Complement activation-‐ good or evil in HIV-‐1 infection?

Linköping University Medical Dissertations No. 1281

Complement activation-‐ good or evil in

HIV-‐1 infection?

Interaction of Free and Complement Opsonized HIV-‐1 with

Monocyte Derived Dendritic Cells and Immune Cells in the

Cervical Mucosa

Veronica Tjomsland

Department of Clinical and Experimental Medicine

Linköping University, Sweden

1

Copyright © Veronica Tjomsland, 2011

Division of Molecular Virology

Department of Clinical and Experimental Medicine

Linköping University

SE-581 85 Linköping

Cover: The Human immunodeficiency virus

The cover is designed by Caroline Dennerqvist, Pixeltown Arts, all rights reserved.

The pictures in this thesis are illustrated by Rada Ellegård. Published articles have been

reprinted with permission from respective copyright holder.

Printed by LiU-Tryck, Linköping, Sweden, 2011

ISBN: 978-91-7393-010-9

ISSN: 0345-0082

2

”They don't actually see the real world, where 95% of the people with HIV are not treated

and are dying. And even though we have some blue sky now in our country, the sky could

become cloudy again very soon”

Luc Montagnier

“The world needs people who dare to think differently, you don’t change anything by

walking in other peoples footsteps”

Veronica Tjomsland

Dedicated to my husband and children for their unending love and

support

3

Linköping 2011

Supervisor Marie Larsson, Associate Professor Division of Molecular Virology Department of Clinical and Experimental Medicine Linköping University, Sweden

Faculty opponent Barbara L. Shacklett, Associate professor Department of Medical Microbiology and Immunology University of California, Davis, USA

Co-‐supervisors Committee Board Jorma Hinkula, Professor Kristina Broliden, Professor Division of Molecular Virology Unit of Infectious Diseases Department of Clinical and Department of Medicine Experimental Medicine Karolinska Institute, Sweden Linköping University, Sweden Karl-‐Eric Magnusson, Professor Maria Jenmalm, Associate professor Division of Molecular Virology AIR/Clinical Immunology Department of Clinical and Department of Clinical and Experimental Medicine experimental Medicine Linköping University, Sweden Linköping University, Sweden

Sven Hammarström, Professor Division of Cell Biology Department of Clinical and Experimental Medicine Linköping University, Sweden

4

PREFACE

This thesis describes the results of my research carried out during my PhD study at the

University of Linköping. The thesis gives you first a general introduction to the world of

HIV-‐1, the complement system, dendritic cells (DCs), and antigen presentation. This is

followed by a presentation of the papers. Not much is known about the MHC class I and II

antigen presentation pathways used by immature and mature DCs to present antigens from

whole HIV-‐1 particles and the first project focused on this topic. In the second project we

studied the initial interactions of free and opsonized HIV-‐1 with DCs with the focus on

receptor families involved in the viral binding. Since our results had shown that opsonized

HIV-‐1 interacted with DCs in a unique way we continued in the third project to study the

receptors and pathways used by DCs to process and present antigens derived from both

free and complement opsonized HIV-‐1. In addition, this project also studied the effects

these viral sources had on the antigen presentation machinery. In the final project we used

the knowledge acquired from our in vitro experiments with free and complement

opsonized HIV-‐1 and applied it on an ex vivo study. The HIV-‐1 interactions and infection of

immune cells located in cervical mucosa were studied using an explant model and we

examined if infection could be prevented by targeting different receptors expressed by

immune cells and mucosa. Finally, I want to thank my supervisor Marie Larsson for making

this thesis possible.

Veronica Tjomsland November 2011

5

ABSTRACT

Worldwide, the heterosexual route is the most common mode of sexual transmission of

HIV-‐1 and women are particularly susceptible to this infection. After penetration of the

mucosal epithelium HIV-‐1 interacts with potential target cells, i.e. dendritic cells (DCs)

and CD4+ T cells. The complement system, a key component of the innate immune

system, is immediately activated by HIV-‐1 in vivo. However, HIV-‐1 can resist

complement mediated lysis and become coated with complement fragments and this

opsonization influences the viral interaction with immune cells. The DCs are the most

potent antigen presenting cell. This cell effectively links the innate recognition of viruses

to the generation of an adaptive immune response. However, HIV-‐1 exploits the function

of the DCs to facilitate viral spread and infection. HIV-‐1 interacts with a range of

receptors expressed by the DCs including C-‐type lectins, integrins and complement

receptors (CRs). The uptake of virions by DCs leads to their activation and migration to

the lymph nodes. At this site DCs present HIV-‐1 derived antigen on MHC class I and II

molecules and trigger an HIV-‐1 specific T cell response. The interplay between the virus

and the DCs is complex and the initial receptor binding may affect antigen uptake,

infection, and antigen presentation.

The fundamental questions of this thesis are the following: How is free and opsonized

HIV-‐1 internalized, processed, and presented on MHC class I and II molecules by DCs

and how do free and opsonized HIV-‐1 particles interact with immune cells in the

cervical mucosa?

