigma-S2501),

100 µg/ml Ribonuclease A (Sigma-R4875) and 100 µg/ml BSA (Promega-R396D).

Reactions were incubated at 37°C overnight. Digests were run alongside a standard

ladder on a large 1% agarose TAE gel for 18 hours at 45V. Prior to blotting, the gel

was photographed alongside a ruler to allow determination of band size on the final

blot. Size markers and excess gel were trimmed away and the remaining gel incubated

in depurination solution (appendix 1) for 10 minutes at room temperature. Following

a brief wash in distilled water the gel was incubated in denaturing buffer (appendix 1)

for 45 minutes at room temperature and then in neutralising buffer (appendix 1) for a

further 45 minutes. The DNA was blotted overnight onto Hybond N+ (Amersham

Biosciences RPN203B), a positively charged nylon transfer membrane, using 20X

SSC (appendix 1) as the transfer buffer. DNA was irreversibly bound to the

membrane by exposure to UV light for 2 minutes. The membrane could then be

washed briefly in distilled water to remove any excess agarose, air-dried and stored at

4°C.

2.21 Southern blot Digoxigenin (DIG) probe labelling, hybridisation and detection

DNA fragments to be DIG labelled and used as Southern probes were generated by

PCR from plasmid DNA using primers specific to each transgene. PCR products were

purified by gel extraction and quantitated by running on a 1% agarose TAE gel

alongside a quantitative DNA ladder. 1-3 pg of DNA template was made up to a

volume of 16 0 with sterile water and boiled for 10 minutes to separate strands before

placing immediately on ice. 4 1A1 of DIG high prime mix (Roche-1 585 606)

containing digoxygenin labelled dUTP was added and the reaction incubated at 37°C

for 20 hours. The mixture was cleaned using a spin column (Bioline-BIO-28030) after

103

labelling to remove any unincorporated labelled nucleotides that could cause high

background. The amount of probe successfully labelled was quantitated by dot

blotting a dilution series of the probe, along with a dilution series of control labelled

DNA (Roche-1 585 738) onto a nylon membrane, cross linking with UV and then

carrying out the detection procedure described below.

The Southern blotted genomic DNA membrane was incubated with 10 ml of pre-

warmed DIG Easy Hyb hybridisation solution (Roche-1 796 895) with gentle rotation

at 42°C for 30 minutes. This solution was then discarded and the membrane incubated

at 42°C overnight with 8 ml of pre-warmed hybridisation solution containing 20 ng/ml

of denatured probe. The probe was denatured by boiling in 20 iil of water for 5

minutes and then placing immediately on ice. This hybridisation solution could be re-

used by storing at -20°C and heating to 68°C for 10 minutes immediately before use.

Following hybridisation the membrane was washed twice for 5 minutes in 2X SSC

containing 0.1% SDS at room temperature and then twice for 20 minutes in 0.5X SSC

containing 0.1% SDS at 65°C with gentle agitation.

For detection of the labelled probe, the membrane was rinsed briefly in maleic acid

wash buffer (appendix 1) before incubation in blocking solution (Roche-1 096 176) for

30 minutes with gentle agitation at 37°C. This blocking solution was discarded and

the membrane incubated for a further 30 minutes in blocking solution containing a

1:10,000 dilution of an anti-digoxigenin alkaline phosphatase conjugated antibody

(Roche-1 093 274). The membrane was washed twice for 15 minutes in maleic acid

wash buffer and then the membrane adjusted to the correct pH by equilibration for 5

minutes in detection buffer (appendixl). The membrane was then placed between two

sheets of soft plastic and 1 ml of the alkaline phosphatase chemiluminescent substrate,

CSPD, (Roche- 1 755 633) applied drop wise to the surface of the membrane. The

membrane was covered with the plastic sheet to distribute the substrate evenly over

the surface and then incubated for 5 minutes at room temperature before removing all

air bubbles and sealing the edges of the plastic by heat. The membrane was exposed

104

to light sensitive film (Sigma-Z37351650EA) for 15-120 minutes to achieve the

desired signal strength.

2.22 Tamoxifen induction

Cre nuclear localisation in MerCreMer (Bm70) transgenic animals was induced using

Tamoxifen (Sigma-T5648). Tamoxifen was administered at a dose of 1

mg/mouse//day for 5 consecutive days and was prepared by combining 10 mg

tamoxifen with 100 d EtOH and 2 ml of sunflower oil (Tesco pure sunflower oil).

The mixture was incubated in a sonic water bath at room temperature for 30 minutes.

200 [1,1 per mouse of the sonicated mixture was administered by i.p injection each day.

2.23 Footpad /flank peptide immunisation

The peptide PLP 56-70 (DYEYLINVIHAFQYV) was used for immunisation and in

vivo priming of T cell responses in NOD.E mice (Lund et al., 1990 and Takacs et al.,

1995). 50 IA.g of peptide, as an emulsion with either Complete Freund's Adjuvant

(CFA) (Sigma-F5881) or Incomplete Freund's Adjuvant (IFA) (Sigma-F5506), was

administered per immunisation in the footpad or the flank. 50 µ.1 or 100 pi of peptide

emulsion were injected into the footpad or flank respectively. Emulsions were a 1:1

mixture of peptide solution and CFA or IFA and were prepared by vortexing for 30

minutes, followed by incubation in an iced sonic water bath for 1 -2 hours.

2.24 Immediately ex vivo T cell proliferation assay

10 days (unless otherwise stated) after immunisation with peptide, the draining lymph

node (s) (DLN) and spleen were harvested. The popliteal lymph node drains from the

footpad and the inguinal lymph node drains from the flank. Single cell suspensions

were prepared in HL-1 medium (Cambrex-BE344017) supplemented with 2 mM L-

glutamine (Gibco-25030024), 25 Wm' Penicillin G (Gibco-15070063) and 25 µg/m1

Streptomycin sulfate (Gibco-15070063). Cells were counted using a haemocytometer

and were cultured in triplicate in 96- well plates (SLS-161093) with 3 x 105 cells per

well in the presence of different concentrations of peptide PLP 56-70

(DKEKLINVIHAFQYV). A substituted (Y-'K) version of the peptide PLP 56-70

105

was used for in vitro assays as the un-substituted peptide is very insoluble. After 2

days of culture, 100 Ill of supernatant was removed from each well. Supernatants

from triplicates were pooled and stored at —20 °C for subsequent cytokine analysis by

ELISA. 100 µ1 of fresh HL-1 culture medium was added to each well along with 5

[Xi of [3H] Thymidine (MP-2405905). Cells were cultured for an additional 18 hours

before plates were harvested (MACH III M Harvester 96) for beta scintillation

counting (Wallac 1450 microbeta TRILUX).

2.25 Culture of T cell lines

T cell lines from immunised footpad draining lymph nodes were set up with 3 x 106

DLN cells / ml in the presence of 25 [tg/ ml of PLP 56-70 (substituted). After 48

hours, IL-2 (10 IU/ml) (R&D systems-402-ML-020) was added to the culture medium

and the cultures incubated for an additional 8 days. Cells were then resuspended in

RPMI 1640 medium containing 10% Foetal Calf Serum (FCS) (Globepharm-batch

F30610), 50 I_LM beta mercaptoethanol (Sigma-M7522), 2 mM L-Glutamine 50 U/ml

Penicillin G and 50 µg/ml Streptomycin sulfate, and restimulated with 25 µg/ml of

peptide in the presence of 3 x 106 /ml of irradiated NOD.E splenocytes. The ratio of T

cells to irradiated APC in restimulation cultures was 1:25. Splenocytes were irradiated

by exposure to 3000 RADs of gamma radiation (Gammacell® 3000 Elan, Nordion

International Inc.). All T cell lines were repeatedly restimulated every 9-11 days and

maintained in 10 IU/ml IL-2. Proliferation assays were set up for each line at each

restimulation to check peptide specificity and cytokine production. Proliferation

assays were performed as described above with 3 x 105 irradiated splenocytes and 2 x

104 T cells in RPMI 1640 medium per well. RNA was also isolated from each cell

line at each restimulation.

2.26 Measurement of bronchial hyperreactivity (BHR)

Measurement of BHR was performed by recording respiratory pressure curves by

whole body plethysmography, calculated as Penh (Enhanced Pause, Buxco, USA).

Mice were placed, fully conscious, into individual plethysmograph chambers and

changes in air pressure between these chambers and a control chamber measured by a

106

differential pressure transducer to give a lung function measurement for each

individual mouse. Penh is a calculated value, which correlates with measurement of

airway resistance, impedance, and intrapleural pressure in the same mouse

(Hamelmann et al., 1997). Penh = ((Te-Tr)/Tr x PEP/PIP (Te = expiration time, Tr =

relaxation time, PEP = peak expiratory pressure, PIP= peak inspiratory pressure).

Following a baseline measurement, Penh measurements were taken for each mouse in

response to increasing doses (3, 10, 30 and 100 mg/ml) of methacholine (Sigma-

A2251).

2.27 Measurement of Resistance (R) and Compliance (Cdyn)

Mice were anaesthetised by sub-cutaneous (s.c) injection of 100 ILL1 of a 1:0.55:0.42

mix of PBS, Medetomidine hydrochloride (Pfizer-Domitor®) and ketamine (Fort

Dodge Animal Health Ltd-Ketaset®). After 15 minutes, a further 150 RI of anaesthetic

was administered by intra-muscular (i.m) injection before cannulation of the trachea.

Mice were placed onto an artificial ventilator and, following a baseline reading,

resistance and compliance measurements taken (Buxco, USA lung function

equipment) in response to PBS and increasing doses (3, 10, 30 and 100 mg/ml) of

methacholine.

2.28 Collection of Bronchoalveolar lavage (BAL) and serum

Unless mice had already undergone resistance and compliance lung function

measurement, in which case they were already unconscious, mice were anaesthetised

by intra-peritoneal (i.p) injection of 80 pi of a 2:1 mix of ketamine (Fort Dodge

Animal Health Ltd-Ketaset®) and xylazine (Bayer-Rompune). Mice were

exsanguinated by cardiac puncture followed by cannulation of the trachea. Lungs

were lavaged 3 times with 0.4 ml Phosphate Buffered Saline (PBS) (Invitrogen-10010-

015) using a 1 ml syringe and all BAL fluid for each mouse was pooled. BAL fluid

was centrifuged at 260 x g for 8 minutes. The supernatant was removed to a new tube

and stored at —80 °C for subsequent cytokine analysis by ELISA (see below). Blood

collected from cardiac puncture was left to coagulate at room temperature overnight

107

before centrifugation at 12,000 x g and collection of the serum fraction. Serum was

stored at -80 °C for subsequent analysis by ELISA.

2.29 Cell extraction from whole lung lobe

One lung lobe was finely chopped and placed into 5 ml of RPMI 1640 medium

(Gibco-31870-025) containing 0.15 mg/ml Collagenase D (Roche-11088858001) and

25 lig/m1DNase I (Roche-11284932001). The lung tissue was incubated with shaking

for 1 hour at 37°C and the mixture passed through a 70 pan cell strainer. Cells were

washed twice in RPMI 1640 medium before differential cell counting (see below).

2.30 Differential cell counting

Following centrifugation of BAL fluid or lung cells, cells were re-suspended in 250 .d

of RPMI culture medium. A small aliquot of this cell suspension was diluted 1:2 in

Turks stain (appendix 1) to lyse red blood cells, and white blood cells were counted on

a haemocytometer. Samples were adjusted to 5 x 105 cells / ml and 100 1A,1 (5 x 104

cells) spun onto a polysine glass microscope slide (VWR-6310107). Cytospins were

performed by centrifugation (Shandon-Cytospin 3) at 400 rpm for 4 minutes. Slides

were allowed to air dry for 5 minutes before fixing in methanol for 3 minutes. Wright

Giemsa staining of BAL cytospins allowed different cell types to be distinguished on

the basis of morphology (appendix 9). Slides were stained by immersing in Wright

Giemsa stain (Sigma-WG16) for 1 minute, followed by distilled water for 6 minutes

without agitation. Slides were rinsed briefly in running distilled water to stop stain

development and then allowed to air dry before addition of a cover slip and cell

counting. A total of 300 cells were counted on each slide and the total number of each

cell type recovered calculated by multiplying the percentage of each cell type by the

total number of cells isolated in each sample.

2.31 Lung homogenate

One lung lobe was weighed and placed into Hank's Balanced Salt Solution (HBSS)

without MgC12 or CaC12 (Invitrogen-14175-046), containing a mixture of protease

inhibitors (Roche-11836153001), at a concentration of 100 mg of lung per 1 ml of

108

HBSS solution. The lung was homogenised using an electric homogeniser before

centrifugation (1500 x g) and collection of the supernatant for analysis by ELISA. If

it was not possible to process the lung tissue immediately after harvesting, lung lobes

were snap frozen using liquid nitrogen and stored at -80 °C.

2.32 Lung histology - Tissue preparation

Lungs and heart were removed from the chest cavity en bloc prior to lung inflation or

harvesting of lung for RNA / cell extraction. If lung tissue was required for RNA or

cell extraction it was necessary isolate and prevent inflation of several lobes by tying

main bronchi with sewing cotton. Remaining lobes were inflated by intra-tracheal

infusion of a 1:1 mixture of PBS and Optimal Cutting Temperature (OCT) embedding

medium (Cell Path-KMA010000A). Inflated lung tissue was snap frozen in OCT

using a dry ice/isopentane (BDH-103614T) slurry and stored at -80°C. Sections were

cut on a cryostat (Bright) at a thickness of 5 IAM and mounted onto polysine glass

microscope slides. Sections were allowed to air dry for 15 minutes before staining or

storage at -20°C.

2.33 Haematoxylin and Eosin (H+E) staining

Slides were immersed in Harris Haematoxylin (VWR-351945S) for 20 seconds and

then held in running tap water until the sections were visibly blue. Slides were then

immersed in 1% Eosin (VWR-341973R) for 5 seconds before dehydration and cover

slip mounting. Sections were dehydrated by immersing, consecutively, for 30 seconds

with gentle agitation, in 70%, 90%, 100% and 100% Industrial Methylated Spirit

(IMS) (Fisher Scientific-M/4400/17) followed by 3 x 5 minute incubations in

Histoclear (National Diagnostics-HS200). Cover slips were applied with DPX

mountant (BDH-360294H).

2.34 Periodic Acid Schiff (PAS) staining and scoring

Immediately after sectioning, lung tissue was fixed by incubating slides in ice-cold

acetone for 15 minutes. Slides were allowed to air dry at room temperature before

oxidising for 5 minutes in 1% periodic acid (MP Biomedicals-102595). Slides were

109

rinsed briefly in distilled water and then immersed in Schiff's reagent (Fisher

Scientific-J/7300/PB08), in a fume hood, for 15 minutes. Slides were rinsed again in

distilled water and then held under running tap water for 5 minutes to terminate the

reaction. Nuclei were counterstained with Haematoxylin before dehydration and cover

slip mounting (see above). To score PAS stained sections, the percentage of cells

surrounding the lumen of each airway that were positively staining with PAS was

calculated. Each airway was assigned a score from 0 to 4 where:

0 = less than 5% of cells are positively stained

1 = 5-25% of cells are positively stained

2 = 26-50% of cells are positively stained

3 = 51-75% of cells are positively stained

4 = greater than 75% of cells are positively stained

The average airway score in each section was then calculated.

2.35 Immunohistochemistry - tissue fixation

A hydrophobic barrier (Vector-H4000) was drawn around each tissue section to allow

incubation of the section in small volumes (100-150 µl per section) of staining

solutions. Tissue sections were fixed using 4% paraformaldehyde (Sigma-P6148)

made up in PBS (Sigma-P4417). Sections were incubated with this fixative for 10

minutes before washing twice for 10 minutes in PBS. Peroxidase based or

fluorescence antibody staining was then performed.

2.36 Peroxidase based immunohistochemistry

Following fixation with paraformaldehyde, sections were incubated in 0.5% H202

(BDH laboratory supplies-101284N), made up in PBS, for 10 minutes. Sections were

washed twice for 10 minutes with PBS and then incubated in blocking solution

(appendix 1) for 30 minutes before rinsing in PBS. Sections were incubated for 15

minutes in Avidin block (Vector—SP2001) and 15 minutes in Biotin block (Vector-

SP2001), rinsing with PBS in between, before application of the primary antibody.

The primary and biotinylated secondary antibodies used for the detection of Cre,

CC10, IL-8 and myeloperoxidase proteins are shown in appendix 2. Antibodies were

110

diluted in block solution and sections incubated at room temperature overnight.

Sections were washed twice for 10 minutes in PBS and the secondary antibody applied

for 40 minutes at room temperature. Sections were washed twice for 10 minutes in

PBS, followed by application of an avidin/biotinylated peroxidase enzyme complex,

solution ABC (Vector-SP2001) for 30 minutes, and two further PBS washes. To

visualise antibody staining, solution AEC (Vector-SP2001) containing a peroxidase

enzyme substrate was applied and colour development monitored under a light

microscope. When the desired staining intensity was achieved, AEC solution was

removed and slides washed several times in distilled water to terminate the reaction.

Sections were then counterstained with hematoxylin to allow visualisation of tissue

morphology and cover slips mounted using a glycerol based mounting medium

(DAKO-00563). Images of tissue sections were captured using a light microscope

and digital camera (Leica MPS 60 with IM1000 software).

2.37 Immunofluorescence tissue staining

Following fixation in paraformaldehyde, sections were incubated in block solution for

30 minutes. Sections were washed twice for 10 minutes in PBS before application of

the primary antibody overnight at room temperature. Primary antibodies used for

fluorescence staining were as described for peroxidase staining. Sections were washed

twice for 10 minutes in PBS and then incubated with secondary antibody for 1 hour at

room temperature. For Cre staining, a TRITC conjugated donkey anti-rabbit antibody

(SantaCruz-SC2095) was used at a dilution of 1:400. For CC10 staining, a FITC

conjugated donkey anti-goat antibody (Jackson-705-095-147) was used at a dilution of

1:50. When double staining the same section for both Cre and CC10, the CC10

primary antibody was applied for 4 hours before incubation with the Cre primary

antibody overnight. Secondary antibodies were also applied sequentially for 1 hour

each, with the CC10 secondary antibody being applied first. Sections were washed

twice for 10 minutes in PBS and cover slips mounted using Vectashield (Vector-

H1200) to preserve the fluorescence lifetime. Images of fluorescence staining were

captured using a fluorescence microscope and digital camera (Leica MPS 60 with

IM1000 software).

111

2.38 Inflammatory cell scoring

The same inflammatory scoring system was used to assess levels of inflammation

around bronchi and blood vessels in both H+E and MPO stained lung sections. Each

airway and blood vessel in a section was assigned a score from 0 to 3 where:

0 = Bronchi or blood vessel is not surrounded by any inflammatory cells

1 = Bronchi or blood vessels is surrounded by the occasional inflammatory cell

2 = Inflammation completely surrounds and is 1-4 cells in diameter from the edge of

the bronchi or vessel

3 = Inflammation completely surrounds and is 5 or more cells in diameter from the

edge of the bronchi or vessel

Examples of bronchi and blood vessels assigned each score are shown in appendix 30.

The average score for all bronchi and the average score for all blood vessels in a

section was then calculated.

2.39 Induction of allergic airway disease using ovalbumin (OVA)

Groups of hIL-8 transgenic and littermate control mice were sensitised by i.p injection

of OVA (Sigma -A5503) (0.01 mg / mouse) in 0.3 ml of alum (Au-Gel-S, Serva

Electrophoresis, Heidelberg, Germany) (AMS Biotechnology-12261.01) on days 0 and

12. Control mice received the same volume of PBS in alum. All mice were

challenged on days 19, 20, 21, 22 and 23 with a 5% solution of OVA in PBS,

administered as a 20 minute aerosol (PART Medical Ltd — TurboBoyN compressor).

BHR was measured on day 24. Resistance and compliance measurements were taken

on day 25 and lung tissue and peripheral blood were harvested for analysis.

2.40 Enzyme Linked ImmunoSorbant Assay (ELISA)

Levels of cytokine production in cell culture supernatants and BAL were determined

by ELISA, measuring against linear standard curves. 100 1A,1 of capture antibody

(appendix 2) were added to wells of an ELISA plate (SLS-442404), the plate sealed

and incubated overnight at room temperature. The next day, wells were washed 3

times with 400 [11 of wash buffer (appendix 1) and plates blotted against paper towel to

112

remove all liquid. 300 11,1 of block solution (appendix 1) were added to each well and

plates incubated at room temperature for 1 hour. Wells were washed again and 50 pi

of the appropriate dilution of standard (appendix 2) or sample added. Plates were

covered and incubated at room temperature for 2 hours. Wells were washed again and

100 µ1 of the appropriate biotinylated detection antibody (appendix 2) added to each

well. Plates were covered and incubated at room temperature for another 2 hours

before another wash and addition of 100 !al of a 1/200 dilution of streptavidin Horse

Radish Peroxidase (HRP) (R&D systems-DY998). Plates were incubated with

streptavidin HRP for 20 minutes at room temperature before another wash step and

addition of 100m1 of substrate solution (R&D systems-DY999). Plates were incubated

at room temperature in the dark for 20-25 minutes before terminating the reaction with

of 50 µl of stop solution (1M H2SO4). The optical density of each well at 450nm was

determined using an ELISA plate reader (TECAN Sunrise) with wavelength correction

set to 570 nm.

2.41 Flow cytometric analysis

Fluorescence activated cell sorting (FACS) was performed on a BD Biosciences

FACSCalibur, with CellQuest software (Becton Dickinson UK LTD) and

fluorochrome-labelled antibodies. Single cell suspensions of thymocytes and

splenocytes were prepared and washed once with PBS containing 10% FCS. Red

blood cells in splenocyte preparations were lysed by resuspending briefly in distilled

water followed by an additional PBS wash. Cells were stained with Phycoerythrin

(PE) conjugated anti-mouse CD4 (eBioscience-12004183) and Fluorescein

isothiocyanate (FITC) conjugated anti-mouse CD8 (eBioscience-11008182) or with

FITC conjugated anti-mouse TCR (eBioscience-115961-81). Isotype matched

controls, FITC Rat IgG2a (eBioscience 11-4321-81) and PE Rat IgG (eBioscience 12-

4301-81) were also used. Cells were stained for 45 minutes in the dark at 4 °C and

washed twice with PBS containing 10% FCS before analysis.

113

Chapter 3

Generation of Thl and Th2 TCR a chain transgenic lines

Results

3.1 Determination of a and /3 TCR gene usage in Thl and Th2 lines specific for PLP

56-70

Short term Thl and Th2 polarised T cell lines derived from NOD.E mice and specific

for the peptide epitope PLP 56-70 had been previously generated in our laboratory.

The Thl line was maintained in IL-2 (330 IU/ml), IL-12 (100 IU/ml) and anti-IL-4 (10

µg/ml). The Th2 line was maintained in IL-2 (330 IU/ml), IL-4 (100 IU/ml) and anti-

IFN-y (10 µgimp. The lines were strongly polarised and highly peptide specific and

both had undergone three restimulations with NOD.E splenocytes and PLP 56-70.

cDNA from both lines was available for analysis.

TCR gene usage in the Thl and Th2 cell lines described above was determined using a

PCR strategy first described by Candeias et al. (Candeias et al., 1991). This strategy

uses sets of degenerate primers, designed to bind all known TCR a and TCR p sequences, in combination with primers specific for the TCR constant regions. The

strategy allows all TCR a or TCR 13 genes expressed by a population of T cells to be

identified from a single nested PCR experiment. The PCR strategy and PCR products

obtained from the Thl and Th2 T cell line cDNA are shown in figure 3.1.

114

z Z'

z 1--i

H bp

750 —III. 500 --110 250-0-

(b)

bp Z Q o

0 0

1000—* 750-10- 500-0-

250

(c)

Rearranged TCR a chain cDNA Rearranged TCR 13 chain cDNA

(a)

NW36 NK121 NW37 NK122 NW38 NK123 —IP- --110- --IP- -110. ___00. —110-

V J C V D J C

111111W/7 A I

4— 4— 4—NJ110 NJ109 NJ108

if-- Al—MS175 MQ284

(adapted from Candeias et al., 1991)

Figure 3.1 The PCR strategy used to determine a and p TCR gene usage in Thl and Th2 line cDNA. (a) A nested PCR strategy was used, involving 3 rounds of amplification for the TCR a chain and 2 for the TCR 13 chain. V region primers for both TCR a and TCR (3 chains are degenerate oligonucleotides designed to bind all known V genes. These primers were used in all PCR rounds. For the (3 chain, the C region primer MQ284 was used in the 1st round of amplification, followed by MS175. For the a chain, C region primers NJ108, NJ109 and NJ110 were used successively. The gel photos in (b) and (c) show the PCR products obtained from Thl and Th2 line cDNA using the TCR a chain and TCR p chain PCRs respectively. These PCR products are approximately 300bp in length and were TA cloned and sequenced in order to determine TCR gene usage for each T cell line.

115

TCR gene sequences amplified by PCR were identified by TA cloning and

sequencing. 29 TCR a clones were sequenced for both the Thl and Th2 T cell lines.

30 and 31 TCR p clones were sequenced for the Thl and Th2 lines respectively.

Dominant TCR a sequences were found in both cell lines. 48% of a clones sequenced

from the Thl line were identified as using the a chain variable region gene TRAV1OD

with the a chain joining region gene TRAJ58 (table 3.1). The CDR3 region of this

TCR a chain was 12 amino acids in length (CAASREGTGSKLSF). 31% of a clones

sequenced from the Th2 line were identified as using the a chain variable region gene

TRAV17 with the a chain joining region gene TRAJ50 (table 3.2). The CDR3 region

of this TCR a chain was 14 amino acids in length (CALEGIASSSFSKLVF). These

dominant Thl and Th2 TCR a chains were selected for transgenic expression.

Although both lines had a dominant TCR a chain sequence, the gene usage and CDR3

region sequences were more heterogeneous in the Th2 line. 6 different combinations

of variable and joining a chain genes and 6 different CDR3 a sequences were

identified in the Thl line compared with 9 different gene combinations and 12

different CDR3 a sequences in the Th2 line (figure 3.2). In keeping with the data

previously published by our laboratory (Boyton et al., 2002), there was a statistically

significant difference in CDR3 a length between Thl and Th2 derived sequences. The

Thl line had a mean CDR3 a length of 11.4 + 0.2 compared to 12.5 + 0.2 for the Th2

line. Comparison of these two mean values gives a P value of <0.0005.

There was no statistical difference in CDR3 p length between Thl and Th2 derived

sequences, which had mean values of 12.3 + 0.2 and 12.4 + 0.3 respectively (P =

0.974). No TCR p chain appeared dominant in either line (tables 3.3 and 3.4),

although as seen for the a chain genes, p gene usage and CDR3 region sequences were

more heterogeneous in the Th2 line (figure 3.2). The lack of a dominant TCR p chain

in either line made it impossible, at this stage, to determine which 13 chain was pairing

with the dominant Thl and Th2 TCR a chains. Indeed the heterogeneity of the 13

chains, particularly in the Th2 line, suggests that the dominant a chains are able to pair

with more than one (3.

116

Previously published data (Boyton et al., 2002) suggested that the dominant 14 amino

acid CDR3 a motif identified in the Th2 line would not be present in the Thl line, but

that the shorter, Thl derived, 12 amino acid CDR3 a motif would be permissible in

both Thl and Th2 cells. PCR primers specific to these CDR3 regions were used to

confirm this finding in these cell lines (figure 3.3). This data confirmed the selection

of these Thl and Th2 derived dominant TCR a chains as good candidates for

transgenic expression. No beta chain pair for either TCR a chain had been

determined. Therefore, only transgenes for expression of TCR a chains were

constructed. The intent was to identify beta chain pairs for each a chain in vivo and

also to use these animals to answer questions about the impact of the structurally

different Thl and Th2 a chains alone on T cell responses and beta chain selection.

117

Table 3.1 TCR a sequences from a Thl polarised NOD.E T cell line against PLP (56-70)

TA clone TRAV CDR3a amino acid sequence TRAJ CDR3 length

Th1 A1.5 10D CAASREGTGSKLSF 58 12 A1.10 A1.11 A1.16 A1.19 A1.20 A1.33 A1.42 A1.43 A1.44 A1.47 A1.48 A1.61 A1.62 A1.23 CAASKPNNRIFF 31 10 A1.4 17 CALEGRGGRALIF 15 11 A1.15 A1.18 A1.34 A1.45 A1.46 A1.49 A1.51 A1.9 4D-3 CAALPGTGSNRLTF 28 12 A1.17 A1.55 A1.8 CAAETNSAGNKLTF 17 A1.27 CAADSNYQLIW 33 9 A1.60

Mean Thl CDR3a length = 11.4 + 0.2 (SE)

118

Table 3.2 TCR a sequences from a Th2 polarised NOD.E T cell line against PLP (56-70)

TA clone TRAY CDR3a amino acid sequence TRAJ CDR3 length Th2

A2.13 17 CALEGIASSSFSKLVF 50 14 A2.15 A2.16 A2.20 A2.21 A2.33 A2.39 A2.41 A2.54 A2.10 12-2 CALSDANNYAQGLTF 112-2 13 A2.31 A2.38 A2.42 A2.32 13-4 CAMERQDNYAQGLTF A2.3 6-6 CALGEDTNAYKVIF 30 12

A2.17 CALVRDTGYQNFYF 49 A2.49 A2.53 CALGFQGGRALIF 15 11 A2.48 CALGSQGGRALIF A2.22 10 CAASRGNMGYKLTF 9 12 A2.25 A2.58 A2.14 3-3 CAVRGTNAYKVIF 30 11 A2.40 A2.55 A2.34 CAAGDTNTGKLTF 27 A2.35 A2.36 CAALNTNTGELTF A2.43 CAALNTNTGKLTF

Mean Th2 CDR3a length = 12.5 + 0.2 (SE)

119

Table 3.3 TCR 3 sequences from a Thl polarised NOD.E T cell line against PLP (56-70)

TA clone TRBV CDR3P amino acid sequence TRBJ CDR3 length

Thl B1.14 2 CASSQGPLSNERLFF 1.4 13 B1.36 B1.41 B1.81 B1.82 B1.83 B1.98 B1.57 CASSKAGTGEDTQYF B1.1 CASSQEMQGQDTQYF B1.94 B1.95 B1.37 CASSQQGDQDTQYF 12 B1.60 CASSQEGTGVQDTQYF 14 B1.3 , CASSRDWGDTQYF 11 B1.21 B1.85 B1.90 B1.70 CASSPWGVQDTQYF 2.5 12 B1.93 B1.10 : CASSQAGTDTQYF 11 B1.18 19 CASSRSGDQDTQYF 12 B1.58 B1.88 B1.92 B1.96 B1.97 B1.77 16 CASSNQNYAEQFF 2.1 11 B1.84 CASSLAGQGARSQNTLY] 2.4 16 B1.89 CASSSRDWGDEQYF 2.7 12 B1.8 15 CASSLVGAEQFF 2.4 10

Mean Thl CDR3p length = 12.3 + 0.2 (SE)

120

Table 3.4 TCR 13 sequences from a Th2 polarised NOD.E T cell line against PLP (56-70)

TA clone TRBV CDR3[3 amino acid sequence TRBJ CDR3 length

Th2 B2.2 16 CASSFQTGGAETLYF 2.3 13 B2.7 B2.5 CASSVRDWGDTQYF 2.5 12 B2.84 CASSLRGHTEVFF 1.1 11 B2.53 CASSLRNTEVFF 10 B2.4 : CASSQEAGGVDTQYF 13 B2.8 B2.56 CASSQEGWGPYEQYF 2.7 B2.61 CASSQGLGNYAEQFF 2.1 B2.66 CASSQEGTGGYAEQFF 14 B2.82 CASSQDGTGGYAEQFF B2.78 CASSLGTGDAEQFF 12 B2.30 CASSPANSDYTF 1.2 10 B2.46 CASSQGIYEQYF 2.7 B2.74 CASSLRDNGDTQYF 2.5 12 B2.13 19 CASSPRTTSGNTLYF 1.3 13 B2.71 B2.31 CASSSPTGGWNAWQFF 2.1 14 B2.50 CASSSPTGGWNAEQFF B2.86 B2.76 CASSPPTGSPNERLFF 1.4 B2.79 B2.85 CASSIEDSGTEVFF 1.1 12 B2.26 4 CASSPRGLYAEQFF B2.43 CASSNYAEQFF 9 B2.68 13-3 CASSGRTTANTEVFF 13 B2.75 20 CGARDLWGGKNTLYF 2.4 B2.83 B2.80 1 CTCSADGSYEQYF 2.7 11 B2.24 17 CASSSGGFAETLYF 2.3 12 B2.63 CASRDWGETLYF 10

Mean Th2 CDR3(3 length = 12.4 + 0.3 (SE)

121

(a) Thl line TCR a gene usage TCR 13 gene usage

VlOD - 358 V17 - 315 V4D3 - 328 V4D3 - 333 V4D3 - 317 V10 - 358

V2 - 31.4 V19 - 32.1 V4 - 31.4 V4 - 32.5 V5 - 32.5

_ V16 - 32.1 • V16 - 32.4 E V16 - 32.7 • V15 - 32.4

(b) Th2 line

TCR a gene usage

TCR 13, gene usage

E V17 - 350 'V12-2 - 3112-2 V10 - 39 V3-3 - 330 V6-6 - 349 V3-3 - 327

• V6-6 - 315 V6-6 - J30

E V13-4 - 3112-2

EV16 - 32.3 riV2 - 31.1

V19-31.3 :7,V19 - 32.1 EV19 -31.4 ,V20 - 32.4 E V2 -32.1 EV16 - 31.1 E V2 - 32.7 55V17 - 32.3 E V4 - 31.1

V2 - 31.2 EIV2 - 32.5

V16 - 32.5 EIV19 - 32.1 E V19 - 31.1

V13-3-31.1 E V1 - 32.7

Figure 3.2 A comparison of TCR a and gene usage in Thl and Th2 T cell lines in order to select chains for transgenic expression. TCR gene usage in a Thl and Th2 T cell line are shown in (a) and (b) respectively. Each segment of a pie chart represents an individual CDR3 sequence, while V-J gene combinations are differentiated by colour. The percentage of each a repertoire made up by the most dominant TCR chain is also shown. The dominant a chains for both the Thl and Th2 lines were selected to be expressed transgenically, but diverse gene usage and CDR3 regions in TCR 13 chain repertoires meant that (3 chain pairs could not be identified.

122

bp

Thl CDR3a motif Th2 CDR3a motif

a.) .0 .0 .0 .0 —

H H. 4 0 4 z

500

250-'10.

Figure 3.3 Presence or absence of the dominant Thl and Th2 CDR3 a motifs in Thl and Th2 lines. Primers specific for the dominant Thl 12 amino acid CDR3 motif and Th2 14 amino acid CDR3 a motif were used with C region primer NJ108 to determine presence of these motifs in Thl and Th2 line cDNA. The shorter Thl motif is present in both Thl and Th2 lines, while the longer Th2 motif is only present in the Th2 line.

123

3.2 Construction of Thl and Th2 TCR a chain transgenes.

Several different cassette vectors have been used to achieve transgenic expression of

TCR genes. Promoter sequences that have been used in these vectors to drive TCR

expression include CD2 (McHugh et al., 2001) and MHC class I (Kb) (Mendel et al.,

2004). However, as expression of TCR genes is not an endogenous function of these

promoters, TCRs under the control of these vectors can be subject to abnormal timing

and regulation of expression. The vector used in this study (pTa) was designed by

Kouskoff et al. (Kouskoff et al., 1995) and utilises endogenous mouse TCR

promoter and enhancer elements. As such, the vector contains many kilobases of non-

coding sequence cloned from regions upstream and downstream of the mouse TCR

locus. The aim is to ensure that TCR expression is, as far as possible, under the control

of its natural regulatory elements. The vector also contains the mouse TCR Ca gene

and associated regulatory sequences. To express a TCR a chain transgenically it is

necessary to clone into the vector the rearranged V-J coding sequence of interest.

Many TCR transgenics have been created using TCR a or I chain cDNA. However,

although in the genome of a T cell, TCR genes have been rearranged and spliced

together, some intronic sequences remain. Intronic sequence is present at the 5' and 3'

ends of the rearranged V-J segment, but also separates a coding leader sequence from

the remainder of the V gene (figure 3.4). To express a TCR a chain using the pTa

cassette vector it is essential to include a short section of intronic sequence 3' to the

rearranged J gene. This allows splicing of the V-J coding segment to the Ca gene also

present in the vector. It is unknown exactly what role the other intronic sequences

may play in the regulation and processing of the TCR, but it is thought advisable to

include them in the construct. Therefore, the T cell genomic configuration of the V-J

segment, rather than that found in cDNA should be used to create the TCR transgene.

124

fa) Non-T cell genome

Intron LP1 V J C // //

(b) T cell genome

Intron LP1 V C

umoma:zzzmu---// CDR3

(c) T cell mRNA

LP1

V

C

CDR3

(d) Required TCR gene arrangement in transgenic expression vector

Intron LP1 V

il= r af i I- '—r--)

CDR3

Figure 3.4 The different states of arrangement of TCR a genes. (a) No rearrangement of TCR a genes occurs in non-T cells. Germ-line configuration is maintained. (b) In the thymus, rearrangement of TCR a genes occurs in T cells such that a V gene is spliced to a J gene at the level of genomic DNA, to create a variable CDR3 a region. Intronic sequence remains between the V leader region (LP1) and the rest of the V gene and also between the J and C regions. (c) These intronic sequences are removed by splicing at the level of mRNA prior to expression of a functional TCR a chain on the surface of the T cell. (d) To express a TCR a chain transgenically, the arrangement of TCR a genes should resemble that found in the T cell genome, as intronic sequences present in this configuration facilitate correct processing and expression of the TCR.

125

For the T cell lines and T cell receptor sequences being used in this study, only cDNA

samples were available. It was therefore not possible to obtain the correct T cell

genomic configuration of the Thl and Th2 TCR V-J segments by a single PCR

strategy.

The only region of each required TCR a V-J segment not present in non-T cell

genomic DNA was the junction between the rearranged V and J gene (CDR3 region).

The CDR3 region is present in cDNA and so it was possible to create the required

configuration of each V-J segment by cloning together fragments derived from non-T

cell NOD.E genomic DNA and Thl or Th2 line cDNA. The cloning strategies,

including PCR primers and restriction enzyme sites, used for both the Thl and Th2

TCR a chain constructs are shown in figures 3.5 and 3.6.

Three PCR fragments were used to create each TCR a V-J segment. The 2 intron

containing fragments were derived from non-T cell NOD.E genomic DNA and

fragments containing the rearranged 12 amino acid and 14 amino acid CDR3 regions

were derived from Thl and Th2 cDNA respectively. The sequences of all PCR

primers used for these cloning strategies, along with the PCR conditions and product

sizes are shown in appendix 6. The PCR products cloned, and the joining together of

these PCR fragments to create the Thl and Th2 TCR a transgenes, are shown in figure

3.7. For both the Thl and Th2 strategies it was necessary to create a Hind III

restriction site in the J region in order to join the PCR fragments. In both cases the

change was a single base pair mutation from G to T, introduced using the PCR primers

used to obtain the PCR fragments. Mutations were silent and resulted in the creation

of an alternative codon for the amino acid leucine. All fragments generated by PCR

were verified by sequencing prior to proceeding with subsequent stages of the cloning

strategy. The nucleotide and amino acid sequences of the Thl and Th2 TCR a V-J

segments are shown in appendices 17 and 18 and appendices 19 and 20 respectively.

126

Xma I 11..2MAI

V Nco I Hind III

VIO J58 LP1

(b)

Fragments joined using Hind III

(ii) (iii)

NOD.E genomic DNA Thl line cDNA NOD.E genomic DNA

TRA V10

Xma 1

Nco I

ThlintronF

Nco- I Hind III

HindIIITh IF

Hind - III

Sac II

LP1 V10 V10 J58 J58

Th1LP1/2IntronR

ThlHindIIIR TRAJ58

Fragments joined using Nco I

(c) Xma I Nco I

Iimmi Hind III Sac II

LP1 V10 J58

Complete Thl VI 0/J58 fragment cloned into the pTa expression vector using Xma I and Sac II

(d) Sal I

Xma I

Sac II

pTa Sal I cassette vector --......,

Thl a construct is linearised for injection using Sal I Figure 3.5 A schematic diagram showing the cloning strategy used to create the Thl a chain construct. It was only possible to obtain the correct CDR3 a region from Thl line cDNA. However, cDNA derived TCR sequence does not contain the required intronic regions (figure 3.4). (a) The TCR a chain was therefore created using 2 intron containing fragments (i) and (iii) derived from NOD.E genomic DNA and one CDR3 a containing fragment (ii) obtained by PCR from Thl line cDNA. (b) The 5' intronic fragment and that containing the CDR3 region were cloned together using a unique and endogenous NcoI site. (c) It was necessary to introduce a unique HindIII site into the sequence of J58 by PCR in order to join the 3' intronic fragment. The G-*T mutation introduced does not alter the amino acid sequence of the final protein. (d) XmaI and SacII sites were introduced by PCR at the 5' and 3' ends of the a chain respectively to allow directional cloning into the pTa cassette vector. Bacterial sequences were removed from the vector, by digestion with Sall, prior to pronuclear injection. All PCR primers and conditions are shown in appendix 6.