Our results indicate that opsonization of HIV-‐1 plays a critical role in the interaction

with immune cells. Complement opsonization of HIV-‐1 (C-‐HIV) significantly enhanced

the internalization by the DCs compared to free HIV (F-‐HIV). Both C-‐HIV and F-‐HIV

interacted with the CD4 receptor, C-‐type lectins and integrins. In addition, opsonization

of HIV-‐1 favored an MHC class I presentation by DCs compared to F-‐HIV. However, the

endocytic receptors macrophage mannose receptor, β7 integrin, and CR3 guided the

antigens to different compartments with distinct properties and efficiencies for

degradation and MHC class I and II presentation of viral antigens. MHC class I

presentation of F-‐HIV and C-‐HIV was dependent of viral fusion in a CD4/coreceptor

dependent manner. Moreover, MHC class II presentation of antigens derived from HIV-‐1

required endocytosis and proteolysis in acidified compartments. HIV-‐1 infection of

cervical mucosa immune cells and tissue was assessed in a cervical tissue explant model.

6

C-‐HIV significantly enhanced infection of DCs compared to F-‐HIV, whereas C-‐HIV

decreased the infection of CD4+ T cells. Blocking the viral use of integrins in the cervical

tissue explants significantly decreased the HIV-‐1 infection of both emigrating DCs and

CD4+ T cells and the establishment of founder populations in these tissues. This thesis

work has brought forward new facts that can be used to facilitate the development of an

effective vaccine or microbicide.

7

LIST OF PAPERS INCLUDED IN THE THESIS

I Pathways utilized by dendritic cells for binding, uptake, processing

and presentation of antigens derived from HIV-‐1.

Sabado RL, Babcock E, Kavanagh DG, Tjomsland V, Walker BD, Lifson JD,

Bhardwaj N, Larsson M.

Eur J Immunol. 2007 Jul; 37(7):1752-‐63.

II Complement Opsonization of HIV-‐1 Enhances the Uptake by Dendritic

Cells and Involves the Endocytic Lectin and Integrin Receptor

Families. Tjomsland V, Ellegård R, Che K, Hinkula J, Lifson JD, Larsson M.

PLoS One. 2011; 6(8):e23542. Epub 2011 Aug 11.

III Complement opsonization of HIV-‐1 results in a different intracellular

processing efficiency and pattern leading to an enhanced MHC I class

presentation by dendritic cells.

Tjomsland V, Ellegård R, Burgener A, Hinkula J, Lifson JD, Larsson M.

Manuscript

IV Blocking of integrins significantly inhibits HIV-‐1 infection of human

cervical mucosa immune cells and development of founder

populations.

Tjomsland V, Ellegård R, Kjölhede P, Hinkula J, Lifson JD, Larsson M.

Manuscript

8

ABBREVIATIONS

Ab Antibody

ABC Avidin biotin complex

AIDS Acquired immunodeficiency syndrome

APC Antigen presenting cell

APOBEC3G Apoplipoprotein B mRNA-‐editing, enzyme-‐catalytic, polypeptide-‐like 3G