127

Sac II Sac I

V17

Hind III

J50

Fragments joined using Sac I

Xma I

LP1 Complete Th2 V17/J50 fragment is cloned into the pTa expression vector using Xma I and Sac II

(c)

Sac II

Xma I I 11011=.11 1

V17 J50 Fragments joined using Hind III

(b) V Sac I Hind III

LP1

Xma I

(ii) (iii)

NOD.E genomic DNA Th2 line cDNA NOD.E genomic DNA TRAV 17

Th2intronF

Sac I Hind III

Adomr2=1]

Th2HindIIIF

Hind III

Xma I

Sac I

Sac II

LP1 V17 V17 J50 J50

Th2LP 1 /2IntronR Th2HindIIIR TRAJ50

Th2 a construct is linearised for injection using Sal I Figure 3.6 A schematic diagram showing the cloning strategy used to create the Th2 a chain construct. It was only possible to obtain the correct CDR3 a region from Th2 line cDNA. However, cDNA derived TCR sequence does not contain the required intronic regions (figure 3.4). (a) The TCR a chain was therefore created using 2 intron containing fragments (i) and (iii) derived from NOD.E genomic DNA and one CDR3 a containing fragment (ii) obtained by PCR from Th2 line cDNA. (b) The 5' intronic fragment and that containing the CDR3 region were cloned together using a unique and endogenous Sad site. (c) It was necessary to introduce a unique HindIII site into the sequence of J50 by PCR in order to join the 3' intronic fragment. The G---*T mutation introduced does not alter the amino acid sequence of the final protein. (d) XmaI and SacII sites were introduced by PCR at the 5' and 3' ends of the a chain respectively to allow directional cloning into the pTa cassette vector. Bacterial sequences were removed from the vector, by digestion with Sall, prior to pronuclear injection. All PCR primers and conditions are shown in appendix 6.

128

To enable each complete V-J segment to be inserted into the pTa cassette vector,

XmaI and SacII restriction sites were introduced by PCR at the 5' and 3' ends

respectively (figure 3.7). Screening for successfully ligated clones by restriction

enzyme digest was possible due to the difference in size between the V-J a segment

being removed and that being introduced (figure 3.8). The large size of the pTa

cassette vector, and the tendency of this plasmid to undergo rearrangements and

deletions during manipulation, meant that it was also necessary to check patterns of

digest with other restriction enzymes, such as EcoRl (figure 3.8). Observation of the

same patterns of digest before and after manipulation meant that the cassette vector

was intact. That the Thl and Th2 TCR a V-J segments were correct within the final

constructs was also confirmed by sequencing. Plasmid maps of the Thl and Th2 TCR

a constructs are shown in appendices 10 and 11 respectively.

Introducing bacterial sequences from the cloning vector into the mouse genome during

transgenesis is not recommended and can result in greatly reduced levels of transgene

expression or even no expression of the transgene at all. (Kjer-Nielsen et al., 1992).

Bacterial sequences of the pTa cassette vector were therefore removed from both the

Thl and Th2 TCR a constructs, by digestion with Sal I, prior to pronuclear injection.

The 15,000 bp fragment of each construct purified for injection is shown in figure 3.8.

129

Th2

< < < 0 z z .z :. . 0 .... . :. ....

z

750 —10 500-0.

250

(a)

750 —* 500 —0.

250 —*

bp

(b)

bp

Thl

z z z

z z z

(c) (d)

(EcoR1) (EcoR1)

bp c (X

maI

/ Sa

cII)

1000-0' 1000-0 750-10 750 500 500

Figure 3.7 Assembly of Thl and Th2 TCR a chain constructs. All gel lane labels refer to fragments described in figures 3.5 and 3.6 for Thl and Th2 constructs respectively. (a) and (b) show the 3 PCR fragments required for assembly of the Thl and Th2 TCR a chains respectively. (c) and (d) show the joining of these fragments and the successful introduction of XmaI and SacII sites at either end of these constructs. All construct fragments shown in (c) and (d) are in the cloning vector pCRe2.1, and within this vector are flanked by EcoR1 sites.

130

cd bp

Thl

tu

10000 6000

3000

2000

Original pTa Thl

cd o cn v) $... --- --.. N T' ' r4 bp "0 X"0 c.71

E o X

10000 6000 3000

1000 750

500

Th2

Th2

o o 8 a

(a)

(b)

Figure 3.8 Complete Thl and Th2 TCR a constructs. Thl and Th2 VJ fragments were inserted into the pTa cassette vector by directional cloning using XmaI and SacII. (a) XmaI/SacII digests of the final Thl and Th2 constructs show the presence of the respective VJ regions (661 and 622 bp) replacing a larger VJ fragment (approx. 950 bp) used previously in this vector. The pTa vector is very large (20,000 bp) and so can be prone to rearrangements during manipulation. EcoRI digests of the Thl and Th2 constructs, and of the original vector, demonstrate that no rearrangements or deletions have occurred. (b) It was necessary to remove the 5000 bp bacterial sequence from the Thl and Th2 vectors prior to pronuclear injection. This was achieved for both constructs by digestion with Sall and purification of the upper 15,000 bp band.

131

3.3 Identification of Thl and Th2 TCR a chain transgenic founder animals.

The inbred mouse strains used most frequently for pronuclear injection are FVB/N and

C57BL/6. These strains respond well to super-ovulation and pronuclear embryos are

generally larger and less fragile than those of other mouse strains, making them

technically easier to inject and giving better rates of embryo survival and transgene

integration (Brinster et al., 1985). However, the Thl and Th2 cell lines, from which

the transgenic TCR a chains were identified, were originally derived from NOD.E

mice. In NOD and NOD.E mouse strains, the peptide epitope for which the TCR a

chains are specific, PLP 56-70, is presented in the context of the MEIC class II

molecule I-Age. Transgenic mice carrying either TCR a chain transgene must

therefore also express I-Age in order for T cell responses to PLP 56-70 to be studied,

and TCR I chain pairs to be identified in these animals. To circumvent the need to

cross the transgenic founder animals onto a NOD.E background, the Thl and Th2

TCR a chain transgenes were initially injected into NOD oocytes. Despite their

fragility, NOD oocytes have successfully been used to generate transgenic lines

(O'Shea et al., 2006 and Birk et al., 1996). However, multiple attempts to inject the

TCR constructs into these pronuclear embryos failed to generate any litters.

Therefore, to achieve transgenesis, it was necessary to use C57BL/6 or FVBN oocytes.

48 potential founder animals obtained from pro-nuclear injection of FVB/N oocytes at

the Nuffield department of Medicine in Oxford were screened for the presence of the

Th2 TCR a chain transgene. An initial PCR screen, using genomic DNA extracted

from a tail biopsy, identified 2 founder animals (figure 3.9). These founder animals

are subsequently referred to as Th2 a line 20 and Th2 a line 34, based on numbers

assigned to each animal at the point of tail biopsy. The transgenic status of these two

founder animals was confirmed by Southern blotting. The entire rearranged V-J

segment present in the construct was used to probe genomic DNA that had been

digested using the restriction enzyme EcoRI (figure 3.9). EcoRl does cut within the

construct itself, but sites are only 3' to the V-J segment with the most 5' site 3013 bp

from the 5' end of the construct. Integration of a transgene into the genome can occur

132

at one or multiple sites and at each site, multiple copies of the transgene are often

found as head to tail concatamers. On the Th2 TCR a line Southern blot, bands

present in transgenic and littermate samples are due to binding of the probe to

endogenous, unrearranged, TRAV17 and TRAJ50 sequences. Bands that are

differential between the two transgenic lines represent different sites of integration

into the genome, while the most intense band, which is 6 kilobases in size, is due to

the head to tail concatamers present at one or more of the integration sites.

23 potential founder animals obtained from pronuclear injection of C57BL/6 oocytes

at the MRC Clinical Sciences Centre, Imperial college, were screened for the presence

of the Thl TCR a chain transgene. An initial PCR screen identified 1 founder animal,

Thl a line 30 (figure 3.10). The transgenic status of this animal was confirmed by

Southern blot using the same strategy as that described for the Th2 TCR a chain, but

using the Thl rearranged V-J segment as a probe (figure 3.10).

The sequence and product sizes of all PCR and Southern primers used for screening of

Thl and Th2 TCR a chain transgenic founder animals are shown in appendix 6.

NOD.E mice were used as breeding partners for subsequent breeding of all TCR a

chain transgenic founder animals. New generations of each line were also backcrossed

onto NOD.E. As NOD.E mice are homozygous for the MHC class II molecule I-M7,

all TCR a chain transgenic animals subsequent to the founder will express the

transgene and I-Ag7.

133

LP I V17

Sac II (a)

TRA,150

J50

Th2HindIIIR

TRAV 17 Th2lntronF

Xma I

1000 500 250

(c)

35-48 Potential founders 1-19 bp

rn

21-28 29-33 •2

r-A---•

(b) z . 8 0 a, 3

kb

10 8 —0. 6 —+ 5-4 4-4

3 2.5—+

2--+

E E

V

DIG labelled probe

unde

r

Line

34 fo

unde

r

Figure 3.9 Identification of Th2 TCR a chain transgenic founder animals. Transgenic founders were identified by PCR and by Southern blot. The location of primers used for PCR screening (Th2lntronF and Th2HindIIIR) and for generation of the Southern probe (TRAV17 and TRAJ50) are shown in (a). Two transgenic animals were identified from an initial PCR screen of 48 potential founders (b). The transgenic status of these two animals (numbers 20 and 34) was then confirmed by Southern blot (c). The restriction enzyme used to digest genomic DNA for Southern blotting was EcoRI (E). This enzyme only cuts the integrated construct on the 3' side of the V-J region being used as a probe (c), giving information about construct integration sites and copy number.

134

1000 500 250

(c) kb

Lin

e 30 fo

unde

r

12-11. 8 —IP. 6—O. 5 4 —111

3 —110.

C J

EEE E

V

DIG labelled probe

(a)

TRAV 1 0 ThllntronF

Xma I

Sac II

LP1 TRAVIO TRAJ58

ThlHindIIIR

TRA,158

4::, M <

Z cli .,18 Z q Potential founders 16-29 1 31-39 2 r.._____—•1/4—._._Th 1 r______—A_____‘

Figure 3.10 Identification of the Thl TCR a chain transgenic founder animal. The transgenic founder was identified by PCR and by Southern blot. The location of primers used for PCR screening (ThllntronF and Thl HindIIIR) and for generation of the Southern probe (TRAV10 and TRAJ58) are shown in (a). One transgenic animal was identified from an initial PCR screen of 23 potential founders (b). The transgenic status of this animal (number 30) was then confirmed by Southern blot (c). The restriction enzyme used to digest genomic DNA for Southern blotting was EcoRI (E). This enzyme only cuts the integrated construct on the 3' side of the V-J region being used as a probe (c), giving information about construct integration sites and copy number.

135

(b)

bp

3.4 Determination of TCR a chain transgene expression.

The TCR a chain transgenes contain multiple regions of intronic sequence as well as

coding regions for the Thl and Th2 rearranged V-J segments and the mouse Ca gene.

For correct expression, all intronic regions should be removed and the V-J coding

segment spliced to the Ca gene.

A PCR strategy was used to demonstrate that correct processing of the transgenic TCR

a chain, by removal of intronic sequence, and expression of the TCR a chain coding

sequence, at the level of mRNA, was occurring in T cells from both of the Th2 TCR a

chain transgenic lines (figure 3.11) and the Thl TCR a chain transgenic line (figure

3.12). Removal of the intronic sequence at the 5' end of the V gene results in a shorter

V-J segment and therefore a smaller PCR product when cDNA is used as a template

compared to genomic DNA.

Correct splicing of the TCR a V-J segments to the Ca gene present in the transgene

was demonstrated by using the nested PCR strategy described in figure 3.1 to look at

TCR a chain usage in these animals. The strategy uses primers specific for mouse V

genes in combination with a primer specific for the mouse Ca gene. In the transgene,

the J region and C region are separated by many kilobases of intronic sequence. A

PCR product of the correct size will only be obtained if splicing has occurred.

Splenocyte cDNA was used as a template for PCR and the sequences obtained from

each line are shown in tables 3.5 and 3.6. From Th2 a line 20, all a sequences

obtained were transgenic. From Th2 a line 34, most sequences were transgenic but a

small number of endogenous TCR a sequences were also seen. From Thl a line 30

the percentage of the repertoire made up by the transgenic TCR alpha chain was more

variable. In one animal 16% of the sequences obtained were transgenic compared to

54% for another. This data suggest that in the Th2 a chain transgenic lines levels of

expression of the transgenic TCR are high, but are not completely exclusive of

endogenous TCR. In the Thl a chain transgenic line expression levels may be more

136

variable and a significant number of T cells bear endogenous T cell receptors in these

animals.

Although data presented thus far confirms correct processing and high levels of

expression of the TCR a chain transgenes at the level of mRNA, they do not provide

evidence for expression of the TCR chains at the surface of the T cell. Direct evidence

for this is difficult to obtain as no antibodies exist against either TRAV10 or TRAV17.

Other single TCR a chain transgenic models do exist and studies with these mice have

demonstrated that mice expressing a TCR a chain transgene have an increased

percentage of double negative (DN) thymocytes compared to wild-type mice (Huang

and Kanagawa, 2004). During normal thymocyte development, the TCR p locus is the

first to rearrange (Mombaerts et al., 1992b). Successfully rearranged p chains then

pair with a surrogate a chain known as the pre-TCR a. This pre-TCRa/r3 complex

mediates the differentiation of the thymocyte from CD4-CD8- (double negative) to

CD4+CD8÷ (double positive) (Fehling et al., 1995). Studies of TCR a chain

transgenic mice suggest that premature expression of a rearranged a chain at the DN

stage of thymocyte development precludes the formation of pre-TCRa/f3 complexes.

This results in less efficient transition of thymocytes from DN to DP and an increased

percentage of DN thymocytes. Staining of thymocytes with antibodies against CD4

and CD8 showed this to be true for both lines of Th2 TCR a chain transgenic mice

(figure 3.13). Additional evidence for altered TCR expression at the cell surface also

comes from the observation that Th2 TCR a chain transgenic animals have a reduced

CD4+: CD8+ ratio in the thymus. However, this difference in ratio was not observed in

splenocytes (P = 0.9977 and P = 0.1999 for lines 34 and 20 compared to littermates

respectively) and so the CD4+: CD8+ ratio of T cells in the periphery seems to be

unaffected by Th2 TCR a chain transgene expression. An increased percentage of

double negative thymocytes was also observed in Thl TCR a chain transgenic mice

(figure 3.14). However, there was no change in the CD4+: CD8+ ratio in the thymi or

splenocytes of these animals.

137

Despite high levels of transgene expression in Th2 TCR a chain transgenic animals,

and significant levels of expression in some Thl TCR a chain transgenics, staining of

splenocytes with an antibody directed against the constant region of the TCR

demonstrated normal levels of a/13 TCR at the cell surface (figure 3.15).

138

(b) Transgenic T cell cDNA Th2lntronF —1

LP1

Th2HindIIIR

V17 J50

(c)

,-c) -,:, by ,-,.:, ct _..1

1000 750 500 250

-i-R 71-

< 0 N cn :4 4 '1) a) a) *., CI =

..-. a.'+

(d)

N c.) - 1- cn

,- ,4.

bp

1000 750 500

250

527bp

(a) Transgenic genome Th2lntronF Th2HindIIIR

—.111111-1111 LP1 V17 J50

391bp

Figure 3.11 Demonstration of correct Th2 TCR a chain transgene processing and expression by PCR. Splicing out of intronic sequence should occur during correct processing and expression of the TCR a chain transgene. PCR using primers shown in (a) and (b) should demonstrate a size difference in PCR product between the genomic configuration of the construct (a) and that obtained from transgenic T cells where intronic sequence has been removed (b). An HPRT control PCR demonstrates successful reverse transcription from transgenic and littermate control splenocyte RNA (c). Expression of the transgene and correct splicing of intronic sequence has been demonstrated by PCR from splenocyte cDNA in both Th2 TCR a chain transgenic lines (d).

139

(b) Transgenic T cell cDNA

LP1 V10

372bp

V10

J58

► ThlHindIHR

J58

4111 ThlIntronF

LP1

-rt bp -cf ccs

1000 750

500

250

Tra

nsge

nic

gDN

A

0 •

0

Cyd

538bp

(a) Transgenic genome ThlIntronF ThlHindIIIR

-111=-- 77/7/7/A

(c)

Figure 3.12 Demonstration of correct Thl TCR a chain transgene processing and expression by PCR. Splicing out of intronic sequence should occur during correct processing and expression of the TCR a chain transgene. PCR using primers shown in (a) and (b) should demonstrate a size difference in PCR product between the genomic configuration of the construct (a) and that obtained from transgenic T cells where intronic sequence has been removed (b). (c) Expression of the transgene, and correct splicing of intronic sequence, has been demonstrated by PCR in the Thl TCR a chain transgenic line using splenocyte cDNA. A low level of expression of this V10/J58 TCR sequence was also detected in the littermate cDNA sample.

140

Table 3.5 TCR a chain sequences from splenocytes of Th2 TCR a chain transgenic and littermate control animals.

TA clone TRAV CDR3a amino acid sequence TRAJ CDR3 length

Th2 a line 20 20-2 Al 17 CALEGIASSSFSKLVF 50 14 20-2 A3 20-2 A4 20-2 A8 20-2 A9 20-2 A10 20-2 All 20-2 Al2 20-2 A13 20-2 A14

Th2 a line 34 34-20 AI 17 CALEGIASSSFSKLVF 50 14 34-20 A2 34-20 A4 34-20 A5 34-20 A7 34-20 A10 34-20 All 34-20 A6 4D-3 CAAVNYGGSGNKLIF 32 13 34-20 A9 CASSGSWQLIF 22 9

Littermate 34-21A15 4D-3 CAAETNTDKVVF 34 10 34-21A16 CAAPDSNYQLIW 33 34-21A17 CAAYIASSSFSKLVF 50 13 34-21 A3 CAADNTNTGKLTF 27 11 34-21 A2 4D-4 CAAGLPNYNVLYF 21 34-21Al2 CAARNSGYNKLTF 11 34-21 A5 4-4 CAAENTGNYKYVF 40 34-21 A9 CAAEITGNTRKLIF 37 12

141

Table 3.6 TCR a chain sequences from 2 Thl TCR a chain transgenic animals.

TA clone TRAY CDR3a amino acid sequence TRAJ CDR3 length

Thl a line 30 Th1-56 Al4 10D CAASREGTGSKLSF 58 12 Th1-56 A8 Th1-56 A5 CAALGAGNTRKLIF 37 Thl-56 A6 Thl-56A4 4D-3 CAAPPNYGNEKITF 48 Th1-56 Al6 CAAETGTGGYKVVF 12 Th1-56 Al7 CAAEATRGNNKLTF 56 Th1-56 A3 4-3 CAAEIMGTYQRF 13 10 Th1-56 Al5 CAAEGVNTGNYKYVF 40 13 Th1-56 Al 4D-4 CAAVNTGNYKYVF 11 Th1-56 A7 4-4 CAAEAKNTGYQNF 49 13 Thl-56A10 4-2 CAAEDANNRIFF 31 10

Th1-63 A2 10D CAASREGTGSKLSF 58 12 Thl-63 A3 Thl-63 A8 Thl-63 All Thl-63 Al2 Thl-63 A13 Thl-63 A16 Th1-63 Al 4-4 CAAEAGGADRLTF 45 11 Th1-63 A20 Th1-63 Al9 CAAEKDSGTYQRF 13 Th1-63 A4 4D-3 Th1-63 Al8 CAAEEDMGYKLTF 9 Th1-63 Al0 CVAEDYGGSGNKLIF 32 13

142

Littermate Line 20 Tg Line 34 Tg

16.4% 71.7% O

O

TV

• • •

•• • •

LL

:i•N .... a. 4:1.,f .,:•• .....'..5;.,

N.--4- • • •••••.. • ...n” o .0 10o 101 102 3 1 0 10 10 —.10 103 101 ^100 101 102 10 104 101 102 3

FL 1-H -

O

O

Ct".0 LL

0

FL1-H FL I-H

CD8

40

46 10

0

(b)

• •

* *

littermates line 34 line 20

(c) (d) 5 •

•

CD4

/CD

8 ra

tio

littermates line line 34 line 20

4 • •

3 • • • ■

2- • •

1 • •• • ■

0 littermates line 34 line 20

0 TS2 3 co O 0 2 -4-

0

0

Figure 3.13 An increased percentage of double negative thymocytes and a decreased CD4:CD8 ratio suggests expression of the Th2 a chain transgene at the cell surface early in thymocyte development. (a) Thymocytes from both lines of Th2 TCR a chain transgenic animals and littermate control animals were stained with CD4-PE and CD8-FITC antibodies. FACS analysis showed an increased percentage of double negative thymocytes in transgenic animals compared to controls (b). The CD4:CD8 ratio was also decreased in thymocytes from transgenic animals (c) but no change in CD4:CD8 ratio was observed in splenocytes (d). Data points for individual animals are shown and mean values + SD are also marked. The statistical significance of differences between transgenic and littermate groups were tested using a non-paired t test, ***P < 0.001, **** P < 0.0001, and data shown is representative of two independent experiments.

143

30-o

.c 20-z 46 10-

3

2

•

•

CD

4/CD

8 ra

tio

3

CD4J

CD

8 ra

tio

2-

1-

litterinates Th1 he 30 0 0

littermates Thl line 30

4 4-

1-

(a) Littermate Thl 30 Tg

A

o

6.66% . . .'72.76%

f: •

.. .. ..

)5.90% 3.68%

7.94% ... 65.92% • :./

•sA. .. , ''' '

• e: .

'' ...

: . 3.396/0 - . 2.76%

FL1-H 161

2 3

10 10 FL1-1-1

104

CD8

(b) 40 **

0

(c)

litterrnates Th1 line 30

(d)

Figure 3.14 An increased percentage of double negative thymocytes suggests expression of the Thl a chain transgene at the cell surface early in thymocyte development. (a) Thymocytes from Thl TCR a chain transgenic and litter mate control animals were stained with anti CD4 PE and anti CD8 FITC antibodies. FACS analysis showed an increased percentage of double negative thymocytes in transgenic animals compared to controls (b). There was no difference in the CD4:CD8 ratio observed between the two groups in either thymocytes (c) or splenocytes (d). Data points for individual animals are shown and mean values + SD are also marked. The statistical significance of differences between transgenic and littermate groups were tested using a non-paired t test, **P < 0.01, and data shown is representative of a single experiment.

144

102 FL1.H

0

Isotype control Littermate

Th2 line 20 Th2 line 34

10 10

Isotype control Littermate

Thl line 30

(a) Th2

(b) Thl

...... 100 10 10 2 '103 -104

FL I -H

Figure 3.15 Transgenic expression of Thl or Th2 T cell receptor a chains does not affect the levels of TCR at the cell surface. Splenocytes from TCR oc chain transgenic and littermate control animals were stained with an anti TCR [3 chain FITC conjugated antibody. No differences in levels of TCR at the cell surface were observed in either Th2 TCR a chain transgenic line (a) or the Thl TCR a chain transgenic line (b) when compared to littermate control animals.

U

145

Discussion

Many factors are known to influence CD4+ T lymphocyte differentiation and there is a

growing body of evidence to suggest that the avidity and affinity of the TCR/pMHC

interaction is important. Most studies suggest that a low affinity interaction between

the TCR and pMHC complex will favour a Th2 response, while a high affinity

interaction promotes Thl development (Malherbe et al., 2000). A study from our

laboratory in 2002 observed that CD4+ T cells selected under Th2 polarising

conditions favoured an elongated TCR CDR3a chain and predicted that these

elongated TCR a chains may result in less optimal (lower affinity) interactions

between the class II pMHC complex and CDR loops of the TCR (Boyton et al., 2002).

As a continuation of this work, the aim of this study was to express these elongated

Th2 and shorter Thl TCR a chains transgenically to determine if TCR structure can

cause an inherent Thl or Th2 bias in CD4+ effector cell phenotype. This chapter has

described identification of TCR a chains derived from polarised T cell lines and the

generation of Thl and Th2 TCR a chain transgenic mice.

A PCR strategy was used to determine the profile of TCR a and TCR r3 gene usage in

the cDNA of short term polarised NOD.E Thl and Th2 cell lines, both of which were

specific for an antigen (PLP 56-70) used in the previous study. Dominant TCR a

chains were present in both cell lines and there was a statistically significant difference

in CDR3a length between Th 1 and Th2, with the CDR3a regions of the Th2 line

being longer than those of the Thl. This data is in keeping with the findings of the

original study from which these lines derive, as was the observation that there was no

significant difference in CDR3 (3 length and that the Th2 CDR3 motif could not be

identified in the Thl line by PCR. No clonal expansion of a dominant TCR p chain

was observed in either line, suggesting that the dominant TCR a chains were able to

form peptide specific heterodimers with more than one p chain. However, as the

dominant TCR a chains identified made up a significant proportion, but the not the

entirety, of each line no single TCR (3 chain partner for either a chain could be

146

confidently identified. Selective expansion of TCR sequences, which then become

dominant during the course of an immune response has been well documented in

studies of both mice and humans and for both CD4 and CD8 responses (Wedderburn

et al., 1993, Fasso et al., 2000 and Zhong and Reinherz, 2004). However, these

studies mostly report the presence of dominant TCR a and p chain pairs or have

observed narrowing of the TCR (3 chain repertoire. That a TCR a chain, using a

conserved CDR3 a region, i.e. not just a V gene bias, should come to dominate a TCR

repertoire in the absence of any simultaneous TCR p sequence expansion is perhaps

unexpected. During normal T cell development in the thymus, TCR p chain

rearrangement and cell surface expression occurs before that of the a chain (Kishi et

al., 1991). Transition of thymocytes from double negative to double positive denotes

successful (3 chain rearrangement and selection and is accompanied by cell

proliferation (Hoffman et al., 1996). Many T cells bearing the same TCR p chain with

an identical CDR3 (3 region will therefore be present in the thymus, and populations of

mature T cells exiting the thymus with the same TCR (3 chain, but pairing with

different TCR a chains are likely. However, that exactly the same TCR a chain will

be generated by different T cells as they undergo TCR a chain rearrangement is a

random event and therefore T cells exiting the thymus carrying the same TCR a chain

but different TCR p chains are likely to occur at a much lower frequency. The TCR a

chain bias observed in the lines used for this study may therefore be of great

significance, in terms of providing peptide specific structural features to the TCR

heterodimer that are selected for under Thl or Th2 polarising conditions, and lends

further weight to the argument that an inherent bias may be observed upon transgenic

expression of these TCR a chains. However, transgenic expression of the TCR a

chain alone is unlikely to yield an inherent peripheral repertoire of T cells with the

correct antigen specificity. One approach to finding TCR (3 chain partners that formed

antigen specific receptors with the expanded a chains would have been to use a T cell

transfection technique. In vitro expression and analysis of TCRs has been described

by many other groups (Sugiyama et al., 2004) and transfecting T cells lacking

endogenous TCR with the Thl and Th2 TCR a chains, along with TCR (3 chains

147

identified from the diverse repertoire of each line, would have allowed identification

of [3 chain partners which were able to form stable heterodimers with the required

specificity for the antigen PLP 56-70. However, given the unexpected, but striking,

TCR a chain bias observed in both Thl and Th2 cell lines, further questions arise

about whether indeed the dominant TCR a chains alone could be responsible for any

observed phenotype bias. As such it was decided that the Thl and Th2 TCR a chains

should be expressed transgenically in the absence of an identified antigen specific

TCR 13 chain pair, and that pairing would be subsequently established from in vitro

and in vivo studies of these TCR a chain transgenic mice.

Following construction of transgenes for both Thl and Th2 TCR a chains, mice

transgenic for each chain were identified by both PCR and Southern blot. 2 lines of

mice for the Th2 TCR a chain were established, and one line of mice carried the Thl

TCR a chain. Founder animals were crossed, and then each subsequent generation

backcrossed, onto the NOD.E strain of mice as this strain expresses the MHC class II

molecule, I-Ag7, to which the peptide specificity of the original Thl and Th2 cell lines

was restricted. At each generation, mice for subsequent breeding were identified by

both PCR and Southern blot, as germline transmission of transgenes can sometimes

result in loss of transgene copies (Kim et al., 2004). As it was not practical to examine

levels of transgene expression in each line at each new generation, transgene copy

number by southern blot was taken as a surrogate marker that the new generation was

the same as the previous.

The most straightforward way to determine expression of a TCR transgene would be

to use a V region specific antibody. However, although many antibodies exist against

V(3 sequences, very few Va antibodies are available. Cell surface staining of T cells

from transgenic animals was therefore not possible as a means of determining

transgene expression in these mice. Direct observation of transgene expression at the

level of cDNA was possible however, and a PCR strategy demonstrated transgene

expression, and indeed correct splicing out of intronic sequences present in the V

region of each transgene, for each line of transgenic mice. Transgenes for both Thl

148

and Th2 TCR a chains were derived from endogenous mouse TCR a promoter and

enhancer elements contained within the transgenic cassette vectors designed by

Kouskoff et al. (Kouskoff et al., 1995), NOD.E genomic DNA and Thl or Th2 cell

line cDNA. In using these endogenous elements from the TCR a locus, as far as

possible, expression patterns of the transgenic TCR chains should be normal.

However, it is well documented that mice expressing a TCR a chain transgene have an

increased percentage of DN thymocytes compared to wild-type mice (Huang and

Kanagawa, 2004). This is a consequence of the necessity to include pre-rearranged V

and J gene segments in the transgenic construct and altered percentages of thymocyte

populations result from early expression of the transgenic TCR a chain compared to

endogenous a chains. This consequence of TCR a chain transgenesis was alluded to

in the original paper by Kouskoff et al. where the transgenic vectors used in this study

were first described. In this study, high expression levels of transgenes were

documented in double negative thymocytes. The impact of this premature TCR

expression on T cell development and selection was addressed in a recent study by

Baldwin et al. In this study, correct timing of TCR a chain expression was achieved

using the Cre/lox system of conditional gene targeting where Cre was expressed under

the control of the CD4 promoter/enhancer and a TCR a chain transgene was under the

control of a constitutively active promoter, but preceded by a foxed stop sequence

(Baldwin et al., 2005). TCR a chain expression was therefore restricted to DP and SP

thymocytes. The authors report that, when the TCR a chain was expressed in DP

thymocytes, no perturbations of thymocyte DN and DP populations were observed

and, by using a well characterised HY TCR transgenic line, they demonstrated that in

male mice negative selection of HY specific thymocytes occurred at the single positive

(SP) stage of thymocyte development rather than at the DP stage reported in

conventional HY transgenic animals. Thus, the unphysiological expression pattern of

TCR a chains observed in studies using conventional TCR expression vectors are non-

ideal and potential consequences for normal T cell development and selection should

be born in mind. However, in this study, the expected block in thymocyte

development observed serves to answer an important question regarding transgene

expression. Increased percentages of double negative thymocytes were observed in all

149

3 lines of TCR a chain transgenic animals and because thymocyte development is

perturbed by early expression of the TCR a chain at the cell surface, that the Thl and

Th2 TCR a chain transgenes are indeed being expressed at the cell surface can be

surmised.

As both the Thl and Th2 TCR a chains were derived from CD4+ T cells, which are

restricted to antigen recognition in the context of MHC class II molecules, a shift

towards the dominance of CD4+ T cells in the periphery may have been predicted.

Other studies using a13 transgenic mice have seen a large shift in ratio towards CD4+

or CD8+ T cells both in the thymus and the periphery depending on whether the TCR

in question was MHC class II or MHC class I restricted respectively (Lobito et al.,

2002). This was observed in the study by Lobito et al. using an MHC class II

restricted TCR, although they reported a small increase in CD8+ T cells when the TCR

a chain of the receptor was expressed alone. In our study, no statistically significant

shift in CD4/CD8 ratio was observed in the periphery of either Th 1 or Th2 TCR a

chain transgenic animals, although a small apparent shift towards CD8+ T cells in the

Th2 TCR a transgenics, particularly line 20, may have become more apparent if more

mice were included in the analysis. There is however, a significant shift towards

CD8+ T cells in the thymi of these mice. That the same shift is not observed in the

Thl TCR a chain transgenic line suggests that this difference is inherent to the

different TCR a chains being expressed rather than a consequence of transgene

expression per se, but the lack of CD4+ T cell bias in either line suggests that MHC

class restriction is not mediated by either TCR a chain alone and that the transgenic

TCR a chains are likely to be present on the surface of both CD4+ and CD8+ T cells.

In the absence of an antibody and despite some evidence that both TCR a chain

transgenes were being expressed at the cell surface, levels of transgene expression in

the periphery could only be assessed by TCR sequence analysis. However, for all 3

transgenic lines, normal levels of TCR expression on peripheral T cells, whether

endogenous or transgenic, were demonstrated using an antibody specific for the

constant region of the TCR. This finding is particularly important in light of findings

150

about T cell phenotype and is discussed in chapter 4. Sequence analysis of TCRs

expressed by peripheral T cells from splenocytes of both Th2 TCR a chain transgenic

lines and the Thl TCR a chain transgenic line revealed the presence of transgenic

sequences in all samples. However, the percentage of the repertoire comprising the

transgenic sequence was very different between Thl and Th2 animals. From Th2

TCR a chain transgenic animals, almost all sequences obtained derived from the

transgene, with only the occasional endogenous sequence identified. In contrast, no

more than 50% of sequences from Thl TCR a chain transgenic animals were

transgenic and percentages between animals were highly variable. There are several

possible reasons for these variations. Expression levels of transgenes are known to be

highly variable depending on the site of transgene integration (Robertson et al., 1995)

and so the Thl TCR a chain transgene may be integrated into a locus of the genome

permitting weaker expression of the transgene than those in which the Th2 a chain

transgene has integrated. However, if the block at the DN stage of thymocyte

development is any indication of the level of transgene expression, then the

thymocytes of Thl and Th2 transgenic animals appear to be very similar. Another

possibility is that many T cells bearing the transgenic Thl TCR a chain are negatively

selected in the thymus and therefore do not proceed to become part of the peripheral T

cell repertoire. The Thl a chain was originally identified from a highly specific, but

also highly polarized T cell line, whose TCRs were predicted to display a high affinity

for pMHC. T cells with a high affinity for self pMHC are known to be negatively

selected in the thymus (Alam et al., 1996) and it may be that T cells bearing this

shorter CDR3 a region of the Thl TCR a chain are more likely to make high affinity

interactions with self pMHC compared to those bearing the longer CDR3 a region of

the Th2 TCR a chain. This hypothesis would be most easily tested by sorting

different populations of T cells as they proceed through the different stages of T cell

development, i.e. DN, DP, SP and mature T cells and analysing the repertoire of TCR

a chains at each stage, although the picture will be complicated by the appearance of

endogenous TCR a at a different time point to that of the transgene. Perhaps a cleaner

system in which to analyse thymic selection of this TCR a chain would be to use mice

151

in which the endogenous TCR a locus is disrupted. Also a possibility is transgene

deletion as there is some evidence that TCR transgenes can become deleted in

peripheral T cells (Komori S et al., 1993). This issue could be resolved by single cell

sorting of peripheral T cells and using PCR to detect the presence or absence of the

transgene at the level of genomic DNA and cDNA. A final explanation for apparent

low levels of expression in the Thl TCR a chain transgenic line is that many T cells

are expressing the transgenic a chain as well as endogenous a chains, i.e. that there

are many dual a expressing cells in these animals. This may equally apply to

endogenous TCR a sequences detected in samples from Th2 TCR a chain transgenics.

Again, single cell sorting could determine whether some T cells from these animals

were expressing two TCR a chains. These latter two possibilities would also help to

explain the variability observed between animals of the same line.

Data in this chapter has described the identification of dominant Th 1 and Th2 cell

derived TCR a chains and the strategy used to express these TCR chains

transgenically. In vitro and in vivo studies using these animals to try to determine

antigen specific TCR 13 chain pairing, and to determine whether T cells expressing

these chains display any inherent effector phenotype bias are described in the next

chapter.

152

Chapter 4

TCR 6 chain selection and cytokine phenotype in Thl and Th2 TCR

a chain transgenics

Results

4.1 Analysis of TCR /3 chain usage in unprimed Th2 TCR a chain transgenics.

Previous analysis of TCR a chain usage in the Th2 TCR a chain transgenic animals

had shown that, in both transgenic lines, most T cells in the periphery bear the

transgenic TCR a chain (table 3.5). Using the PCR strategy described previously

(figure 3.1) splenocytes from unprimed mice were used to examine TCR 13 chain usage

in these animals (figure 4.1). The aim was to determine whether dominance of this

transgenic TCR a chain generates any innate bias in the TCR (3 chain repertoire of

these animals in the absence of exogenous priming with the antigen (PLP 56-70) for

which the TCR, from which the a chain derives, was specific. However, no

preference for a particular TCR (3 chain was observed in either line, and the pattern of

TCR (3 chain genes used was very similar between transgenic and littermate samples.

There were also no differences in the length of CDR3 regions seen in the TCR (3

chains. From this sequence data it seems that the peripheral TCR (3 chain repertoire is

unaffected by the dominant expression of the transgenic Th2 TCR a chain. However,

as the number of sequences obtained for any one sample represent just a tiny fraction

of TCR (3 chains present in the periphery, any subtle impact of the Th2 TCR a chain

on the 13 chain repertoire may be hard to detect using this approach. CDR3 spectratype

analysis was therefore used in order to build a more global picture of TCR (3 chain

usage in the periphery of these animals. CDR3 spectratype analysis was originally

developed and described by Pannetier et al. (Pannetier et al., 1993). It uses TRBV

gene specific primers in combination with either a fluorescently labelled constant

153

region primer or fluorescently labelled TRBJ gene specific primers to amplify all TCR

p chain sequences in a given sample. Products from each PCR reaction are then

analysed according to size. A typical profile of product sizes for any given primer

combination usually consists of 4 or more discrete peaks, spaced 3 nucleotides apart,

that follow a Gaussian (normal) distribution. Clonal expansion or bias towards a

particular TCR p chain is observed as a dominant peak or skewing of the peak profile

away from a normal Gaussian distribution. To analyse TCR p chain usage in the

periphery of the Th2 TCR a chain transgenic animals, TRBV gene specific primers

were used in combination with a fluorescently labelled constant region primer.

Profiles obtained for each TRBV gene were then compared between transgenic and

littermate animals (figure 4.2). No major differences were observed between the

spectra of transgenic and littermate animals. There was also no evidence of clonal

expansion for any TRBV gene, with all spectra displaying a normal distribution of

product sizes. Good spectra were not obtained for all TRBV genes. These genes (e.g.

TRBV 23, TRBV 21, TRBV 30 and TRBV 24) were also never observed from

sequence analysis of transgenic or littermate splenocytes samples and so as a

consequence of genetic background are probably poorly represented in the periphery

of these animals. It is not possible from this spectratyping data to determine the CDR3

lengths represented by individual peaks as they represent combinations of each TRBV

gene with many different TRBJ genes, but the similarity in spectra between transgenic

and littermate samples suggests that there are no differences in average TCR I CDR3

length or TRBV gene usage as a consequence of transgenic Th2 TCR a chain

expression.

154

TA clone TRBV CDR3(3 amino acid sequence TRBJ CDR3 length

Th2 a line 20 20-2 B1 20-2 B7 20-2 B9 20-2 B2 20-2 B5 20-2 B4 20-2 B6 20-2 B8

20-2 B10 Mean CDR3 length

2 CASSQDSQNILYF 2-4 11 CASSPDRGRNTLYF 12 CASSQGQGSYEQYF 2-7

20 CGARLGLNQDTQYF 2-5 13-2 CASGDAGFNQDTQYF

13

CASGDWGSGNTLYF 1-3

12 16 CASSFRDRGF 2-7

8

17 CASSPDNYEQYF

10 19 CATLGQNYAEQF F 2-1

11 11.2

Th2 a line 34 34-23 B1 34-23 B9 34-23 B3

34-23 B10 34-23 B8 34-23 B5 34-23 B6 34-23 B2 34-23 B11 34-23 B7

Mean CDR3 length

17 CASSRTGGQDTQYF 2-5 12 CASSRLGTGDTQYF

2 CASSQERRINQDTQYF 14 13-2 CASGDARWGGSQDTQl 15 19 CASSILTGSNQDTQYF 14

CASSILSQNTLYF 2-4 11 15 CASSSGQQNTLYF 29 CASGTNTEVFF 1-1

9

CASSPPTGRSAETLYF 2-3

14 4 CASSSGQGDNQAPLF 1-5

13 12.5

Littermate 20-31 B1

20-31 B12 20-31 B6 20-31 B2 20-31 B3

20-31 B11 20-31 B4 20-31 B8 20-31 B9

20-31 B10 Mean CDR3 length

29 CASSSRQGMDSPLYF 1-6 CASRDQGANTGQLYF 2-2 CASSRTGYEQYF 2-7

15 CASSLDRDEQYF 13-1 CASSLWGNQDTQYF 2-5

CASSDWGASYEQYF 2-7 2 CASSQDRDYEQYF

13-3 CASGVLGGGAETLYF 2-3 16 CASSLGTGGYEQYF 2-7

13

10

12

11 13 12 11.8

Figure 4.1 TCR 13 chain sequences from splenocytes of unprimed Th2 TCR a chain transgenic and littermate control animals. A PCR strategy (figure 3.1) was used to determine TCR (3 chain usage in Th2 TCR a chain transgenic and littermate splenocytes.

155

10 170 150 147 160 170 200 271

Th2 TCR transgenic

, A

ft 1'11

Littermate

IAA ))

I\A

ft\

TRBV 1

TRBV 2 TRBV 4

TRBV 5 TRBV 12-1 TRBV 13-1 TRBV 13-2 TRBV 13-3

Th2 TCR a transgenic

Littermate

TRBV 14 TRBV 15 TRBV 16 TRBV 17 TRBV 19 TRBV 20 TRBV 29 TRBV 31

Figure 4.2 Spectratype analysis of TCR 13 chain gene usage in splenocytes of unprimed Th2 TCR a chain transgenic and littermate control animals. TRBV gene specific primers were used with a fluorescently labelled constant region primer to amplify TCR (3 chain sequences from splenocyte cDNA. Analysis of product sizes obtained from each PCR reaction showed no major differences in spectra obtained for transgenic compared to littermate control animals and no evidence of clonal expansion for any TCR (3 chain in the presence of a dominant Th2 TCR a chain. Numbers at the top of each spectra indicate the size of PCR product in base pairs. Data shown is representative of 2 animals per group.

4.2. Analysis of TCR fi chain usage in Th2 TCR a chain transgenics in the context of

antigen specificity.

The data from unprimed animals suggests no bias in TCR 13 chain repertoire in the

context of a dominant transgenic Th2 TCR a chain and that the transgenic a chain is

able to form a functional TCR with many different TCR 13 chains with different

lengths of CDR3. However, the TCRs present in the periphery of the animals used to

generate this data have an enormous range of antigen specificities. Structural

constraints on which TCR 13 chains may be able to pair with the transgenic Th2 TCR a

chain may be more apparent in the context of an antigen specific response. PLP (56-

70) is the antigen for which the original TCR, from which the transgenic Th2 TCR a

chain was derived, was specific. Th2 TCR a chain transgenic and littermate animals

were immunised in the footpad with PLP 56-70. The peptide was administered as an

emulsion with Complete Freund's Adjuvant (CFA) in order to generate an immune

response and 10 days after immunisation, popliteal draining lymph nodes from these

animals were harvested and peptide specificity and TCR [3 chain usage determined.

Good proliferation in response to antigen was seen from both transgenic and littermate

lymph node T cells, but neither sequence analysis (figure 4.3) nor CDR3 spectratype

analysis (figure 4.4) showed any evidence of clonal expansion, TRBV gene bias or

differences in TRBV gene usage and CDR3 length between Th2 TCR a chain

transgenic and littermate animals.

157

20-131311 13-2 CASGSGGPNQDTQYF 2-5 13 20-13B13 CASGDAGGRDTQYF 12 20-13 B5 CASGGLGDEQYF 2-7 10 20-13 B9 CASGDALGASAETLYF 2-3 14 20-13 B1 19 CASSPDSAETLYF 11 20-13 B4 CASRQTGGDQDTQYF 2-5 13 20-13 B7 13-1 CASSDGGGPEVFF 1-1 11

20-13 BIO CASSPGENTLYF 2-4 10 20-13 B6 31 CAWTTGGTTGWLYF 2-2 12 20-13 B8 4 CASSWDWEQDTQYF 2-5

Mean CDR3 11.8

Th2 a line 34 34-3 BIO 16 CASSPRQGENTGQLYF 2-2 14 34-3 B11 CASSLVDAANSDYTF 1-2 13 34-3 B7 CASSLTGGVQDTQYF 2-5 34-3 B8 15 CASSLAPGLGKDTQYF 14 34-3 B9 CASSRQGNTEVFF 1-1 11 34-3B12 13-1 CA SSDAYRGGTEVFF 13 34-3 B1 CASSDEGYTLYF 2-4 10 34-3 B3 5 CASSQQGEQYF 2-7 9 34-3 B5 19 CASSIEDQGARTERLFF 1-4 15

Mean CDR3 12.4

Littermate 34-1 B7 13-2 CASGGRDRASERLFF 1-4 13 34-1B11 1-4 34-1 B2 13-1 CASSDVWDWGYEQYF 2-7

34-1 B15 CASSDGQGDNQAPLF 1-5 34-1 B8 CASSDSRLGGAETLYF 2-3 14

34-1 B13 CASSDQGANTEVFF 1-1 12 34-1 B3 2 CASSQDLSNQAPLF 1-5 34-1 B5 CASSQQNSDYTF 1-2 10 34-1 B6 20 CGARDWGGAYAEQFF 2-1 13

34-1 B14 19 CASSILGGEETLYF 2-3 12 Mean CDR3 12.5

M 70000—g 80000-. 50000-

40000.

30000. 2 20000.

10000-g. 0

10 20 30 40 50 peptide concentration (p.almli

8000 700 800 5000 4000o

E 3000 E zoos

1000

10 20 30 40 50 peptide concentration (p0/m1)

5000

S 4000

3000

2000

a 1000

10 20 30 40 50 peptide concentration (µ0/m1)

TA clone TRBV CDR3p amino acid sequence TRBJ CDR3 length

Th2 a line 20

Figure 4.3 TCR I chain sequences from primed draining lymph node of Th2 TCR a chain transgenic and littermate control animals. Mice were primed with the peptide PLP 56-70 by footpad immunisation. The draining popliteal lymph node was harvested at day 10 and peptide specificity of T cells determined by proliferation assay. TCR (I chain usage was determined by PCR. The proliferation data shown corresponds to the draining lymph node from which each set of sequence data is derived.

158

_A m

170 80 130 140

A A V

TRBV 4 TRBV 5 TRBV 12-1 TRBV 13-1 TRBV 13-2 TRBV 13-3 1.

TRBV 1 160

TRBV 2 00 000 210 220

I I)

180 130 ISO

110

TRBV 14 TRBV 15 TRBV 16 TRBV 17 TRBV 19 TRBV 20 TRBV 29 TRBV 31 14) 150 170 100 150 160 160 200 510

A )

Th2 TCR a transgenic

Littermate

Th2 TCR transgenic

Littermate

Figure 4.4 Spectratype analysis of TCR 13 chain gene usage in primed draining lymph node T cells of Th2 TCR a chain transgenic and littermate control animals. TRBV gene specific primers were used with a fluorescently labelled constant region primer to amplify TCR 13 chain sequences from draining lymph node cell cDNA. Analysis of product sizes obtained from each PCR reaction showed no major differences in spectra obtained for transgenic compared to littermate control animals and no evidence of clonal expansion for any TCR [3 chain in the context of a dominant Th2 TCR a chain and PLP peptide specificity. Numbers at the top of each spectra indicate the size of PCR product in base pairs. Data shown is representative of 2 animals per group.

4.3. Analysis of PLP (56-70) specific Th2 TCR a chain transgenic T cell lines.

The lack of a dominant TCR (3 chain in the draining lymph nodes of PLP 56-70

immunised transgenic animals suggests that a single priming with antigen and 10 days

between priming and harvesting of the lymph node was not sufficient for a clonal

expansion to occur. Antigen specific T cell lines were therefore established from Th2

TCR a chain transgenic and littermate draining lymph nodes and grown in vitro over

many weeks with repeated exposure to antigen and APC in growth medium containing

10U/m1 IL-2. T cell lines were restimulated with antigen (PLP 56-70) every 9-11 days

and splenocytes from NOD.E mice were used to present the peptide at each

restimulation, as APCs from this mouse strain carry the MHC class II molecule I-Ag7,

against which the original Th2 TCR was restricted. TCR p chain usage in these PLP

56-70 specific cell lines was analysed over successive restimulations. Figure 4.5

shows TCR p chain sequence data for Th2 TCR a chain transgenic (derived from

transgenic line 20) and littermate T cell lines grown over 6 restimulations. A clonal

expansion of a TCR (3 chain was apparent at the 4th restimulation of the transgenic T

cell line and this 13 chain (TRBV 31 / TRBJ 2-3) with a CDR3 p length of 12 amino

acids, comprised 100 % of all sequences obtained at the 6th restimulation. In contrast,

the littermate derived line showed no evidence of clonal expansion at the 4th

restimulation and the most significant expansion at the 6th restimulation comprised just

21% of the sequenced repertoire. However, despite the major differences in TRBV

gene usage between the two lines, CDR3 (3 lengths remained similar throughout.

The rapid narrowing of the TCR p chain repertoire in the transgenic line compared to

the littermate line was also seen from CDR3 spectratyping data (figure 4.6). For each

of the TRBV genes shown, even at the 2nd restimulation, the number of peaks in each

spectra was greater in the littermate line than in the transgenic line where all spectra

rapidly narrow or disappear completely. Narrowing of the repertoire was also evident

in the littermate line by the 6th restimulation but for this line the kinetics of this change

in (3 chain usage was much slower.

160

2°d restimulation 4th restimulation 6th restimulation

TA clone TRBV CDR3IO amino acid sequence TRBJ CDR3 length TA clone TRBV CDR30 amino acid sequence TRBJ CDR3 length TA clone TRBV CDR313 amino acid sequence TRIM CDR3 length

Th2 a transgenic 2°20814 2° 20 1316 2° 20 135

2°20 B13 2°20818 2° 20 B8

2° 20021 2° 20 B4

2°20 B23 2°20 B17 2° 20 Ell

2° 20 1312 2° 20 B20 2° 2082

2° 20 B22 2° 20 B7

2

13-2

13.1

4 31 29

CAS SQRL VNQDTQYF

CA S S LGT ANTGQLYF

CAS S LGT ANTGQP YF CASSQEERTNSDYTE CAS SQDGQFLNERLFF CASGDPD W TETLY F

CASGDRDRGNNSPLYF CAS SAGTNSNERLFF CAS S DAGD 1NNQAPLF CASSDAWGQGNTEVFF CAS SGTGGNTLYF CALGHRINERLFF CA S SPGTGVDTQYF

2-5

2-2

1-2 1-4 2-3

1-6 1-4 1-5 1-1 2-4 1-4 2-5

13

14 12

14 13 14

12

Th2 a transgenic 4° 20 131 4°2003 4° 20 136 4° 20 B8 4120E19 4° 20 1310 4° 20811 4°20013 020B14 4° 20E16 4° 20 B21 4°20137 4° 20 1317 020 1318 4°20E123 4°20 B2 4°2004

(201320

31

13-1

13-3

5

CAWS LGGGAETLYF

CASSDAWGQGNTEVFF

CAS SG PPGQSSGNTLYF CAS RRDNQAPLF

CASSQTFLNSDYTF

2-3

1.1

1-3 1-5

1-2

12

14

15 10

12

Th2 a transgenic 6°20131 e20B2 6°20 B3 6°2084 6° 20 B5 6° 20 B6 e 20 87 6'2088 6° 20 B9 (201310 6°20611 (20 B12 e 20 513 6°20814 6°20E115 e 20 1316 6°201317 6°20 BIS 6°201320 6'20521

31 CAWSLGGGAETLYF 2.3 12

Mean CDR3 12.8 Mean CDR3 12.6

Littermate 2° WT B3 2° WT1320 2°WTB1 2° WT B2 2° WT B13 2° WT B17 2° WT B4 2° WTB11 2° WT B14 2° WT B6 2° WT B12 21 WT 139 2° WT B21 2° WT B20" 2° WT B24 2° WT 137 2° WT B18 rwr 819

16

13-1

2

19 24 31

CASSTTSYEQYF

CASS LDGT TNSDY TF CAS S LQGA 1 S NEGLEF CAS SLVGSNERLF F CASSSDWGSTLYF CAS S DEWGETDTQYF CAS S DAGTGSYEQY F CASSALGGRSYEQYF CAS RPDWG GAGAEQFF CASSDAGGARDAETLYF CASSQDRDFYAEQFF CAS SQEGGVG AEQFF CAS S PRDNQ NTLY F CA S S QDM GGAHQDTQYF CASTPGQGNSPLYF CA SSPSGTGGNTL YF CAWSPGGGAETLYF

2-7

1-2 1-4

2-4 2-5 2-7

2-1 2-3 2-I

2-4 2-5 1-6 2-4 2-3

10

13 14 12 11 13 13 13 14 15 13 13 12 15 12 13 12

Littermate e wrm

WT B12 4° WT131 4° WT 86

WT Bit 4° WT 820

WT BIO 4° WT BI6 4° WT B2 4°WT 114 4° WT 135

WT B21 4° 337 813 4° W1"1314 4°WT 87

WT 89 4° WT 1317 4° s/17 B15 (WT1119

WT BR

13-1

15

17

16

12-2 2 14 31 20

13-2

CASSDAGGARDAETLYF 2-3 15

CAS SPRTTSQNTLYF 2-4 13 CAS R PDWGGAGAEQ IF 2-1 14 CASSLGGSGNTLYF 1-3 12

CASSSRDNQDTQYF 2-5 12

CAS SLELGQSNQDTQYF 15 CAS RDWGSNTGQLYF 2-2 13 CASSLGGVTEVFF 1-1 1 CASSLDDANTEVFF 12 CASSRDWGDEQYF 2-7 II CASSLVIFSNE RIFF 1-4 13

2 CASSLDNNQDTQYF 2-5 CASSQEGLGETQYF CASSSREDTQYF 10 CAWSSRDWGDKQYF 2-7 12 CGARDRDWGDEQYF CASGGDWGLALYF 2-4 II

Mean CDR3 12

Littermate (IVT134 6° WT 136 6° Wr 07 ewTBI9 6°Avr 85 6° WT 03 6" WT139 6° WT BIO 6° 18T1315 6° WT 1318 6" WT1320 eurrI311 6° WT 82

6° WT 822 6° 1118817

WT 131 ex7521 61%71316 6° WT138

16

2

13-1

13-2

29

CASSRDWGDEQYF

C AS SLE WG DEQ VF CASSRDWGDTQYF CASSLDDANTEVFF C AS S FQG YNE RL FF CASSLv1FSNF.RLFF CASSQEGTGGAGAEQFF

CASSDAGGARDAETLYF

CAS SD VG KDTE VFF CASGESANTEVFF CAS CPT ANTE VFF C AS GDAGG AET L CF CASSFSGTGENERLFF

2.7

2.5 1-1 1-4

2.1

2-3

1-1

2-3 1-4

12

13 15

1121

12 14

Mean CDR3 12.8 Me. CDR3 12.4 Mean CDR3 123

Figure 4.5 Rapid clonal expansion of TCR over successive restimulations of a Th2 TCR a chain transgenic T cell line. T cell lines were established from PLP 56-70 primed draining lymph nodes of transgenic and littermate control animals. T cells were restimulated with peptide in the presence of NOD.E splenocytes every 9-11 days. RNA was extracted from cell lines at each restimulation and TCR beta usage determined by PCR. A clonal expansion in the transgenic T cell line is apparent from the 4th restimulation and this TCR comprises 100% of the repertoire by the 6th restimulation. No significant expansion was observed in the littermate line.

IAA % \.-

U:

Transgenic

Littermate

kr'

TRBV 31

TRBV 16

TRBV 13-1

TRBV 2

restimulation 4th restimulation 6th restimulation

Figure 4.6 Spectratype analysis of Th2 a chain transgenic and littermate derived T cell lines through successive restimulations. T cell lines were restimulated every 9-11 days with peptide (PLP 56-70) and NOD.E splenocytes. RNA was extracted from cell lines at each restimulation. TRBV gene specific primers were used with a fluorescently labelled constant region primer to amplify TCR 13 chain sequences from T cell line cDNA. Analysis of product sizes obtained from each PCR reaction showed narrowing of the TCR 13 chain repertoire in both T cell lines over successive restimulations. Narrowing of the repertoire was more rapid in the Th2 a chain transgenic line with complete disappearance of some TRBV genes by the 6th restimulation.

162

Given the enormous differences observed in TCR usage, proliferation in response to

antigen and cytokine phenotype of the two lines were examined. Proliferation assays

with multiple doses of peptide at each restimulation showed that both cell lines were

highly peptide specific and that rates of proliferation in response to antigen were

roughly equivalent between the two lines (figure 4.7). However, cytokine profiles

revealed marked differences. In the Th2 TCR a chain transgenic line levels of the Thl

cytokine IFN-y were decreased between the 2❑d and the 6th restimualtion while levels

of the Th2 cytokines IL-4 and particularly IL-5 were increased. In the littermate

control line IFN-y production remained high and IL-4 and IL-5 remain low throughout

the timecourse. This apparent phenotype difference between the two lines was also

seen at the level of Thl and Th2 transcription factors by real time PCR for Tbet (Thl)

and GATA3 (Th2) transcripts (figure 4.8). At all time points, levels of Tbet

transcripts were higher in the littermate line than in the transgenic line and, from the

4th restimulation, levels of GATA3 transcripts show the opposite trend. Taken

together, the sequence, cytokine, and real time PCR data suggest that, by the 6th

restimulation, the Th2 TCR a chain transgenic T cell line has undergone a rapid clonal

expansion such that the line now comprises a dominant TCR and that the line has a

Th2 type phenotype compared to the more heterogeneous TCR repertoire of the

littermate line which has a predominantly Thl type phenotype.

The original hypothesis of this study asked whether the structure of the TCR could

cause an inherent Thl or Th2 bias in T cell phenotype. As it was not possible to

identify a TCR (3 chain pair for the Th2 TCR a chain used to make the transgenics in

this study, it would be necessary to use the a chain only transgenic lines to identify a 13

chain that pairs with this alpha chain in the context of peptide specificity and a Th2

phenotype. The data for the transgenic cell line described here suggested that the

clonally expanded TRBV 31/TRBJ 2-3 TCR (3 chain may be a suitable P chain pair for

transgenic expression. The picture of TCR usage and cytokine profile over 6

restimulations of this transgenic T cell lines is a rapidly changing one. However, if

cytokine phenotype here is linked to the clonal expansion of the TCR you would

expect that once a single TCR is present, the cytokine phenotype should remain stable.

163

The cell lines were therefore grown over many more restimulations. Figure 4.9 shows

the cytokine phenotype and relative abundance of Tbet and GATA3 transcripts for the

transgenic and littermate T cell lines after 15 restimulations with antigen. The

transgenic T cell line was still producing high levels of Th2 cytokines IL-4 and IL-5,

but low levels of IFN-y while the littermate line produced only IFN-y. Levels of Tbet

transcripts were still higher in the littermate line while GATA3 transcripts were more

abundant in the transgenic line. The Th2 type phenotype of this Th2 TCR a chain cell

line therefore appears to be stable.

164

7 00

q xp 10 20 10 40 50

1.13.0 DOnc0114011011 0.111D

10 /0 30 40 00001. .002.11.

10 20 00 40 50 000000.110n (..11

0 10 20 30 40 0.00010 a c.ntr tb. (.5.1)

2nd restimulation 4th restimulation 6th restimulation

(a)

3

20110

proliferation

IFN-y

IL-4

0 10 20 10 40 50 pop1101..c.o00o• h/p/m1) 1.011115 Danc•ntratIon 0.101m1)

0 20 10 000112. moo...1110n 0..1)

500

400

300

100

IL-5

10 10 30 40 50 0.1101. c.t.ntrtbn 1,400,09

20 50 40 50 p0011011100Kontra11011 (11020)

(b)

10 20 31 40 50 .21. Conan.. W.)

0 10 20 30 40 1.10110•100nCentr1000n 100121101)

0 10 21 30 40 pe0010 C00.101111011 2021110

SO

proliferation

IFN-y

IL-4 1

IL-5

200

1 100

10 10 10 40 (p09.)

200

/D 10 40 11•010111Coneentr.n (01/1101)

20 . 40s0 10 10 40 00 gollde.n0entrzt100 200029 got. 00nc«wtlon (1.1119

Op 2. S

100

1D 20 JO 40 50 0000. concontrotlen 419.1)

Figure 4.7 Cytokine phenotype of Th2 TCR a chain transgenic and littermate T cell lines over successive restimulations. Th2 TCR a chain transgenic (a) and littermate (b) T cell lines were restimulated every 9-11 days with peptide (PLP 56-70) and NOD.E splenocytes. At each restimulation, proliferation assays were performed with multiple doses of antigen. After 48 hours of culture, supernatants were harvested and wells pulsed with [3H] thymidine to determine proliferation. Cytokines in cell culture supernatants were quantitated by ELISA.

165

(a) (b)

2nd restimulation

c

• 4 ;3<• 2

u • 3

re re 2 E 1 D

1 c

22 0 0

4th restimulation

e 0

a - 2 - z z cc E -

d o

0 I ; • 4 a. o 3 z cc 2

> ̀

o n

6th restimulation

Figure 4.8 Real time PCR analysis of GATA3 and Tbet transcripts in Th2 TCR a chain transgenic and littermate T cell lines over successive restimulations. T cell lines were restimulated every 9-11 days with peptide (PLP 56-70) and NOD.E splenocytes. RNA was extracted from cell lines at each restimulation. Real time PCR analysis was used to compare levels of GATA3 (a) and Tbet (b) transcripts between the Th2 TCR a transgenic line (grey bars) and the littermate line (black bars) at each restimulation. RNA levels in each sample were normalised to 18s. At each time point, and for each gene of interest, the sample with the lowest level of gene expression was assigned a value of 1 and gene expression levels in other samples expressed as a value relative to 1.

166

10 ID ID so 10 le 30 40 a. pe....entrellen(.1.1) peptide conceninition

0 10 20 30 40 60 p.600 conoentration (p2/m11

310

10 30 20 dO pisptide Doncentralion(i./211)

IFN-y

IL-4

10 20 30 40 60 peptide concentration 1.1.0)

10 20 . 10 50 pepIcit concentration (pyng)

IL-5

proliferation 1.00

(a)

(b)

10 20 30 60 10 20 40 50 acnoentration pap.. concentration (µ0/01)

(c)

(d)

c 14-0 7n 1 2- .42 o. 10-

rt 6 4- 2-

e

rela

tive

mRN

A ex

pres

sion

Figure 4.9 Cytokine phenotype of Th2 TCR a chain transgenic and littermate T cell lines after 15 restimulations. T cell lines were restimulated every 9-11 days with peptide (PLP 56-70) and NOD.E splenocytes. At the 15th restimulation RNA was extracted from each line and proliferation assays were performed with multiple doses of antien. After 48 hours of culture, supernatants were harvested and wells pulsed with [ thymidine to determine proliferation. Cytokines in the cell culture supernatants of the Th2 TCR a chain transgenic line (a) and the littermate line (b) were quantitated by ELISA. Relative abundance of GATA3 (c) and Tbet (d) transcripts in the Th2 TCR a chain transgenic line (grey bars) compared to the littermate line (black bars) were determined by real time PCR. RNA levels in each sample were normalised to 18s. For each gene of interest the sample with the lowest level of gene expression was assigned a value of 1 and gene expression levels in other samples expressed as a value relative to 1.

167

The transgenic T cell line described above was derived from the primed draining

lymph nodes of Th2 TCR a chain line 20 transgenic animals. Another example of a

Th2 TCR a chain transgenic T cell line with a rapid TCR (3 chain clonal expansion

and a Th2 type cytokine phenotype was derived from line 34 transgenic animals. TCR

(3 chain sequence data in figure 4.10 shows that, over 9 restimulations, a single TCR (3

chain expanded to comprise the majority of the T cell line, with this TCR already

making up 50% of the repertoire by the 5th restimulation. The TCR 13 chain expanded

in this line (TRBV 13-1/TRBJ 2-7) had a long CDR3 (3 of 15 amino acids. This T cell

line produced no IFN-y (figure 4.11) but made high levels of Th2 cytokines IL-4 and

IL-5. A switch in cytokine phenotype towards a Th2 type profile between the 1St and

5th restimulation was also apparent from Tbet and GATA3 real time PCR data with

Tbet transcripts more abundant at the 1st restimulation and low levels of Tbet

transcripts but high levels of GATA3 transcripts at the 5th and 9th restimulations.

Cytokine phenotype data from this line 34 derived T cell line therefore confirmed

previous findings from the line 20 derived T cell line discussed earlier.

Although in the line 34 derived transgenic T cell line the kinetics of TCR (3 chain

expansion are slightly slower, in both examples one TCR ultimately dominates the

line, which displays a predominantly Th2 type phenotype with low levels of the Thl

type cytokine IFN-y and high levels of the Th2 type cytokines IL-4 and IL-5. Both

expanded TCR (3 chains would therefore be good candidates for transgenic expression.

Transgenic constructs for both Th2 TCR 13 chains were created, but technical

difficulties verifying the final line 34 derived TRBV 13-1/TRBJ 2-7 construct meant

that only the line 20 derived TRBV 31/TRBJ 2-3 (3 chain was subsequently used for

transgenesis. The generation of transgenic animals carrying this Th2 TCR (3 chain are

described below.

168

TA clone TRBV CDA3p amino acid sequence TRBJ CDR3 length

1° 34132 1° 34 B3 1° 34 135 I° 34 B7

16 CASSPDWGETLYF

CASSLDWGSDYT F

2-3

1-2 1° 34 1316 1° 34 BI CASSEGKGTNTEVFF I-1 13

1° 34 BIS CASSLDPAGGYEQY F 2-7 I ° 34 B21 CASSGDWGDEQYF 11 1° 34 1317 13-1 CASSGDSTEVFF 1-1 10 1° 34 BI9 I ° 34 B4 CASSDAGGTEVF F 11 I° 34 B6 CASSDAGTGRDTEVFF 14

I ° 34 BI2 CASSDAGTSANTEVFF I ° 34 013 CASTQGPTNERLFF 1-4 12 I' 34 BI5 CASSQTNNYAEQFF 2-1 I° 34 B20 CASSDVGRGNAF.TLYF 2-3 14 1° 34 B8 12-2 CASSLEGWGGAGDTQYF 2-5 15

I ° 34 1111 19 CASSPPGTTYSGNTLYF 1-3 Mean CDR3 12.6

TA clone

5° 34 132 5° 34 B3 5' 34 B4 r 34 135 5" 34 06 r 34 B14 5° 34 1315 r 34 B16 5° 34 1318 5° 34 1321 r 34 BI9 5° 34 B9 5° 34 B20 5° 14 B1 r 34 1310 5° 34 013 r 34 BI2 r 34 Bl7 5° 34 BI8

Mean CDR3

10t restimulation 5th restimulation 9th restimulation

TRBV CDR33 amino acid sequence TRBJ CDR3 length TA clone TRBV CDR3tI amino acid sequence TRBJ CDR3 length

13-I CASSDTGGAQSSYEQYF 2-7 15 9°34 131 9° 34 B2 9° 34 133 9° 34 B7 r 34 I38 9° 34 119

9° 34 1310 r 34 B12 r 34 BI3 9° 34 B25

I3-1 CASSDTGGAQSSYEQYF 2-7 15

2 CASSQDGLGGLDTQYF 2-5 14 9° 34 B26 r 34 B27

CASSQPPTI1NQDTQYF 9° 34 13213 r 34 B29 r 34 B30 r 34 B31

16 CASSLDGLVDSNSDYTF 1-2 15 9° 34 B32 CASSLEW GAEQF F 2-I II 9° 34 1333 ON

5 CASSRHRADTEVFF I-I 12 r 34 B34 .f:3 13.5 9° 34 BI4 13-2 CASGDAGGEQDTQY F 2-5 13 i--1

Mean CDR3 14

Figure 4.10 Clonal expansion of a TCR over successive restimulations of a Th2 TCR a chain transgenic T cell line. The T cell line was established from PLP 56-70 primed draining lymph nodes from line 34 Th2 TCR a chain transgenic animals. The line was restimulated with peptide and NOD.E splenocytes every 9-11 days. RNA was extracted at each restimulation and TCR beta chain usage determined by PCR. A clonal expansion was apparent at the 5th restimulation of this line and this TCR comprises the majority of the TCR beta chain repertoire by the 9th restimulation.

1000

.0 A 30 40 A p•ptitle 509290.19n (..9

IL-4

3000

HOD I

1000 I 31

111 20 30 40 sp 10 30 30 40 50

990105 conemlnklon (I00 90511510 9999050011On 929/2110

15009 15004 13400 12500

.1 10,000.

5000 .rj 4000 2500 3504

0 10 20 30 40 0

MOO

1/1

0 10 20 30 40 GO 0. 11 A A MI 10

IL-5

.2 50- ra ct)

0.2 40- x 0

< 30-z

E 20-

9111

c 90-

•N • 80- (5 a) 70-

cx̀ 60-

< • 50-

Z re

40•

E

> 20. ri co 10-a.)

0 r.) • 0 ""T""

1st 5th 1;t 5th 9th

(a)

proliferation

IFN-y

15t restimulation 5th restimulation 9th restimulation

#14000..

6000.

1 2SOOD.

Slr ID 0 39 40 SO

ccccc .410on

10 20 30 40 40 110910.11 000000.900 1102009

0 10 39 30 40 10 peptide concenbattOn (WM

10 20 31 40 SO 20121151 ConCantratIon ( 2001)

ppIldeconcomtrstlan 002/02) peptide concrnien "Okla concsalretIon 950/0111

(b) (c)

Figure 4.11 Cytokine phenotype of a line 34 Th2 TCR a chain transgenic T cell line. The T cell line was restimulated with peptide (PLP 56-70) and NOD.E splenocytes every 9-11 days. At each restimulation a proliferation assay was performed with multiple doses of antigen. After 48 hours of culture, supernatants were harvested and plates pulsed with [3H] thymidine to determine proliferation (a). Cytokines in cell culture supernatants were quantitated by ELISA (a). Relative abundance of GATA3 (b) and Tbet (c) transcripts in cDNA samples from successive restimulations were determined by real time PCR. RNA levels in each sample were normalised to 18s. For each gene of interest the sample with the lowest level of gene expression was assigned a value of 1. Gene expression levels in all other samples are expressed as a value relative to 1.

170

4.4. Construction of a Th2 TCR p chain transgene.

The cassette vector used in the construction of the Thl and Th2 TCR a chain

transgenes has already been described. The TCR p chain vector (pT(3) was also

designed by Kouskoff et al. (Kouskoff et al., 1995) and is based upon the same

principle of using endogenous mouse TCR [3 promoter and enhancer elements to drive

gene expression. To express a TCR (3 chain transgenically it is necessary to insert into

the vector the rearranged TCR p V-J coding region of interest. Also described in the

previous chapter was the importance of using the genomic DNA configuration rather

than the cDNA configuration of the rearranged TCR chain. However, unlike the

situation described for the generation of the TCR a chain constructs, genomic DNA

was available from the T cell line from which the TCR p chain of interest was

identified. It was therefore possible to obtain the full sequence of the required TCR (3

chain V-J segment by a single PCR strategy, whereby PCR was used to generate

restriction sites at either end of the V-J segment which could then be inserted directly

into the cassette vector (figure 4.12). The nucleotide and amino acid sequence of the

Th2 TCR p chain used to make the transgene are shown in appendices 21 and 22

respectively. PCR primers used to clone the V-J segment from cell line genomic DNA

are listed in appendix 8.

The PCR product that was cloned into the pT(3 cassette vector and verification of the

final construct by restriction digest are shown in figure 4.13. The sequence of the

TCR (3 chain V-J segment in the final construct was also verified by sequencing prior

to preparation for pronuclear injection. Bacterial sequences were removed from the

vector prior to pronuclear injection by digestion of the construct with Kpnl and

purification of the upper 17,700 bp band (figure 4.13). A plasmid map of the complete

Th2 TCR (3 chain construct is shown in appendix 12.

171

VB31F

Xhol L_. ME

LP1 -

LP1 VB3I JB2-3 \-1 SacII JB2-3R

Xho I I NMI

7.777,77 Sac II

LP1 VB31 JB2-3

(c) Xho I

(a)

(b)

Kpn I

Kpn I

Th2 (3 construct is linearised for injection using Kpn I

Figure 4.12 A schematic diagram showing the cloning strategy used to create the Th2 13 chain construct. (a) Genomic DNA from the Th2 a transgenic cell line was used to isolate the required VB31/JB2-3 fragment containing the appropriate intronic regions for correct transcript processing. PCR primers (VB31F and VJ2-3R) were used to introduce Xho I and Sac II restriction sites to the 5' and 3' ends of the VJ fragment respectively. (b) The Th2 p VJ fragment was then inserted into the pTp cassette vector by directional cloning using Xho I and Sac II. (c) Bacterial sequences were removed from the vector, by digestion with Kpn I, prior to pronuclear injection.

172

Z rearranged TRBV31-TRBJ2-3 bp

1000 750 500 —111"

bp

10000

,—, 0 at

—••, $.4 a) V)

^c1 0 0 ,—] 0 W Ca at >,.. 0 ad = U bp

(b) o.

(c)

10000 5000 3000 2000

1000 --Po-

500 —110.

(a)

Figure 4.13 Cloning of the rearranged Th2 TCR f chain into the TCR 13 expression cassette. (a) The rearranged configuration of the Th2 TCR p chain was cloned from T cell line genomic DNA by PCR. The cloned VJ fragment was sequenced and then inserted into the TCR (3 expression cassette using restriction enzymes XhoI and SacII. (b) Prior to digestion and purification for pronuclear injection, the complete construct was checked by restriction digest to ensure that no rearrangement of the plasmid had occurred during cloning. (c) Bacterial sequences were removed from the construct before injection by digestion with KpnI and purification of the upper 17,000 bp band.

173

4.5. Identification of a Th2 TCR fl chain transgenic founder animal

The Th2 TCR (3 chain transgene was injected into C57BL/6 oocytes at the MRC

Clinical Sciences Centre of Imperial College, London. 20 potential founder animals

were screened for the presence of the Th2 TCR (3 chain transgene. An initial PCR

screen, using genomic DNA extracted from a tail biopsy, identified 1 founder animal

(figure 4.14). This founder was subsequently referred to as Th2 TCR [3 line 4 based

on the number assigned to the animal at the point of tail biopsy. Technical difficulties

with the technique have meant that, so far, the transgenic status of this animal has not

been confirmed by Southern blot.

The founder animal (Th2 TCR [3 line 4) was male. Th2 TCR a chain line 20

transgenic females were therefore used as breeding partners to obtain the next

generation of this line, which may be transgenic for both a and p chains of the Th2

TCR. Normal rates of germline transmission of the Th2 TCR a chain were observed

in the progeny of this mating. However, transmission of the Th2 TCR p chain

transgene was very poor. Of 54 progeny screened by PCR for the Th2 TCR a and p chain transgenes, 27 animals carried the a chain compared to just 3 carrying the p

chain. 2 mice carrying both transgenes have now been set up as the next generation

breeding pair for this transgenic line, but the small number of Th2 TCR (3 chain

positive animals identified means that experiments to characterise these animals are

not reported here as they are not within the timecourse of this thesis.

174

(a) 20TCRI3F1 Xho I Sac II

LP1 TRBV31 TRBJ2-3 20TCRPR1

(b) < 6 Potential .1. O.) I- 4 founders -0

bp '-i) -t , E-, 1-3 = 0 ,fi r—A---, PZ

5-20

1000 —111,'

750 —0. 500 —11. 250 —IP.

Figure 4.14 Identification of the Th2 TCR 13 chain transgenic founder animal. The transgenic founder was identified by PCR. The location of primers used for PCR screening (20TCR13F1 and 20TCR(3R1) are shown in (a). One transgenic animal was identified from an initial PCR screen of 20 potential founders.

175

4.6 Analysis of a PLP (56-70) specific Thl TCR a chain transgenic T cell line.

To determine whether rapid clonal expansion of a TCR p chain would also occur in T

cell lines derived from Thl TCR a chain animals, T cell lines were established from

PLP 56-70 primed draining lymph nodes of these mice. As described for the Th2 TCR

a chain transgenic lines, T cells were grown in vitro in growth medium containing

10U/m1 IL-2 by restimulation with peptide and NOD.E splenocytes every 9-11 days.

Proliferation in response to antigen, and cytokine phenotype, were determined at

successive restimulations. Figure 4.15 shows that over 6 restimulations the Thl TCR

a chain transgenic line was highly antigen specific and displayed a predominantly Thl

type phenotype, producing high levels of IFN-y. Sequence analysis of TCR (3 chain

usage at the 6th restimulation showed a small clonal expansion, comprising 26% of the

repertoire. However, sequence analysis of TCR a usage by this line failed to identify

the transgenic Thl TCR a chain (figure 4.15). A PCR for the rearranged Thl TCR a

chain in cDNA from the line at the 6th restimulation suggested that the transgenic a

chain was being expressed by this line. However, sequence data suggests that T cells

bearing this transgenic a chain are not present at any significant level and are not

likely to be pairing with the expanding TCR p chain. This finding is consistent with

the low level of expression of the Thl TCR a chain transgene found in unprimed

splenocytes of this TCR transgenic line (table 3.6). Further T cell lines derived from

Thl TCR a chain transgenic animals are currently being characterised.

176

proliferation

IFN-y

IL-4

IL-5

(a)

40000—

o

1:0

10000. 1000

10 20 34 40 10 0,35154 conoentr•tion 100/m1)

10 20 30 4 0 peptide concentration inonnI)

4000

10 20 30 10 SO peptide COnCentratiOn 04/010

15000 1500

12500 1250

tl "1;00 I 7° 50

3 5050 1 500

2500 260

pryal100 001.0ntrallon 04003) 0 10 30 30 40 60

peptide concentration (WM 3•0010a concanIntlan 101111.0 To 2.0 30 411 60 10 20 30 40 05

2300

100e

30001

io A io io 50 concontra000 (1410110 0.110000ncentratIon 040001)

1

,.,.:000

20 30

2' restimulation 4th restimulation 6th restimulation

(b) (c) TA clone TRBVCDR313 amino acid sequence TRBJ CDR3 length

6°Thl 6°Thl

1311 1311

13-1 CASSDAPGQGAGNTLYF 1-3 15 TA clone TRAY CDR3a amino acid sequence TRAJ CDR3length 6°Thl B2( 6°Thl B24

6thThl Al 3-3 CAVTNTNAYKVIF 30 11 6"Thl 1321 6thThl Al( 6°Thl B1( CASSDLGTAGNTLYF 13 6thThl A21 6°Thl 131. 6thTh1A22 6°Thl B2 CASSEPGTGRSGNTLYF 15 6thThlA2 4D-3 CAAPSGSFNKLTF 4 6°Thl 134 2 CASSQEGWGAWEQYF 2-7 13 6thThIA6 6°Thl Blf CASSQDGGGAYEQYF 13 6thThIA17 6°Thl B3 CASSQVDRPYEQYF 12 6thThlAl9 CAAMNYNQGKLIF 23 6°Thl B8 CASSQEGTGGYAEQFF 2-I 14 6thTh1 A20 6°Thl B21 19 CASSILGGHTGQLYF 2-2 13 6thThlA3 CAAGARNNNNRIFF 31 12 6°Thl B21 CASSLGNTGQLYF 11 6thThIA4 CAAEKHNNRIFF 10 6"ThI B5 4 CASSLRDWGDEQYF 2-7 12 6thThlA5 4D-4 CAAEGENNNAPRF 43 11 6°Thl B7 1 CTCSGRDWGDEQYF

6°Thl B9 31 CAWSLGGGAETLYF 2-3 12 6°Thl 1311 16 CASSFGTDYEQYF 2-7 11 6°Thl 1323 17 CASS RAGDSNERLFF 1-4 13

Figure 4.15 Analysis of a Thl TCR a transgenic derived T cell line. A T cell line was established from PLP 56-70 primed draining lymph nodes of Thl TCR a transgenic animals. The T cell line was restimulated every 9-11 days with peptide (PLP 56-70) and NOD.E splenocytes. At each restimulation a proliferation assay was performed with multiple doses of antigen and supernatants harvested for cytokine analysis by ELISA (a). RNA was also extracted at each restimulation and TCR alpha chain (b) and beta chain (c) usage at the 6th restimulation determined by PCR.

177

4.7. Altered cytokine phenotype of an in vivo memory response to antigen in Th2 TCR

a chain transgenic animals.

The Th2 TCR a chain transgenic T cell lines described in this chapter were grown in

vitro in growth medium containing 10 U/ml IL-2. This growth medium should be

non-polarising (i.e. should not promote a Thl or Th2 phenotype bias). Not all Th2

TCR a chain transgenic cell lines grown in vitro during the course of this study

developed a Th2 type phenotype, or indeed demonstrated a rapid TCR clonal

expansion. However, the expansions and Th2 type phenotype of the 2 lines described

here, which were derived from different transgenic lines, are quite striking and quite

surprising given the lack of any exogenous polarising cytokines in the growth

medium. The implication is that expression of the Th2 TCR a chain can, in the

context of specificity to antigen (PLP 56-70) predispose to selection of a TCR 1 chain

pair, which can then bias the T cell response towards a Th2 phenotype. A peptide

priming experiment was therefore designed to test whether the same bias, observed so

far in an in vitro system, could also be observed in vivo. Figure 4.16 outlines the

experimental protocol. Mice were primed with 50 [ig of peptide (PLP 56-70), which

was administered in the footpad as an emulsion with CFA. Mice were then re-primed

in the flank with the same dose on day 28. Splenocytes were harvested from groups of

animals (4 transgenics and 4 littermates) at each time point shown in figure 4.16. At

each time point, proliferation in response to antigen and cytokine phenotype were

determined. The proliferative response of splenocytes to peptide (PLP 56-70) at each

time point was similar between transgenic and littermate groups and demonstrated a

good memory response to antigen (figure 4.16). At day 10 there was no major

difference in IFN-y production between transgenics and littermates. However at days

28 and 32 animals in the transgenic group produced very little, or no IFN-y in response

to antigen. At day 28 the difference in IFN-y production between the transgenic and

littermate groups was statistically significant. This was not the case at day 32, but

animal numbers in groups were small and the difference is still apparent. Neither

group produced IFN-y in any significant amount at day 38. No splenocytes produced

IL-4 or IL-5 at any time point of this experiment. From this data it appears that the in

178

vivo memory response to antigen in Th2 TCR a chain transgenic animals is shifted to

a less Thl type profile than for littermate animals, but that this shift does not extend to

appearance of a Th2 type profile.

CDR3 spectratype analysis of TCR l chain usage in splenocyte samples from days 28

and 32 of this experiment was used to try to determine whether any clonal expansions

had occurred. Figure 4.17 shows that for a few TRBV genes there was some evidence

of possible clonal expansions in Th2 TCR a chain transgenic compared to littermate

samples. These clonal expansions are suggested by the presence of a dominant peak

in the spectra for these TRBV genes, where the dominant peak comprises greater than

35% of the total area. However, sequence analysis of TCR 13 chains in these

splenocytes samples failed to find any evidence of these expansions (figure 4.17).

Although the spectra for some TRBV genes show the emergence of a dominant peak,

this does not mean that this expansion within a particular TRBV gene family equates

to an expansion in the context of all TRBV genes being used in that splenocytes

sample. To detect these expansions it may be necessary to just sequence TCR 1

chains using the TRBV gene in question. A further complication is the problem of

using a whole splenocyte cDNA sample to detect a peptide specific clonal expansion.