ART Antiretroviral therapy

AT-‐2 Aldrithiol-‐2

AZT Azidothymidine

CCR5 CC chemokine receptor 5

CXCR4 CXC chemokine receptor 4

C-‐HIV Complement opsonized HIV-‐1

C-‐IgG-‐HIV Complement opsonized HIV-‐1 in combination with immune complex

DAPI 4’,6’-‐diamidino-‐2-‐phenylindole

DCs Dendritic cells

DC-‐SIGN Dendritic cell-‐specific ICAM-‐3-‐grabbing non-‐integrin

dsDNA Double stranded DNA

EDTA Ethylene-‐diamine-‐tetra-‐acetic acid

ER Endoplasmic reticulum

F-‐HIV Free-‐HIV

fH factor H

FITC Fluorescein isothiocyanate

gp41 HIV-‐1 glycoprotein 41

gp120 HIV-‐1 glycoprotein 120

HAART Highly active anti-‐retroviral therapy

HIV-‐1 Human immunodeficiency virus-‐1

ICAM Intercellular adhesion molecule

IgG-‐HIV IgG opsonized HIV-‐1

IDCs Immature dendritic cells

IFN Interferon

IFRs Interferon regulatory factors

IL Interleukin

ISG IFN-‐stimulatory genes

9

LFA-‐1 lymphocyte function-‐associated antigen 1

LCs Langerhans cells

LTR Long terminal repeats

MAC Membrane attack complex

MHC Major histocompatibility complex

MDC Mature dendritic cells

MDDC Monocyte derived dendritic cells

MMR Macrophage mannose receptor

Nef Negative factor

PAMPS Pathogen associated molecular patterns

PBMC Peripheral blood mononuclear cells

PBS Phosphate-‐buffered saline

PDCs Plasmacytoid dendritic cells

PE Phycoerythrin

PFA Para formaldehyde

PHS Pool human serum

PIC Pre-‐integration complex

PR HIV-‐1 protease

RNA Ribonucleic acid

RT Reverse transcriptase

SIV Simian immunodeficiency virus

SAMHD-‐1 SAM domain and HD domain containing protein 1

ssRNA Single stranded RNA

TAR Transactivation response element

TLR Toll like receptor

TRIM Tripartite motif-‐ containing protein

Vif Viral infectivity factor

10

TABLE OF CONTENTS

PREFACE………………………………………………………………………………….................................................I

ABSTRACT…………………………………………………………………………...................................................... II

LIST OF PAPERS…………………………………………………………………………..........................................III

ABBREVIATIONS……………………………………………………………………………………………………... IV

CHAPTERS

1. INTRODUCTION……………………………………………………………………………………………………...1

2. HIV-‐1……………………………………………………………………………………...............................................3

2.1 Life cycle.………………………………………………………………………........................................4

2.2 Relevant aspects of HIV-‐1 innate and adaptive immunity……………………………..7

3. THE COMPLEMENT SYSTEM…………………………………………………………………………………10

3.1 Overview………………………………………………………………………………………………….10

3.2 Complement opsonization of HIV-‐1…………………………………………….....................12

3.3 Outcomes after complement activation by HIV-‐1……………………………................15

4. DENDRITIC CELLS.......................................................................................................................................16

4.1 The role of dendritic cells in immunity…………………………………………..................16

4.2 Dendritic cell lineages and subsets …………………………………………….....................17

4.2.1 Plasmacytoid dendritic cells (PDCs)……………..…………………….................17

4.2.2 Myeloid dendritic cells (MDCs)…………………………………….........................18

4.2.3 Monocyte derived dendritic cells (MDDCs)…………………………………….19

4.3 HIV-‐1 capture by dendritic cells………………………………………………………………..19

4.4 Intrinsic antiretroviral factors…………………………………………………………………..21

5. ANTIGEN PRESENTATION BY DENDRITIC CELLS…………………………………………………..23

5.1 Overview………………………………………………………………………………………………….23

5.2 MHC class I restricted antigen presentation……………………………………...............24

5.3 MHC class II restricted antigen presentation……………………………………………...27

11

6. MUCOSAL IMMUNITY AND HIV-‐1…………………………………………………..................................30

6.1 Transfer of HIV-‐1 through the female genital tract…………………………….............30

7. AIMS OF THESIS……………………………………………………………………………………………………33

8. METHODS………………………………………………………………………………..........................................34

8.1 Propagation of monocyte derived DCs………………………………………..……………..34

8.2 Virus propagation and opsonization…..………………………………………………………34 8.3 ELISPOT assays………………………...……………………………………….................................35

8.4 Quantification using Real-‐time PCR…………………………………………….....................35

8.5 Preparation of cervical tissue samples…………………………………………...................36

8.6 Flow Cytometry………………………………………………………………………………………..37

8.7 Immunofluorescence and confocal microscopy………………………………………….38

8.8 Immunohistochemisty (IHC)……………………………………………………………………..38

8.9 Statistical analysis…………………………………………………………………………………….39

9. RESULTS AND DISCUSSION…………………………………………………………....................................40

9.1 Paper I……………………………………………………………………………………………………..40

9.1.1 Background…………………………………………………………………………………..40

9.1.2 Principal findings………………………………………………………...........................41

9.1.3 Discussion/ Conclusion…………………………………………………………………41

9.2 Paper II…………………………………………………………………………………………………….42

9.2.1 Background…………………………………………………………………………………..42

9.2.2 Principal findings………………………………………………………...........................42


9.3 Paper III………………………………………………………………….…….………………………….43

9.3.1 Background…………………………………………………………………………………..43

9.3.2 Principal findings……………………………………………………...…………………..44


9.4 Paper IV…………………………………………………………………………………………………...45

9.4.1 Background…………………………………………………………………………………..45

9.4.2 Principal findings…………………………………………….……………………………46

9.4.3 Discussion/ Conclusion………………………………………………………………....47

12

10. CONCLUSIONS AND FUTURE DIRECTIONS ………………………………………………………….48

10.1 Complement activation-‐ good or evil in HIV-‐1 infection?......................................48

10.2 Future Challenges……………………………………………………………................................49

11. POPULÄRVETENSKAPLIG SAMMANFATTNING………………………………..............................50

12. ACKNOWLEDGEMENTS…………………………………………………………….....................................53

13. REFERENCES…………………………………………………………………………........................................57

14. REPRINTS OF ORIGINAL PAPERS AND MANUSCRIPT

14.1 Paper I

14.2 Paper II

14.3 Paper III

14.4 Paper IV

13

Introduction

1

1. INTRODUCTION In 1981 a new syndrome appeared in the United States. The patients had an acquired

immune deficiency with a marked depletion of the CD4+ T cell count. Two years later

HIV-‐1 was identified by Luc Montagnier and Françoise Barré-‐Sinoussi as the causative

agent of acquired immune deficiency syndrome (AIDS) (1). Currently more than 30

million people are infected with HIV-‐1 and an estimated 2.6 million are newly infected

every year in the world and millions have died from AIDS (2). This makes this infection

one of the worst epidemics of this century. Moreover, the HIV/AIDS epidemic is

accompanied by many tragic and difficult social challenges like discrimination, stigma,

denial and a growing number of children who have lost parents to AIDS (3). In 2005,

thirteen million children younger than 15 years of age had already lost one or both of

their parents to AIDS (4).