The memory T cells in the spleens of these animals will have many different

specificities, and although the proliferation and cytokine data can be attributed to PLP

56-70 specific T cells, the splenocyte sequence and CDR3 spectratype data cannot.

179

(a) 50 ug of PLP 56-70 given as a flank immunisation with CFA

50 ug of PLP 56-70 given as a footpad immunisation with CFA

40000

:_-... 30000 E 1M a 20000 t- z l'-`- 10000

*

Day 10 Day 28 Day 32 Day 38

(b)

day 6

I10

1 r I 28 32 38

+ + + + Splenocyte Splenocyte Splenocyte Splenocyte

assay assay assay assay

-I.

I i I 1 1 1 1 Day 10 Day 28 Day 32 Day 38

3 H t hym

idin

e in

corp

ora

tion

(Acp

m)

10000

7500

5000

2500

0

(c)

Figure 4.16 An in vivo memory response to peptide demonstrates a difference in cytokine profile between Th2 TCR a chain transgenic and littermate animals. (a) Mice were primed with a peptide (PLP 56-70) / CFA emulsion on day 0 and day 28. Splenocytes were harvested at days 10, 28, 32 and 38 and proliferation assays performed with multiple doses of antigen. After 48 hours of culture, supernatants were harvested and wells pulsed with [3H] thymidine to determine proliferation. (b) Proliferation of splenocytes to 50 µg/m1 of peptide from Th2 TCR a chain transgenic (grey bars) and littermate (black bars) animals at each time point are shown. Data shown are mean values (minus background) for the 4 animals in each group + SE (c) Levels of IFN-y in supernatants from cells cultured with 50 µg/m1 of peptide were quantitated by ELISA. Data shown are mean values for the 4 animals in each group ± SE. The statistical significance of differences between transgenic (grey bars) and littermate (black bars) groups were tested using a Mann-Whitney U test. *P <0.05.

180

TRBV 3 TRBV 17

0 180

150

Th2 TCR a chain transgenic

\ • P-• A, A, ik-fl •V kA,r‘

(a)

Littermate

(b)

TA clone TRBV CDR3I3 amino acid sequence TRBJ CDR3 length

34-196B2 13-2 CASGEQGGTGQLYF 2-2 12 34-196 B4 34-196 B5 CASGDTDWGDPNTGQLYF 16

34-196B13 CASGDATGVYEQYF 2-7 12 34-196B6 2 CASSQGLDYEQYF 11 34-196 B8 CASSQELGPSAETLYF 2-3 14 34-196B10 CASSQERRADTQYF 2-5 12 34-196B15 CASSPQISNERLFF 1-4 34-196B21 CASSQVRGRRGNTLYF 2-4 14 34-196B3 13-1 CASRDSSNERLFF 1-4 11 34-196B14 CASSDDRTGQLYF 2-2 34-196B17 CASSPGETLYF 2-3 9 34-196B19 CASSGNWEQDTQYF 2-5 12 34-196B11 5 CASSRDWGYAEQFF 2-1 34-196B18 CASSRRDWGYEQYF 2-7 34-196 B7 17 CASSPGTGGDTQYF 2-5 34-196B12 20 CGARDRKNTGQLYF 2-2

Figure 4.17 TCR analysis of splenocytes from Th2 TCR a transgenic and littermate animals at day 32 after priming. (a) Spectratype analysis using splenocyte cDNA obtained from animals at day 32 of the in vivo priming protocol (figure 4.16) showed some evidence of possible clonal expansions for some TRBV genes in Th2 TCR a chain transgenic compared to littermate samples. However, these expansions were not apparent when TCR (3 chains were sequenced from these samples (b).

181

Discussion

A previous study demonstrated that CD4+ T cells selected under Th2 polarising

conditions favoured an elongated CDR3a region and that this longer CDR loop may

result in less optimal (lower affinity) interactions with the pMHC complex (Boyton et

al., 2002). This finding led to the hypothesis that, under Th 1 or Th2 polarising

conditions, different TCR structural motifs are preferred, and that if TCRs bearing

these motifs were to be expressed transgenically, an inherent Thl or Th2 bias in T cell

phenotype would be observed. The previous chapter of this thesis described the

identification of TCR a chains for transgenic expression, and the generation of

animals expressing these chains on peripheral T cells. Data presented in this chapter

has described analysis of the TCR p chain repertoire of these animals and experiments

to try to determine an antigen specific TCR (3 chain partner, as well as any phenotype

bias that may result from expression of structurally distinct Thl and Th2 derived TCR

a chains.

As antigen specific TCR (3 chain partners for the dominant a chains had not been

identified, TCR transgenic animals, described in chapter 3, were expressing the Thl or

Th2 TCR a chain only. To exit the thymus as a mature T cell, positive selection

events dictate that the cell must be expressing an a13 heterodimer at the cell surface

(Mombaerts et al., 1992b). Cells expressing the transgenic TCR a chain in the

periphery must, therefore, be pairing with endogenous TCR p chains. To determine

whether expression of the Th2 TCR a chain during T cell development had any impact

on the peripheral repertoire of TCR (3 chains, in the absence of peptide specificity,

sequence analysis and spectratype analysis were employed to look at TCR usage in

splenocytes. TCR a chain transgenics made by other groups have previously been

reported to show normal V(3 usage in the periphery (Huang and Kanagawa, 2004)

implying that these transgenic a chains are capable of pairing with a broad spectrum

of endogenous 13 chains. In addition, other studies have implied that it is the CDR3a

region rather than the CDR3(3 region that dictates TCR repertoire diversity in the

182

recognition of foreign antigens and the formation of the preimmune TCR repertoire

(Yokosuka et al., 2002). In the study by Yokosuka et al, an in vitro transfection

system was used to analyse hundreds of potential ap heterodimers for their ability to

recognise foreign antigen. It was found that for a given antigen, very few a chains

were permissible, but that any one particular a chain could use a wide variety of TCR

13 chains to achieve peptide specificity. Consistent with these findings, no particular

bias in VP repertoire, either in terms of the V[3 genes used, or the CDR3f3 region

lengths observed, were seen in either Th2 TCR a chain transgenic line by sequence

analysis or by spectratyping. The VP gene families identified by sequencing were

similar between littermate animals and transgenics and the CDR313 lengths were also

comparable. No obvious bias in the amino acids used in the CDR3P regions were seen

and spectra obtained for each VP family showed a normal Gaussian distribution,

suggesting that no significant expansions of any one sequence were present. This

analysis suggests that the transgenic Th2 TCR a chain is able to form stable a(3

heterodimers with a wide variety of different endogenous P chains that bind self-

pMHC with sufficient affinity to be positively selected, although this data does not

provide evidence that the diverse receptors are able to respond normally to a diverse

array of foreign antigens.

The concept of repertoire diversity, however, is not a straightforward one and is highly

dependent upon the peptide antigen in question. For some peptide antigens, the TCR

a chain has been seen to be highly restrictive in determining which TCR 13 chains are

permissible (Turner et al., 1996). Therefore, although in the peripheral T cell

repertoire of the Th2 TCR a chain transgenic mice a diverse array of TCR

sequences are observed, this is in the context of many self pMHC having been

presented in the thymus and the situation may be different in the context of specificity

to single antigen. The antigen of interest in this study is the proteolipoprotein peptide

PLP 56-70. The T cells from which the transgenic TCR a chains derive were highly

specific for this peptide. In order to generate a peptide specific immune response,

mice were primed with an emulsion of PLP 56-70 and CFA. However, sequence and

183

spectratype analysis of T cells in draining lymph nodes of these animals revealed a

similar picture to that seen in unprimed mice, where many different TCR (3 chains

from different gene families were identified and symmetrical spectratype profiles

revealed no obvious clonal expansions within each V(3 gene family. V(3 gene usage

and spectratype profiles were comparable between transgenic and littermate animals

and it is also worth noting that proliferative responses of DLN cells to antigen in vitro

were also similar between transgenics and non transgenics, indicating that T cells

bearing the transgenic TCR a chain are able to form PLP peptide specific

heterodimers with endogenous TCR (3 chains and mount an effective response to this

antigen.

Although no obvious TCR 13 chain bias was observed in DLN T cells of primed mice,

it should be born in mind that in order to provoke an immune response to antigen,

mice are primed with CFA. The ability of CFA to provoke an immune response is

mediated by the mycobacterial components it contains and as such, although many T

cells in the DLN will be specific for the peptide, others are also likely to be specific

for these bacterial antigens. To look at TCR (3 chain usage in a population of truly

peptide specific T cells, T cell lines derived from the DLN cells of PLP 56-70 primed

mice were grown in vitro. This approach also allowed selection and change in the V13

repertoire to be examined over successive restimulations with antigen. The data from

two Th2 TCR a chain transgenic T cell lines were presented in this chapter. One line

was derived from mouse line 20 and the other from mouse line 34. Both T cell lines

demonstrated a clonal expansion of a dominant TCR (3 chain through successive

peptide restimulations. The expansion was particularly rapid in the T cells derived

from mouse line 20 where, by the 6th restimulation, 100% of TCR (3 sequences

obtained were identical. Both transgenic T cell line expansions were in contrast to the

absent or very limited expansions observed in a littermate derived T cell line. The

marked rate of clonal expansion in the TCR a chain transgenic T cell lines could also

be observed from spectratype profiles of selected V13 families. Small quantities of

cDNA available from each line at each restimulation meant that a full spectratype

analysis across all V(3 gene families was not possible. However, using V(3 families

184

known from sequence analysis to be present in the repertoire at very early

restimulations, spectra reveal that even as early as the 2nd restimulation, the diversity

of the V13 repertoire in the littermate derived T cell line is far greater than that seen for

the TCR a chain transgenic line. This data suggests that, in the context of specificity

to the cognate antigen PLP 56-70, the diversity of the TCR p chain repertoire is

limited by the expression of the transgenic Th2 TCR a chain.

The dominant TCR (3 chain sequences selected in the two transgenic lines presented

here are not the same. They use different V[3 genes, have different CDR3(3 regions

and indeed have different CDR3 (3 region lengths. Therefore, although influencing

TCR (3 chain diversity, the transgenic TCR a chain appears not to mediate selection of

a common, or "public" TCR (3 sequence. A "public" TCR sequence is one that is

identified as common between different individuals and is probably the least common

type of TCR bias. One of the most well studied public TCRs is LC13, a human TCR

specific for an antigen of the Epstein-Barr virus (EBV) (Argaet et al., 1994). In many

individuals, CD8+ T cells recognising this antigen use the same TRAV26-2/TRBV 7-6

TCR with identical CDR3 regions. The sequence of this TCR is not the only one able

to recognise antigen, as individuals expressing the MHC class I molecule HLA-

B*4402 are found to use other receptors different from LC13 (Burrows et al., 1995),

but structural studies of this TCR have revealed exquisite complimentarity between

the antigen and peptide binding pockets formed by CDR loops of the TCR (Kjer-

Nielsen et al., 2003). However, aside from complete sequence homology, other forms

of public TCR bias have also been documented. Some studies document preference of

particular V(3 genes, for example, the use of V(38 and V(37 genes in murine responses

to different antigens of the influenza virus (Belz et al., 2000). Others have reported

the selection of conserved amino acid sequences within CDR3 regions (Trautmann et

al., 2005). When reviewing the large numbers of TCR (3 sequences obtained during

the course of this thesis, it is of note that one amino acid motif in the CDR3 region is

particularly common in the context of antigen specific T cells. Sequences containing

the amino acid motif, SRDWG, in the CDR3(3 region were observed in both of the

original Thl and Th2 T cell lines from which the transgenic TCR a chains were

185

cloned. This motif was then also sequenced in peptide specific lines from littermate

animals and indeed is part of the CDR3I3 region most dominant in this line following 6

restimulations with peptide. The motif becomes even more common if seen as

SXDWG although this motif was also isolated from T cells of unprimed mice. The

SRDWG motif in the CDR3(3 region of a TCR may therefore be predicted to make

some important contribution towards pMHC recognition to account for this bias,

although it was not observed in either of the TCR p chains seen to rapidly clonally

expand in the Th2 TCR a chain transgenic lines. The contributions that the CDR3a

and CDR3p regions of a TCR make to peptide recognition are known to be dependent

upon the peptide being recognised and to involve different residues of the peptide. In

a study by Jorgensen et al. (Jorgensen et al., 1992) mice transgenic for either an a or a

p chain were immunised to peptide variants in order to determine the relative

contribution of each chain to the binding of specific residues. This study found that

the a and p chains contact independent parts of the peptide and so that preferred

motifs are observed in one chain in conjunction with more heterogeneity in the other is

not an unexpected finding.

One of the main aims of this study was to explore the possibility that the transgenic

expression of structurally distinct Thl and Th2 TCRs results in an inherent Thl or Th2

bias in T cell phenotype. Transgenic expression of peptide specific Thl and Th2 TCR

heterodimers has not yet been achieved to permit the ultimate test of this hypothesis,

but, as discussed in chapter 3, the dominance of the Thl and Th2 TCR a chains in the

original T cell lines, in the absence concomitant TCR (3 expansion, raises interesting

questions about the role of the TCR a chain alone. Cytokine production by the

antigen specific Th2 TCR a chain transgenic and littermate T cell lines was therefore

determined. As expected, particularly from T cells originally primed using CFA, the

dominant cytokine produced by the littermate derived T cell line was IFN-y. This was

in stark contrast to the cytokine profile of both Th2 TCR a chain transgenic lines,

where high levels of the Th2 cytokines, IL-4 and IL-5 were observed. Production of

these cytokines was seen to increase through progressive restimulations with cytokine

186

and indeed this phenotype was stable, with the line 20 derived T cells producing

significant amounts of IL-4 and IL-5, although also a small amount of IFN-y, after 15

restimulations. At 15 restimulations, the littermate derived T cell line was still

producing only IFN-y. That T cells expressing the transgenic Th2 TCR a chain had

spontaneously acquired a Th2 like phenotype was further supported by real time PCR

data of Tbet and GATA3 transcription factor expression. The enormous number of

variables involved in CD4+ T cell differentiation can make the relative contribution of

individual factors hard to assess. However, in this system, transgenic and littermate

mice were primed identically and T cells restimulated at identical time points using the

same APCs and the same dose of peptide. Additionally, data presented in chapter 3

demonstrated that levels of TCR heterodimers on the surface of peripheral T cells were

similar between transgenic and littermate animals. T cells were cultured in the

absence of any polarising cytokines, with only a low dose of IL-2 added to the culture

medium and so as far as possible, the only differences between the T cells present in

the different lines are the TCR a chains that they express.

This cytokine and real-time PCR data, while convincing in its demonstration of a Th2

phenotype, is derived from T cell lines grown over a long period of time in vitro. In

vitro studies will always be open to criticism surrounding the role of a particular

variable in normal physiological conditions where interactions and signals are more

complex. More convincing would be a demonstration of phenotype differences

between Th2 TCR a chain transgenic and littermate animals in vivo. To begin to

address this, data presented in this chapter described reduced IFN-y production by

immediately ex vivo T cells bearing the transgenic TCR a chain, in an experiment

designed to investigate the consequences of antigen restimulation in vivo. This data

suggests that, similar to results obtained from in vitro culture, successive restimulation

of transgenic T cells with antigen results in an altered cytokine phenotype, with

respect to littermate animals, with a shift away from a Thl phenotype and IFN-y

production. No Th2 type cytokines were detectable in the immediately ex vivo

responses of transgenic T cells seen to be making less IFN-y, but given the Th 1

promoting environment of CFA immunisation, this is perhaps not surprising. It may

187

be that in these animals, spontaneous induction of a Th2 type phenotype will not occur

in vivo but rather T cell phenotype bias will manifest as a shift away from a polarised

Thl phenotype as the outcome. If this is the case, it may be of interest to determine

whether this inherent bias will have any impact in the context of an IFN-y driven

disease model, such as EAE. In a previously study, Thl T cell responses to the

antigen PLP 56-70 were associated with susceptibility disease, while deviation away

from Thl cytokine responses were associated with protection (Takacs et al., 1995).

Data presented in this chapter appears to support a role for the TCR a chain in

determining CD4+ T cell phenotype and when put back into context with the original

study (Boyton et al., 2002), where molecular modelling predicted that elongated

CDR3a regions of Th2 TCR a chains would make lower affinity interactions with the

pMHC complex, fits with other data suggesting that lower affinity TCR/pMHC

interactions favour Th2 type responses (Malherbe et al., 2000) and that interventions

that impede optimal TCR/pMHC binding, such as the use of APLs, can result in a

switch in T cell phenotype from Thl to Th2 (Nicholson et al., 1995). Altered

signalling through the TCR, as explained by the serial triggering hypothesis previously

discussed, remains the most likely explanation for the link between TCR structure /

affinity and effector outcome. A model where the elongated CDR3a loops of the

transgenic Th2 TCR a chain forces a lower affinity interaction with the pMHC

regardless of the TCR p chain sequence, although where only a limited number of 13

chains can functionally pair to give good antigen specificity, would suggest that this

lower affinity interaction then results in suboptimal signalling, and an altered pattern

of phosphorylation events downstream of the TCR which ultimately result in

preferential induction of Th2 differentiation pathways. Altered patterns of

phosphorylation signalling molecules, for example ZAP70 and Lck, have been

observed upon TCR ligation with APLs (Boutin et al., 1997) and more prolonged

signalling, perhaps via a higher affinity interaction, has been shown to be required for

full mitogen-activated protein kinase (MAPK) activation and Thl differentiation

(Badou et al., 2001). More recent data looking at thymic selection, another T cell

signalling scenario where the affinity of the TCR/pMHC interaction is proposed to

188

play a major role in dictating the outcome of TCR signalling, has shown that the

signalling proteins Ras and MAPK are segregated into different subcellular regions

depending on the affinity of the TCR/ligand interaction (Daniels et al., 2006).

Efforts to identify whether any bias in the TCR [3 chain repertoire had occurred during

antigen restimulation in vivo were limited and the experimental system not sensitive

enough to detect this. Although spectratype analysis appeared to show some evidence

of clonal expansions this was not supported by sequencing data. However, sequencing

data was obtained from whole splenocytes and so is highly unlikely to reflect the

antigen specific TCR repertoire. Further experiments are currently underway to try to

select antigen specific T cells from the spleen following antigen restimulation. The

ideal way to achieve this would be through the use of pMHC tetramers, although

selecting T cells activated in response to peptide presentation on the basis of activation

markers such as CD69, may also be of use. The ultimate test for the role of TCR

structure in CD4+ T cell phenotype bias will be to look at the T cell responses of Thl

and Th2 TCR ar3 transgenic animals, which have been crossed onto a RAG"'"

background and thus do not express any endogenous TCR. The Th2 phenotype of the

antigen specific transgenic T cell lines described in this chapter and the concomitant

clonal expansion of TCR p chains observed through successive restimulation makes

these TCR 13 chains good candidates for transgenic expression. The construction of a

transgene for one of these chains and identification of a founder animal were described

in this chapter and a programme of breeding is currently ongoing to generate mice

carrying both the Th2 TCR a and I chains.

Chronologically, the two independent lines of Th2 TCR a chain mice were established

before the Thl TCR a chain transgenic line and as such, much of the data obtained to

date refers to the Th2 TCR a chain animals. However, expression data presented in

the previous chapter and towards the end of this chapter highlight potential pitfalls in

the analysis of the Thl TCR a chain line. Sequence analyses of TCR sequences from

peripheral T cells of Thl TCR animals were presented in the previous chapter and

showed that less than 50% of sequences obtained were derived from the transgene.

189

Percentages were also highly variable between animals and in one sample, transgenic

sequences made up less than 10% of the repertoire. To examine whether the

transgenic a chain may be more favoured and thus become more dominant in the

context of antigen specificity, mice were primed with antigen (PLP 56-70) and T cell

lines derived from the primed draining lymph nodes (DLNs) of these animals grown in

vitro. This was also with the view to identifying an antigen specific TCR (3 chain

partner for this Thl TCR a chain. However, following 6 restimulations with antigen,

TCR sequence analysis revealed a complete lack of transgenic TCR a chains in the

line. This finding has now been reproduced for 4 separate Thl TCR a chain

transgenic derived T cell lines and suggests that in the context of antigen specificity,

the transgenic TCR a chain is highly disfavoured in preference for endogenous chains.

The reason for this bias is unclear and needs further investigation. From previous

studies it has been predicted that Th 1 TCRs bind pMHC with high affinity (Malherbe

et al., 2000). Given that high affinity TCRs show a preference for expansion during

immune responses (Busch and Pamer, 1999), why would a TCR a chain from a high

affinity receptor be disfavoured in responses to its cognate antigen? One possibility is

that the answer may lie in the relationship between the polarising environment and

costimulation. The T cell line from which the TCR a chain was originally identified

was a short term T cell line, grown in vitro under highly Thl polarising conditions

(Boyton et al., 2002) i.e. high levels of the Thl polarising cytokine IL-12, and an anti-

IL-4 monoclonal antibody (mAb) were present in the culture medium. Cytokines

secreted during the course of an immune response, whether particularly polarising or

not, will influence expression of costimulatory molecules (Creery et al., 1996) and

whether through modulating the avidity of the T cell-APC interaction, or through

signalling, costimulatory molecules can have an enormous impact on the outcome of T

cell activation (Graf et al., 2007). It may be that under non-polarising conditions, such

as those used in this study where only a low concentration of IL-2 was added to the

culture medium, conditions of costimulation are such that high affinity interactions are

disfavoured e.g. by increasing the dwell time of the TCR/pMHC complex such that

rates of TCR signalling and T cell activation are suboptimal (Kalergis et al., 2001).

Under these conditions, T cells bearing TCRs with lower affinity endogenous TCR a

190

chains may proliferate faster and consequently become dominant. To circumvent the

problem of endogenous TCR a chain expansion, Thl TCR a chain animals could be

crossed with animals carrying a disrupted TCR a chain locus. These animals would

not express any endogenous TCR a chains and so questions of selection,

costimulatory requirements and phenotype bias could more easily be addressed.

Alternatively, it would be of interest to grow transgenic T cell lines in the same highly

polarising culture medium used to grow the short term Thl cell line in the first place,

to determine whether the transgenic TCR a chain is more favoured under these

conditions.

With data presented in this chapter serving to confirm the hypothesis that TCR

structure can influence T cell phenotype, also of interest is whether TCR transgenic

mice could be used to develop models of Th 1 and Th2 mediated disease. The lung is

one site at which populations of polarised Thl and Th2 T cells are found associated

with disease pathology and the following chapter describes the generation of a novel

model, using lung targeted expression of the antigen PLP 56-70, which could be

combined with the TCR transgenics discussed in this chapter to create new models of

Thl and Th2 mediated lung disease.

191

Chapter 5

Generation of transgenic lines for inducible, lung targeted, antigen

expression.

Results

5.1 Design and construction of transgenes for inducible, lung targeted, expression of

the antigen PLP56-70.

The antigen PLP 56-70 is the peptide epitope for which the Thl and Th2 TCRs,

described in chapter 3, are specific. One long-term aim is to use these TCR

transgenic lines to study inflammatory responses in the lung, by expressing antigen

(PLP56-70) in a tissue specific and inducible manner. The inducible system chosen

for this study is the Cre/Lox system (figure 1.5). Figure 5.1 outlines the overall

expression strategy.

In an uninduced animal, expression of the peptide is prevented by the presence of a

stop sequence in the transgene. The stop sequence is flanked by two loxP sites and

was first described by Lakso et al. (1992) as a way of temporally controlling oncogene

expression in mice. Transgenic mice were generated that carried a transgene

containing the simian virus 40 large tumor antigen gene driven by a murine lens

specific promoter but where the tumor antigen gene was separated from the promoter

by a floxed stop sequence. These mice showed no sign of tumor development, but

when crossed with another transgenic line carrying the Cre recombinase protein under

the control of the same lens specific promoter, Cre mediated deletion of the stop

sequence resulted in all double transgenic offspring developing lens tumors (Lakso et

al., 1992). The stop sequence comprises the 550 bp C-terminal sequence of the yeast

His3 gene, 823 bp of the SV40 polyadenylation signal region and a DNA

192

oligonucleotide that contains a false translational initiation signal. The sequence was

kindly supplied by Brian Sauer for use in this study.

Upon administration of the drug tamoxifen the bacteriophage enzyme Cre

recombinase excises the stop sequence by catalysing recombination between the two

flanking loxP sites. This allows expression of the peptide, driven by the lung specific

promoter CC10. The vector used for this strategy means that the peptide will be

expressed within the protein Hen Egg Lysozyme (HEL). Antigen processing within

APCs will cleave the HEL protein to yield the peptide PLP 56-70 which can then be

presented in the context of the MHC class II molecule I-Age. Correct expression of the

HEL/PLP fusion protein is aided by the presence of rabbit 13 globin intronic and PolyA

sequence at the 5' and 3' ends of the coding region respectively. This HEL expression

cassette has been used successfully by Vowles et al. (Vowles et al., 2000) to express a

peptide of Myelin Basic Protein (MBP 84-105). In that study they showed that the

HEL/MBP fusion protein could be expressed as a secreted or membrane bound

protein, depending on whether the membrane domain of the influenza virus A

hemagglutinin protein (HA) was present at the C terminus of the coding region and

that, particularly in the case of the membrane bound version of the protein, transgenic

expression resulted in resistance of these mice to EAE induction using the MBP

peptide. It is not known whether membrane bound or secreted forms of the HEL/PLP

protein will result in the PLP peptide being processed and presented most efficiently

and so both membrane bound and secreted forms of the expression cassette were

kindly supplied by Helen Bodmer for use in this study.

Several different promoters have been used to target transgene expression to the lung.

The three most common are CC10, K18 and SP-C. The promoter of pulmonary

surfactant protein C (SP-C) drives lung specific expression in mature alveolar type II

cells (Glasser et al., 2000) with little or no expression observed in more proximal

bronchiolar structures. Conversely, CC10 and K18 promoters direct transgene

expression to the trachea and all levels of the bronchial tree with no expression

observed in alveoli (Stripp et al., 1992 and Chow et al., 1997). As many chronic lung

193

diseases affect larger airway structures, the CC10 promoter was selected for this study.

It is well characterised and has been more widely used than the K18 promoter, for

example in targeting expression of the Th2 type cytokines IL-4 and IL-13 to the mouse

lung in order to study the role of these cytokines in Th2 type lung inflammation

(Zheng et al., 2000 and Rankin et al., 1996). The DNA sequence of the CC10

promoter used for this study is derived from rat. This DNA sequence was

characterised as the genetic element responsible for cell specific regulation of the

CC10 gene by linking stretches of DNA sequence upstream of the CC10 coding region

to a reporter gene and transfecting a lung cell line in vitro. Transgenic mice carrying a

transgene containing this regulatory sequence and the reporter gene were then

generated and lung specific expression using this promoter was confirmed (Stripp et

al., 1992). The DNA sequence used in this study was kindly supplied by Jeffrey

Whitsett.

194

Hen egg lysozyme (HEL)

(3 globin 'HI globtn Intron PolyA

Transmembrane anchor

(a)

CC10 lung promoter STOP

PLP (56-701

IQ4erCreMer Tamoxifen

J V

PLP (56-70) expressed within HEL

CC 10 lung promoter

Figure 5.1 The strategy for inducible, lung specific, expression of antigen PLP (56-70). Lung specific expression is achieved using the clara cell specific promoter, CC10. Clara cells are found only in the bronchi and bronchioles of the lung. The peptide PLP (56-70) is expressed within the protein Hen egg lysozyme (HEL), and correct mRNA processing is achieved using intronic and poly A sequence from rabbit p globin. 2 constructs were created. One construct contains a transmembrane anchor sequence and results in the expression of membrane bound HEL/PLP (56-70). In the construct not containing this anchor the HEL/PLP protein is secreted. (a) In an uninduced animal, expression of HEL and PLP (56-70) is prevented by the presence of a translational stop sequence flanked by 2 loxP sites. (b) On administration of tamoxifen, Cre recombinase previously sequestered in the cytoplasm (figure 1.5) moves to the nucleus and excises the stop sequence by recombination between the two loxP sites. Expression of HEL and PLP (56-70) is therefore induced only on administration of tamoxifen and only in clara cells of the lung.

195

(b) loxP

The expression strategy outlined in figure 5.1 depends upon both the Cre recombinase

and the CC10 promoter being active within the same cell. The strain of MerCreMer

transgenic mouse available in our laboratory ((3m70) (created by L. Bugeon and M.

Dallman) expresses the MerCreMer protein under the control of the chicken 13 actin

promoter. The MerCreMer protein expressed in these animals is known to be

functional (unpublished data), but expression in the lung is not characterised. In an

uninduced mouse the MerCreMer protein should be present diffusely in the cell

cytoplasm. On administration of tamoxifen the Cre protein translocates to the cell

nucleus where it should be detectable by immunohistochemistry. Inflated lung

sections from uninduced and tamoxifen induced (3m70 mice were stained with an

antibody specific for the Cre protein (figure 5.2). No specific staining was observed in

the uninduced mouse, but widespread nuclear staining can be seen upon induction

with tamoxifen. Not all cells are positive for nuclear localised Cre protein, but some

staining was observed in all structures of the lung. An antibody specific for the CC10

protein was also used (figure 5.2). As expected, staining was only observed in the

bronchiolar structures of the lung. Staining of the CC10 protein was the same in

uninduced and induced animals indicating that the activity of the CC10 promoter is

unaffected by the tamoxifen induction protocol.

Although, from the biotin based immunohistochemistry shown in figure 5.2, the

nuclear Cre protein appears to be present in CC10 positive bronchiolar structures of

the lung, it was important to establish that both proteins were present within exactly

the same cell. Inflated lung sections from induced animals were therefore stained with

Cre and CC10 specific antibodies at the same time, and co-localisation of these

proteins visualised using fluorescently labelled secondary antibodies (figure 5.3). The

data shows that some, but not all, CC10 positive cells also contained nuclear localised

Cre protein and that activity of the CC10 promoter was unaffected by the presence of

the Cre protein within the nucleus. Excision of the stop sequence in the transgene and

expression of PLP 56-70 in the lung should therefore be possible using the (3m70

transgenic line.

196

(a) CC10 (b) Cre

Uninduced

bronchi

alveoli Tamoxifen

induced

blood vessel

Secondary antibody only

Figure 5.2 The localisation of Cre and CC10 proteins in uninduced and tamoxifen induced lung sections from iii m70 MerCreMer mice. Mice were induced with tamoxifen i.p. for 5 days prior to harvesting of the lung. The binding of primary antibodies specific for Cre and CC10 proteins was visualised using biotin labelled secondary antibodies. (a) CC10 staining is confined to the bronchi and bronchiole structures of the lung and expression of the CC10 protein is not affected by the tamoxifen induction protocol. (b) No nuclear Cre staining can be seen in the uninduced lung, but on induction with tamoxifen, nuclear localised Cre is observed in both bronchiolar and alveolar structures of the lung. All photos are at x20 original magnification and the main structures of the lung are marked.

197

(a) x40 magnification x20 magnification

(b)

(c)

bronchi

Figure 5.3 Co-localisation of Cre and CC10 in the lung of a tamoxifen induced 13m70 MerCreMer mouse. (a) CC10 was visualised using a FITC labelled secondary antibody and is confined to the bronchi and bronchioles of the lung. (b) Cre was visualised using a TRITC labelled antibody and nuclear staining is observed in both alveolar and bronchiolar structures. (c) Co-localisation of CC10 and Cre stains shows that some but not all cells expressing CC10 also contain nuclear localised Cre protein. Photos are at x20 or x40 original magnification and main lung structures are marked.

198

The cloning strategies used to create the membrane bound and secreted versions of the

inducible, lung targeted, PLP 56-70 constructs are shown in figure 5.4. A synthetic

oligonucleotide sequence of PLP 56-70 was inserted into both the membrane and

bound and secreted HEL expression cassettes by directional cloning, replacing the

MBP peptide used previously in the constructs. PCR was used to confirm correct

insertion of the PLP 56-70 oligonucleotide into both constructs and presence of the

HA transmembrane domain in the membrane bound version of the transgene (figure

5.5). To join the HEL expression cassettes to the floxed stop component it was

necessary to introduce SfiI sites, by PCR, to the 5' and 3' ends of the rabbit 13 globin

sequences. The SfiI PCR fragments and restriction digests used to confirm successful

joining of HEL and stop sequence fragments are shown in figure 5.5.

The final step in the assembly of each transgene was the joining of the lung specific

CC1 0 promoter using NotI. The final sequence of each transgene was contained

within the cloning vector pBluescript II. A PCR using primers specific for the CCI 0

promoter and rabbit 13 globin sequence, and a XhoI restriction digest, demonstrate that

the transgenes are complete (figure 5.6). Both transgenes were also verified by

sequencing. Complete secreted and membrane bound, inducible, lung PLP constructs

are subsequently referred to as SIPLP and MIPLP respectively. Full nucleotide

sequences of membrane bound and secreted PLP transgenes are shown in appendices

23 and 25. Coding nucleotide and amino acid sequences are shown in appendices 24

and 26.

Bacterial sequence was removed from the pBluescript II vectors, by digestion with

BssHII, prior to pronuclear injection. The 6000 bp fragments of each transgene

purified for injection are shown in figure 5.6. Final plasmid maps of the MIPLP and

SIPLP constructs are shown in appendices 13 and 14 respectively.

199

PLP (56-70) 122

Nar I S Sac II

MBP peptide exchanged for PLP (56-70) oligonucleotide using Narl and SacII

(a)

loxP

II" STOP

Sf I N loxP ot I

PI" Intron HEL PLP HEL PolyA

BodXhoSfiR

Not I

(b)

Sfi I sites introduced at either end of PLP construct by PCR and the construct joined to foxed stop sequence using Sfi I

(c)

—I CC I 0 promoter

SfiIBamHIBodF

Not I

BssHII Not I PLP loxP

BssHII Not I

CCIO promoter

loxP

STOP 0101-1

(d)

Floxed stop-PLP fragment joined to CC10 promoter using Not I

Intron HEL MBP HEL PoIyA

Constructs linearised for injection using BssHII

Figure 5.4 A schematic diagram of the cloning strategy used to create the inducible, lung specific PLP (56-70) transgenes. (a) The peptide PLP (56-70) was introduced into the HEL expression cassette (Vowles et al., 2000) using Nan and SacII, replacing an MBP peptide used previously. (b) SfiI sites were introduced at either end of the HEL construct by PCR and SfiI then used to join this fragment to the floxed stop sequence (Lakso et al., 1992) (c). The lung specific promoter of CC10 (Stripp et al., 1992), was joined using NotI (d). Bacterial sequences were removed from the vector by digestion with BssHII prior to pronuclear injection.

200

z

ZD v)

a. a, 6

P.0

cip

bp Z O 8 Z VD

a. cc a.

HELA / PLPB HELA / TMCYTOB

1000 750-0-500

250

(a)

3000 2000

1000-0. 750 500

EcoRI r_.A....___

S fi I PCR ,.)a r—A—AT.

at4 al. ..-1

P-I P. 6 C) 6

CU ▪ a. .4• 6 v) bp -o

-o 6 o

— s-, o 0

)-- I) .q a. ia

CID CIO

Figure 5.5 Assembly of the inducible, lung specific, membrane bound and secreted PLP (56-70) transgenes. (a) PCRs using primers specific to HEL and PLP (56-70) demonstrate successful introduction of this peptide into the HEL expression cassette. The presence of the transmembrane anchor is also demonstrated using a primer specific to this region. (b) PCR was used to introduce SfiI sites at either end of the PLP constructs and products are 1755 bp and 1888 bp for the secreted and membrane forms respectively. These products were TA cloned and then joined to the floxed stop sequence using SfiI. EcoRI digests demonstrate correct joining of these two fragments and also the presence of the transmembrane anchor, as EcoRI sites flank the HEL coding sequence (see appendices 13 and 14). HEL fragments are 548 bp and 681 bp for secreted and membrane forms respectively.

(b)

201

CCI0/13globin PCR Xho I

I-I f A ---774 a t r__..k— bp t: -c

tu

j 'ci) cil

4000—* 3000 2500 2000-*

6000 --Po.

3000 2500-0.

(a)

(b) SIPLP MIPLP

-0 a) . k: .f) E 0 E 0

4:1

Figure 5.6 Complete inducible, lung specific, PLP constructs (membrane [MIPLP] and secreted [SIPLP]) (a) A PCR using primers specific to the CC10 promoter and rabbit 13 globin sequence demonstrates complete assembly of the construct. Products are 3239 bp and 3372 bp for the secreted and membrane constructs respectively. A XhoI digest also demonstrates that all components are present in the correct configuration. Fragments should be (3505 bp, 3006 bp, and 2175 bp) or (3505 bp, 3006 bp and 2378 bp) for SIPLP and MIPLP respectively (see appendices 13 and 14). (b) Prior to pronuclear injection bacterial sequence was removed from each vector. This was achieved by digestion with BssHII and purification of the upper 6000 bp band.

202

bp

5.2 Identification of an MIPLP transgenic founder animal.

The MIPLP construct was injected into FVB/N oocytes at the Nuffield department of

Medicine in Oxford. 24 potential founder animals were screened for the presence of

the MIPLP transgene. An initial PCR screen, using genomic DNA extracted from a

tail biopsy, identified 1 founder animal (figure 5.7). This animal is subsequently

referred to as MIPLP line 11, based on a number assigned to the animal at the point of

tail biopsy. Southern blot also confirmed the transgenic status of this animal (figure

5.7). A single intense band seen on the Southern blot for this transgenic line suggests

that multiple copies of the transgene have integrated at a single locus. The sequence

and product sizes of all PCR and Southern primers used for screening of the MIPLP

transgenic founder animal are shown in appendix 7.

Prior to generation of the MIPLP transgenic line, Pm70 MerCreMer transgenic

animals had been backcrossed for at least 3 generations onto a NOD.E genetic

background. The MHC class II molecule I-Ag7 expressed by the NOD.E strain is

required for presentation of the peptide PLP 56-70. These NOD.E backcrossed I3m70

transgenic animals were used for subsequent breeding of MIPLP line 11.

203

3000 2000

1000 750 500 250

CC101500F

CC l0 promoter

CC10210OR

3rdexonF

loxP loxP PLP STOP

(a)

2 Potential founders 1-10 "c 'CS bp 3233

(b)

(c)

kb Line

11 fo

unde

r

EcoRV

600bp DIG labelled probe

BetaglobinB

12-18

4 --11o.

3 —*

Figure 5.7 Identification of an MIPLP transgenic founder animal. The transgenic founder was identified by PCR and by Southern blot. The location of primers used for PCR screening (3rdexonF and BetaglobinB) and for generation of the Southern probe (CC101500F and CC102100R) are shown in (a). One transgenic animal was identified from an initial PCR screen of 24 potential founders (b). The transgenic status of this animal (number 11) was then confirmed by Southern blot (c). The restriction enzyme used to digest genomic DNA for Southern blotting was EcoRV. This enzyme only cuts the integrated construct once, on the 3' side of the CC10 promoter region being used as a probe (c), giving information about construct integration sites and copy number.

204

5.3 Determination of stop sequence excision and MIPLP transgene expression.

Lung expression of the antigen PLP 56-70 requires that the stop sequence present in

the transgene be removed. Cre recombinase is activated to excise the stop sequence

following administration of the drug tamoxifen. Animals carrying both the MIPLP

and Cre transgenes were identified. Following administration of tamoxifen, lung

tissue was harvested, and a PCR strategy used to determine whether stop sequence

excision had occurred (figure 5.8). A change in size of PCR product in tamoxifen

induced animals carrying both the MIPLP and Cre transgenes demonstrated that

removal of the stop sequence had occurred in the lung tissue of these animals and that

the CC10 promoter and HEL expression cassette were subsequently juxtaposed. No

stop sequence excision was observed in animals that had not received tamoxifen or in

induced animals carrying the MIPLP transgene alone. PCR was also used to

demonstrate expression of the HEL/PLP protein at the level of cDNA in lung tissue

following tamoxifen induction (figure 5.8). To detect expression of the HEL/PLP

transgene at the level of protein, immunohistochemistry of inflated lung sections from

induced and uninduced animals using an anti-HEL antibody was attempted. However,

no positive staining was observed (data not shown).

Lung tissue was harvested from transgenic animals at different time points following

tamoxifen induction. PCR demonstrates that removal of the stop sequence from the

transgene does start to occur immediately following administration of tamoxifen, but

takes many days to be removed from all copies in the lung (figure 5.9). 2 days after

tamoxifen induction, many copies of the transgene still contain the stop sequence. No

HEL/PLP protein was detectable in lung mRNA from these animals. Removal of the

stop sequence from the transgene is irreversible. However, following clearance of

tamoxifen from the circulation, new cells formed as a consequence of normal lung cell

turnover will carry the full length transgene and will therefore not express the

HEL/PLP protein. Estimates of the rate of turnover of lung epithelial cells are quite

varied and probably depend upon many factors such as the age, strain and pathogen

status of the animal. The most consistent studies have used pulse/chase experiments

205

with tritiated thymidine ([3H]) to calculate airway cell turnover and the consensus is

that the turnover time of tracheal-bronchial epithelium of adult rodents is more than

100 days (4 months) (Kauffman, 1980). The PCR data, shown in figure 5.9, show that

most copies of the stop sequence in the lung are still deleted more than 100 days after

tamoxifen induction. This suggests a very slow rate of cell turnover.

206

4— 1647bp 2000 --It. 1500 P

500 —111,- 250 --110- I 264bp

110-264hp

PLPB

U

a., a., 5 2 a. CI.)