The natural history of HIV-‐1 infection involves a long period of clinical latency with a

gradual loss of CD4+ T cells before the infection progresses to AIDS. AIDS are defined by a

CD4+ T cell count below 400cells/µl blood and without treatment this will lead to

opportunistic infections, the appearance of rare malignancies and ultimately death.

The most prevalent route of sexual transmission is by heterosexual intercourse. Women

are particularly at high risk to acquire HIV-‐1 infection due to social and biological factors

and therefore bear the greatest burden (5). However, much is still unknown about the

biological factors in the female genital tract contributing to resistance against HIV-‐1

infection.

HIV-‐1 is a retrovirus that belongs to the genus Lentiviridae. Lentivirus is characterized by

a long incubation period, however it is now clear from studies in Macaques that local

events important to establish an systemic infection take place quickly in the early stages

of simian immunodeficiency virus (SIV) infection (6). Following entry of HIV-‐1 through

the mucosa epithelium founder populations are established in the submucosa and the

dendritic cells (DCs) will transfer the virus to CD4+ T cells in the mucosal stroma and

lymph nodes (7). In the lymph nodes the DCs will efficiently present HIV-‐1 antigens to T

cells via MHC class I and II restricted pathways and mount a specific immune response

against HIV-‐1. MHC class I and II presentation and activation of CD4+ and CD8+ T cells

are important events that will determine the outcome of the infection. Most individuals

control the viremia poorly in the absence of antiretroviral therapy. Today the only

14

Introduction

2

effective approach against HIV-‐1 infection is antiretroviral therapy but many limitations

exist such as toxicity, costs, distribution in developing countries, and resistance.

Unfortunately strategies to prevent HIV-‐1 transmission have had limited success over the

past three decades (6). Vaccines or microbicides have not proven efficient and have even

in some cases enhanced HIV-‐1 infection (8, 9). An effective HIV-‐1 vaccine will probably

require activation of CD4+ and CD8+ T cell responses directed against crucial HIV-‐1

epitopes (10).

There exists an urgent need today for an HIV-‐1 vaccine or microbicides to prevent HIV-‐1

transmission and constrain the ongoing pandemic.

15

HIV-‐1

3

2. HIV-‐1

HIV-‐1 belongs to the genus Lentivirus and is further divided into the family Retroviridae.

HIV-‐1 has a spherical morphology with a diameter of 100-‐120 nm and is surrounded by a

lipid bilayer, an envelope. This envelope is acquired from the host cell during the process

of viral budding and contains approximately 72 spikes of the viral receptor gp120 bound

together with the transmembrane spanning glycoprotein gp41(11). The envelope may

also express many other receptors like ICAM-‐1 and HLA class I and II molecules, acquired

from the infected cell during the budding process (12). The nucleocapsid, which has a

conical shape, contains a viral protease (PR), reverse transcriptase (RT), integrase (IN),

and two copies of a single stranded RNA (ssRNA) molecule (13) (Fig. 1).

Figure 1. Structure of the HIV-‐1 particle.

The HIV-1 is composed of two copies of positive ssRNA encoding the 9 viral genes. The viral

genome is enclosed by a conical nucleocapsid composed of 2000 copies of the viral protein gag

p24 (14). In the nucleocapsid are the pol encoded enzymes, integrase (IN), reverse transcriptase

(RT), and protease (PR), all needed by the virus for infection. Surrounding the nucleocapsid is a

matrix composed of the p17 gag protein and the matrix is in turn surrounded by a viral envelope.

The HIV protein Env protrudes from the viral envelope and is composed of gp120 and gp41

proteins. gp41 is an anchor protein, attaching gp120 to the viral envelope and HIV-1 uses this

glycoprotein complex to attach and fuse with target cells (15).

16

HIV-‐1

4

2.1 HIV-‐1 life cycle The infection begins with the binding of HIV-‐1 to the target cells by the viral receptor

gp120 to a 58kDa glycoprotein, the CD4 receptor. The CD4 receptor is expressed on T

cells, monocytes, macrophages, DCs, eosinophils, and microglia cells (16). Upon binding

to CD4, gp120 undergoes a conformational change and is able to bind the coreceptor CC-‐

chemokine receptor 5 (CCR5) or CXC-‐chemokine receptor 4 (CXCR4). The binding of

gp120 to both CD4 and coreceptor leads to further conformational changes that allow

gp41 to penetrate the cell membrane (17, 18). Following membrane fusion the virus

capsid is uncoated in the cytoplasm of the host cell and the viral RNA is released. The

capsid undergoes a progressive destabilization during its transport towards the nucleus

to ensure productive infection as uncoating should not occur too early or too late in the

process (19) (Fig. 2). The viral RNA is transcribed into a double stranded DNA (dsDNA)

by RT, but this transcription is negatively affected by the presence of the host cell protein

APOBEC3G. However, the HIV-‐1 protein Vif counteracts the cell’s antiviral effect by down

regulation of APOBEC3G and prevents incorporation of this protein into progeny virions

(20). The pre-‐integration complex navigates through the pores of the nucleolus. In the

nucleus the viral DNA can be found in three different forms, linear, a circular form of 2-‐

long terminal repeats (LTR), or a circle of 1-‐LTR (21). None of the circular forms lead to

the production of infectious virus but the viral genes Tat and Nef can be transcribed from

them (22). The linear dsDNA of the pre-‐integration complex is integrated in the host cell

genome and this is mediated by IN (23). The integration might lead to a latent infection,

i.e. nonproductive (24), but if cellular proteins bind to the viral LTR, transcription of Nef,

Tat, and Rev can occur and these HIV-‐1 proteins are normally expressed very shortly

after infection. When sufficient amount of Tat protein has been produced, Tat proteins

start to control further transcription of HIV-‐1 genes by binding to the TAR site

(Transactivation response element). In the early phase of replication only multiply

spliced mRNA are produced, but when sufficient amounts of Rev proteins are produced,

non-‐spliced or single spliced mRNA can be generated as well (25) (Fig. 2). The core of the

maturing HIV particle is formed by the gene products pol and gag. The gene products

coded by the env gene form the glycoprotein 120/41 spikes in the viral envelope (Fig. 3).

The proteins Gag and Pol are also derived from a big precursor polyprotein. The

formation of a new viral particle occurs in several steps; two copies of ssRNA associate

together with the RT enzymes, while core proteins assemble around them forming the

viral capsid. The immature particles migrate toward the cell surface and assemble, the

17

HIV-‐1

5

large precursor polyproteins are then cleaved resulting in the viral budding from the cell

plasma membrane and thereby the acquiring of a lipid envelope. The budding of HIV-‐1

virions is believed to occur through areas in the host cell membrane rich in cholesterol

(26). During the budding it is essential that the expression of CD4 receptors are

downregulated in the host cell membrane to avoid the interaction with gp120 (27). Nef

(negative factor) is important for replication and the pathogenesis of HIV. Many functions

have been described for Nef, including the down regulation of CD4, coreceptors, MHC

class I and II molecules by inducing endocytosis of these molecules, consequently

affecting antigen presentation and recognition by the HIV-‐1 specific immune response

(28-‐30). Later in the replication cycle the env gene product trap CD4 in the endoplasmic

reticulum (ER) (31).

18

HIV-‐1

6

Figure 2. Life cycle of HIV-‐1

The life cycle of HIV-‐1 begins when the virus binds to CD4 and coreceptor on a target cell.

When HIV-‐1 have bound to the infection receptors the envelope complex undergoes a

structural change resulting in fusion with the cell membrane and the virus inject its

contents into the cytosol (17, 18). The viral genetic material is transcribed from ssRNA into

dsDNA by the use of the HIV-‐1 enzyme RT. The viral dsDNA is then integrated into the host

genome by the help of IN. From the integrated DNA the cell produces RNA and viral proteins

(32). The HIV-‐1 protease cleaves the newly synthesized proteins, enabling them to join the

RNA and assemble by the cell membrane. Finally, new viral particles bud off from the cell

membrane and can infect new target cells (32).

19

HIV-‐1

7

Figure 3. Organization of the HIV-‐1 genome. HIV-‐1 has nine genes coding for 15 viral proteins. The structural genes gag, pol and env are

the same in all retroviruses and these genes contain information necessary to make new

viral particles. The other six genes, tat, rev, nef, vif, vpr, and vpu, are regulatory genes for

proteins that control the ability of HIV-‐1 to infect and replicate in a host cell. Long terminal

repeats (LTR) are regions controlling the production of new virions and is triggered by HIV-‐1 proteins or host cell proteins (16).

2.2 Relevant aspects of HIV-‐1 innate and adaptive immunity It is well established that HIV-‐1 infection results in strong activation of the immune

system (6). The innate immunity conducts the first line of defense followed by the

adaptive immunity. The innate and adaptive responses are closely interlinked and a

strong initial innate response is likely to lead to potent adaptive immunity. Several

components of the innate defense are activated by HIV-‐1, e.g. the complement cascade,

type I IFNs, and inflammatory cytokines (33). HIV-‐1 is transmitted through the mucosa

and targets specific immune cells, i.e. CD4+CCR5+ T cells and DCs (34, 35). The adaptive

immune response is incapable to mount a defense sufficient to clear the infection and the

onset is too late to stop the massive destruction of the CD4+CCR5+ T cells that occurs

within two weeks after onset of infection (34, 35).