1647bp 10-

(a)

stopfloxedF loxP loxP

stooiloxedR

PLP

lax])

HELA

PLP

CC10 promoter

(b)

(c)

bp z C C z

a a U;?..

a 2 -+-+

a a a. (d)

,_.1 i_a a a P. 5 2 2

9 U U 0 _ + + + ':' z MIP

LP g

DN

A

U

a. a a a a a a a 2 5 a a >< a) X o x

2 d 6' U + + + +

bp

2000 —IP. 1000 500 -÷

500 250

Figure 5.8 Removal of the foxed stop sequence and expression of MIPLP in lung tissue following induction with tamoxifen. (a) PCR strategies were used to demonstrate removal of the fluxed stop sequence (using primers stopfloxedF and stopfloxedR) and expression of PLP (using primers HELA and PLPB) in lung tissue from transgenic animals that had been induced with tamoxifen for 5 days. (b) Following tamoxifen induction, lung genomic DNA was used as a template to demonstrate removal of the floxed stop sequence in animals carrying both the Cre and MIPLP transgenes. No change in size of PCR product was observed in animals carrying the MIPLP transgene alone. Lung cDNA was then used to demonstrate low levels of expression of HEL/PLP. (c) HPRT was used as positive control for successful cDNA synthesis and cDNA reactions with (+) and without (-) the reverse transcriptase enzyme were used to control for any contaminating genomic DNA in lung RNA preparations. PLP expression was observed only in those animals shown to have deleted the stop sequence (d).

207

(a)

a) bp 7:1

Cre x

MIP

LP

(c)

bp z 9 O z Cr

e x M

un i

nduc

ed

a. a.

a. V

5U

2000 —0.- 1600

500 —OP-220

Figure 5.9 Complete removal of the foxed stop sequence occurs over many days and persists for many weeks following induction with tamoxifen. (a) Genomic DNA from lung tissue harvested 2 days after completion of the tamoxifen induction protocol contains copies of the transgene in which the stop sequence has been removed, but many copies of the transgene still remain unfloxed. (b) 2 weeks after completion of the tamoxifen induction protocol, almost all copies of the stop sequence in the lung have been deleted. (c) Copies of the transgene that do not contain the stop sequence remain present in the lung at least 100 days after tamoxifen induction.

208

5.4 Re-design of the SIPLP transgene.

Initial data from the MIPLP line 11 founder animal demonstrates that the lung

expression constructs designed are functional, i.e. correct deletion of the stop sequence

is observed and that HEL/PLP 56-70 mRNA transcripts can be detected in the lung.

However, levels of expression seen are very low (figure 5.8). One possible

explanation for the low levels of expression observed is that the sequence of the loxP

site, which remains in the transgene following stop sequence excision, contains an

ATG triplet and may act as a false translational start site (figure 5.10). The simplest

solution is to invert the stop cassette such that the loxP sites are still functional but are

in the reverse orientation.

No SIPLP transgenic founders were generated from an initial round of pronuclear

injections. As re-injection of the construct was required, a new SIPLP construct was

generated containing an inverted stop cassette. The cloning strategy used to create the

new SIPLP transgene is shown in figure 5.11. The complete nucleotide sequence and

final plasmid map of the new SIPLP construct are shown in appendices 27 and 15

respectively.

Bacterial sequence was removed from the construct prior to pronuclear injection as

described previously.

209

CC10 promoter WE

x ► loxP

(b) loxP PLP

CC 10 promoter

(a) fi x► loxP

Cre mediated recombination

loxP I PLP CC 10 promoter

ATAACTTCGTATAATGTATGCTATACGAAGT

loxP PLP

Cre mediated recombination

loxP r:,P

ATAACTTCGTATAGCATACATTATACGAAG

Figure 5.10 A schematic diagram to demonstrate the potential importance of loxP site orientation. (a) Recombination between loxP sites in a forward orientation results in the presence of an ATG triplet upstream of the coding region, which has the potential to act as a false translational start site. Use of this false start site may affect the efficiency of translation from the intended start site and protein expression levels may be low. Potential start sites are marked (*). (b) If loxP sites are in the reverse orientation, no ATG triplet will remain following recombination and translation of the protein of interest should be unaffected by the remaining loxP sequence.

210

HindIII KpnI

BamHI

XhoI

Mr/A. Intron HEL PLP HEL PolyA

XbaI XbaI

HindIII BssHII BssHII

I I I

WAN CC I 0 promoter

CC 10 promoter joined using HindIII

Nod Nod KpnI

HindIII HindIII BssHII

•

KpnI BamHI XhoI XbaI BssHII

(c)

Stop sequence cloned, using NotI, into a pBluescriptll vector that has been modified, using an oligonucleotide linker, to contain two KpnI sites. Stop sequence then joined to CCIO promoter and PLP fragment using KpnI

HindIII KpnI

CC I 0 promoter __LAI STOP

V

BamHI KpnI

HindIII BssHII XhoI XbaI BssHII

(d)

KpnI

4 STOP 4

CCIO promoter

PBluescriptll vector modified to destroy the KpnI site using T4 DNA polymerase

(a) InI

Rabbit 13 globin /PLP fragment cloned into the shuttle vector pCle2.1 using BamHI and XhoI

Directional cloning using HindIII and XbaI

(b) HindIII BssHII KpnI

BamHI V XhoI

1 1.4.1 1 Intron HEL PLP HEL PolyA

XbaI BssHII

HindlII HindlII

PLP inducible lung construct linearised for injection using BssHII

Figure 5.11 A schematic diagram of the cloning strategy used to invert the foxed stop sequence of the SIPLP construct. (a) A pBluescriptll shuttle vector was modified to remove a KpnI restriction site and the Rabbit 13 globin/PLP fragment inserted into this vector using HindIII and XbaI (b). The CC10 promoter was joined using HindIII (c). The floxed stop sequence was cloned into a modified pBluescriptll vector such that it was flanked by KpnI sites which were then used to clone the stop sequence into the final vector, with loxP sites in the opposite direction to those in the previous inducible PLP constructs (d). Bacterial sequence was removed from the vector by digestion with BssHII prior to pronuclear injection.

211

5.5 Identification of SIPLP transgenic founder animals.

The new SIPLP construct was injected into C57BL/6 oocytes at the MRC Clinical

Sciences Centre of Imperial College, London. 56 potential founder animals were

screened for the presence of the SIPLP transgene. An initial PCR screen, using

genomic DNA extracted from a tail biopsy, identified 4 founder animals (figure 5.12).

These animals are subsequently referred to as SIPLP line 48, SIPLP line 50, SIPLP

line 60 and SIPLP line 69, based on numbers assigned at the point of tail biopsy. The

transgenic status of each of these animals was then confirmed by Southern blot (figure

5.12). No positive band was seen on the Southern blot of SIPLP line 50. Based on

this data, SIPLP line 50 was not selected for further breeding. Positive bands on the

Southern blot for SIPLP line 69 are more intense than those for lines 48 and 60. This

suggests that the number of copies of the transgene integrated into the genome is the

greatest in line 69. Two bands are visible on the blot for SIPLP line 48 suggesting

several sites of integration of the construct in this line.

All SIPLP transgenic founders were crossed with (3m70 MerCreMer transgenic

animals that had been backcrossed for at least 6 generations onto a NOD.E genetic

background. 3 litters derived from SIPLP line 60 were screened by PCR for the

presence of the SIPLP transgene. From a total of 18 pups, no positive mice were

identified. The transgenic status of the breeding founder animal was confirmed by

PCR from a 2nd tail biopsy but, as no germ line transmission of the transgene was

observed, the line was terminated.

To date, a single transgene positive animal has been identified from a total of 20 mice

derived from the breeding of SIPLP line 69. Therefore, no further analysis of this line

has so far been possible.

The frequency of germ line transmission of the SIPLP transgene has been the greatest

for litters derived from the breeding of SIPLP line 48 (19 positive animals from a total

of 40). Litters derived from SIPLP line 48 were screened by PCR for the presence of

212

the SIPLP and Cre transgenes. To demonstrate expression of the SIPLP construct in

lung tissue of these animals, mice were induced with tamoxifen and after 14 days, lung

tissue was harvested and RNA and DNA extracted. As described for the MIPLP

transgenic line, administration of tamoxifen to animals carrying both the Cre and PLP

transgenes should result in excision of the foxed stop sequence, which can be detected

by PCR. Figure 5.13 shows removal of the stop sequence in a tamoxifen induced

animal carrying both the SIPLP and Cre transgenes. This demonstrates that, despite

inversion of the stop cassette during the re-design of the SIPLP transgene, the loxP

sites are still functional. Additionally very low levels of HEL/PLP transcripts were

detected in lung cDNA from these mice by PCR and as such expression levels

detected from the MIPLP or SIPLP transgenes appear to be similar regardless of loxP

site orientation. The SIPLP line from which this data derives (line 48) had the lowest

number of transgene copies by Southern blot. SIPLP line 69 contains many more

copies of the transgene in the genome than line 48 and experiments are currently

ongoing to determine floxing and expression upon tamoxifen induction in these mice.

213

bp 70-75 61-68

(e)

EcoRV

CC 10 STOP HEL/PLP —

600bp DIG labelled probe

1000 500

0

1000 HO. 500 —1111.

(a) loxP loxP

CC10210OR

3rdexonF

PLP

BetaglobinB

CC101500F

CC 10 rornoter STOP 4

-,,rcc u-) (b) ¢ 7..

V 6? 51-59 a.)

Z 0-, Potential founders 39-47 -= = ,... e =

1::: 2 . .

Figure 5.12 Identification of SIPLP transgenic founder animals. The transgenic founders were identified by PCR and by Southern blot. The location of primers used for PCR screening (3rdexonF and BetaglobinB) and for generation of the Southern probe (CC101500F and CC102100R) are shown in (a). Three transgenic animals were identified from an initial PCR screen of 56 potential founders (b). The transgenic status of these animals (numbers 48, 50, 60 and 69) was then confirmed by Southern blot (c). The location of bands on the Southern blot are marked by grey arrows. The restriction enzyme used to digest genomic DNA for Southern blotting was EcoRV. This enzyme only cuts the integrated construct once, on the 3' side of the CC10 promoter region being used as a probe (c), giving information about construct integration sites and copy number. Based on the result of Southern analysis, founder animal 50 was not selected for further breeding.

214

Qa 5

bp

(b)

bp

2000 1600

500 —P.- 500 —111,. 340 —IP'

z O z IP

LPxC

re u

nind

uced

SIPL

PxC

re

(c)

SIPL

PxC

re

1736 bp

stopfloxe loxP loxP PLP

cciop noter 4 STOP 4 —E111 SUPLPstoptioxedR

(a)

CC1CproMoter

HELA loxP PLP

4 —1=1/11 44-00. PLPB

353 bp

Figure 5.13 Removal of the floxed stop sequence and expression of SIPLP in lung tissue following induction with tamoxifen. (a) PCR strategies were used to demonstrate removal of the foxed stop sequence (using primers stopfloxedF and stopfloxedR) and expression of PLP (using primers HELA and PLPB) in lung tissue from transgenic animals that had been induced with tamoxifen for 5 days. (b) Following tamoxifen induction, lung genomic DNA was used as a template to demonstrate removal of the foxed stop sequence in animals carrying both the Cre and SIPLP transgenes. No change in size of PCR product was observed in animals carrying the MIPLP transgene alone. Lung cDNA was then used to demonstrate very low levels of expression of HEL/PLP. (c) cDNA reactions with (+) and without (-) the reverse transcriptase enzyme were used to control for any contaminating genomic DNA in lung RNA preparations. PLP expression was observed only in those animals shown to have deleted the stop sequence.

215

Discussion

Polarised populations of Thl or Th2 CD4+ T cells, and the cytokines that they

produce, are an important inflammatory component of a number of different lung

diseases. Thl type cytokines such as IL-12 and IFN-y, and Thl mediated immune

responses, have been found associated with chronic lung diseases such as sarcoidosis,

while Th2 type responses, involving cytokines such as IL-4, IL-5 and IL-13, are

thought to be involved in the pathogenesis of lung diseases such as cryptogenic

fibrosing alveolitis and asthma. One of the main aims of this thesis was to explore the

hypothesis that the structure of the TCR could lead to an inherent Thl or Th2 bias in

CD44- T cell phenotype, and chapters 3 and 4 have described the generation and

characterisation of TCR transgenic animals to test this hypothesis. An extension of

this main hypothesis however is an additional question of whether TCR transgenic

animals, displaying inherent bias towards Thl or Th2 type immune responses, could

be used to generate new models of Thl and Th2 mediated lung disease. Data

presented in this chapter has described the generation of inducible transgenic mice

with lung targeted expression of the Thl and Th2 TCR cognate epitope, PLP 56-70.

The inducible system selected for this study was the Cre/lox system (figure 1.5) and

lung targeted expression was achieved using the lung specific CC10 promoter. Two

versions of the transgene were created. In both versions the PLP peptide is expressed

as part of the hen egg lysozyme (HEL) protein, but one version contains a membrane

anchor sequence at the 3' end of the coding region and so results in the antigen being

membrane bound rather than secreted. Administration of the drug tamoxifen to

transgenic animals results in nuclear translocation of a ubiquitously expressed Cre

recombinase, which catalyses the removal of a translational stop sequence situated 5'

to the start of the coding sequence. Immunohistochemistry was used to demonstrate

that the Cre recombinase would become nuclear localised in Clara cells of the lung,

the cell type in which the CC10 promoter is specifically activated, validating the use

of the pm70 line of Cre mice selected for this study. One important issue of this

transgenic approach, mentioned previously in discussions of the Cre/lox system, is the

216

issue of Cre toxicity. Cre toxicity has been widely reported in mammalian cells

(Loonstra et al., 2001) and while in this inducible Cre system toxicity is unlikely to be

an issue until administration of tamoxifen, the possibility that any lung pathologies

observed in these mice might be due to the Cre protein itself must be considered.

Indeed the ubiquitous expression pattern of the Cre protein means that effects in

tissues other than the lung are also possible. While the issue of Cre toxicity is

unavoidable in this system and will persist for the lifetime of the drug within the

mouse, it is straightforward to control for by using animals carrying the Cre transgene

but not the inducible lung constructs. Any secondary effects of the Cre protein on

lung tissue and in causing lung pathology will also be observed in these animals.

Data presented in this chapter have described the cloning of secreted (SIPLP) and

membrane bound (MIPLP) lung constructs and the identification of transgenic animals

carrying each of these transgenes. A PCR strategy was used to demonstrate that

tamoxifen induction resulted in correct excision of the floxed stop sequence in lung

tissue and low levels of transgene expression were detected in lung cDNA from the

MIPLP line and SIPLP line 48. Poor germline transmission of the transgene in SIPLP

line 60 meant that this line was not carried forward for characterisation and

experiments are currently ongoing to determine transgene floxing and expression

levels in SIPLP line 69. Yet to be demonstrated in any transgenic line is the presence

of the HEL/PLP in the lung at the protein level. Attempts to visualise the protein

intracellularly using immunohistochemistry and an anti-HEL antibody have been

unsuccessful. One likely explanation for this is that the HEL protein present in the

lungs of these animals is not the native form. The coding sequence of PLP 56-70 is

inserted into the centre of the protein and as such is likely to affect the structure of this

molecule. The antibody used was polyclonal, but if the structural impact of PLP 56-70

addition to the protein is severe enough, normal antibody binding epitopes may be

destroyed. Another approach currently ongoing is to try to demonstrate the presence

of antigen using antigen specific T cell lines. If the HEL/PLP antigen is being

expressed in the SIPLP lines then protein should be present in the BAL of induced

animals. However, the presence of the membrane anchor in the MIPLP transgene

217

means that this will not be true for MIPLP transgenic mice and disruption of the lung

tissue to release intracellular protein would be required. Indeed, membrane bound

expression of antigen, and data presented in this chapter showing large numbers of

foxed transgene copies present in the lung more than 100 days following tamoxifen

induction, raises important questions about how efficiently this protein will be

presented to T cells in the lung. Presentation of PLP 56-70 in the lungs of induced

MIPLP animals will rely on natural breakdown of the airway epithelium to release the

protein. As turnover of the airway epithelium is low (Kaufmann, 1980), the amount of

antigen presented by lung APCs to resident T cells may be tiny. It may be that in this

system, infliction of mild lung injury, for example by low dose LPS exposure (Rojas et

al., 2005), may be required in order to potentiate death of airway epithelial cells,

release of antigen and initiation of an inflammatory response in the lung.

From PCR data obtained to date for both MIPLP and SIPLP lines, levels of expression

appear to be low although, from PCR data of the floxed transgene, expression should

persist for many days. In previous discussion of different lung diseases, difficulties in

modelling the chronic nature of many of these diseases, and also the unknown nature

of the antigens to which the immune system is responding, were mentioned. When the

SIPLP and MIPLP transgenic lines have been crossed with the TCR transgenic

animals bearing Thl or Th2 antigen specific TCRs, it may be that the low levels of

expression of the TCR antigen over an extended period of time that can be induced in

these animals is an effective model for some of these disease processes, particularly if

some of the antigens yet to be identified in these diseases derive from endogenous

lung proteins. Immune responses to weakly expressed endogenous antigens in the

lung, perhaps following an initial mild insult or injury, would provide an explanation

for the chronic and progressive nature of many of these lung pathologies. However,

an alterative outcome of low levels of peptide expression in the lung may turn out to

be tolerance rather than inflammation. Central tolerance to the PLP 56-70 antigen

would not be expected as the transgene is inducible and as such, especially given the

presence of the translational stop sequence 5' to the coding sequence, should not be

presented as part of the repertoire of self antigens during thymic selection. However,

218

as was noted in the original OVA TCR transgenic mice, subsequent presence of the

peptide antigen, in the case of the OVA study, by giving peptide intraperitoneally, can

result in clonal deletion of T cells bearing the OVA specific TCR in the thymus

(Murphy et al., 1990). Additionally, following tamoxifen induction and removal of

the floxed stop sequence in all tissue, i.e. not just in the lung, the potential for Aire

protein mediated tolerance induction in the thymus would exist, and recent evidence

for the existence of gene microclusters expressed by Aire suggest that this may depend

on the site of transgene integration in the genome (Derbinski et al., 2005). In the

periphery, it is well established that TCR ligation in the absence of APC-derived

costimulatory signals, such as those of the CD28 or CD4OL pathways, leads to

functional inactivation and anergy (Howland et al., 2000). This idea has been

explored in the field of allergen immunotherapy, where administration of therapeutic

doses of protein or peptide can lead to tolerance and attenuation of the normal immune

response to allergen. Peptide studies have administered antigens subcutaneously

(Norman et al., 1996), intradermally (Alexander et al., 2005) orally (Takagi et al.,

2005) and intranasally (Hall et al., 2003) and have all demonstrated improvements in

outcome measures of allergic airways disease, suggesting that the peptides have

indeed induced tolerance to the allergen. However, although many of these studies

have described using low doses of peptide, in the study of peptide tolerance by

intranasal peptide administration, therapeutic doses of peptide were quite high (100

!Ag), and such levels are unlikely to be reached in this inducible system, especially

given the low levels of transcription observed from PCR data. However, as there are

currently no reports in the literature of inducible peptide antigens in the lung, the

outcome of either low or high levels of expression on potential tolerance induction is

unknown. Peptide expression levels are likely to be a key determinant of which

immune mechanisms are initiated in these mice and the time course of any response

will also be of interest. In mouse models of lung inflammation, chronic exposure to

antigen actually decreases inflammatory responses and a suppressed, or more tolerant,

immune response is observed (Yiamouyiannis et al., 1999). Again, antigen dose is

probably a key issue and in mouse models of asthma, doses used to initiate disease are

very high. It would be interesting to observe whether the chronic stages of any

219

inflammation induced by peptide expression in the lung in this model are different and

perhaps better reflect the ongoing chronic inflammation observed in many human

diseases.

The nature of the inflammation induced using this model, in combination with the Thl

and Th2 TCR transgenic models, will depend very much on the phenotype of the TCR

transgenic T cells that traffic to the site of antigen expression. Whether prior priming

of TCR transgenic T cells with antigen and adjuvant, or as previously mentioned, the

generation of some form of mild inflammation in the lung, before T cells will traffic

there, remains to be seen. Ideally no adjuvant or other inflammatory stimulus would

be required as this will inevitably bias the Thl/Th2 nature of the subsequent

inflammatory reaction. If the TCR structural hypothesis holds true then Thl and Th2

TCR transgenic T cells in vivo will have an inherent phenotype bias. If phenotypes

observed in vitro are similar to those subsequently seen in vivo, then you would expect

T cells from the Th2 TCR transgenic line, chains for which have now both been

expressed transgenically and mice lines of which are currently being crossed together,

to express predominantly IL-5, with some IL-4 and low levels of IFN-y. In the context

of lung inflammation, one major action of the cytokine IL-5 is in the recruitment of

eosinophils. Eosinophilic inflammation may therefore be a predicted outcome in these

mice, as might several other Th2 immune pathologies, as mice constitutively

expressing high levels of IL-5 in the lung have evidence of goblet cell hyperplasia,

collagen deposition, eosinophil infiltration and airway hyperresponsiveness (Lee et al.,

1997).

As cytokine signalling pathways both antagonise and synergise in the context of

immune responses in the lung, exactly what pathologies may arise in the transgenic

mice and models of this study are unknown. However, that polarised CD4+ T cell

responses may be generated without having to artificially manipulate one cytokine,

e.g. IL-5 (Lee et al., 1997) or component of T cell differentiation, e.g. Tbet (Finotto et

al., 2002) in the context of continuous antigen presentation in the lung without having

to administer a biasing adjuvant or exceptionally high dose of antigen, means that the

220

transgenics described in this chapter and in chapters 3 and 4 of this thesis may lead to

valuable new models of Thl and Th2 mediated pulmonary pathology, particularly in

the context of chronic lung disease.

221

Chapter 6

Generation and initial characterisation of transgenic lines with lung

targeted expression of human interleukin 8.

Results

6.1 Design and construction of a transgene for lung targeted expression of human

interleukin 8.

A schematic diagram of the cloning strategy used to construct a transgene for lung

targeted expression of human interleukin 8 (hIL-8) is shown in figure 6.1. The lung

specific promoter of CC10, and the rabbit beta globin expression cassette, are the same

as those used for the inducible lung targeted antigen transgenes discussed in chapter 5.

Use of the CC10 promoter means that the human IL-8 protein will only be expressed

within bronchial epithelial cells. In human cells, the IL-8 protein is expressed as a 99

amino acid precursor (Matsushima et al., 1988). Following removal of a 20 amino

acid signal peptide, this precursor is secreted, and then cleaved extracellularly at the

N-terminus into several possible biologically active forms of the protein (Padrines et

al., 1994). To ensure secretion and correct subsequent processing of the hIL-8 protein

it was therefore necessary to use the coding sequence for the entire 99 amino acid

precursor protein in this transgene. Low levels of hIL-8 cDNA transcripts are found in

normal human peripheral blood mononuclear cells (PMBCs) (Matsushima et al.,

1988). RNA was therefore isolated from a human PBMC sample and, following

cDNA synthesis, PCR was used to clone the coding sequence of hIL-8 (figure 6.2).

The cloned PCR product was verified by sequencing prior to proceeding with the

cloning strategy. The nucleotide and amino acid sequence of hIL-8 used in this

construct is shown in appendix 29. The hIL-8 coding sequence was inserted into an

expression cassette that uses rabbit beta globin derived intronic and polyA sequences,

at the 5' and 3' ends of the cassette respectively, to aid correct processing and mRNA

222

stability of the gene of interest. The hIL-8 sequence replaced the coding sequence of

Hen Egg Lysoszyme (HEL) and the antigen PLP 56-70, used in a previous construct

(chapter 5). Removal of the HEL/PLP sequence and insertion of hIL-8 required the

use of the restriction enzyme EcoRl. An endogenous EcoR1 restriction site is present

at the 3' end of the hIL-8 sequence and so a single base pair mutation (T-->C) was

introduced by PCR at the time of cloning from cDNA such that there was no change in

amino acid sequence of the protein but that the EcoRl site was destroyed.

Joining of the lung specific CC10 promoter to the hIL-8 expression cassette was by

directional cloning using BamHI and XbaI restriction enzymes. The final construct

was checked by restriction digest (figure 6.2) and also by sequencing. The complete

sequence of the lung targeted hIL-8 transgene is shown in appendix 28. A plasmid

map of the construct is shown in appendix 16.

Bacterial sequences were removed from the construct, prior to pronuclear injection, by

digestion of the construct with Xho I and purification of the upper 3930 bp band. This

injection fragment is shown in figure 6.2.

223

PolyA tail \-7(-1

EcoR1 Rabbit beta HEL cDNA globin intron

(c) B amH 1 Xho 1

Endogenous EcoR1 site destroyed by point Xba 1 BamHl EcoRl EcoRl mutation Xho 1

I I

EcoRl hIL-8

(a) cDNA 40-• • •

hIL-8 cDNA cloned into rabbit beta globin expression cassette using EcoR1

Fragments joined using BamHl and Xbal

EcoRl IL-8

Xbal EcoRl Xhol

CC I 0 promoter

(b) Xhol BamH1 Xbal

BamHl

I CC 10 promoter

EcoR1 EcoRl Xba 1 Xho 1

I IL-8 I MA I

Construct linearised for injection using Xhol

Figure 6.1 A schematic diagram of the cloning strategy used to create the lung targeted hIL-8 transgene. (a) Human IL-8 cDNA was cloned from human PBMC cDNA using PCR primers specific for IL-8 (IL-8F and IL-8R) modified to contain EcoR1 sites at each end of the transcript and to destroy an endogenous EcoRl site. The point mutation (T—>C) did not alter the amino acid sequence of the protein. (b) hIL-8 cDNA was inserted into a rabbit beta globin expression cassette using EcoRl. (c) The rabbit beta globin expression cassette containing hIL-8 cDNA was joined to the lung specific promoter of CC10, contained in the shuttle vector pBluescriptII, using BamHl and Xbal. Bacterial sequences were removed from the vector, by digestion with Xhol, prior to pronuclear injection.

224

(a)

bp

o re

vers

e tr

ansc

ript

ase

2000 —010- 1000-10. 750 500

250

(c)

bp 0 ..z

6000 —Ow 4000 —11,- 3000 —10'

2000 --110- 1500

1 000 --Ow

Inje

ctio

n fr

agm

ent

bp

(b)

4000 2000

Figure 6.2 Cloning of human IL-8 cDNA into a lung targeted expression construct (a) The coding sequence of human IL-8 was cloned by PCR from human PBMC cDNA. The cloned PCR product inserted into a lung expression construct containing the rat lung specific promoter, CC10, and intronic sequence from rabbit beta globin. (b) The final construct was verified by restriction digest. A BamHI/XbaI digest gives expected restriction fragments of 5516 bp and 1500 bp. A XhoI digest gives expected restriction fragments of 3930 bp and 3086 bp. (c) Bacterial sequences were removed from the construct prior to pronuclear injection by digestion of the construct with XhoI and purification of the upper 3930 bp band.

225

6.2 Identification of lung targeted hIL-8 transgenic founder animals.

The lung targeted hIL-8 construct was injected into FVB/N oocytes at the Nuffield

department of Medicine in Oxford. 36 potential founder animals were screened for the

presence of the hIL-8 transgene. An initial PCR screen, using gDNA extracted from

tail biopsy, identified 2 founder animals (figure 6.3). These animals are subsequently

referred to as hIL-8 line 19 and line 33 based on numbers assigned to each animal at

the point of tail biopsy. Following the PCR screen, the transgenic status of animals 19

and 33 was confirmed by Southern blotting (figure 6.3). EcoRV was the restriction

enzyme selected to cut genomic DNA used for Southern blotting, as this enzyme

cleaves the transgene only once. The Southern blot for hIL-8 lines 19 and 33 show

that in both lines the transgene has integrated at more than one locus. These

integration sites are represented by bands that are differential between the two

transgenic lines. The EcoRV restriction site is located 2473 bp from the 5' end of the

transgene. The intense band present in both lanes represents multiple copies of the

transgene that have inserted as head to tail concatamers at one or more of the

integration loci. The sequence and product sizes of all PCR and Southern primers used

for screening of lung targeted hIL-8 transgenic founder animals are shown in appendix

8.

Both transgenic founders were subsequently bred with C57BL/6 animals to obtain

lung targeted hIL-8 transgenics for further analysis.

226

CC I 0 IL-8

600bp DIG labelled probe

EcoRV

-o Potential founders 20 - 32 o

34-36 bp

z oc

(c)

1000 750 --10- 500 —10- 250 —÷

Line

19 f

ound

er

Line

33

foun

der

CC101500F 1L-8F

IL-8 (a)

CCIO promoter

CC1021001R. IL-8R

(b)

Figure 6.3 Identification of lung targeted hIL-8 transgenic founder animals. Transgenic founders were identified by PCR and by Southern blot. The location of primers used for PCR screening (IL-8F and IL-8R) and for generation of the Southern probe (CC101500F and CC102100R) are shown in (a). Two transgenic animals were identified from an initial PCR screen of 36 potential founders (b). The transgenic status of these animals (numbers 19 and 33) was then confirmed by Southern blot (c), using rat genomic DNA as a positive control for the CC10 sequence. The restriction enzyme used to digest genomic DNA for Southern blotting was EcoRV. This enzyme only cuts the integrated construct once, on the 3' side of the CC10 promoter region being used as a probe (c), giving information about construct integration sites and copy number.

227

6.3 Determination of lung specific hIL-8 transgene expression.

The lung specific promoter used in the transgene is derived from the rat CC10 gene

and should direct expression of the gene of interest (hIL-8) in epithelial cells lining the

trachea, bronchi and bronchioles of the murine lung (Stripp et al., 1992). PCR was

used to determine the presence of hIL-8 cDNA transcripts in lung tissue from both

hIL-8 transgenic lines (figure 6.4). No hIL-8 cDNA transcripts were detected in lung

tissue from littermate control animals. In line 33 animals, hIL-8 cDNA transcripts

were only detected in the lung. In line 19, very low levels of hIL-8 transcripts were

also detected in brain tissue. The CC 10 sequence used in this transgene is well

characterised as a promoter for lung specific expression of a target gene. Expression

of hIL-8 cDNA transcripts in brain tissue is therefore unlikely to be driven by this

promoter. The observation of hIL-8 transcripts in brain tissue from line 19 but not line

33 suggests that in line 19, one or more copies of the transgene has integrated close to

a strong promoter that is active in the brain. The data shown in figure 6.4

demonstrates that hIL-8 cDNA transcripts are present in lung tissue of both hIL-8

transgenic lines. Real-time PCR was then used to investigate whether there was a

difference in relative levels of transcripts between line 19 and line 33 (figure 6.5). The

data shows that line 19 animals have a greater number of hIL-8 cDNA transcripts in

the lung compared to line 33 animals (P = 0.0005). A difference in level of hIL-8

expression between the two lines was also observed at the level of protein. hIL-8

levels in bronchoalveolar lavage (BAL) fluid (P = 0.0005) and in lung homogenate (P

= 0.0098) were higher in line 19 than in line 33. The BAL data also confirms that the

hIL-8 protein is being secreted by the lung cells expressing the transgene, and failure

to detect any hIL-8 protein in serum samples from these animals suggested that the

secreted hIL-8 protein remained in the local lung environment and was not present in

the peripheral circulation. Activity of the rat CC10 promoter has been shown to be

localised to bronchial epithelial cells of the lung rather than lung alveoli. This pattern

of expression for the transgenic hIL-8 protein was confirmed for both lines of mice

using immunohistochemistry (figure 6.6). In keeping with the cDNA transcript and

protein data shown in figure 6.5, more IL-8 staining was observed in lung sections

228

from line 19 animals. However, for both lines, IL-8 staining was patchy suggesting

that, at any one time, the transgene is not being expressed by all bronchial epithelial

cells in the lung.

229

18s IL-8

bp

750 —10. 500-0.

250-0.

(a) Line 19

Smal

l int

estin

e

r=4 0 0

0 0

0 a. V)

by z

>, 0 $... • 0 0 ▪ . - cat -0 6-..i

(b) Line 33

-4—I8s

bp

o o O 0 • —• • •4-.> o

• - • —•

.0 •—• tao 77-1) E "

E

(c) Litterm ate

Figure 6.4 Tissue specific expression of hIL-8 in both transgenic lines. (a) and (b) RNA was isolated from a range of different tissues, and primers specific for hIL-8 cDNA (IL-8F and IL-8R) were used to demonstrate lung specific expression of the transgene in both transgenic lines. cDNA reactions where no reverse transcriptase (RT) enzyme had been included were used as negative controls to ensure that any PCR product seen was not due to genomic DNA carried over during the RNA isolation procedure. (c) No hIL-8 transcripts were detectable in littermate control samples. Primers specific for 18s RNA (Ambion-1716) were used as an internal control for each sample. (a) Low levels of hIL-8 expression were also detected in brain tissue from line 19. Gels shown are representative of 3 animals per group.

230

(a)

rela

tive

mRN

A ex

pres

sion

* * * * *

10000-

Line 19

_ 7500-E. a) a. 5000-

c° J_ 2500-

0 Line 33 Littermates

I

1200

1000 --.. E 800

7:1) ra. 600

_1 400

200

Line 19 Line 33 Littermates 0

(b)

* * *

Line 19 Line 33 LittermI ates

(c)

Figure 6.5 Levels of hIL-8 cDNA transcripts and hIL-8 protein in the lung of both lines of hIL-8 transgenic mice. (a) Real-time PCR was used to compare levels of hIL-8 cDNA transcripts in lung tissue from hIL-8 line 19 and line 33 transgenic animals. Levels of IL-8 protein in BAL fluid (b) and lung homogenate (c) were determined by ELISA. hIL-8 cDNA transcripts and protein were both shown to be more abundant in lung tissue from line 19 animals. Data shown represent means + SE for each group. n = 9 for line 19, n = 11 for line 33, n = 8 for littermate controls. Statistical significance of differences between line 19 and line 33 were tested using a Mann-Whitney U test. ** P < 0.01, *** P < 0.001.

231

Secondary antibody only hIL-8 CC10

Line 19

bronchi

Line 33

Littermatc

alveoli

Figure 6.6 hIL-8 expression is confined to bronchial epithelial cells of the lung in both lines of transgenic mice. Inflated lung sections from line 19, line 33 and littermate control mice were stained using primary antibodies specific for the CC10 protein and hIL-8. Staining was visualised using a peroxidase based immunohistochemistry protocol. CC10 staining was observed in bronchiolar epithelial cells of all mice. hIL-8 staining was observed in some bronchiolar epithelial cells of lungs from both lines of transgenic animals while no staining was observed in littermate control samples. More staining was observed in samples from line 19 animals than from line 33. Images shown are representative of staining observed for more than 5 animals per group and are x20 normal magnification. The main structures of the lung are marked.

232

6.4 Lung function and lung cell infiltrate in lung targeted hIL-8 transgenics.

Having established lung specific expression of hIL-8 in both lines of transgenic mice,

the impact of constitutive expression of hIL-8 in the murine lung on lung function and

lung cell infiltrate was investigated. hIL-8 is a neutrophil chemoattractant and the

ability of hIL-8 to be chemotactic for the neutrophils of other species, including

mouse, has previously been demonstrated (Rot, 1991). Analysis of the numbers and

types of cells infiltrating both the BAL and lung of hIL-8 transgenic and littermate

animals revealed that while the total number of cells in the lung was not elevated in

the presence of hIL-8, the percentage of infiltrating cells that were neutrophils was

significantly increased (figure 6.7). The observed increase in the percentage of

neutrophils infiltrating the lung was greatest in line 19 animals (P = 0.0002), the line

previously shown to be expressing the highest levels of the hIL-8 protein, although a

significant difference was also seen in the lavage of line 33 animals (P = 0.0345). The

percentage increase in neutrophils was most apparent in the BAL, as in normal healthy

animals most cells recovered from BAL are macrophages and the percentage of cells

that are neutrophils is very low. However, in the lung, where normally at least 50 %

of cells are neutrophils, the percentage increase in neutrophils in the lungs of hIL-8

line 19 transgenic animals was still significant (P = 0.0091). Staining of inflated lung

sections with an antibody against myeloperoxidase (MPO), an enzyme that serves as a

specific marker for neutrophils (Schmekel et al., 1990), confirmed increased numbers

in line 19 animals, but also showed the location of neutrophils in the lung tissue of

these animals to be clustered around bronchi and blood vessels compared to a more

diffuse pattern of staining seen for line 33 and littermates (figure 6.8). When

consecutive lung sections from line 19 animals were stained with antibodies against

hIL-8 and myeloperoxidase, areas of neutrophil clustering next to bronchi were seen to

be associated with areas of intense hIL-8 staining (figure 6.8). The clustering of cells

around some airways and blood vessels in the lungs of line 19 animals was also

evident from the staining of lung sections with Haemotoxylin and Eosin (H+E). This

stain does not allow the different types of cells infiltrating the lung to be distinguished,

but when a general inflammatory scoring system was used, mild inflammation was

233

evident around some bronchi and vessels in the lungs of line 19 animals, compared to

very little or no inflammation in the lungs of line 33 or littermate mice (figure 6.9).

Mice used to examine cell infiltrates and lung pathology in hIL-8 transgenics

compared to littermates were 20 to 30 weeks in age and despite some evidence of mild

inflammation around bronchi and blood vessels of the transgenic line expressing the

highest levels of hIL-8, lung histology by H+E staining showed no evidence of other

lung pathology.

Lung diseases associated with high levels of IL-8 such as asthma and chronic

obstructive pulmonary disesase (COPD) (Nocker et al., 1996) are also invariably

associated with altered lung function (Cherniack, 1956). The three main

measurements of lung function used routinely in other mouse models of lung disease,

bronchial hyperreactivity (Penh), resistance and compliance were measured in the hIL-

8 transgenic lines and littermate animals. Neither hIL-8 transgenic line showed any

difference, compared to littermates, in bronchial hyperreactivity or airway resistance

in response to the muscarinic receptor agonist methacholine (figure 6.10). However,

line 19 animals and to a lesser extent, line 33 animals showed decreased airway

compliance compared to littermates although the difference was not statistically

significant.

234

Lung

Line 19 Line 33 Littermates

cell

num

ber (

x10 4

)

100-,

75-

50-

25-

0

BAL

Line 19 Line 33 Liftamates

(a)

cell

num

ber (

x104 )

10.0

7.5-

5.0-

2.5-

0.0

(b)

40- -J Ca 30- •_

.c 20-C. 0

10-C

Line 19 FTTI Line 33 Littamates

*

* * *

0 Line 19 Line 33 Littermates

* *

*

75- 01 C

50-o a. 0 0

25-o

Figure 6.7 Baseline cell infiltrates in the lungs of IL-8 transgenics and littermate animals. (a) The total number of cells recovered from the bronchoalveolar lavage (BAL) and whole lung lobe of IL-8 transgenic and littermate mice were determined by cell counting. (b) Differential cell counts of cytospins from BAL and lung were used to determine the percentage neutrophils in each sample. Mice used were 20-30 weeks of age and the data shown represents the mean values for 9 animals for line 19, 11 animals for line 33 and 8 littermate animals + SE. The statistical significance of differences between line 19, line 33 and littermate groups were tested using a Mann-Whitney U test. * P <0.05, ** P < 0.01, *** P < 0.0005

235

(a)

aver

age

MPO

score

3

2

=lbronchi =vessels

0

Line 19 Line 33 Littermate

(b) Line 19

Line 33 Littermate

(c)

IL-8

Myeloperoxidase

Figure 6.8 Neutrophil staining by immunohistochemistry of lung sections from IL-8 transgenic and littermate animals. Inflated frozen lung sections from IL-8 transgenic and littermate control animals aged 20 -30 weeks were stained using a primary antibody specific for the neutrophil marker myeloperoxidase. Staining was visualised using a peroxidase based immunohistochemistry protocol. (a) The number of neutrophils surrounding bronchi and blood vessels in each section were assessed using an inflammatory score system (appendix 30). The data is presented as a mean value + SE for each group and represents 9 line 19, 9 line 33 and 3 littermate animals. The statistical significance of differences between transgenic and littermate groups was tested using a Mann-Whitney U test. *P <0.05, **P <0.005. (b) Line 19 animals were found to have a greater number of neutrophils around both bronchi and blood vessels compared to line 33 and littermate animals. (c) Staining of consecutively cut sections with myeloperoxidase and IL-8 antibodies showed a relationship between clustering of neutrophils around bronchi and secretion of IL-8 by bronchial epithelial cells in line 19 animals. All photos are x20 normal magnification.

236

(a)

aver

age

infla

mm

atio

n sc

ore

=J bronchi =vessels

3

2

0 line 19 line 33 littermate

(b) Line 19

Line 33

Littermate

Figure 6.9 Mild inflammation in line 19 transgenics compared to line 33 and littermate animals. Inflated frozen lung sections from IL-8 transgenic and littermate animals aged 20-30 weeks were stained with Haematoxylin and Eosin (H+E). (a) Cell infiltrates around bronchi and blood vessels were assessed using an inflammatory score system. Data is presented as mean values per group + SE and represents 9 line 19, 9 line 33 and 3 littermate animals. The statistical significance of differences between groups was tested using a Mann-Whitney U test. ** P < 0.01. (b) Lung sections from line 19 animals showed evidence of mild cell infiltrates around some bronchi and vessels. Very few or no cell infiltrates were present in the lung sections from line 33 and littermate animals. All photos are x20 normal magnification.

237

25 50 75 100 Methacholine (mg/ml)

(c)

(a)

(b)

0.020

6 o cu 0 0.015 c cs • 4

0.000 25 50 75 100 0 Methacholine (mg/ml)

a 0.010 o

U ct 2 0.005

25 50 75 Methacholine (mg/ml)

100

Figure 6.10 Lung function of IL-8 transgenic and littermate animals. Mice aged 20-30 weeks were used to obtain Penh (a), resistance (b) and compliance (c) lung function measurements for IL-8 transgenic and littermate animals. Penh data shown is shown as mean values for 14 line 19 (0), 16 line 33 (A) and 8 littermate animals (0) + SE and is representative of three independent experiments. Resistance and compliance data is shown as mean values for 4 line 19, 5 line 33 and 3 littermate animals + SE and is representative of a single experiment.