The first line of defense does not require previous antigen encounter and may if strong

enough limit replication of the microbe giving the adaptive immunity enough time to

mount a potent and efficient immune response (36). The innate immune response can be

divided in to three groups; cellular, intracellular, and extracellular (37). The cellular

20

HIV-‐1

8

components of the innate immunity are for instance Langerhans cells (LCs), DCs,

monocytes, γδ T cells, and natural killer cells (NK cells) (38). To begin with these cells

have innate effector functions but later they may play a part in the induction of adaptive

immunity (36). For instance, DCs produce factors important for the initial innate defense

but they also prime the naïve T cells in the lymph nodes and activate the adaptive

immune response (39). In the initial immune response two families of transcription

factors play a major role in the innate anti-‐viral defense, the NFkB family and the

interferon regulatory factors (IRFs). The IRFs play a central role in the induction and

regulation of proteins, type I IFNs, and chemokines mediating antiviral responses. The

production of type I IFNs has an important role in the innate antiviral response, they

attract immune cells to the site of infection, increase the function of macrophages, T cells,

NK cells, and B cells and induce maturation of plasmacytoid DCs (PDCs) (40-‐42). IRF-‐3

plays a central role in the induction of antiviral response. The viral activation of this

factor leads to production of IFNβ, which stimulates the transcription of IRF-‐7 that

further augments the synthesis of IFNβ. The antiviral effect of IFN is mediated by the

induction of a large amount of cellular genes, i.e. IFN-‐stimulatory genes (ISG), ISG15 was

one of the first ISG identified and has been shown to have antiviral effects (43).

Toll like receptors (TLRs) is a family of receptors important in the innate immune

response. TLRs detect microbes and induce antimicrobial host defense responses by

recognizing conserved regions on pathogens, denoted as pathogen-‐associated molecular

patterns (PAMPS) (44). TLRs are involved in the destruction of pathogens, coordinating

the immune response, and regulating the functionality of DCs (42). The presence of

ssRNA activates TLR7/8 while dsRNA activates TLR3 (45). HIV-‐1 is recognized mainly

through TLR7 on PDCs and TLR8 on blood myeloid DCs (MDCs) and monocyte derived

DCs (MDDCs). PDCs are an important component of the innate immune defense and a

main producer of type I IFNs (46). Another part of the innate immune defense is the

restriction factors including, tripartite motif-‐containing protein (TRIM), 5α, 1, 19 and 22,

tetherin, SAM domain and HD domain-‐containing protein 1 (SAMHD-‐1), and

apoplipoprotein B mRNA-‐editing, enzyme-‐catalytic, polypeptide-‐like 3G (APOBEC3G)

(47-‐50). APOBEC3G is found in T cells, monocytes, macrophages, and DCs. The

incorporation of APOBEC3G into the HIV-‐1 genomes leads to extensive mutations in the

viral DNA, rendering them nonfunctional and inhibiting viral replication (51). However,

HIV-‐1 counteracts this defense mechanism by the production of the viral protein Vif. Vif

decrease the synthesis of APOBEC3G and enhances the 26S proteasome mediated

21

HIV-‐1

9

degradation making APOBEC3G unavailable for budding virions (47). Innate factors that

exert their effects in an extracellular manner are produced as a part of the innate defense

and include large amounts of type I interferons (IFNs), i.e. IFN-‐α and IFN-‐β. Type I IFNs

are produced by mainly by PDCs but also by MDCs, and macrophages during the early

phase of a viral infection and they promote TH1 cell development by activating the

transcription factor STAT4. In addition IFNs also prevent activated T cells from

undergoing apoptosis (52, 53). The CC chemokines CCL5 (RANTES), CCL3 (MIP-‐1α) and

CCL4 (MIP-‐1β) are secreted by activated DCs, macrophages, NK cells, and γδ T cells and

these factors can block the CCR5 coreceptors and prevent HIV-‐1 infection (54). However,

some cellular proteins downregulate the antiviral response, among them are the cellular

DNAse TREX1, which degrades unintegrated proviral DNA and thereby helping the virus

to be undetected by TLR9 or cytoplasmic DNA sensors (55). Defensins are extracellular

innate peptides that can contribute to protection against HIV-‐1 infection in the mucosa.

Another essential component of the innate immune response is the complement system

(56) and this part of the innate immunity is described and discussed in depth below.

Figure 4. Approaches by HIV-‐I to circumvent the cell mediated antiviral responses.

Complement factors, type I IFNs and the intrinsic cellular proteins TRIM, tetherin, APOBEC3G, and SAMHD-‐

1 contribute to the inhibition of viral replication inside the host cells. On the other hand, some of host cell

proteins, e.g. TREX1, contribute to the down regulation of the antiviral response. In addition, the virus has

genes encoding for proteins that can impair the antiviral defense.

APOBEC3G

SAMHD1

TRIM5!, 1, 19, 22

Tetherin

ISG15

Type I interferons

Vif

Vpx

Vpu, NefIRF-3

Vpr, Vif

TREX1

Complement

22

The complement system

10

3. THE COMPLEMENT SYSTEM

3.1 Overview The complement system is composed of more than 30 cell surface and serum

components (57) and around 90% of them are produced by hepatocytes but complement

proteins can also be produced by monocytes, macrophages, endothelial, and epithelial

cells (58, 59). The human complement system is the first line of the defense against

pathogens by inducing complement mediated lysis and tagging targets for phagocytosis.