238

6.5 Investigation of the impact of hIL-8 expression in the murine lung during

ovalbumin induced lung inflammation.

Lung data obtained for the hIL-8 transgenic lines thus far was obtained in the absence

of any external inflammatory stimulus. To examine the effect of hIL-8 expression in

the lung, in the context of an inflammatory response, an ovalbumin (OVA) model of

lung inflammation was used. Mice used for this experiment were 20-30 weeks in age

and were sensitised to OVA antigens by intra-peritoneal (i.p) injection of OVA, using

alum as an adjuvant. Control mice received PBS and alum. All mice were then

exposed to aerosolised OVA for 5 consecutive days before analysis of lung function

and lung tissue. Lung function measurements showed greater bronchial

hyperreactvity, greater resistance and reduced compliance in OVA sensitised mice

compared to controls, but differences were small and not statistically significant

(figure 6.11). In the OVA sensitised groups no significant differences were observed

between transgenic and littermate control animals for Penh, resistance or compliance

measurements. Line 33 OVA sensitised animals appeared to show increased

resistance compared to line 19 and littermate controls but this difference did not reach

statistical significance. However, of note was an apparent difference in bronchial

hyperreactivity between hIL-8 transgenic and littermate animals that had received just

alum as part of the sensitisation protocol. hIL-8 transgenic animals showed increased

bronchial hyperreactivity compared to littermate controls with the difference reaching

statistical significance in the case of line 33 animals (P=0.0159) (figure 6.11).

Despite the lack of significant changes in lung function between OVA sensitised and

alum control groups upon antigen challenge, there were differences in lung cell

infiltrates and pathology. Figure 6.12 shows differential cell counts for BAL and lung

in all groups. In the BAL of control mice, dominant cell types were macrophages and

lymphocytes although some neutrophils and eosinophils were also present. The

percentage of neutrophils in the BAL of these animals followed the same pattern as

seen previously in the absence of any antigen challenge, with the greatest percentage

in the line 19 hIL-8 transgenic group. This was also true in the BAL of mice

239

sensitised to OVA, although in all groups of OVA sensitised mice, the dominant cell

type was the eosinophil and actual neutrophil numbers found in the lung were not

greatly increased. In the lung tissue of control mice the most dominant cell type was

the neutrophil. Again, the greatest percentage was in the line 19 hIL-8 transgenic

group, although this was not the case in lung tissue of mice sensitised to OVA. In all

OVA sensitised groups, the dominant cell type was the eosinophil, numbers of which

were greatly increased compared to controls. The number of neutrophils in the lungs

of these mice remained largely unchanged even in hIL-8 transgenic lines.

Inflammation scores for H+E and myeloperoxidase (MPO) (figure 6.13) stained lung

sections from all groups of mice revealed significant differences in severity of

inflammation and pathology between control and OVA sensitised groups, but very

little difference between hIL-8 transgenic groups and littermates. The biggest,

although not statistically significant difference, was in the H+E inflammation scores of

bronchi and vessels for the hIL-8 line 33 transgenic control group which appeared to

show a greater degree of mild inflammation. However, the same trend was not

observed in the MPO inflammation score and so any differential inflammation that

may be present in this group is not due to neutrophils. Equivalent inflammation by

H+E and MPO staining was seen around the bronchi and blood vessels in all groups of

OVA sensitised animals.

Periodic Acid Schiff (PAS) staining was also performed. PAS is used to identify cells

containing high levels of glycoproteins. Glycoproteins known as mucins are the major

component of mucus which is secreted by airway epithelial goblet cells (Rogers 1994).

Unlike in humans, goblet cells are not found in murine airways in the absence of an

inflammatory stimulus (Pack et al., 1981), however, goblet cell mucus hypersecretion

is a common characteristic of airway inflammation in both species. PAS staining of

lung tissue from hIL-8 transgenic and littermate animals following OVA challenge

showed increased numbers of mucus secreting globlet cells in the airways of hIL-8

transgenic animals in both control and OVA sensitised groups compared to littermates

(figure 6.14). No mucus secreting cells at all were found in the airways of littermate

240

control animals and while OVA sensitisation and challenge did induce globlet cell

mucus secretion in some airways of littermate animals, the number of airways

secreting mucus and the average number of globlet cells in each airway was greater in

hIL-8 transgenic animals. Statistical significance was not reached with this data

however, and so further experiments are necessary.

241

0.020

0.015 c., c co Q 0.010 E 0 0 0.005

0.000 0 25 50 75

Methacholine (mg/ml)

(c)

100

(a) —N— Line 19 OVA —o— Line 33 OVA —*— Littermate OVA —a— Line 19 Alum —o— Line 33 Alum —it— Littermate Alum

0 25 50 75

100 Methacholine (mg/ml)

0 25 50 75 100 Methacholine (mg/ml)

(b)

Res

ista

nce

10

8

6

4

2

0

Figure 6.11 Lung function measurements in IL-8 transgenic and littermate animals following ovalbumin sensitisation and challenge. Mice were sensitised to ovalbumin by i.p injection of ovalbumin and alum on days 0 and 12. Control mice received PBS and alum. All mice received an ovalbumin aerosol on days 19, 20, 21, 22 and 23. (a) Penh lung function measurements were obtained on day 24. Resistance (b) and compliance (c) measurements were obtained on day 25. Data shown represents mean values for a minimum of 3 animals per group + SE.

242

PBS OVA PBS OVA

'Lung

Neutrophils =Eosinophils

Macrophages 5553 Lymphocytes

Line 19 Line 33 Littermate Line 19 Line 33 Littermate

PBS OVA

Neutrophils =Eosinophils 71- =Macrophages cV

5553 Lymphocytes

300

200

E _o

e • 100 a) V

Line 19 Line 33 Littermate Line 19 Line 33 Littermate Line 19 Line 33 Littermate Line 19 Line 33 Littermate

Figure 6.12 Differential BAL and lung cell counts from IL-8 transgenic and littermate animals following ovalbumin sensitisation and challenge. Cytospins of BAL and lung cells were stained with Wright Geimsa and numbers of neutrophils, eosinsophils, macrophages and lymphocytes counted in each sample. The percentage of each cell type (a) was calculated and converted to a total number of cells in BAL or lung (b) by multiplying the cell percentage by the total number of cells isolated for each sample. Data shown represents a minimum of 3 animals per group and is presented as a mean value for the group ± SE.

(a) 100-

co 80-

0 111111 .111

BAL

I

— Line 33

NM.

Littermate 4111

Line 19 Line 33 Littermate Line 19

PBS

400

300

-0 200 E

= 100

OVA (b)

(a) (b)

PBS OVA PBS OVA

bronchi Line 33

(c) Littermate Line 19

PBS

OVA

blood vessels

aver

age in

flam

mat

ion s

core

3- 3

2

1

0

2-

Ave

rage

MPO

sco

re =lbronchi

=vessels

1-

0 Line 19 Line 33 Littermate Line 19 Line 19 Line 33 Littermate Line 19 Line 33 Littermate Line 33 Littermate

Figure 6.13 Lung inflammation in OVA sensitised and challenged IL-8 transgenic and littermate animals. IL-8 transgenic and littermate animals were sensitised by i.p injection of OVA and alum on days 0 and 12. Control mice received PBS and alum. All mice were challenged with aerosolised OVA on days 19 to 23. Lung tissue was harvested on day 25 and frozen lung sections stained with H+E (a) or an antibody specific for the neutrophil marker myeloperoxidase (b). Cell infiltrates around bronchi and blood vessels were assessed using an inflammatory score system (appendix 30). Data is presented as mean values per group ± SE and represents a minimum of 3 animals per group. The statistical significance of differences between groups was tested using a Mann-Whitney U test. (c) H+E stained lung sections from line 33 PBS control animals showed evidence of mild cell infiltrates around some bronchi and vessels but equivalent inflammation was seen in lung sections from all groups of OVA sensitised animals.

244

(a)

PBS OVA

Line 19 Line 33 Littermate Line 19 Line 33 Littermate

aver

age

PAS s

core

2

1

0

(b) Line 19

Line 33

Littcrmate

PBS

OVA

Figure 6.14 Prevalence of mucus secreting goblet cells in inflated lung sections from OVA sensitised and challenged IL-8 transgenic and littermate animals. IL-8 transgenic and littermate animals were sensitised by i.p injection of OVA and alum on days 0 and 12. Control mice received PBS and alum. All mice were challenged with aerosolised OVA on days 19 to 23. Lung tissue was harvested on day 25 and frozen lung sections stained with PAS to identify mucus secreting goblet cells (bright purple staining). (a) A score system based on the percentage of each airway containing goblet cells was used to assess the prevalence of goblet cells in each sample. Data is presented as mean values per group + SE and represents a minimum of 3 animals per group. The statistical significance of differences between groups was tested using a Mann-Whitney U test. (b) Small numbers of goblet cells were found in the airways of PBS control IL-8 transgenic animals. In animals that had been sensitised to OVA, goblet cells were most prevalent in the airways of line 19 animals and the least prevalent in the airways of littermate animals.

245

Discussion

IL-8 is a CXC chemokine with potent chemoattractant properties. It is secreted by

many different cell types including T cells, neutrophils and NK cells, as well as by

somatic cells such as endothelial cells, fibroblasts and epithelial cells. It acts

predominantly to recruit and activate neutrophils at sites of infection or injury and

high levels of IL-8 have been detected in a variety of disease states including lung

diseases such as cystic fibrosis, bronchiecstasis, COPD and asthma. There are

currently no reports in the literature of human IL-8 having been overexpressed in the

mouse lung, but the reported associations between high levels of this protein and

disease and increasing evidence for neutrophil independent roles of this cytokine in

disease processes make IL-8 an attractive target for transgenesis. Data presented in

this chapter has described the generation of two mouse lines with lung targeted

expression of human IL-8 and initial basic characterisation of their phenotype.

The IL-8 transgene created for this study contained the human IL-8 (hIL-8) cDNA

sequence, intronic and polyA sequences from the rabbit beta globin protein and the rat

lung specific CC10 promoter. Two founder animals carrying this transgene were

identified by PCR and southern blot, which revealed both lines to be carrying multiple

copies of the transgene at multiple integration loci. Transgene expression levels in

each line were assessed by PCR, ELISA and antibody staining. Each revealed line 19

animals to be expressing higher levels of hIL-8 than those of line 33 and in both lines

expression was restricted to lung tissue, although in line 19 low levels of expression

were also detected in the brain. The CC10 promoter has been used extensively in lung

targeted transgenesis and is well characterised as a lung specific promoter whose

activity is restricted to Clara cells of the airway epithelium (Hagen et al., 1990). The

promoter regions surrounding the CC10 gene that confer lung specific expression have

been characterised (Stripp et al., 1992) and while exactly how this tissue specific

expression is achieved is not yet completely understood, negative regulators thought to

repress CC10 expression in non-Clara cells have been identified (Navab et al., 2002).

There are no reports in the literature of the CC10 protein being expressed, or the CC10

246

promoter being active, in brain tissue and so the low levels of expression found in the

brain tissue of line 19 animals are most likely to be due to position effects at the site of

transgene integration in this line. Brain expression of IL-8 in line 19 animals was not

explored further in this study, but it may be worthwhile to do so in the future. IL-8 is

known to be expressed by neural cells, such as microglia, and upregulation has been

associated with some inflammatory pathologies in the brain, such as that mediated by

the amyloid beta peptide in Alzheimer's disease (Walker et al., 2006). Whether

constitutive hIL-8 expression in brain tissue of these mice causes any kind of

inflammatory cell recruitment or other pathology to be observed may therefore be of

interest. Different integration loci between the two lines are also the most likely

explanation for the higher levels of expression seen in line 19 compared to line 33 and

that transgene expression can be affected by the flanking 5' and 3' chromosomal

regions at an integration locus has been well documented (Cranston et al., 2001).

Although immunohistochemical staining of inflated lung sections demonstrated that

lung expression in both lines was restricted to the bronchial epithelium, expression

patterns in both lines were patchy. This may reflect the impact of 5' and 3'

chromosomal regions on the activity of the transgenic CC10 promoter or perhaps

suggests that some elements that control endogenous CC10 gene expression are

missing from the sequence used in the transgene. The CC10 protein, also known as

the Clara cell secretory protein (CCSP) is the predominant protein product of Clara

cells (Patton et al., 1986) and has been proposed to function as a downregulator of

airway inflammation. Indeed, altered levels of this protein have been associated with

some lung diseases, such as sarcoidosis, where lower levels of the CC10 protein in

serum and in BAL were found in patients with progressive compared to regressive

disease (Shijubo et al., 2000). However, inflammatory cytokines are also major

secretory products of the bronchial epithelium with lung insults or infection being

potent inducers of production. For example, exposure to cigarette smoke, the main

known cause of COPD, is known to induce high levels of IL-8 production by human

bronchial epithelial cells (Mio et al., 1997) and recently the bacterial compound LPS

was found to induce production of the murine IL-8 homologue KC in Clara cells

247

(Elizur et al., 2007). IL-8 expression therefore constitutes a normal function of the

airway epithelium.

As discussed previously, the major function of IL-8 is in mediating chemoattraction

and activation of leukocytes, particularly neutrophils. That hIL-8 is able to mediate

chemotaxis of mouse neutrophils has been described (Rot, 1991) and although mice do

not possess an IL-8 gene, murine proteins with potent neutrophil chemoattractant

properties which bind the murine CXCR2 receptor have been identified (Bozic et al.,

1994). The murine CXC chemokine KC has been overexpressed in the murine lung

under the control of the CC10 promoter and has been reported to result in enhanced

neutrophil migration within the lungs of transgenic animals, although the neutrophils

were not activated and no tissue damage was apparent (Lira, 1996). Given that mouse

neutrophils can respond to hIL-8 and the similarities in function between KC and IL-8,

it might be predicted that overexpression of hIL-8 in the lungs of the two mouse lines

described in this study would also result in elevated numbers of neutrophils in the

lungs of these animals. Differential cell counts from BAL and lung tissue of IL-8

transgenic and littermate animals showed that IL-8 expression in the mouse lung did

result in an increased percentage of neutrophils in the lungs of transgenic animals and

this increase was observed to be the greatest in line 19 animals, the line also reported

to have the highest levels of IL-8 protein expression. However, perhaps unexpectedly,

no real differences were observed in terms of absolute cell numbers, although scoring

of transgenic and littermate inflated lung sections did suggest that some bronchi and

vessels showed signs of inflammation with the degree of inflammation being the

greatest in the lungs of line 19 animals. Although no large infiltrates of neutrophils

were observed in either line, the increased percentage of neutrophils in both BAL and

lung tissue suggests that the hIL-8 expressed by the lung epithelial cells in this model

is biologically active. This was also implied by immunohistochemistry staining for

neutrophils in inflated lung sections where areas of neutrophil clustering in the lungs

of transgenic mice were found to be associated with areas of greatest IL-8 protein

expression. This suggests that hIL-8 can indeed bind to and activate the CXCR2

receptor expressed by murine neutrophils and as expression is limited to bronchial

248

epithelial cells, the IL-8 protein secreted into airways of these mice is likely to be

capable of endothelial transcytosis in order to mediate neutrophil recruitment from

vessels into the lung tissue and BAL fluid. This is also supported by studies in which

recombinant hIL-8 has been administered directly into an organ and neutrophil

infiltration reported (Singer and Sansonetti 2004). However, this study by Singer and

Sansonetti, which reports colitis type pathology in mice given intracolonic rhIL-8

concomitantly with bacterial infection, also reports a lack of pathology or infiltrate

when rhIL-8 was administered alone. The authors speculate that lack of neutrophilic

infiltrates in mice given rhIL-8 alone may be due to the short half life of the protein in

the gut or due to a low affinity for murine CXCR2 compared to the murine

homologues, KC and MIP-2. In relation to the data presented in this chapter, where

expression of hIL-8 in the lung results in only mild inflammatory cell infiltrates, short

half-life of the protein is an unlikely explanation as IL-8 expression is constitutive.

However, it is possible that low affinity binding of hIL-8 to the mouse CXCR2

receptor means that much higher levels than those reported in lines 19 and line 33 of

this study would be required to mediate infiltration of large numbers of neutrophils

into the lung. This hypothesis is supported by the increased percentage of neutrophils

found in BAL and lung of line 19 animals compared to the lower expressing line 33

and the increased numbers of neutrophils and greater levels of lung inflammation

found in BAL and lung of some, although not all, line 19 animals. However, an

alternative explanation, which also has some support in the literature, is that

constitutive expression of the IL-8 protein results in downregulation of trafficking,

perhaps though altered adhesion molecule expression or reduced expression of the

chemokine receptor itself on the surface of circulating leukocytes. In support of

reduced neutrophil chemotaxis, a recent study of neutrophils from COPD patients who

also display high levels of IL-8 protein in the lung over long periods of time, found

COPD patient neutrophils to have reduced chemotactic responsiveness compared to

cells from healthy controls (Yoshikawa et al., 2007). Although there are no previous

reports of hIL-8 overexpression in the mouse lung, hIL-8 has been targeted to other

organs. The first report of a hIL-8 transgenic mouse described hIL-8 under the control

of a liver specific promoter (Simonet et al., 1994). In this study by Simonet et al. high

249

levels of circulating neutrophils in the peripheral blood were observed, but neutrophil

migration in response to localised administration of rhIL-8 was impaired. It is of note

that concentrations of rhIL-8 used in this study to induce local neutrophil trafficking

when given intraperitoneally are similar and often lower than those used by Singer and

Sansonetti, (2004) intracolonically, where no infiltrates were reported, suggesting that

mechanisms dictating levels of neutrophil trafficking may be different depending on

the site of IL-8 administration. In the transgenic study by Simonet et al. where

impaired neutrophil migration was reported, peripheral blood neutrophils were found

to have reduced levels of the adhesion molecule L-selectin. L-selectin is an important

mediator of neutrophil recruitment as it supports rolling of leukocytes along the

vascular endothelium. Downregulation of this molecule was proposed to be mediated

by the long term elevation of hIL-8 in the serum of these transgenic mice. With

constitutive expression of hIL-8 in the lungs of the two IL-8 transgenic lines described

in this chapter, that some IL-8 protein may reach the peripheral circulation is not

implausible and may have meant that a similar mechanism of adhesion molecule

downregulation was occurring. However, there were no detectable levels of hIL-8

protein in serum from either transgenic line described in this study. Other studies have

described receptor desensitisation as a mechanism of reducing neutrophil migration

(Wiekowski et al., 2001b). In this study by Wiekowski et al. inducible expression of

KC was targeted to many different tissues using the chicken beta actin promoter. No

inflammatory cell infiltrates were observed in any organ expressing KC and CXCR2

mediated signals were found to be attenuated in peripheral blood neutrophils

compared to cells from non transgenic animals. In keeping with the study by Simonet

et al. high levels of KC were detectable in serum, a finding not consistent with data

obtained from the lung targeted hIL-8 mice described in this study. However IL-8 is

known to regulate receptor expression on the cell surface (Samanta et al., 1990) and

the observation of downmodulation of CXCR1 and CXCR2 on peripheral blood and

also sputum neutrophils from COPD and asthmatic patients suggests that high levels

of local as well as systemic IL-8 protein may be able to mediate this mechanism of

regulation (Pignatti et al., 2005). To determine the reason for the lack of

inflammatory cell infiltrates in the mice described in this chapter several different

250

approaches are possible. If concentration of hIL-8 is the issue then intranasal

administration of rhIL-8 should induce neutrophil trafficking into the lungs of these

mice. However, additional evidence against this hypothesis includes a report into the

effects of rhIL-8 in guinea pigs where intranasal administration of this protein induced

bronchoconstriction but with no evidence of leukocyte accumulation (Fujimura et al.,

1999). Additionally, peripheral blood neutrophils and indeed neutrophils from the

lungs of these animals should migrate normally to rhIL-8 in in vitro chemotaxis

assays. If neutrophil migration has in some way become impaired, through the

constitutive expression of IL-8 over a long period of time, then in vitro neutrophil

chemotaxis should also be impaired. In addition you would expect neutrophils

transferred from a non transgenic animal to traffic normally to the lung in response to

IL-8 expression there. Analysis of the cell surface expression levels of adhesion

molecules such as L-selectin, CD11 b and CD11c and the CXCR2 receptor on

neutrophils from these transgenic animals would also be of interest as would intranasal

administration of KC or MIP-2 to determine whether impaired migration is also in

response to endogenous CXC chemokines.

Although at a baseline level, neutrophil accumulation in the lungs of both line 19 and

line 33 transgenic animals is not induced or is impaired, other studies have reported

that even when neutrophils are recruited in response to overexpression of CXC

chemokines, they do not appear to be activated (Lira, 1996 and Kucharzik et al.,

2005). Further experiments presented in this chapter attempted to determine whether

patterns of neutrophil recruitment would be different between transgenic and littermate

animals in the context of an inflammatory response. The well characterised

ovalbumin model of allergic airway inflammation was used in this study as it is a

potent inducer of inflammation in the lung and although neutrophilic infiltrates and

elevated IL-8 levels are a common feature of chronic asthma, the inflammation

observed in the ovalbumin model is predominantly eosinophilic. Any subtle changes

in neutrophil infiltration into the lungs of the IL-8 transgenic mice of this study should

therefore be more apparent, but the effect of hIL-8 on the other disease outcome

measures of this model of asthma could also be measured. However, despite seeing

251

the same increased percentage of neutrophils in the BAL of transgenic animals

compared to littermates as was reported previously in the baseline data, no significant

increase in neutrophil numbers was observed in any group following sensitisation and

exposure to ovalbumin. In both transgenic and littermate groups the lung infiltrate

was predominantly and equally eosinophilic and no major differences in general

inflammation scores or neutrophilic inflammation scores were found. From this data,

any impairments in neutrophil accumulation in the lung targeted hIL-8 transgenic mice

cannot be overcome by inflammatory signals in the ovalbumin lung disease model and

the migration of eosinophils to the lung is not impaired or indeed augmented by the

presence of the IL-8 protein. However, impaired neutrophil migration is difficult to

assess here as no significant neutrophilic infiltrate is expected in normal non-

transgenic animals. It would be of interest to determine whether migration in response

to a well characterised neutrophil activating stimulus, such as LPS, is perturbed.

However, a study by Remick et al. showing normal recruitment of neutrophils in

response to acid induced lung injury in transgenic mice with high levels of serum IL-8,

previously described as having impaired neutrophil migration, suggests that it might

not be (Remick et al., 2001). In that study, neutrophil migration was found to be

mediated normally by the endogenous murine chemokines KC and MIP-2.

If neutrophil migration is indeed impaired in the lung targeted hIL-8 transgenic mice

described in this chapter, then this effect could be circumvented with the use of

conditional transgenic mice. There are now several reports in the literature of

conditional IL-8 expression. In one study an adenoviral vector was used to

overexpress hIL-8 in the cornea (Oka et aL, 2006) and in another the tetracycline

system was used to induce hIL-8 expression in the intestine (Kucharzik et al., 2005).

In both cases, significant infiltration of neutrophils to the site of hIL-8 expression was

induced. However, it may be of interest to study the impact of hIL-8 in the lung in the

absence of large numbers of neutrophils and neutrophil driven inflammation and

damage as there is increasing evidence for potential neutrophil independent roles for

IL-8 in human lung pathology. One important study recently published concerned a

potential role for IL-8 in impaired lung function and lung remodelling with evidence

252

that IL-8 could induce Ca2+ release in human airway smooth muscle cells as well as

causing cell contraction and inducing cell migration (Govindaraju et al., 2006). Lung

responses to methacholine, measured as Perth, resistance and compliance are the most

commonly used markers of lung function in the mouse and so these parameters were

determined in the two lung targeted hIL-8 transgenic lines and compared to littermate

animals. No differences in lung function by any of these measures were observed

however, except perhaps for lower compliance measurements particularly in line 19

compared to littermates, although differences were not significant. From this data it

appears that constitutively high levels of IL-8 in the mouse lung do not affect lung

responses to methacholine, i.e. there is no apparent increase in bronchial

hyperreactivity, as might have been suggested from studies with guinea pigs (Fujimura

et al., 1999). However, in the study by Fujimura et al. IL-8 was not expressed by the

lung but delivered as a single dose at a single time point and the constitutive nature of

IL-8 expression in this study may have a major impact, for example by decreasing

CXCR2 expression levels on the surface of murine airway smooth muscle. Of interest

however were the findings of differences in bronchial hyperreactivity between hIL-8

transgenic mice and littermate controls that had been sensitised with alum alone but

then exposed to aerosolised OVA. Reasons for increased bronchial hyperreactivity in

transgenic animals compared to littermate controls in this situation, but not in the

absence of antigen challenge, are not clear but it may be that chronic exposure to hIL-

8 over long periods of time in these mice somehow predisposes to impaired lung

function upon challenge with antigen in a way that is independent of a T cell mediated

immune response e.g. by directly impacting on the lung tissue itself in terms of airway

remodelling or smooth muscle reactivity, the consequences of which are more

pronounced in the context of aerosolised exposure to a foreign protein

Despite this small difference in lung function between alum treated transgenic and

littermate control groups, differences in lung function measurements between

transgenic and littermate control disease groups were not observed. Also importantly,

although in all transgenic and littermate groups sensitisation and exposure to

253

ovalbumin was associated with slightly worse lung function measurements,

differences between disease and alum only control groups were very slight. As other

parameters, such as eosinophilic lung inflammation and goblet cell scoring suggested

that disease had indeed been successfully induced, the lack of large changes in lung

function is most likely due to the genetic background of mice used in this study.

Ovalbumin protocols involving lung function measurements most commonly use

BALB/c mice as they show good susceptibility and marked outcomes of disease

compared to other strains. Strain dependent variations in lung function measurements

are well reported (Reinhard et al., 2002) and it is likely that larger differences between

disease and control groups, and perhaps even at a baseline as a consequence of IL-8

expression, may be more evident on a different genetic background.

Despite no observed differences between IL-8 transgenic and littermate disease groups

with respect to cell infiltrates and lung function, one disease parameter was found to

be altered. The disease groups of both lines of lung targeted hIL-8 transgenic animals

showed an increased prevalence of mucus secreting goblet cells in the airways of the

lung when compared to littermate control animals. Goblet cell mucus hypersecretion

and goblet cell metaplasia is a common characteristic of allergic airway inflammation

in both humans and mice and is associated with airway remodelling. Metaplasia is the

process by which goblet cells arise from normal airway epithelial cells, such as Clara

cells, and once formed, produce mucins in response to many factors including Th2

type cytokines IL-13 and IL-4 (Kuperman et al., 2005). Mucus hypersecretion is not

just a feature of asthma, but is found in association with many other lung diseases such

as cystic fibrosis, chronic bronchitis, bronchiectasis and COPD, and excessive mucus

plays a major part in the pathology of these diseases by plugging airways and causing

recurrent infections. Neutrophils have been implicated in pathways leading to goblet

cell hyperplasia (Shao and Nadel, 2005) but aside from their role in neutrophil

recruitment, there have been no reports directly implicating CXC chemokines in this

process. Data presented in this chapter suggests that there is no difference in the

number of goblet cells or mucus secretion in the lungs of transgenic compared to

control mice in the absence of disease induction. This argues against a hypothesis

254

where IL-8 is acting directly on the airway epithelium in the absence of other

inflammatory stimuli to mediate hyperplasia. The lack of a neutrophil infiltrate in

these animals also argues against a neutrophil mediated mechanism and so perhaps

one more likely hypothesis is that IL-8 may be affecting the IL-4 and IL-13 mediated

pathways of goblet cell production and activation. Mice that overexpress IL-4 or IL-

13 show evidence of increased goblet cell hyperplasia and mucus production (Temann

et al., 1997 and Zhu et al., 1999). It would therefore be of great interest to look at

levels of IL-4 and IL-13 in the BAL and lung of all disease groups in the ovalbumin

experiment described in this study to try to determine whether presence of the hIL-8

protein in the lung has affected endogenous cytokine production in the context of this

disease model. If no differences in IL-4 or IL-13 levels are observed then possible

novel mechanisms involving IL-8, probably in conjunction with another inflammatory

mediator(s) should start to be explored and, as Clara cells themselves can both become

goblet cells and are constitutively expressing the IL-8 protein, the possibility that any

mechanism could be mediated by IL-8 protein inside the cell should also be borne in

mind. As goblet cell hyperplasia and mucus secretion is one measure of airway

remodelling, other markers of this process, such as collagen deposition and fibrosis

should also be considered. Angiogenesis is one part of the fibrotic process in which

CXC chemokines are heavily implicated (Strieter et al., 1995) and in a broader picture

of fibrosis, neutralisation of the murine IL-8 homologue, MIP-2, was found to be

protective in a mouse model of fibrotic lung disease (Keane et al., 1999). Of interest

would be assessment of fibrosis in lung tissue from the ovalbumin disease experiment

described in this chapter, but also important would be characterisation of any fibrotic

pathologies in lungs of these transgenic animals prior to disease induction. Indeed

subtle changes in airway structure may be able to account for the small changes

observed in baseline airway compliance and increased bronchial hyperreactivity upon

aerosol exposure to OVA of these lines.

255

Overview

Transgenesis, CD4+ T cells and lung inflammation have been the main themes of this

thesis, but while much has been achieved, much work still remains in characterising

the new transgenic lines that have been created, answering questions surrounding

observed phenotypes and in creating new models to try to answer key questions about

disease pathogenesis and basic T cell biology.

One of the main aims at the start of this thesis was to address a possible role for the T

cell receptor in determining CD4+ T cell effector phenotype. Evidence from previous

studies suggested that preferred structural motifs may expand during the course of Thl

or Th2 type immune response and that these motifs may convey TCR/pMHC binding

characteristics that inherently skew the process of differentiation and Thl or Th2 T

cell development. This thesis described identification of Th 1 and Th2 derived TCR a

chains that were successfully expressed transgenically, although levels of Thl TCR a

chain expression were much lower than those seen in the Th2 TCR a transgenic lines.

Ongoing work is needed to establish the cause of this apparent low transgene

expression level in the Thl TCR a chain transgenic mice as this low level of

expression and lack of expansion of the transgenic TCR sequence in the context of

peptide specificity in in vitro culture is precluding the use of this line to determine

inherent T cell phenotype and p chain pairing. It may be necessary to cross this mouse

line with mice carrying a disrupted TCR a locus in order to achieve these aims. High

levels of expression in the Th2 TCR a chain transgenic lines meant that experiments

to determine 1 chain pairing and inherent effector cell phenotype were possible. It

was found that transgenic expression of this Th2 TCR a chain, with an elongated

CDR3a region, did not bias the naïve TCR [3 chain repertoire of these mice, but that in

the context of peptide specificity a [3 chain pair could quickly be established. A

dominant a13 TCR in peptide specific T cell lines derived from these animals was also

associated with a Th2 type cytokine and transcription factor profile suggesting that

expression of the elongated Th2 derived TCR a chain is sufficient to bias TCR p chain

selection and pMHC binding such that development of a Th2 phenotype is favoured.

256

In vivo data supported this argument by demonstrating that during a memory response

to peptide, T cells bearing the elongated transgenic TCR a chain produced less IFNI.

Experiments are currently ongoing to determine whether these cells that make less

IFN-y are associated with a dominant TCR 13 chain, and further work to establish an

inherent Thl or Th2 bias in vivo could include a disease model such as EAE, to

demonstrate increased resistance or susceptibility to disease as a consequence of

inherent T cell phenotype. It will also be of interest to look at Th17 and regulatory T

cell phenotypes in these animals. The question of inherent phenotype bias will

nevertheless be most easily and most conclusively answered in mice expressing a

transgenic TCR a and 13 chain that have been subsequently crossed onto a RAG

knockout background and thus do not express any endogenous TCR at all. One TCR

[3 chain pair identified from in vitro studies has now been expressed transgenically and

breeding of these animals with Th2 TCR a chain animals is now currently underway.

Basic characterisation of these animals in terms of normal proliferative and cytokine

responses to peptide stimulation will be required both in vitro and in vivo, leading to

the ultimate test of the original hypothesis using a RAG knockout background and

comparing these animals to those with a Thl TCR a and 13 chain pair. At this point,

comparisons of TCR binding affinity for pMHC can be achieved using MHC class II

tetramers and signalling experiments to determine differential downstream

consequences of TCR/pMHC ligation will also be possible. The predicted outcome

from these studies would be that the Th2 TCR makes lower affinity interactions with

the pMHC ligand and consequently has an altered pattern of phosphorylation and

downstream signalling events that lead to a Th2 rather than Thl profile of gene

expression.

A second hypothesis leading from these studies concerned Thl and Th2 responses in

the context of lung inflammation. If transgenic expression of Thl and Th2 derived

TCRs can lead to inherent T cell phenotype bias then these transgenic animals could

be used to generate Thl and Th2 type models of lung inflammation. As a starting

point for this question this thesis has described the generation of an inducible

transgene that confers lung specific, but inducible expression of the TCR cognate

257

peptide, PLP 56-70, in the mouse lung. Results obtained to date have shown the

inducible mechanism of this approach to be functioning correctly and low levels of

transgene expression have been detected using both membrane bound and secreted

versions of the construct. Trafficking of T cells to the lung in response to this peptide

expression has yet to be demonstrated and should become the immediate focus of

work with these lines. In the absence of a complete TCR transgenic animal,

experiments to try to prime and induce T cell trafficking in mice transgenic for the

lung construct and the Cre protein have been started. Priming with peptide followed

by induction of lung expression has been the first approach, but it may be that due to

peptide induced tolerance mechanisms priming with whole PLP protein will be

required. Whether any priming at all will be required and whether Thl or Th2 biased

T cells will successfully traffic to the lung upon transgene induction awaits the

existence of complete Thl and Th2 TCR transgenic animals and so at present this

second hypothesis remains far from being properly addressed. However, if transgenic

T cells do successfully traffic in this model, there is great potential for modelling some

of the chronic inflammatory processes in the lung that have so far eluded many others.

An alternative approach to lung inflammation and the last section of this thesis

described overexpression of the human interleukin 8 cytokine in the bronchial

epithelial cells of the mouse lung. High levels of IL-8 protein and concomitant

neutrophilia in the lung are found in association with a number of different lung

diseases but, despite much evidence that the human protein is biologically active in the

mouse, there are as yet no reports in the literature of this protein having been

overexpressed in the lung. This thesis has described the generation of two lines of IL-

8 expressing mice and lung specific expression has been demonstrated in both.

Despite well known functions of IL-8 as a chemoattractant for leukocytes, especially

neutrophils, no significant inflammatory cell infiltrates were observed in these animals

in response to the constitutive IL-8 expression, although that the protein was

biologically active was suggested by altered neutrophil percentages and distribution in

the lung. Levels of IL-8 protein, as well as mechanisms of impaired recruitment such

as receptor desensitisation have been suggested to explain these findings and further

258

work is required to establish the reason for the lack of neutrophil trafficking into the

lungs of these mice. Lack of neutrophilic infiltrates in these animals could aid the

analysis of neutrophil independent IL-8 mediated disease mechanisms in the lung.

The finding of increased goblet cell numbers and mucus production in the lungs of

transgenic animals compared to littermate controls, in the context of ovalbumin

induced lung inflammation, is of particular interest and leads to further questions

about potential roles for IL-8 in lung remodelling and fibrosis. Further work to

establish whether there are any changes in lung structure as a consequence of IL-8

overexpression, in the absence of lung inflammation, is required as well as

experiments to try to determine the mechanism of increased mucus production in the

context of disease. To establish a model of lung neutrophilia and the pathologies

associated with these infiltrates it may be necessary to generate mice carrying an

inducible version of this hIL-8 transgene, but despite the absence of these cells in the

lungs of mice described here, this model still holds much potential for dissecting

mechanisms of IL-8 mediated pathology in the lung.