However, lately it has been shown that complement also plays an important role in

induction and maintenance of the adaptive immune responses, i.e. antigen presentation,

and T cell activation (60). In addition, the complement system is involved in the

enhancement of the antibody induced responses via complement receptors (CRs) and Fc

receptors (FcRs) (60).

The complement system can be activated in three different ways dependent on the

trigger. All pathways; the classical pathway, the lectin pathway, and the alternative

pathway converge at the activation and triggering of complement component 3 (C3).

The classical pathway is sometimes also referred to as the antibody dependent classical

pathway and is activated by the binding of complement component 1q (C1q), a

subcomponent of the C1 complex, to IgG/IgM clusters bound to cell walls of pathogens or

apoptotic cells, or by the pentraxin family members. Alternatively, direct interaction by

C1q with some types of pathogens can also trigger this pathway. The C1 complex attracts

C2 and C4 and generates the C2C4 convertase, which is able to cleave the C3 protein and

results in C3a and C3b (61, 62).

The lectin binding pathway or the mannose binding pathway (MBP) is initiated by the

recognition of characteristic carbohydrate patterns expressed on the surface of

microorganisms. Binding occurs via the mannose-‐binding lectin (MBL) protein family and

ficolins and activates MBP associated serine proteases (MASPs) (62). The different MASPs

are similar to C1r and C1q, therefore the following cascade resembles the classical

pathway and will converge at the activation and cleavage of C3 (63, 64).

The alternative pathway of the complement cascade represents a process that needs no

exogenous trigger. By spontaneous C3 hydrolysis, new binding sites are exposed and

factor B binds to hydrolyzed C3 and is cleaved by factor D and results in formation of C3

23


11

convertase, which is cleaved into C3a and C3b. C3b interacts with factor B and this factor

in turn is cleaved by factor D, creating a full C3 convertase (C3bBb) that is stabilized by

the binding of properdin (65, 66). Subsequently, more and more C3b is drawn to this

multiprotein complex attached to the surface of the microbe leading to an effective

opsonization (60).

After opsonization of the pathogen, the terminal complement pathway is triggered

resulting in formation of a terminal membrane attack complex (MAC). The MAC is a pore

like structure created in the membrane of the pathogen leading to its lysis and

destruction (60). The complement system is strictly controlled to protect the host from

complement mediated damage. This is mediated by soluble and cell bound complement

regulators.

Among the regulators is C1 esterase inhibitor (C1-‐INH). This inhibitor have an effect on

several proteases in the classical and lectin binding pathway. The abundantly expressed

factor H (fH) acts on the C3 convertase or serve as a cofactor for degradation of C3b, but

can also prevent self attack. The C3 convertase is also regulated by factor I (fI), factor H

like protein, and C4 binding protein. In addition, most cells in the body express receptors

that function as convertase regulators, e.g. complement receptor 1 (CR1) and CD55, but

they also express receptors working as cofactors for fI, e.g. CR1 and CD49. The plasma

membrane bound protein protectin (CD59), a complement regulatory protein, inhibits

the formation of the MAC complex (67, 68). The inactivation and degradation of C3b leads

to the production of inactivated C3 fragments iC3b, iC3dg, and iC3d and these

complement fragments do not have any further function in the lytic cascade but are

ligands to complement receptors.

Complement receptor 1 (CR1: CD35) is a cell membrane receptor expressed on

leucocytes, erythrocytes, and podocytes. CR1 binds C3b and C4b and plays an important

role in the regulation of the complement cascade but CR1 also binds immune complexes

coated with C3b and remove them from circulation by transporting them to the liver or

spleen (69). Complement receptor 2 (CR2: CD21) is predominantly expressed on B cells,

T cells, and follicular dendritic cells (FDCs) and interacts mainly with C3dg and C3d.

Complement receptor 3 (CR3: MAC-‐1) and complement receptor 4 (CR4: pl 150,95) are

both members of the β2-‐integrin family. CR3 consists of two chains, an 165 kDa αM-‐chain

(CD11b) and an 95 kDa β-‐chain (CD18) and is expressed primarily on myeloid cells but

also on NK cells , microglia, osteoblasts, and some epithelial cells (70). CR4 has the same

β2-‐chain but instead this chain is linked to a 150 kDa αX-‐chain (CD11c) and the CR4 is

24


12

basically found on the same cell types as CR3. CR3 has been shown to be involved in

many coordinating and adhesion functions in the immune system, e.g. adhesion and

migration of leucocytes during homing, and the binding and phagocytosis of opsonized

particles (70-‐72). CR3 can bind to several ligands with high affinity including iC3b, ICAM-‐

1, fibrinogen, and clotting factor X and with low affinity to C3b and C3bg (70, 73). The

binding site for iC3b, C3b, and C3bg are located on the α-‐chain (CD11b) and the binding is

Ca2+ dependent (73). Several studies have reported that cells expressing CR3 and CR4

have an enhanced HIV-‐1 replication. The CR3 and CR4 expressed by DCs are involved in

trans infection of HIV-‐1 (74). In addition, an increasing amount of evidence indicates that

CR3 and CR4 also play a role in antigen presentation and CD8+ T cell activation (75).