259

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Appendices

Appendix 1. Stock solutions and Buffers

TE

■ 10 mM Tris (pH 8.0) (Sigma-T1503)

■ 1 mM EDTA (pH 8.0) (Sigma-E4884)

TAE

■ 40 mM Tris-acetate

■ 1 mM EDTA (pH 8.0)

Tissue digestion buffer for genomic DNA extraction

■ 100 mM Tris (pH 8.0)

■ 5 mM EDTA

■ 200 mM NaC1 (Sigma-S7653)

■ 0.2 % w/v SDS (Calbiochem-428015)

Competent cell preparation solution A

■ 10 mM MgC12 (Sigma-M2670)

■ 0.2 mM MES (Sigma-M5287)

■ 0.05 mM CaC12 (Sigma-C7902)

Competent cell preparation solution B

■ 15 % glycerol (BDH-101186M)

■ 50 mM CaC12

■ 10 mM MgC12

■ 2 mM MES

Southern blot depurination solution

■ 11 mM HC1 (BDH-101254M)

307

Southern blot denaturing buffer

■ 1.5 M NaC1

■ 0.5 M NaOH (BDH-102524X)

Southern blot neutralisation buffer

■ 1.5 M NaC1

■ 0.5 M Tris (pH 7.5)

1 x SSC

■ 0.015 M Tri-sodium citrate (Sigma-S4641)

■ 0.15 M NaC1

Maleic acid wash buffer

■ 0.1 M Maleic acid (Sigma-M0375)

■ 0.15 M NaC1

adjust to pH 7.5 with concentrated NaOH

■ 0.3 % v/v Tween 20 (Sigma-P7949)

DIG detection buffer

■ 0.1 M Tris

■ 0.1 M NaC1

adjust to pH 9.5

Immunohistochemistry block solution

Solution should be made up with PBS

■ 1 % BSA (Sigma-A9647)

■ 0.2 % TritonX100 (Sigma-X100)

■ 0.1 % NaN3 (BDH-10369)

■ 10 % Normal mouse serum (Sigma-M5905)

■ 10 % goat serum (Sigma-G9023) or 10 % donkey serum (Sigma-D9663) depending

on species origin of secondary antibody

308

ELISA block solution

Solution should be made up with PBS

■ 1% BSA

■ 5% sucrose (Sigma-S7903)

■ 0.05% NaN3

ELISA wash buffer

■ 0.05% Tween 20 in PBS

Turks stain

■ 0.01% crystal violet

■ 1% acetic acid (BDH-10001)

309

Appendix 2. Antibodies and recombinant proteins

Antibody ELISA capture antibodies Anti human IL-8 Anti-mouse IFN-y Anti-mouse IL-4 Anti-mouse IL-5

Product details Concentration used

R&Dsystems-MAB208 R&Dsystems-MAB785 R&Dsystems-MAB404 R&Dsystems-MAB405

0.5 µg/m1 4 vg/m1 2 vg/m1 1 µg/m1

ELISA detection antibodies Biotinylated anti human IL-8 Biotinylated anti-mouse IFN-y Biotinylated anti-mouse IL-4 Biotinylated anti-mouse IL-5

R&Dsystems-BAF208 R&Dsystems-BAF485 R&Dsystems-BAF404 R&Dsystems-BAM705

20 ng/ml 400 ng/ml 100 ng/ml 50 ng/m1

Immunohistochemistry primary antibodies Rabbit anti-Cre Goat anti-mouse CC10 Rabbit anti-human IL-8 Rabbit anti-human myeloperoxidase Rabbit anti Hen Lysozyme

Covance-PRB-106C SantaCruz-SC9773 Serotec-AHP781 DakoCytomation-A0398 Abcam-ab391-100

1/2500 1/1000 1/500 1/500

1/200— 1/1000

Immunohistochemistry secondary antibodies Biotinylated goat anti-rabbit Jackson-111-066-047

1/500

Biotinylated donkey anti-goat Jackson-705-065-003

1/500

Recombinant protein

Product details

Human IL-8 eBioscience-14-8089 Mouse IFN-y R&Dsystems-485-MI Mouse IL-4

R&Dsystems-404-ML Mouse IL-5 R&Dsystems-405-ML-005

310

Appendix 3. Restriction enzymes and buffers

Enzyme BamHI BssHII EcoR1 EcoRV HindIII KpnI Nan NcoI NotI Sad SacII Sall SfiI XbaI XhoI XmaI

Product details Promega-R6021 Promega-R6831 Promega-R6011 Promega-R6351 Promega-R6041 Promega-R6341 Promega-R6861 Promega-R6513 Promega-R6431 Promega-R6061 Promega-R6221 Roche-348-783 Promega-R6391 Promega-R6 181 Promega-R6161 Promega-R6491

Cleavage site (A) (5'-3') G A GATCC G A CGCGC G A AATTC GAT A ATC A A AGCTT GGTAC A C GG A CGCC C A CATGG GC A GGCCGC GAGCT A C CCGC A GG G A TCGAC GGCCNNNN A NGGCC T A CTAGA C A TCGAG C A CCGGG

Optimal buffer

E H H D E J G D D J C

suRE/cut H B D D B

Temperature (°C) 37.0 50.0 37.0 37.0 37.0 37.0 37.0 37.0 37.0 37.0 37.0 37.0 50.0 37.0 37.0 37.0

Buffer (1x) B C D E G H J suRE/cut H

Tris (mM) MgC12 (mM) NaC1 (mM) KC1 (mM) DTT (mM) 6 6 50 1 10 10 50 1

150 1 100 1

50 50 1

150

pH 7.5 7.9 7.9 7.5 8.2 7.5 7.5 7.6

6 6 50 90 10 100

6 6 5 10 7 10

311

Appendix 4. PCR primers for analysis of TCR a and 13 gene usage

Primer Sequence (5' - 3') TCR a V region primers NW36 (F) GTCCTGACCTCGCATGCTHCMMBWMYYTCYYSTGGTAY NW37 (F) GTCCTGACCTCGCATGCTTCDWYTRYHTCYTYTGGTAY NW38 (F) GTCCTGACCTCGCATGCRYSASRRYYGTSCWGTGGTTY

TCR a C region primers NJ108 (R) GGCCCCATTGCTCTTGGAATC NJ109 (R) CGGCACATTGATTTGGGAGTC NJ110 (R) TCTCGAATTCAGGCAGAGGGTGCTGTCC

TCR f3 V region primers NK121 (F) GTCCTGACCTCGCATGCCAYMAYDMYRTKKAYTGGTAY NK122 (F) GTCCTGACCTCGCATGCCAYCMADMWRTKTWYTGGTAY NK123 (F) GTCCTGACCTCGCATGCMCYWMSRTMRMYTGGTAY

TCR p C region primers MQ284

(R) AGCACACGAGGGTAGCCTT MS175

(R) GACAGAACTTTGAATTCCTCTGCTTTTGATGG

M13 (sequencing primer) (F) CTGGCCGTCGTTTTAC

B=CorGorT R=AorG

D=AorGorT S=CorG

H=AorCorT W=AorT

K=GorT Y=CorT

M=AorC

(F)= forward primer (R) = reverse primer

312

Appendix 5. TCR CDR3I3 spectratyping primers

primer Sequence (5' - 3') TCR V ri (TRBV) primer V(3 1 TRBV 5 vp 2 TRBV I Vi3 3.1 TRBV26 VI3 4 TRBV 2 Vi3 5.1 TRBV 12-2 V(3 5.2 TRBV 12-1 V13 5.3 TRBV 12-3 Vi3 6 TRBV 19 VP 7 TRBV 29 VI3 8.1 TRBV 13-3 V(3 8.2 TRBV 13-2 V(3 8.3 TRBV 13-1 V(3 9 TRBV 17 V13 10 TRBV 4 V(3 11 TRBV 16 V(3 12 TRBV 15 V(3 13 TRBV 14 vp 14 TRBV 31 VI3 15 TRBV 20 V13 16 TRBV 3 V13 17 TRBV 24 VI3 18 TRBV 30 V13 19 TRBV 21 Vi3 20 TRBV 23

(F) CTGAATGCCCAGACAGCTCCAAGC (F) TCACTGATACGGAGCTGAGGC (F) CCTTGCAGCCTAGAAATTCAGT (F) GCCTCAAGTCGCTTCCAACCTC (F) CATTATGATAAAATGGAGAGAGAT (F) AAGGTGGAGAGAGACAAAGGATTC (F) AGAAAGGAAACCTGCCTGGTT (F) CTCTCACTGTGACATCTGCCC (F) TACAGGGTCTCACGGAAGAAGC (F) CATTACTCATATGTCGCTGAC (F) CATTATTCATATGGTGCTGGC (F) TGCTGGCAACCTTCGAATAGGA (F) TCTCTCTACATTGGCTCTGCAGGC (F) ATCAAGTCTGTAGAGCCGGAGGA (F) GCACTCAACTCTGAAGATCCAGAGC (F) GATGGTGGGGCTTTCAAGGATC (F) AGGCCTAAAGGAACTAACTCCCAC (F) ACGACCAATTCATCCTAAGCAC (F) CCCATCAGTCATCCCAACTTATCC (F) CACTCTGAAAATCCAACCCAC (F) AGTGTTCCTCGAACTCACAG (F) CAGCCGGCCAAACCTAACATTCTC (F) CTGCTAAGAAACCATGTACCA (F) TCTGCAGCCTGGGAATCAGAA

Constant 3 region primer C(3 - FAM labelled (R) 6FAM-CTTGGGTGGAGTCACATTTCTC

6FAM = 6-carboxyflurorescein-amino-hexy fluorescent dye (F) = forward primer (R) = reverse primer

313

Appendix 6. Thl and Th2 TCR a chain primers

Primer Sequence (5' — 3') Annealing Product

temp [MgC12] size Thl a construct

ThlIntronF (F) CAGGAAGAATGATGAAGACATC ThlHindIIIR (R) CAAATGAAAGCTTAGACCCAG

TRAV 10 (F) GTCCCGGGTCCCAGGCAGGAAGAATGATG Th1 LP1/2IntronR (R) CATTTGAACGAATGTCTATCAC

HindIIITh I F (F) CTGGGTCTAAGCTTTCATTTG TRAVJ58 (R) GCCGCGGCCGCGAAGCCACATGCCCATCAGTTGGTG

Thl CDR3 motif (F) TGCAGCAAGCAGGGAAGGCACTGGGTCTAAGC NJ108 (R) GGCCCCATTGCTCTTGGAATC

TRAVIO See above TRAVJ58 See above

55 °C

52 °C

52 °C

56 °C

55 °C

1.5 mM

2.5 mM

3.0 mM

1.5 mM

1.5 mM

538 / 384 bp

403 by

148 bp

250 by

661 by

Th2 a construct

Th2lntronF (F) CTGCCTAGCCATGTTCCTAGTG Th2HindIIIR (R) CTGCCCAAACACAAGCTTGC

TRAV17 (F) GTCCCGGGCTTCTCACTGCCTAGCCATG Th2LP1/2IntronR (R) GGCTCTGGCCAGGATACTGC

Th2HindIIIF (F) GCAAGCTTGTGTTTGGGCAG TRAJ50 (R) GCCGCGGCCGCTTAAAGGACCCTAGTAGTAGCAAGG

Th2 CDR3 motif (F) GCTGTGAGCTGGGATAACTATGCCCAGGGATTAAC NJ108 (R) GGCCCCATTGCTCTTGGAATC

TRAV17 See above TRAVJ58 See above

55°C

52 °C

52 °C

56 °C

55 °C

1.5 mM

2.5 mM

2.5 mM

1.5 mM

1.5 mM

527 / 391 bp

348 by

119 bp

250 by

622 by

(F) = forward primer (R) = reverse primer

314

Appendix 7. MIPLP and SIPLP trans2ene primers and oli2onucleotides

Primer Sequence (5' — 3') Annealing Product size

temp [MgCl2] Sec./Memb. PLP constructs

HELA PLPB

HELA TM/CYTOB

SfilBamHlBodF BodXho I Sfi1R

CC102000F BetaglobinB

3rdexonF BetaglobinB

CC101500F CC10210OR

StopfloxedF StopfloxedR SIPLPStopfloxedR

(F) CTGCTCTGGGGAAAGTCTTT (R) GTTCCATAGATGACATACTG

(F) CTGCTCTGGGGAAAGTCTTT (R) TATTCTGCACTGCAAAGATC

(F) GGCCATTGCGGCCGGATCCTGAGAACTTCA (R) GGCCGCAATGGCCCTCGAGGGATCTCCATA

(F) GCCTCTGGTTCTCCAGGGTT (R) TGATAGGCAGCCTGCACCTG

(F) CTGGGCAACGTGCTGGTTAT See above

(F) ATAAAAGCACTGGAGAAGTA (R) CAGAGGTCGTGGAACTGTAG

(F) CATCAGCCCACATCTACAGAC (R) CAATGGCCAGTACTAGTGAAC (R) CCTGAAGTTCTCAGGATCCG

53 °C

53 °C

68 °C

53 °C

55 °C

53 °C

60 °C

1.5 mM

1.5 mM

1.5 mM

1.5 mM

1.5 mM

1.5 mM

2.5/1.5 mM

214 bp

611 bp

1755 / 1888 by

3239 / 3372 by

625 / 758 by

629 bp

1736/1647 bp

Oligonucleotide Sequence (5' — 3')

PLP 56-70 top

PLP 56-70 bottom

Kpnl linker top Kpnl linker bottom

(T) CGCCAAAAACTACCAGGACTATGAGTATCTCATTAATGTGATTCATGCTTTCC AGTATGTCATCTATGGAACCCGC

(B) GGGTTCCATAGATGACATACTGGAAAGCATGAATCACATTAATGAGATA CTCATA GTCCTGGTAGTTTTTGG

(T) GGAGAGGTACCAACTTGAACGCTATGGGCATCCAATGCATCCATGGAGCT (B) CCATGGATGCATTGGATGCCCATAGCGTTCAAGTTGGTACCTCTCCGC

(F) = forward primer (R) = reverse primer (T) = top strand (B) = bottom strand

315

Appendix 8. PCR primers

Primer Sequence (5' — 3') Annealing

temp [MgCl2] Product size Th2 13 construct

VP3 1 F J(32-3R

20TCRI3F1 20TCROR1

(F) GACTCGAGCCTAGACAAAGACCATCTTGAAC (R) CTCCGCGGTCACAGCCTCTTGGTACAGGAC

(F) CATCTTGAACTATGCTGTACTCTC (R) CCAACTTACCGAGAACAGTCAG

55 °C

55 °C

2.0 mM

1.5 mM

850 by

755 by

hIL-8 construct

IL-8F IL-8R

CC101500F CC10210OR

(F) AGGAATTCTGTAAACATGACTTCCAAGC (R) GCGAATTCTTATGAGTTCTCAGCCCTCTTC

(F) ATAAAAGCACTGGAGAAGTA (R) CAGAGGTCGTGGAACTGTAG

55 °C

53 °C

1.5 mM

1.5 mM

319 by

629 by

Bm70 MerCreMer

Cre 1 Cre 2

(F) CGCAGAACCTGAAGATGTTCGCGA (R) GGATCATCAGCTACACCAGAGACG

60 °C 2.0 mM 500 by

HPRTF HPRTR

(F) TCGTGATTAGCGATGATGAACC (R) CTGGCAACATCAACAGGACTCC

60 °C 1.5 mM 650 by

(F) = forward primer (R) = reverse primer

Real- time PCR

18s Human IL-8 Mouse Tbet Mouse Gata3

Applied Biosystems — Hs-99999901_sl Applied Biosystems — Hs-00174103_ml Applied Biosystems— Mm00450960_m1 Applied Biosystems — Mm00484683_ml

316

Appendix 9. Wright-Giemsa stain — cell morphology

A = Macrophage

B = Lymphocyte

C = Eosinophil

D = Neutrophil

317

Cla 118941 Sal 119941

Not I, Sac 112841

BamH112441

Appendix 10. Thl TCR a chain construct - plasmid map

318

Appendix 11. Th2 TCR a chain construct - plasmid map

BamHI 0 Sal 11990

Cla 1 18956

/mai 2180

pEMBL18 Th2 V17/J50 of I, Sac II 2802

JA2

Sal 1 1510 Th2 pTA cassette

19902 bp BamH1 4947

Bam1-11 1445 constant region

BamH 112456

319

Appendix 12. Th2 TCR 13 chain construct - plasmid map

Kpn 1 20670 BamHI 860

coR1 2190

Kpn 117740

BamHI 17740

EcoRI 17590

_____X ho 14470

Th2 pTB cassette

Th2 VB31/JB2-3 20670 bp

--Sac II 5320 J62.6

amHI 6490

coRl 6690

BamHI 1259

constant region

\coRI 7590

320

PTZ 18

BssH II 601 S

Sac 115971 Not 1595 • Sfi 15958

Xho 15945

EcoR 15410

Rabbit beta globin

TM region HEL

PLP (56-70) HEL

ind III 2465 EcoR 12477 amH 12495 of 1 2514

EcoR 1 2595 ind III 2605

amH 1 2612 ind III 2686

LoxP

Floxed STOP

LoxP Xho 13557

Appendix 13. MIPLP construct — plasmid map

ssH111 hoI53

Hind III 74 amH 18D

EcoR 1340

EcoR 1744

CC10 promoter

MIPLP

8793 bp

Sac II 500 Nar 14926

EcoR I 4729

amH 13995 fi 1 4083

amH 14089

321

Appendix 14. SIPLP construct - plasmid map

-=--1"-Flind III 2465 EcoR 1 2477 BamH 1 2495 Not 1 2514

EcoR 1 2595 ind 111 2605 amH 1 2612

Hind III 2686

BssH II 5877 Sac II 5838 Not 1 5826 Sfi 1 5825

Xho 1 581

amH 1 3995

fi 1 4083 amH 1 4089

EcoR 1 5277

Sac II 5002

Nar 1 492

ssH 111 ho 153 indlIl 74 amH 180

EcoR 1 340

322

ind1112461 pnl 2471

Notl 2485

SacI12479

BamH1 2574 LoxP

SIPLP (LSL rev)

8733 bp

CC10 promoter

BssH 115948

Sacll 5906 Notl 5894

Xbal 5887 Xhol 5875

Rabbit beta globin

HEL PLP (56-70)

HEL

Floxed STOP

Lox P EcoR15340/

Xhol 3012

Sacll 5065-j

Nan 4989

EcoRl 479

hol 4125 pnl 4144

amHI 4152

'nal 3883 BamHI 3957

ind1113964 otl 4055 EcoR1 3974

Xbal 4062 amH14074

coR1 4092 Hind111 4104

Appendix 15. Inverted stop sequence SIPLP construct — plasmid map

BssH111

Xhol 49 ind11170

EcoRl 332

EcoR1 739

323

BasH11405 Not1 4002

Xbal 3995 Xhol 398

\----EcoR1 3135 indlIl 3352

coR1 3448

BssH11 1 Kpnl 42

hol 53 indlIl 74

HindlIl 2465

NN N.-,--NE, csoRV 2473 ---EcoR1 2477

amHi 2495

Appendix 16. Lung targeted hIL-8 construct — plasmid map

324

Appendix 17. Thl TCR a complete V-J insert nucleotide sequence

1 TCCCAGGCAG GAAGAATGAT GAAGACATCC CTTCACACTG TATTCCTATT

51 CTTGTGGCTG TGGATGGACT GTGAGTTGGA AGATTTGGGT CAAGAGAAAA

101 AAATGGTGGA TGTTAGACAT CTATGGGCAA TCTATTTTAT ACAGTTCCTT

151 CTAGTTGTGG ACAAAGAGAT TTAGCAGAGT ACTGCCTGAC TGCTCCTCTC

201 CTTTCTGTTT TTCTTTCTGT GTAGGGGAAA GCCATGGAGA GAAGGTCGAG

251 CAACACCAGT CTACACTGAG TGTTCGAGAG GGAGACAGCG CTGTCATCAA

301 CTGCACTTAC ACAGATACTG CCTCATCATA CTTCCCTTGG TACAAGCAAG

351 AAGCTGGAAA GAGTCTCCAC TTTGTGATAG ACATTCGTTC AAATGTGGAC

401 AGAAAACAGA GCCAAAGACT TACAGTTTTA TTGGATAAGA AAGCCAAACG

451 ATTCTCCCTG CACATCACAG CCACACAGCC TGAAGATTCA GCCATCTACT 501 TCTGTGCAGC AAGCAGGGAA GGCACTGGGT CTAAGCTTTC ATTTGGGAAG

551 GGGGCAAAGC TCACAGTGAG TCCAGGTAAG TGTGAGGCTA TCTAACCATC

601 TTTCTAAATG CGATTTGAGA ATTTTTTTTT CTTTCACACC AACTGATGGG

651 CATGTGGCTT C

Black = intronic sequence. Blue = coding sequence Red = mutated base pair (G—>T)

325

Appendix 18. Thl TCR a rearranged V-J insert nucleotide/protein sequence

1 - ATG AAG ACA TCC CTT CAC ACT GTA TTC CTA TTC TTG TGG CTG GAA 1-M K T S L H T VF L F LW L E

46 - AGC CAT GGA GAG AAG GTC GAG CAA CAC CAG TCT ACA CTG AGT GTT 16-S HGE KVEQHQST L S V

91 - CGA GAG GGA GAC AGC GCT GTC ATC AAC TGC ACT TAC ACA GAT ACT 31-R E G D S A V I N C T YT D T

136 - GCC TCA TCA TAC TTC CCT TGG TAC AAG CAA GAA GCT GGA AAG AGT 46-A S S YFPWYKQE AGK S

181 - CTC CAC TTT GTG ATA GAC ATT CGT TCA AAT GTG GAC AGA AAA CAG 61-L H F V ID I R S N V D R K Q

226 - AGC CAA AGA CTT ACA GTT TTA TTG GAT AAG AAA GCC AAA CGA TTC 76-S Q R LT V L L D K K A K R F

271 - TCC CTG CAC ATC ACA GCC ACA CAG CCT GAA GAT TCA GCC ATC TAC 91-S L H IT A T Q P E D S A I Y

316 - TTC TGT GCA GCA AGC AGG GAA GGC ACT GGG TCT AAG CTT TCA TTT 106-F CAA SR E G T G S K L S F

361 - GGG AAG GGG GCA AAG CTC ACA GTGAGT CCA 121-G K GA K LT VSP

Light blue = Leader sequence of TRAV10 Dark blue = TRAV10 Red = CDR3a region Green = TRAJ58

326

Appendix 19. Th2 TCR a complete V-J insert nucleotide sequence

1 CTTCTCACTG CCTAGCCATG TTCCTAGTGA CCATTCTGCT GCTCAGCGCG

51 TTCTTCTCAC TGAGTAAGTA TTATTTCAGT CCTTGCTTGC TTTTGTTTCC

1 01 ACAGACAGAA CATGTTCCTA GTCAGAATCT ACACAAGGGA CATAGTATCA

151 TTACAGGTAG GTCTATTCTG GTCTCACTGC TGCTACGTTG GTATTACAGG

201 AGGAAACAGT GCCCAGTCCG TGGACCAGCC TGATGCTCAC GTCACGCTCT

251 ATGAAGGAGC CTCCCTGGAG CTCAGATGCA GTTATTCATA CAGTGCAGCA

301 CCTTACCTCT TCTGGTACGT GCAGTATCCT GGCCAGAGCC TCCAGTTTCT

351 CCTCAAATAC ATCACAGGAG ACGCCGTTGT TAAAGGCACC AAGGGCTTTG

401 AGGCCGAGTT TAGGAAGAGT AACTCCTCTT TCAACCTGAA GAAATCCCCA

451 GCCCATTGGA GCGACTCAGC CAAGTACTTC TGTGCACTGG AGGGTATAGC

501 ATCCTCCTCC TTCAGCAAGC TTGTGTTTGG GCAGGGGACA TCCTTATCAG

551 TCGTTCCAAG TAAGTTTGCA GGCTGCCTGT GGGGCAATGG TTCTCAACCT

601 TGCTACTACT AGGGTCCTTT AA

Black = intronic sequence. Blue = coding sequence Red = mutated base pair (G—T)

327

Appendix 20. Th2 TCR a rearranged V-J insert nucleotide/protein sequence

1 - ATG TTC CTA GTG ACC ATT CTG CTG CTC AGC GCG TTC TTC TCA CTG 1-M F L V T I L LL S AF FS L

46 - AGA GGA AAC AGT GCC CAG TCC GTG GAC CAG CCT GAT GCT CAC GTC 16-R G N S A QS V D Q P D A II V

91 - ACG CTC TAT GAA GGA GCC TCC CTG GAG CTC AGA TGC AGT TAT TCA 31-T LYE G A SL E L R CS YS

136 - TAC AGT GCA GCA CCT TAC CTC TTC TGG TAC GTG CAG TAT CCT GGC 46-YS A A P Y L F W Y V Q Y P G

181 - CAG AGC CTC CAG TTT CTC CTC AAA TAC ATC ACA GGA GAC GCC GTT 61-Q SLQFLL KYI T G D A V

226 - GTT AAA GGC ACC AAG GGC TTT GAG GCC GAG TTT AGG AAG AGT AAC 76-V K G T K G F E AEFR K S N

271 - TCC TCT TTC AAC CTG AAG AAA TCC CCA GCC CAT TGG AGC GAC TCA 91-SS F N L K K SPA H W S D S

316 - GCC AAG TAC TTC TGT GCA CTG GAG GUT ATA GCA TCC TCC TCC TTC 106-A K YFCAL E GI A SS SF

361 - AGC AAG CTT GTG TTT GGG CAG GGG ACA TCC TTA TCA GTC GTT CCA 121-S K L V F G Q G T SL S V V P

Light blue = Leader sequence of TRAV17 Dark blue = TRAV17 Red = CDR3a region Green = TRAJ50

328

Appendix 21. Th2 TCR 13 complete V-J insert nucleotide sequence

CCTAGACAAA GACCATCTTG AACTATGCTG TACTCTCTCC TTGCCTTTCT 51

CCTGGGCATG TTCTTGGGTG GGTGCATTCC CCTGTTGAAA ACCTACTTTC 101

AAATTGTTGC TTCCATCTCT CCACCTGTTA AAGGTGTAGA TTCTTTGGTA 151

CTGGCTTGCC TCTCTTGACC ACAGTCCTCC AATTACTGAG ATATGGGCAT 201 TTCACACTCT TTCAGTATTG ACTCTCAGTT TTGTATCTTT TAGAAAGGAT

251 TTGTCTCCCC TTGCCCTGTA ACATTTTTCA TCCCTGGCTC TTCTAGTATA

301 CTCCCTGAGA CTGATTACAT GTAACCCCTT CAGAGGGAAG GCAAGCAAGC

351 TGGTGTGTGT GTATGTGTAG AGGTGTGTGT TTCTACTCTA AATGACTCTA

401 TTTCTTTACA GGTGTTAGTG CTCAGACTAT CCATCAATGG CCAGTTGCCG

451 AGATCAAGGC TGTGGGCAGC CCACTGTCTC TGGGGTGTAC CATAAAGGGG

501 AAATCAAGCC CTAACCTCTA CTGGTACTGG CAGGCCACAG GAGGCACCCT

551 CCAGCAACTC TTCTACTCTA TTACTGTTGG CCAGGTAGAG TCGGTGGTGC

601 AACTGAACCT CTCAGCTTCC AGGCCGAAGG ACGACCAATT CATCCTAAGC

651 ACGGAGAAGC TGCTTCTCAG CCACTCTGGC TTCTACCTCT GTGCCTGGAG

701 TCTGGGGGGA GGTGCAGAAA CGCTGTATTT TGGCTCAGGA ACCAGACTGA

751 CTGTTCTCGG TAAGTTGGGA GCTAGTAATG AAGGGGAGGG AGCATTTCCC

801 AGGCTAAGGA TAGCCAGAGC CAGTTTTTGT CCTGTACCAA GAGGCTGTGA

Black = intronic sequence Blue = coding sequence

329

Appendix 22. Th2 TCR 13 rearranged V-J insert nucleotide/protein sequence

1 - ATG CTG TAC TCT CTC CTT GCC TTT CTC CTG GGC ATG TTC TTG GGT 1-ML YS LL A FLLG M F L G

46 - GTT AGT GCT CAG ACT ATC CAT CAA TGG CCA GTT GCC GAG ATC AAG 16-VS A Q T I H Q W P V A E I K

91- GCT GTG GGC AGC CCA CTG TCT CTG GGG TGT ACC ATA AAG GGG AAA 31-AVG S P L S L GCTIK GK

136- TCA AGC CCT AAC CTC TAC TGG TAC TGG CAG GCC ACA GGA GGC ACC 46-S SP N L Y W Y W Q A T G G T

181- CTC CAG CAA CTC TTC TAC TCT ATT ACT GTT GGC CAG GTA GAG TCG 61-LQQLFYSI T VGQVES

226- GTG GTG CAA CTG AAC CTC TCA GCT TCC AGG CCG AAG GAC GAC CAA 76-VV Q L N L S A SR P K D D Q

271- TTC ATC CTA AGC ACG GAG AAG CTG CTT CTC AGC CAC TCT GGC TTC 91-F IL S T E K L L L S HS G F

316- TAC CTC TGT GCC TGG AGT CTG GGG GGA GGT GCA GAA ACG CTG TAT 106-YLCAWSLGGGA E L Y

361- ITT GGC TCA GGA ACC AGA CTG ACT GTT CTC 121-FGS GTR L T V L

Light blue = Leader sequence of TRBV31 Dark blue = TRBV31 Red = CDR313 region Green = TRBJ2-3

330

Appendix 23. MIPLP construct — complete nucleotide sequence

1 GCGCGCAATT AACCCTCACT AAAGGGAACA AAAGCTGGGT ACCGGGCCCC

51 CCCTCGAGGT CGACGGTATC GATAAGCTTA TCGATTTCGA ACCCTGTAGC

101 AGGGCCTCTG TGCAGCAGAG GGTGCACACT ACAGGTTGTG CACACGCGTG

151 TGCGTGCATG CATGCAAGGG TGCACATGCA CAGGGTGGAG GTCAGAGGTC

201 AACGTCAGGT GTCTTCGTCT GTCACCCACA GTCTTGTTTT TCAGACAGGA

251 CTTCTCACTG AATCTGAATC CTGCCCATTT GATGGGCTCA TCAGTCAGTT

301 TCCAGGTTTC CCTGGTCCCC ACCTCCCCAG GGCTGGAATT CAAGGCATAT

351 CTAGCTTTTT TGCATGAATG TTTGAGAGCC AAATTCAGTT CCTCATACTT

401 GCCCAATAGG CATAGTATAT CCACTGAGCC ATGTCCCCAA CTCATTATGT

451 TCACCTCATT CATTCATTCA TCCATCCATC CATCCATCCA TCCATTCATT

501 CATTCAGGTT TGGTTTGGTT TGGTTTTAGT GTTTTGGGTT TTTTTTCAAG

551 ACAGGGTTTC TCTGTGTAGC CCTGGCTGCC CTGAAACTAA CTTTCTTACT

601 TACTTTCTTT CTTTCTTCCT TCCTTCCTTT CTTTCTTTAA GTTGGTTTGG

651 GTTTTTTGGG GGGGGGAGGG TTTGGGTTTT TTTGTTTGTT TGTATTTTAT

701 TCAAGGAAAG GCTCCTCTGG GTTGCCCTGG CTGCCCTGGA AGGAATTCTA

751 TAGCCAGGCT GACCATGAAC TCAGAAATCT ACCTGCCTCT CCCTCTGCCT

801 CCCAAGTGCT AGGATTAAAG GTGTGTGACA CACACACAAC TGGCTCTACA

851 TTTGACACTC ACAGTATTCT GCACACAGCC ACAGGGCTGT ATCGCTAAGA

901 GGCCATGTCA CAGGTCCAGG AAGGCTTCAC TGTTTAATAA CTGGTTTGGA

951 ATTTGGAAGT GGGAAAGTAT GTAGAGACAA TCTCACTCTA GGACTCAAAG

1001 ACAGAGATAA AATGCTCTTT AGAAACCTAT TCTGTGGGGG AACAGAATCA

1051 GAACTCTATG TCAGCCTCTG TGCTAGCTGG GGTCAGGCCA GAGACACAGA

331

1101 AATGAGCTAA GCCTACAGTG AGAACTGATA CGCAGGACAG TTGAGAACAA

1151 TGGGAAAGTG CATCTGAAGG GCCCAAGGCC ACTAGGAAAA AGGCAACCCA

1201 AAATGCCCAC AACTTTAAAT CCCCTTCCTG ATGTGTCATT AGATGCAAAC

1251 GGCCCTGGGG AGGATGTGAC GTTAAACAAG GCAGTGTTCT GTACAGGGGC

1301 AGATCCTGCA GTTACTGGAA GCTGAAGGCT CCAGGCTGAC CACACTAGGT

1351 AGGAAGTTCA TGAAATGAGC TCTCGAAGCG CCTCTTCAGG TCTTCCCCGA

1401 TGTCCAGCCC CACCAGCACC GTAGCAGGTA CTGGGCATCT ATTGATTGGG

1451 TGGGTAGGTG AACATTTGAG ACAACCTGGA AGTTTAAATG GGATTTGTGG

1501 AAATAGAGAG GGTGTTGGTT CCAGCCAAGC CAGGTTCCAT GCTAAGACAT

1551 AAAAGCACTG GAGAAGTAAA AGAGTTAACG CTGAACATGG CCGTGGGAAG

1601 CGAGGAGACC AAGGTAAAGC CTGGGAATGG CTAACACTTG AGAACTGTCA

1651 ACATCATGAA AGGATAAGAA AGAATGGCCA AGGAAAGAAA ACAGGAAAGA

1701 GCTGAGTGTG GGAGAGGCCT GGAGAGAATG GGGCAAGAAG TGGGAGGCTA

1751 AAGGGTAAGG GCAAGGGAGG GTCAAGTCAG ATGAGGACTG AAGGTGCTTT

1801 CAACTGGTAG GACTTGAGGG CTGCTCTGGG TAGGAGCCAG CCCGGTCTGG

1851 GCCCTGGGTC TCTCATGTGT TCTATGGAGA AGTCTTTATA TTTGTTCATG

1901 TGTATTGTAT GTGGGCTCTA ACCTGGCCAA CAATGCCCAA GAATCAAGTG

1951 ATTTTTGTGG CTTGAAGTCT AGCCACCTTC CTTGGAGGGA GGCAATAGAA

2001 GGAGCCTAGT GACATCTCGG ACGGAGTCTG GGGTCTTTGT CCTTCCCCTT

2051 GATCCCTGAA GGGTCTCCAG CCTCTGGTTC TCCAGGGTTG GCAAGTCTAC

2101 AATTGCTTCC CGGAACCTGG AGTGCTCAGT GCTTGACTTT AAAGAGGACA

2151 CAGGTGCCTA CAGTTCCACG ACCTCTGGGT TCCCCGACGC CCCCCCCCCC

2201 GCACTGCCCA TTGCCCAAAC ACCCCACAAG TGGCCTATTG TGTGAGCTCA

332

2251 GTTTCAATGG GAAAAGAAAC TGGGTTTATG AAAAGAGATT ATTTGCTTAT

2301 TGCATGGAGA TGACTAAGTA AATAGTGCAA TTTCTTGAGT GGAGCACAAT

2351 CCCTGCCCCT ACCTCTTGTG GGCTGCCAGG AACATATAAA AAGCCACACG

2401 CATACCCACA CATACCCACA CATACCCTCA CATTACAACA TCAGCCCACA

2451 TCTACAGACA GCCCAAGCTT GATATCGAAT TCCTGCAGCC CGGGGGATCC

2501 ACTAGTTCTA GAGCGGCCGC ACGTCTAAGA AACCATTATT ATCATGACAT

2551 TAACCTATAA AAATAGGCGT ATCACGAGGC CCTTTCGTCT TCAAGAATTC

2601 CATCAAGCTT AGGATCCGGA ACCCTTAATA TAACTTCGTA TAATGTATGC loxP

2651 f site TATACGAAGT TAT TAGGTCC CTCGACCTGC AGCCCAAGCT TACTTACCATJ

2701 GTCAGATCCA GACATGATAA GATACATTGA TGAGTTTGGA CAAACCACAA

2751 CTAGAATGCA GTGAAAAAAA TGCTTTATTT GTGAAATTTG TGATGCTATT

2801 GCTTTATTTG TAACCATTAT AAGCTGCAAT AAACAAGTTA ACAACAACAA

2851 TTGCATTCAT TTTATGTTTC AGGTTCAGGG GGAGGTGTGG GAGGTTTTTT

2901 AAAGCAAGTA AAACCTCTAC AAATGTGGTA TGGCTGATTA TGATCTCTAG

2951 TCAAGGCACT ATACATCAAA TATTCCTTAT TAACCCCTTT ACAAATTAAA

3001 AAGCTAAAGG TACACAATTT TTGAGCATAG TTATTAATAG CAGACACTCT

3051 ATGCCTGTGT GGAGTAAGAA AAAACAGTAT GTTATGATTA TAACTGTTAT

3101 GCCTACTTAT AAAGGTTACA GAATATTTTT CCATAATTTT CTTGTATAGC

3151 AGTGCAGCTT TTTCCTTTGT GGTGTAAATA GCAAAGCAAG CAAGAGTTCT

3201 ATTACTAAAC ACAGCATGAC TCAAAAAACT TAGCAATTCT GAAGGAAAGT

3251 CCTTGGGGTC TTCTACCTTT CTCTTCTTTT TTGGAGGAGT AGAATGTTGA

3301 GAGTCAGCAG TAGCCTCATC ATCACTAGAT GGCATTTCTT CTGAGCAAAA

3351 CAGGTTTTCC TCATTAAAGG CATTCCACCA CTGCTCCCAT TCATCAGTTC

333

3401 CATAGGTTGG AATCTAAAAT ACACAAACAA TTAGAATCAG TAGTTTAACA

3451 CATTATACAC TTAAAAATTT TATATTTACC TTAGAGCTTT AAATCTCTGT

3501 AGGTAGTTTG TCCAATTATG TCACACCACA GAAGTAAGGT TCCTTCACAA

3551 AGATCCCTCG AGAAAAAAAA TATAAAAGAG ATGGAGGAAC GGGAAAAAGT

3601 TAGTTGTGGT GATAGGTGGC AAGTGGTATT CCGTAAGAAC AACAAGAAAA

3651 GCATTTCATA TTATGGCTGA ACTGAGCGAA CAAGTGCAAA ATTTAAGCAT

3701 CAACGACAAC AACGAGAATG GTTATGTTCC TCCTCACTTA AGAGGAAAAC

3751 CAAGAAGTGC CAGAAATAAC AGTAGCAACT ACAATAACAA CAACGGCGGC

3801 TACAACGGTG GCCGTGGCGG TGGCAGCTTC TTTAGCAACA ACCGTCGTGG

3851 TGGTTACGGC AACGGTGGTT TCTTCGGTGG AAACAACGGT GGCAGCAGAT

3901 CTAACGGCCG TTCTGGTGGT AGATGGATCG ATGGCAAACA TGTCCCAGCT

3951 CCAAGAAACG AAAAGGCCGA GATCGCCATA TTTGGTGTCC CCGAGGATCC

4001 GGAACCCTTA ATIATAACTTC GTATAATGTA TGCTATACGA AGTTATTAGG loxP

4051 site TCCCTCGAAG AGGTTCACTA GTACTGGCCA TTGCGGCCGG ATCCTGAGAA

4101 CTTCAGGGTG AGTTTGGGGA CCCTTGATTG TTCTTTCTTT TTCGCTATTG

4151 TAAAATTCAT GTTATATGGA GGGGGCAAAG TTTTCAGGGT GTTGTTTAGA

4201 ATGGGAAGAT GTCCCTTGTA TCACCATGGA CCCCCATGAT AATTTTGTTT

4251 CTTTCACTTT CTACTCTGTT GACAACCATT GTCTCCTCTT ATTTTCTTTT

4301 CATTTTCTGT AACTTTTTCG TTAAACTTTA GCTTGCATTT GTAACGAATT

4351 TTTAAATTCA CTTTTGTTTA TTTGTCAGAT TGTAAGTACT TTCTCTAATC

4401 ACTTTTTTTT CAAGGCAATC AGGGTATATT ATATTGTACT TCAGCACAGT

4451 TTTAGAGAAC AATTGTTATA ATTAAATGAT AAGGTAGAAT ATTTCTGCAT

4501 ATAAATTCTG GCTGGCGTGG AAATATTCTT ATTGGTAGAA ACAACTACAC

334

4551 CCTGGTCATC ATCCTGCCTT TCTCTTTATG GTTACAATGA TATACACTGT

4601 TTGAGATGAG GATAAAATAC TCTGAGTCCA AACCGGGCCC CTCTGCTAAC

4651 CATGTTCATG CCTTCTTCTC TTTCCTACAG CTCCTGGGCA ACGTGCTGGT

4701 TGTTGTGCTG TCTCATCATT TTGGCAAAGA ATTCTGGCAA CATGAGGTCT

4751 TTGCTAATCT TGGTGCTTTG CTTCCTGCCC CTGGCTGCTC TGGGGAAAGT

4801 CTTTGGACGA TGTGAGCTGG CAGCGGCTAT GAAGCGTCAC GGACTTGATA

4851 ACTATCGGGG ATACAGCCTG GGAAACTGGG TGTGTGCCGC AAAATTCGAG

4901 AGTAACTTCA ACACCCAGGC TACAGGCGCC AAAAACTACC ACIGACTATGA

4951 GTATCTCATT AATGTGATTC ATGCTTTCCA GTATGTC ATC TATGGAACCC

5001 GCGGAAACCG TAACACCGAT GGGAGTACCG ACTACGGAAT CCTACAGATC

5051 AACAGCCGCT GGTGGTGCAA CGATGGCAGG ACCCCAGGCT CCAGGAACCT

5101 GTGCAACATC CCGTGCTCAG CCCTGCTGAG CTCAGACATA ACAGCGAGCG

5151 TGAACTGCGC GAAGAAGATC GTCAGCGATG GAAACGGCAT GAACGCGTGG

5201 GTCGCCTGGC GCAACCGCTG CAAGGGCACC GACGTCCAGG CGTGGATCAG

5251 AGGCTGCCGG CTGGGCTCGT CCAGCTCAGG GATCTATCAG ATTCTGGCGA

5301 TCTACTCAAC TGTCGCCAGT TCACTGGTGC TTTTGGTCTC CCTGGGGGCA

5351 ATCAGTTTCT GGATGTGTTC TAATGGATCT TTGCAGTGCA GAATATGCAT

5401 CTGAGATTAG AATTCACTCC TCAGGTGCAG GCTGCCTATC AGAAGGTGGT

5451 GGCTGGTGTG GCCAATGCCC TGGCTCACAA ATACCACTGA GATCGATCTT

5501 TTTCCCTCTG CCAAAAATTA TGGGGACATC ATGAAGCCCC TTGAGCATCT

5551 GACTTCTGGC TAATAAAGGA AATTTATTTT CATTGCAATA GTGTGTTGGA

5601 ATTTTTTGTG TCTCTCACTC GGAAGGACAT ATGGGAGGGC AAATCATTTA

5651 AAACATCAGA ATGAGTATTT GGTTTAGAGT TTGGCAACAT ATGCCCATAT

PLP(: -70)

335

5701 GCTGGCTGCC ATGAACAAAG GTTGGCTATA AAGAGGTCAT CAGTATATGA

5751 AACAGCCCCC TGCTGTCCAT TCCTTATTCC ATAGAAAAGC CTTGACTTGA

5801 GGTTAGATTT TTTTTATATT TTGTTTTGTG TTATTTTTTT CTTTAACATC

5851 CCTAAAATTT TCCTTACATG TTTTACTAGC CAGATTTTTC CTCCTCTCCT

5901 GACTACTCCC AGTCATAGCT GTCCCTCTTC TCTTATGGAG ATCCCTCGAG

5951 GGCCATTGCG GCCGCCACCG CGGTGGAGCT CCAGCTTTTG TTCCCTTTAG

6001 TGAGGGTTAA TTGCGCGC

Black = pBluescript II vector sequence Brown = CC10 promoter Blue = Floxed STOP sequence Green = Rabbit beta globin intronic and PolyA sequence Red = HEL/PLP (56-70) coding region

336

Appendix 24. Transmembrane HEL/PLP(56-70) nucleotide/protein sequence

1- ATG AGG TCT TTG CTA ATC TTG GTG CTT TGC TTC CTG CCC CTG GCT 1-MR S L L I LV LCF L P L A

46- GCT CTG GGG AAA GTC TTT GGA CGA TGT GAG TTG GCA GCG GCT ATG 16-ALGK V F G R CE LAAA M

91- AAG CGT CAC GGA CTT GAT AAC TAT CGG GGA TAC AGC CTG GGA AAC 31-K R H G L D N Y R G YS L G N

136-TGG GTG TGT GCC GCA AAA TTC GAG AGT AAC TTC AAC ACC CAG GCT 46-WV CA AK FE S N F N T QA

181-ACA GGC GCC AAA AAC TAC CAG GAC TAT GAG TAT CTC ATT AAT GTG 61-T G AKNYQDYE YLINV

226-ATT CAT GCT TTC CAG TAT GTC ATC TAT GGA ACC CGC GGA AAC CGT 76-IHA F Q YV I Y G T R G N R

271-AAC ACC GAT GGG AGT ACC GAC TAC GGA ATC CTA CAG ATC AAC AGC 91-N TDGS T D Y G I L Q I N S

316-CGC TGG TGG TGC AAC GAT GGC AGG ACC CCA GGC TCC AGG AAC CTG 106-R WWCND GRIP GSRNL

361-TGC AAC ATC CCG TGC TCA GCC CTG CTG AGC TCA GAC ATA ACA GCG 121-C N I P C S A L L S SDI TA

406-AGC GTG AAC TGC GCG AAG AAG ATC GTC AGC GAT GGA AAC GGC ATG 136-S V N C A K K IV SD G N GM

451-AAC GCG TGG GTC GCC TGG CGC AAC CGC TGC AAG GGC ACC GAC GTC 151-N A W VA NW RNR C K G T D V

496-CAG GCG TGG ATC AGA GGC TGC CGG CTG GGC TCG TCC AGC TCA GGG 166-Q A W I R G C R L G S SS SG

541-ATC TAT CAG ATT CTG GCG ATC TAC TCA ACT GTC GCC AGT TCA CTG 181-IYQILA I Y S T VAS S L

586-GTG CTT TTG GTC TCC CTG GGG GCA ATC AGT TTC TGG ATG TGT TCT 196-V L L V S L G A I S F W M C S

337

631-AAT GGA TCT TTG CAG TGC AGA ATA TGC ATC 211-N G S L Q C R I CI

Black = HEL Red = PLP (56-70) Green = joining sequence Blue = Transmembrane anchor region

338

Appendix 25. SIPLP construct - complete nucleotide sequence

1 GCGCGCAATT AACCCTCACT AAAGGGAACA AAAGCTGGGT ACCGGGCCCC

51 CCCTCGAGGT CGACGGTATC GATAAGCTTA TCGATTTCGA ACCCTGTAGC

101 AGGGCCTCTG TGCAGCAGAG GGTGCACACT ACAGGTTGTG CACACGCGTG

151 TGCGTGCATG CATGCAAGGG TGCACATGCA CAGGGTGGAG GTCAGAGGTC

201 AACGTCAGGT GTCTTCGTCT GTCACCCACA GTCTTGTTTT TCAGACAGGA

251 CTTCTCACTG AATCTGAATC CTGCCCATTT GATGGGCTCA TCAGTCAGTT

301 TCCAGGTTTC CCTGGTCCCC ACCTCCCCAG GGCTGGAATT CAAGGCATAT

351 CTAGCTTTTT TGCATGAATG TTTGAGAGCC AAATTCAGTT CCTCATACTT

401 GCCCAATAGG CATAGTATAT CCACTGAGCC ATGTCCCCAA CTCATTATGT

451 TCACCTCATT CATTCATTCA TCCATCCATC CATCCATCCA TCCATTCATT

501 CATTCAGGTT TGGTTTGGTT TGGTTTTAGT GTTTTGGGTT TTTTTTCAAG

551 ACAGGGTTTC TCTGTGTAGC CCTGGCTGCC CTGAAACTAA CTTTCTTACT

601 TACTTTCTTT CTTTCTTCCT TCCTTCCTTT CTTTCTTTAA GTTGGTTTGG

651 GTTTTTTGGG GGGGGGAGGG TTTGGGTTTT TTTGTTTGTT TGTATTTTAT

701 TCAAGGAAAG GCTCCTCTGG GTTGCCCTGG CTGCCCTGGA AGGAATTCTA

751 TAGCCAGGCT GACCATGAAC TCAGAAATCT ACCTGCCTCT CCCTCTGCCT

801 CCCAAGTGCT AGGATTAAAG GTGTGTGACA CACACACAAC TGGCTCTACA

851 TTTGACACTC ACAGTATTCT GCACACAGCC ACAGGGCTGT ATCGCTAAGA

901 GGCCATGTCA CAGGTCCAGG AAGGCTTCAC TGTTTAATAA CTGGTTTGGA

951 ATTTGGAAGT GGGAAAGTAT GTAGAGACAA TCTCACTCTA GGACTCAAAG

1001 ACAGAGATAA AATGCTCTTT AGAAACCTAT TCTGTGGGGG AACAGAATCA

1051 GAACTCTATG TCAGCCTCTG TGCTAGCTGG GGTCAGGCCA GAGACACAGA

339

1101 AATGAGCTAA GCCTACAGTG AGAACTGATA CGCAGGACAG TTGAGAACAA

1151 TGGGAAAGTG CATCTGAAGG GCCCAAGGCC ACTAGGAAAA AGGCAACCCA

1201 AAATGCCCAC AACTTTAAAT CCCCTTCCTG ATGTGTCATT AGATGCAAAC

1251 GGCCCTGGGG AGGATGTGAC GTTAAACAAG GCAGTGTTCT GTACAGGGGC

1301 AGATCCTGCA GTTACTGGAA GCTGAAGGCT CCAGGCTGAC CACACTAGGT

1351 AGGAAGTTCA TGAAATGAGC TCTCGAAGCG CCTCTTCAGG TCTTCCCCGA

1401 TGTCCAGCCC CACCAGCACC GTAGCAGGTA CTGGGCATCT ATTGATTGGG

1451 TGGGTAGGTG AACATTTGAG ACAACCTGGA AGTTTAAATG GGATTTGTGG

1501 AAATAGAGAG GGTGTTGGTT CCAGCCAAGC CAGGTTCCAT GCTAAGACAT

1551 AAAAGCACTG GAGAAGTAAA AGAGTTAACG CTGAACATGG CCGTGGGAAG

1601 CGAGGAGACC AAGGTAAAGC CTGGGAATGG CTAACACTTG AGAACTGTCA

1651 ACATCATGAA AGGATAAGAA AGAATGGCCA AGGAAAGAAA ACAGGAAAGA

1701 GCTGAGTGTG GGAGAGGCCT GGAGAGAATG GGGCAAGAAG TGGGAGGCTA

1751 AAGGGTAAGG GCAAGGGAGG GTCAAGTCAG ATGAGGACTG AAGGTGCTTT

1801 CAACTGGTAG GACTTGAGGG CTGCTCTGGG TAGGAGCCAG CCCGGTCTGG

1851 GCCCTGGGTC TCTCATGTGT TCTATGGAGA AGTCTTTATA TTTGTTCATG

1901 TGTATTGTAT GTGGGCTCTA ACCTGGCCAA CAATGCCCAA GAATCAAGTG

1951 ATTTTTGTGG CTTGAAGTCT AGCCACCTTC CTTGGAGGGA GGCAATAGAA

2001 GGAGCCTAGT GACATCTCGG ACGGAGTCTG GGGTCTTTGT CCTTCCCCTT

2051 GATCCCTGAA GGGTCTCCAG CCTCTGGTTC TCCAGGGTTG GCAAGTCTAC

2101 AATTGCTTCC CGGAACCTGG AGTGCTCAGT GCTTGACTTT AAAGAGGACA

2151 CAGGTGCCTA CAGTTCCACG ACCTCTGGGT TCCCCGACGC CCCCCCCCCC

2201 GCACTGCCCA TTGCCCAAAC ACCCCACAAG TGGCCTATTG TGTGAGCTCA

340

2251 GTTTCAATGG GAAAAGAAAC TGGGTTTATG AAAAGAGATT ATTTGCTTAT

2301 TGCATGGAGA TGACTAAGTA AATAGTGCAA TTTCTTGAGT GGAGCACAAT

2351 CCCTGCCCCT ACCTCTTGTG GGCTGCCAGG AACATATAAA AAGCCACACG

2401 CATACCCACA CATACCCACA CATACCCTCA CATTACAACA TCAGCCCACA

2451 TCTACAGACA GCCCAAGCTT GATATCGAAT TCCTGCAGCC CGGGGGATCC

2501 ACTAGTTCTA GAGCGGCCGC ACGTCTAAGA AACCATTATT ATCATGACAT

2551 TAACCTATAA AAATAGGCGT ATCACGAGGC CCTTTCGTCT TCAAGAATTC

2601 CATCAAGCTT AGGATCCGGA ACCCTTAAT A TAACTTCGTA TAATGTATGC

/651 TATACGAAGT TATTAGGTCC CTCGACCTGC AGCCCAAGCT TACTTACCAT

2701 GTCAGATCCA GACATGATAA GATACATTGA TGAGTTTGGA CAAACCACAA

2751 CTAGAATGCA GTGAAAAAAA TGCTTTATTT GTGAAATTTG TGATGCTATT

2801 GCTTTATTTG TAACCATTAT AAGCTGCAAT AAACAAGTTA ACAACAACAA

2851 TTGCATTCAT TTTATGTTTC AGGTTCAGGG GGAGGTGTGG GAGGTTTTTT

2901 AAAGCAAGTA AAACCTCTAC AAATGTGGTA TGGCTGATTA TGATCTCTAG

2951 TCAAGGCACT ATACATCAAA TATTCCTTAT TAACCCCTTT ACAAATTAAA

3001 AAGCTAAAGG TACACAATTT TTGAGCATAG TTATTAATAG CAGACACTCT

3051 ATGCCTGTGT GGAGTAAGAA AAAACAGTAT GTTATGATTA TAACTGTTAT

3101 GCCTACTTAT AAAGGTTACA GAATATTTTT CCATAATTTT CTTGTATAGC

3151 AGTGCAGCTT TTTCCTTTGT GGTGTAAATA GCAAAGCAAG CAAGAGTTCT

3201 ATTACTAAAC ACAGCATGAC TCAAAAAACT TAGCAATTCT GAAGGAAAGT

3251 CCTTGGGGTC TTCTACCTTT CTCTTCTTTT TTGGAGGAGT AGAATGTTGA

3301 GAGTCAGCAG TAGCCTCATC ATCACTAGAT GGCATTTCTT CTGAGCAAAA

3351 CAGGTTTTCC TCATTAAAGG CATTCCACCA CTGCTCCCAT TCATCAGTTC

loxP site

341

3401 CATAGGTTGG AATCTAAAAT ACACAAACAA TTAGAATCAG TAGTTTAACA

3451 CATTATACAC TTAAAAATTT TATATTTACC TTAGAGCTTT AAATCTCTGT

3501 AGGTAGTTTG TCCAATTATG TCACACCACA GAAGTAAGGT TCCTTCACAA

3551 AGATCCCTCG AGAAAAAAAA TATAAAAGAG ATGGAGGAAC GGGAAAAAGT

3601 TAGTTGTGGT GATAGGTGGC AAGTGGTATT CCGTAAGAAC AACAAGAAAA

3651 GCATTTCATA TTATGGCTGA ACTGAGCGAA CAAGTGCAAA ATTTAAGCAT

3701 CAACGACAAC AACGAGAATG GTTATGTTCC TCCTCACTTA AGAGGAAAAC

3751 CAAGAAGTGC CAGAAATAAC AGTAGCAACT ACAATAACAA CAACGGCGGC

3801 TACAACGGTG GCCGTGGCGG TGGCAGCTTC TTTAGCAACA ACCGTCGTGG

3851 TGGTTACGGC AACGGTGGTT TCTTCGGTGG AAACAACGGT GGCAGCAGAT

3901 CTAACGGCCG TTCTGGTGGT AGATGGATCG ATGGCAAACA TGTCCCAGCT

3951 CCAAGAAACG AAAAGGCCGA GATCGCCATA TTTGGTGTCC CCGAGGATCC

4001 GGAACCCTTA ATATAACTTC GTATAATGTA TGCTATACGA AGTTATTAGG} loxP

4051 site TCCCTCGAAG AGGTTCACTA GTACTGGCCA TTGCGGCCGG ATCCTGAGAA

4101 CTTCAGGGTG AGTTTGGGGA CCCTTGATTG TTCTTTCTTT TTCGCTATTG

4151 TAAAATTCAT GTTATATGGA GGGGGCAAAG TTTTCAGGGT GTTGTTTAGA

4201 ATGGGAAGAT GTCCCTTGTA TCACCATGGA CCCCCATGAT AATTTTGTTT

4251 CTTTCACTTT CTACTCTGTT GACAACCATT GTCTCCTCTT ATTTTCTTTT

4301 CATTTTCTGT AACTTTTTCG TTAAACTTTA GCTTGCATTT GTAACGAATT

4351 TTTAAATTCA CTTTTGTTTA TTTGTCAGAT TGTAAGTACT TTCTCTAATC

4401 ACTTTTTTTT CAAGGCAATC AGGGTATATT ATATTGTACT TCAGCACAGT

4451 TTTAGAGAAC AATTGTTATA ATTAAATGAT AAGGTAGAAT ATTTCTGCAT

4501 ATAAATTCTG GCTGGCGTGG AAATATTCTT ATTGGTAGAA ACAACTACAC

342

4551 CCTGGTCATC ATCCTGCCTT TCTCTTTATG GTTACAATGA TATACACTGT

4601 TTGAGATGAG GATAAAATAC TCTGAGTCCA AACCGGGCCC CTCTGCTAAC

4651 CATGTTCATG CCTTCTTCTC TTTCCTACAG CTCCTGGGCA ACGTGCTGGT

4701 TGTTGTGCTG TCTCATCATT TTGGCAAAGA ATTCTGGCAA CATGAGGTCT

4751 TTGCTAATCT TGGTGCTTTG CTTCCTGCCC CTGGCTGCTC TGGGGAAAGT

4801 CTTTGGACGA TGTGAGCTGG CAGCGGCTAT GAAGCGTCAC GGACTTGATA

4851 ACTATCGGGG ATACAGCCTG GGAAACTGGG TGTGTGCCGC AAAATTCGAG

4901 AGTAACTTC A ACACCCAGGC TACAGGCGCC AAAAACTACC AG ACTATGA

4951 PLP(5 GTATCTCATT AATGTGATTC ATGCTTTCCA GTATGTC ATC TATGGAACCC 701

5001 GCGGAAACCG TAACACCGAT GGGAGTACCG ACTACGGAAT CCTACAGATC

5051 AACAGCCGCT GGTGGTGCAA CGATGGCAGG ACCCCAGGCT CCAGGAACCT

5101 GTGCAACATC CCGTGCTCAG CCCTGCTGAG CTCAGACATA ACAGCGAGCG

5151 TGAACTGCGC GAAGAAGATC GTCAGCGATG GAAACGGCAT GAACGCGTGG

5201 GTCGCCTGGC GCAACCGCTG CAAGGGCACC GACGTCCAGG CGTGGATCAG

5251 AGGCTGCCGG CTGTGAGGAG CTGCCGGAAT TCTCTATCAG ATTCACTCCT

5301 CAGGTGCAGG CTGCCTATCA GAAGGTGGTG GCTGGTGTGG CCAATGCCCT

5351 GGCTCACAAA TACCACTGAG ATCGATCTTT TTCCCTCTGC CAAAAATTAT

5401 GGGGACATCA TGAAGCCCCT TGAGCATCTG ACTTCTGGCT AATAAAGGAA

5451 ATTTATTTTC ATTGCAATAG TGTGTTGGAA TTTTTTGTGT CTCTCACTCG

5501 GAAGGACATA TGGGAGGGCA AATCATTTAA AACATCAGAA TGAGTATTTG

5551 GTTTAGAGTT TGGCAACATA TGCCCATATG CTGGCTGCCA TGAACAAAGG

5601 TTGGCTATAA AGAGGTCATC AGTATATGAA ACAGCCCCCT GCTGTCCATT

5651 CCTTATTCCA TAGAAAAGCC TTGACTTGAG GTTAGATTTT TTTTATATTT

343

5701 TGTTTTGTGT TATTTTTTTC TTTAACATCC CCTAAAATTTT CCTTACATGT

5751 TTTACTAGCC AGATTTTTCC TCCTCTCCTG ACTACTCCCA GTCATAGCTG

5801 TCCCTCTTCT CTTATGGAGA TCCCTCGAGG GCCATTGCGG CCGCCACCGC

5851 GGTGGAGCTC CAGCTrITGT TCCCTTTAGT GAGGGTTAAT TGCGCGC

Black = pBluescript II vector sequence Brown = CC10 promoter Blue = Floxed STOP sequence Green = Rabbit beta globin intronic and PolyA sequence Red = HEL/PLP (56-70) coding region

344

Appendix 26. Secreted HEL/PLP(56-70) nucleotide/protein sequence

1- ATG AGG TCT TTG CTA ATC TTG GTG CTT TGC TTC CTG CCC CTG GCT 1-MR S L L I LV LCF L P L A

46- GCT CTG GGG AAA GTC TTT GGA CGA TGT GAG TTG GCA GCG GCT ATG 16-A L G K V F G R CE L A A A M

91- AAG CGT CAC GGA CTT GAT AAC TAT CGG GGA TAC AGC CTG GGA AAC 31-K RHGLDNYR G YS L G N

136-TGG GTG TGT GCC GCA AAA TTC GAG AGT AAC TTC AAC ACC CAG GCT 46-WV CA AK FE S N F N T QA

181-ACA GGC GCC AAA AAC TAC CAG GAC TAT GAG TAT CTC ATT AAT GTG 61-T G A K N YQDYE YLINV

226-ATT CAT GCT TTC CAG TAT GTC ATC TAT GGA ACC CGC GGA AAC CGT 76-IHA F Q YV I Y G T R G N R

271-AAC ACC GAT GGG AGT ACC GAC TAC GGA ATC CTA CAG ATC AAC AGC 91-N T D G S T D Y G I L Q I N S

316-CGC TGG TGG TGC AAC GAT GGC AGG ACC CCA GGC TCC AGG AAC CTG 106-R WWCND G R T P G SRNL

361-TGC AAC ATC CCG TGC TCA GCC CTG CTG AGC TCA GAC ATA ACA GCG 121-C N I P C S A L L S SDI TA

406-AGC GTG AAC TGC GCG AAG AAG ATC GTC AGC GAT GGA AAC GGC ATG 136-S V N C A K K IV SD GNGM

451-AAC GCG TGG GTC GCC TGG CGC AAC CGC TGC AAG GGC ACC GAC GTC 151-N A W YAW N W RNR C K G TD V

496-CAG GCG TGG ATC AGA GGC TGC CGG CTG 166-Q A W I R G C R L

Black = HEL Red = PLP (56-70)

Green = joining sequence

345

Appendix 27. Inverted stop sequence SIPLP construct - complete nucleotide sequence

1 GCGCGCAATT AACCCTCACT AAAGGGAACA AAAGCTGGCG GGCCCCCCCT

51 CGAGGTCGAC GGTATCGATA AGCTTATCGA TTTCGAACCC TGTAGCAGGG

101 CCTCTGTGCA GCAGAGGGTG CACACTACAG GTTGTGCACA CGCGTGTGCG

151 TGCATGCATG CAAGGGTGCA CATGCACAGG GTGGAGGTCA GAGGTCAACG

201 TCAGGTGTCT TCGTCTGTCA CCCACAGTCT TGTTTTTCAG ACAGGACTTC

251 TCACTGAATC TGAATCCTGC CCATTTGATG GGCTCATCAG TCAGTTTCCA

301 GGTTTCCCTG GTCCCCACCT CCCCAGGGCT GGAATTCAAG GCATATCTAG

351 CTTTTTTGCA TGAATGTTTG AGAGCCAAAT TCAGTTCCTC ATACTTGCCC

401 AATAGGCATA GTATATCCAC TGAGCCATGT CCCCAACTCA TTATGTTCAC

451 CTCATTCATT CATTCATCCA TCCATCCATC CATCCATCCA TTCATTCATT

501 CAGGTTTGGT TTGGTTTGGT TTTAGTGTTT TGGGTTTTTT TTCAAGACAG

551 GGTTTCTCTG TGTAGCCCTG GCTGCCCTGA AACTAACTTT CTTACTTACT

601 TTCTTTCTTT CTTCCTTCCT TCCTTTCTTT CTTTAAGTTG GTTTGGGTTT

651 TTTGGGGGGG GGAGGGTTTG GGTTTTTTTG TTTGTTTGTA TTTTATTCAA

701 GGAAAGGCTC CTCTGGGTTG CCCTGGCTGC CCTGGAAGGA ATTCTATAGC

751 CAGGCTGACC ATGAACTCAG AAATCTACCT GCCTCTCCCT CTGCCTCCCA

801 AGTGCTAGGA TTAAAGGTGT GTGACACACA CACAACTGGC TCTACATTTG

851 ACACTCACAG TATTCTGCAC ACAGCCACAG GGCTGTATCG CTAAGAGGCC

901 ATGTCACAGG TCCAGGAAGG CTTCACTGTT TAATAACTGG TTTGGAATTT

951 GGAAGTGGGA AAGTATGTAG AGACAATCTC ACTCTAGGAC TCAAAGACAG

1001 AGATAAAATG CTCTTTAGAA ACCTATTCTG TGGGGGAACA GAATCAGAAC

346

1051 TCTATGTCAG CCTCTGTGCT AGCTGGGGTC AGGCCAGAGA CACAGAAATG

1101 AGCTAAGCCT ACAGTGAGAA CTGATACGCA GGACAGTTGA GAACAATGGG

1151 AAAGTGCATC TGAAGGGCCC AAGGCCACTA GGAAAAAGGC AACCCAAAAT

1201 GCCCACAACT TTAAATCCCC TTCCTGATGT GTCATTAGAT GCAAACGGCC

1251 CTGGGGAGGA TGTGACGTTA AACAAGGCAG TGTTCTGTAC AGGGGCAGAT

1301 CCTGCAGTTA CTGGAAGCTG AAGGCTCCAG GCTGACCACA CTAGGTAGGA

1351 AGTTCATGAA ATGAGCTCTC GAAGCGCCTC TTCAGGTCTT CCCCGATGTC

1401 CAGCCCCACC AGCACCGTAG CAGGTACTGG GCATCTATTG ATTGGGTGGG

1451 TAGGTGAACA TTTGAGACAA CCTGGAAGTT TAAATGGGAT TTGTGGAAAT

1501 AGAGAGGGTG TTGGTTCCAG CCAAGCCAGG TTCCATGCTA AGACATAAAA

1551 GCACTGGAGA AGTAAAAGAG TTAACGCTGA ACATGGCCGT GGGAAGCGAG

1601 GAGACCAAGG TAAAGCCTGG GAATGGCTAA CACTTGAGAA CTGTCAACAT

1651 CATGAAAGGA TAAGAAAGAA TGGCCAAGGA AAGAAAACAG GAAAGAGCTG

1701 AGTGTGGGAG AGGCCTGGAG AGAATGGGGC AAGAAGTGGG AGGCTAAAGG

1751 GTAAGGGCAA GGGAGGGTCA AGTCAGATGA GGACTGAAGG TGCTTTCAAC

1801 TGGTAGGACT TGAGGGCTGC TCTGGGTAGG AGCCAGCCCG GTCTGGGCCC

1851 TGGGTCTCTC ATGTGTTCTA TGGAGAAGTC TTTATATTTG TTCATGTGTA

1901 TTGTATGTGG GCTCTAACCT GGCCAACAAT GCCCAAGAAT CAAGTGATTT

1951 TTGTGGCTTG AAGTCTAGCC ACCTTCCTTG GAGGGAGGCA ATAGAAGGAG

2001 CCTAGTGACA TCTCGGACGG AGTCTGGGGT CTTTGTCCTT CCCCTTGATC

2051 CCTGAAGGGT CTCCAGCCTC TGGTTCTCCA GGGTTGGCAA GTCTACAATT

2101 GCTTCCCGGA ACCTGGAGTG CTCAGTGCTT GACTTTAAAG AGGACACAGG

2151 TGCCTACAGT TCCACGACCT CTGGGTTCCC CGACGCCCCC CCCCCCGCAC

347

2201 TGCCCATTGC CCAAACACCC CACAAGTGGC CTATTGTGTG AGCTCAGTTT

2251 CAATGGGAAA AGAAACTGGG TTTATGAAAA GAGATTATTT GCTTATTGCA

2301 TGGAGATGAC TAAGTAAATA GTGCAATTTC TTGAGTGGAG CACAATCCCT

2351 GCCCCTACCT CTTGTGGGCT GCCAGGAACA TATAAAAAGC CACACGCATA

2401 CCCACACATA CCCACACATA CCCTCACATT ACAACATCAG CCCACATCTA

2451 CAGACAGCCC AAGCTTGGTA CCTCTCCGCG GTGGCGGCCG CAATGGCCAG

2501

f 51 TACGAAGTTA TATTAAGGGT TCCGGATCCT CGGGGACACC AAATATGGCG}

loxP site

2601 ATCTCGGCCT TTTCGTTTCT TGGAGCTGGG ACATGTTTGC CATCGATCCA

2651 TCTACCACCA GAACGGCCGT TAGATCTGCT GCCACCGTTG TTTCCACCGA

2701 AGAAACCACC GTTGCCGTAA CCACCACGAC GGTTGTTGCT AAAGAAGCTG

2751 CCACCGCCAC GGCCACCGTT GTAGCCGCCG TTGTTGTTAT TGTAGTTGCT

2801 ACTGTTATTT CTGGCACTTC TTGGTTTTCC TCTTAAGTGA GGAGGAACAT

2851 AACCATTCTC GTTGTTGTCG TTGATGCTTA AATTTTGCAC TTGTTCGCTC

2901 AGTTCAGCCA TAATATGAAA TGCTTTTCTT GTTGTTCTTA CGGAATACCA

2951 CTTGCCACCT ATCACCACAA CTAACTTTTT CCCGTTCCTC CATCTCTTTT

3001 ATATTTTTTT TCTCGAGGGA TCTTTGTGAA GGAACCTTAC TTCTGTGGTG

3051 TGACATAATT GGACAAACTA CCTACAGAGA TTTAAAGCTC TAAGGTAAAT

3101 ATAAAATTTT TAAGTGTATA ATGTGTTAAA CTACTGATTC TAATTGTTTG

3151 TGTATTTTAG ATTCCAACCT ATGGAACTGA TGAATGGGAG CAGTGGTGGA

3201 ATGCCTTTAA TGAGGAAAAC CTGTTTTGCT CAGAAGAAAT GCCATCTAGT

3251 GATGATGAGG CTACTGCTGA CTCTCAACAT TCTACTCCTC CAAAAAAGAA

3301 GAGAAAGGTA GAAGACCCCA AGGACTTTCC TTCAGAATTG CTAAGTTTTT

TACTAGTGAA CCTCTTCGAG GGACCTAATA ACTTCGTATA GCATACATTA

348

3351 TGAGTCATGC TGTGTTTAGT AATAGAACTC TTGCTTGCTT TGCTATTTAC

3401 ACCACAAAGG AAAAAGCTGC ACTGCTATAC AAGAAAATTA TGGAAAAATA

3451 TTCTGTAACC TTTATAAGTA GGCATAACAG TTATAATCAT AACATACTGT

3501 TTTTTCTTAC TCCACACAGG CATAGAGTGT CTGCTATTAA TAACTATGCT

3551 CAAAAATTGT GTACCTTTAG CTTTTTAATT TGTAAAGGGG TTAATAAGGA

3601 ATATTTGATG TATAGTGCCT TGACTAGAGA TCATAATCAG CCATACCACA

3651 TTTGTAGAGG TTTTACTTGC TTTAAAAAAC CTCCCACACC TCCCCCTGAA

3701 CCTGAAACAT AAAATGAATG CAATTGTTGT TGTTAACTTG TTTATTGCAG

3751 CTTATAATGG TTACAAATAA AGCAATAGCA TCACAAATTT CACAAATAAA

3801 GCATTTTTTT CACTGCATTC TAGTTGTGGT TTGTCCAAAC TCATCAATGT

3851 ATCTTATCAT GTCTGGATCT GACATGGTAA GTAAGCTTGG GCTGCAGGTC

3901 GAGGGACCTA ATAACTTCGT ATAGCATACA TTATACGAAG TTATATTAAG} loxP

3951 site GGTTCCGGAT CCTAAGCTTG ATGGAATTCT TGAAGACGAA AGGGCCTCGT

4001 GATACGCCTA TTTTTATAGG TTAATGTCAT GATAATAATG GTTTCTTAGA

4051 CGTGCGGCCG CTCTAGAACT AGTGGATCCC CCGGGCTGCA GGAATTCGAT

4101 ATCAAGCTTA TCGATACCGT CGACCTCGAG GGGGGGCCCG GTACCGAGCT

4151 CGGATCCTGA GAACTTCAGG GTGAGTTTGG GGACCCTTGA TTGTTCTTTC

4201 TTTTTCGCTA TTGTAAAATT CATGTTATAT GGAGGGGGCA AAGTTTTCAG

4251 GGTGTTGTTT AGAATGGGAA GATGTCCCTT GTATCACCAT GGACCCTCAT

4301 GATAATTTTG TTTCTTTCAC TTTCTACTCT GTTGACAACC ATTGTCTCCT

4351 CTTATTTTCT TTTCATTTTC TGTAACTTTT TCGTTAAACT TTAGCTTGCA

4401 TTTGTAACGA ATTTTTAAAT TCACTTTTGT TTATTTGTCA GATTGTAAGT

4451 ACTTTCTCTA ATCACTTTTT TTTCAAGGCA ATCAGGGTAT ATTATATTGT

349

4501 ACTTCAGCAC AGTTTTAGAG AACAATTGTT ATAATTAAAT GATAAGGTAG

4551 AATATTTCTC CATATAAATT CTGGCTGGCG TGGAAATATT CTTATTGGTA

4601 GAAACAACTA CACCCTGGTC ATCATCCTGC CTTTCTCTTT ATGGTTACAA

4651 TGATATACAC TGTTTGAGAT GAGGATAAAA TACTCTGAGT CCAAACCGGG

4701 CCCCTCTGCT AACCATGTTC ATGCCTTCTT CTCTTTCCTA CAGCTCCTGG

4751 GCAACGTGCT GGTTGTTGTG CTGTCTCATC ATTTTGGCAA AGAATTCTGG

4801 CAACATGAGG TCTTTGCTAA TCTTGGTGCT TTGCTTCCTG CCCCTGGCTG

4851 CTCTGGGGAA AGTCTTTGGA CGATGTGAGT TGGCAGCGGC TATGAAGCGT

4901 CACGGACTTG ATAACTATCG GGGATACAGC CTGGGAAACT GGGTGTGTGC

4951 CGCAAAATTC GAGAGTAACT TCAACACCCA GGCTACAGGC GCCAAAAACT

5001 ACCAGIGACTA TGAGTATCTC ATTAATGTGA TTCATGCTTT CCAGTATGTC11 PLP(56-

5051 701

ATCTATGGAA CCCGCGGAAA CCGTAACACC GATGGGAGTA CCGACTACGG 5101

AATCCTACAG ATCAACAGCC GCTGGTGGTG CAACGATGGC AGGACCCCAG 5151

GCTCCAGGAA CCTGTGCAAC ATCCCGTGCT CAGCCCTGCT GAGCTCAGAC 5201

ATAACAGCGA GCGTGAACTG CGCGAAGAAG ATCGTCAGCG ATGGAAACGG 5251

CATGAACGCG TGGGTCGCCT GGCGCAACCG CTGCAAGGGC ACCGACGTCC 5301

AGGCGTGGAT CAGAGGCTGC CGGCTGTGAG GAGCTGCCGG AATTCACTCC 5351

TCAGGTGCAG GCTGCCTATC AGAAGGTGGT GGCTGGTGTG GCCAATGCCC 5401

TGGCTCACAA ATACCACTGA GATCGATCTT TTTCCCTCTG CCAAAAATTA 5451

TGGGGACATC ATGAAGCCCC TTGAGCATCT GACTTCTGGC TAATAAAGGA 5501

AATTTATTTT CATTGCAATA GTGTGTTGGA ATTTTTTGTG TCTCTCACTC 5551 GGAAGGACAT ATGGGAGGGC AAATCATTTA AAACATCAGA ATGAGTATTT

5601 GGTTTAGAGT TTGGCAACAT ATGCCCATAT GCTGGCTGCC ATGAACAAAG

350

5651 GTTGGCTATA AAGAGGTCAT CAGTATATGA AACAGCCCCC TGCTGTCCAT

5701 TCCTTATTCC ATAGAAAAGC CTTGACTTGA GGTTAGATTT TTTTTATATT

5751 TTGTTTTGTG TTATTTTTTT CTTTAACATC CCTAAAATTT TCCTTACATG

5801 TTTTACTAGC CAGATTTTTC CTCCTCTCCT GACTACTCCC AGTCATAGCT

5851 GTCCCTCTTC TCTTATGGAG ATCCCTCGAG CATGCATCTA GAGCGGCCGC

5901 CACCGCGGTG GAGCTCCAGC TTTTGTTCCC TTTAGTGAGG GTTAATTGCG

5951 CGC

Black = pBluescript II vector sequence Brown = CC10 promoter Blue = Floxed STOP sequence Green = Rabbit beta globin intronic and PolyA sequence Red = HEL/PLP (56-70) coding region

351

Appendix 28. Lung targeted h1L-8 construct - complete nucleotide sequence

1 GCGCGCAATT AACCCTCACT AAAGGGAACA AAAGCTGGG ACCGGGCCCC

51 CCCTCGAGGT CGACGGTATC GATAAGCTTA TCGATTTCGA ACCCTGTAGC

101 AGGGCCTCTG TGCAGCAGAG GGTGCACACT ACAGGTTGTG CACACGCGTG

151 TGCGTGCATG CATGCAAGGG TGCACATGCA CAGGGTGGAG GTCAGAGGTC

201 AACGTCAGGT GTCTTCGTCT GTCACCCACA GTCTTGTTTT TCAGACAGGA

251 CTTCTCACTG AATCTGAATC CTGCCCATTT GATGGGCTCA TCAGTCAGTT

301 TCCAGGTTTC CCTGGTCCCC ACCTCCCCAG GGCTGGAATT CAAGGCATAT

351 CTAGCTTTTT TGCATGAATG TTTGAGAGCC AAATTCAGTT CCTCATACTT

401 GCCCAATAGG CATAGTATAT CCACTGAGCC ATGTCCCCAA CTCATTATGT

451 TCACCTCATT CATTCATTCA TCCATCCATC CATCCATCCA TCCATTCATT

501 CATTCAGGTT TGGTTTGGTT TGGTTTTAGT GTTTTGGGTT TTTTTTCAAG

551 ACAGGGTTTC TCTGTGTAGC CCTGGCTGCC CTGAAACTAA CTTTCTTACT

601 TACTTTCTTT CTTTCTTCCT TCCTTCCTTT CTTTCTTTAA GTTGGTTTGG

651 GTTTTTTGGG GGGGGGAGGG TTTGGGTTTT TTTGTTTGTT TGTATTTTAT

701 TCAAGGAAAG GCTCCTCTGG GTTGCCCTGG CTGCCCTGGA AGGAATTCTA

751 TAGCCAGGCT GACCATGAAC TCAGAAATCT ACCTGCCTCT CCCTCTGCCT

801 CCCAAGTGCT AGGATTAAAG GTGTGTGACA CACACACAAC TGGCTCTACA

851 TTTGACACTC ACAGTATTCT GCACACAGCC ACAGGGCTGT ATCGCTAAGA

901 GGCCATGTCA CAGGTCCAGG AAGGCTTCAC TGTTTAATAA CTGGTTTGGA

951 ATTTGGAAGT GGGAAAGTAT GTAGAGACAA TCTCACTCTA GGACTCAAAG

1001 ACAGAGATAA AATGCTCTTT AGAAACCTAT TCTGTGGGGG AACAGAATCA

352

1051 GAACTCTATG TCAGCCTCTG TGCTAGCTGG GGTCAGGCCA GAGACACAGA

1101 AATGAGCTAA GCCTACAGTG AGAACTGATA CGCAGGACAG TTGAGAACAA

1151 TGGGAAAGTG CATCTGAAGG GCCCAAGGCC ACTAGGAAAA AGGCAACCCA

1201 AAATGCCCAC AACTTTAAAT CCCCTTCCTG ATGTGTCATT AGATGCAAAC

1251 GGCCCTGGGG AGGATGTGAC GTTAAACAAG GCAGTGTTCT GTACAGGGGC

1301 AGATCCTGCA GTTACTGGAA GCTGAAGGCT CCAGGCTGAC CACACTAGGT

1351 AGGAAGTTCA TGAAATGAGC TCTCGAAGCG CCTCTTCAGG TCTTCCCCGA

1401 TGTCCAGCCC CACCAGCACC GTAGCAGGTA CTGGGCATCT ATTGATTGGG

1451 TGGGTAGGTG AACATTTGAG ACAACCTGGA AGTTTAAATG GGATTTGTGG

1501 AAATAGAGAG GGTGTTGGTT CCAGCCAAGC CAGGTTCCAT GCTAAGACAT

1 551 AAAAGCACTG GAGAAGTAAA AGAGTTAACG CTGAACATGG CCGTGGGAAG

1601 CGAGGAGACC AAGGTAAAGC CTGGGAATGG CTAACACTTG AGAACTGTCA

1651 ACATCATGAA AGGATAAGAA AGAATGGCCA AGGAAAGAAA ACAGGAAAGA

1701 GCTGAGTGTG GGAGAGGCCT GGAGAGAATG GGGCAAGAAG TGGGAGGCTA

1751 AAGGGTAAGG GCAAGGGAGG GTCAAGTCAG ATGAGGACTG AAGGTGCTTT

1801 CAACTGGTAG GACTTGAGGG CTGCTCTGGG TAGGAGCCAG CCCGGTCTGG

1851 GCCCTGGGTC TCTCATGTGT TCTATGGAGA AGTCTTTATA TTTGTTCATG

1901 TGTATTGTAT GTGGGCTCTA ACCTGGCCAA CAATGCCCAA GAATCAAGTG

1951 ATTTTTGTGG CTTGAAGTCT AGCCACCTTC CTTGGAGGGA GGCAATAGAA

2001 GGAGCCTAGT GACATCTCGG ACGGAGTCTG GGGTCTTTGT CCTTCCCCTT

2051 GATCCCTGAA GGGTCTCCAG CCTCTGGTTC TCCAGGGTTG GCAAGTCTAC

2101 AATTGCTTCC CGGAACCTGG AGTGCTCAGT GCTTGACTTT AAAGAGGACA

2151 CAGGTGCCTA CAGTTCCACG ACCTCTGGGT TCCCCGACGC CCCCCCCCCC

353

2201 GCACTGCCCA TTGCCCAAAC ACCCCACAAG TGGCCTATTG TGTGAGCTCA

2251 GTTTCAATGG GAAAAGAAAC TGGGTTTATG AAAAGAGATT ATTTGCTTAT

2301 TGCATGGAGA TGACTAAGTA AATAGTGCAA TTTCTTGAGT GGAGCACAAT

2351 CCCTGCCCCT ACCTCTTGTG GGCTGCCAGG AACATATAAA AAGCCACACG

2401 CATACCCACA CATACCCACA CATACCCTCA CATTACAACA TCAGCCCACA

2451 TCTACAGACA GCCCAAGCTT GATATCGAAT TCCTGCAGCC CGGGGGATCC

2501 TGAGAACTTC AGGGTGAGTT TGGGGACCCT TGATTGTTCT TTCTTTTTCG

2551 CTATTGTAAA ATTCATGTTA TATGGAGGGG GCAAAGTTTT CAGGGTGTTG

2601 TTTAGAATGG GAAGATGTCC CTTGTATCAC CATGGACCCC CATGATAATT

2651 TTGTTTCTTT CACTTTCTAC TCTGTTGACA ACCATTGTCT CCTCTTATTT

2701 TCTTTTCATT TTCTGTAACT TTTTCGTTAA ACTTTAGCTT GCATTTGTAA

2751 CGAATTTTTA AATTCACTTT TGTTTATTTG TCAGATTGTA AGTACTTTCT

2801 CTAATCACTT TTTTTTCAAG GCAATCAGGG TATATTATAT TGTACTTCAG

2851 CACAGTTTTA GAGAACAATT GTTATAATTA AATGATAAGG TAGAATATTT

2901 CTGCATATAA ATTCTGGCTG GCGTGGAAAT ATTCTTATTG GTAGAAACAA

2951 CTACACCCTG GTCATCATCC TGCCTTTCTC TTTATGGTTA CAATGATATA

3001 CACTGTTTGA GATGAGGATA AAATACTCTG AGTCCAAACC GGGCCCCTCT

3051 GCTAACCATG TTCATGCCTT CTTCTCTTTC CTACAGCTCC TGGGCAACGT

3101 GCTGGTTGTT GTGCTGTCTC ATCATTTTGG CAAAGAATTC TGTAAACATG

3151 ACTTCCAAGC TGGCCGTGGC TCTCTTGGCA GCCTTCCTGA TTTCTGCAGC

3201 TCTGTGTGAA GGTGCAGTTT TGCCAAGGAG TGCTAAAGAA CTTAGATGTC

3251 AGTGCATAAA GACATACTCC AAACCTTTCC ACCCCAAATT TATCAAAGAA

3301 CTGAGAGTGA TTGAGAGTGG ACCACACTGC GCCAACACAG AAATTATTGT

354

3351 AAAGCTTTCT GATGGAAGAG AGCTCTGTCT GGACCCCAAG GAAAACTGGG

3401 TGCAGAGGGT TGTGGAGAAG TTTTTGAAGA GGGCTGAGAA CTCATAAGAA

3451 TTCACTCCTC AGGTGCAGGC TGCCTATCAG AAGGTGGTGG CTGGTGTGGC

3501 CAATGCCCTG GCTCACAAAT ACCACTGAGA TCGATCTTTT TCCCTCTGCC

3551 AAAAATTATG GGGACATCAT GAAGCCCCTT GAGCATCTGA CTTCTGGCTA

3601 ATAAAGGAAA TTTATTTTCA TTGCAATAGT GTGTTGGAAT TTTTTGTGTC

3651 TCTCACTCGG AAGGACATAT GGGAGGGCAA ATCATTTAAA ACATCAGAAT

3701 GAGTATTTGG TTTAGAGTTT GGCAACATAT GCCCATATGC TGGCTGCCAT

3751 GAACAAAGGT TGGCTATAAA GAGGTCATCA GTATATGAAA CAGCCCCCTG

3801 CTGTCCATTC CTTATTCCAT AGAAAAGCCT TGACTTGAGG TTAGATTTTT

3851 TTTATATTTT GTTTTGTGTT ATTTTTTTCT TTAACATCCC TAAAATTTTC

3901 CTTACATGTT TTACTAGCCA GATTTTTCCT CCTCTCCTGA CTACTCCCAG

3951 TCATAGCTGT CCCTCTTCTC TTATGGAGAT CCCTCGAGCA TGCATCTAGA

4001 GCGGCCGCCA CCGCGGTGGA GCTCCAGCTT TTGTTCCCTT TAGTGAGGGT

4051 TAATTGCGCG C

Black = pBluescript II vector sequence Brown = CC10 promoter Green = Rabbit beta globin intronic and PolyA sequence Blue = Human IL-8

355

Appendix 29. hIL-8 nucleotide/protein sequence

1- ATG ACT TCC AAG CTG GCC GTG GCT CTC TTG GCA GCC TTC CTG ATT 1-MT S K L A V A L L A A F L I

46- TCT GCA GCT CTG TGT GAA GGT GCA GTT TTG CCA AGG AGT GCT AAA 16-SA AL CEGA V L P R SA K

91- GAA CTT AGA TGT CAG TGC ATA AAG ACA TAC TCC AAA CCT TTC CAC 31EL R CQCIK T Y S K P F H

136- CCC AAA TTT ATC AAA GAA CTG AGA GTG ATT GAG AGT GGA CCA CAC 46-P K F I K E L R VIES GP H

181- TGC GCC AAC ACA GAA ATT ATT GTA AAG CTT TCT GAT GGA AGA GAG 61-CAN T E II V K L S D G R E

226- CTC TGT CTG GAC CCC AAG GAA AAC TGG GTG CAG AGG GTT GTG GAG 76-LCLDPK E NWVQR VV E

271- AAG TTT TTG AAG AGG GCT GAG AAC TCA 91-K F L K R A E N S

Red = mutated base pair (T—>C)

356

Appendix 30. Inflammatory cell scoring system

Score Bronchi Blood vessels

0. Bronchi or blood vessel is not surrounded by any inflammatory cells

1. Bronchi or blood vessels is surrounded by the occasional inflammatory cell

2. Inflammation completely surrounds and is 1-4 cells in diameter from the edge of the bronchi or vessel

3. Inflammation completely surrounds and is 5 or more cells in diameter from the edge of the bronchi or vessel

357