3.2 Complement opsonization of HIV-‐1 Several viruses including HIV-‐1, Vaccinia virus, Herpes simplex virus (HSV), and Epstein-‐

Barr virus have been shown to directly activate the complement system (76). HIV-‐1 is

able to activate all three pathways of the complement system already in the initial phase

of infection (76). The lectin pathway is activated by the binding of MBL to high mannose

carbohydrates on HIV-‐1 gp120 (77) and the classical pathway is activated by the binding

of viral gp41 to the A-‐chain of C1q (78). The activation occurs in the absence of

antibodies. However, after seroconversion the presence of HIV-‐1 specific antibodies

further enhances the activation of the classical complement pathway (79, 80). Of note,

due to mechanisms developed by HIV-‐1, virions resist complement mediated lysis and

the activation of the complement cascade result in deposition of inactivated C3 fragments

on the viral surface, i.e. opsonization (81, 82) (Fig. 5 and 6). HIV-‐1 acquires complement

lysis resistance factors during the budding from the host cell plasma membrane and

these receptors are incorporated in the viral envelope. These factors that inhibit the

complement cascade are the membrane cofactor protein (MCP: CD46), decay accelerating

factor (DAF: CD55), and CD59 (83). In addition, HIV-‐1 can bind soluble fH, which further

protects virions from destruction (64, 84). There are many other pathogens besides HIV-‐

1 that have developed different methods to escape the complement system (81, 82, 85).

However, HIV-‐1 is not only spared from lysis it also uses the deposition of complement

fragments on the surface to its own advantage (86).

The interaction of HIV-‐1 with cells is mediated by the viral receptor gp120 binding to

multiple receptors including CD4 and coreceptors (87). However when HIV-‐1 is covered

with C3 fragments the carbohydrates expressed on gp120 may be partly or completely

25


13

covered by complement fragments and thereby poorly accessible for receptor binding.

Experiments in macaques and in vitro T cell experiments have shown that opsonization of

virions by C3 fragments masks epitopes on the viral envelope leading to reduced

infection of T cells, which are CR3 negative (88-‐90). Moreover, virions also use the

complement fragments to increase their infectivity by interacting with cells expressing

CRs. The complement fragment iC3b is the major ligand for CR3, but this receptor also

binds to other ligands like ICAM-‐1, which is an adhesion molecule acquired by the virions

from the host cell plasma membrane during the process of budding. In addition the gp41

part of the HIV-‐1 envelope receptor can also interact with CR3 (91). Finally, complement

opsonized HIV-‐1 have been found throughout the body, e.g. in blood, breast milk, mucosa,

seminal fluid, and lymph nodes (64), and should be taken in consideration when

studying HIV-‐1.

Figure 5. Free and opsonized HIV-‐1.

HIV-‐1 immediately activates the complement cascade but is protected from complement mediated lysis

leading to deposition of C3 fragments on the surface of HIV-‐1 (C-‐HIV) (92). After seroconversion, HIV-‐1 can

be covered with HIV-‐1 specific antibodies (IgG-‐HIV) and HIV-‐1 specific antibodies in combination with

complement fragments (C-‐IgG-‐HIV) (93). Seroconversion enhances the activation of the classical pathway

and increases the amount of C3 cleavage products deposited on the surface of HIV-‐1 (64, 94).

26


14

Figure 6. Complement activation on the

viral surface of HIV-‐1.

HIV-‐1 can activate all three pathways of the

complement system, classical, mannose-‐

binding-‐lectin (MBL) and alternative

pathway. The initiation of the classical

pathway can occur in the absence of HIV-‐

specific antibodies but they enhance the

activation of the classical pathway after

seroconversion (95, 96). The classical

pathway is initiated by the binding of C1q to

gp41 (97). However, activation by the

mannose-‐binding-‐lectin (MBL) pathway is

triggered by the binding of MBL to

carbohydrate side chains expressed on

gp120 (98).

The alternative pathway is independent of

antibodies and starts by the hydrolyzation of

C3 to C3(H2O). All three pathways result in

the formation of C3 convertase, which

cleaves C3 into C3b and C3a. However, HIV-‐

1 escape compliment mediated lysis by MAC

(C5b6789), owing to factors acquired during

the budding from the host cell. These factors

are incorporated in the viral envelope and

include CD55, CD59, and CD46 (99). CD55

dissociates the C3 convertase and CD59

blocks the formation of the MAC complex by

the polymerization of C9. CD46 interacts

with factor I (fI), which cleaves C3b to

inactive C3b (iC3b) and subsequently to C3c

and C3d. Factor H (fH), incorporated in the

viral envelope, interacts with gp120 and

gp41 and this protects the virions from

complement mediated lysis (86, 100-‐102).

However, fH also plays role in the

inactivation of C3b by working as an

additional cofactor for fI (73, 103).

27


15

3.3 Outcomes after complement activation by HIV-‐1 A fraction of the HIV-‐1 particles trigger the terminal activation pathway and are lysed by

the MAC, but a substantial amount of the virions remains opsonized and mediates their

effects on the immune system by interacting with CRs and FcRs (60). The complement

opsonized virions affect the immune system in many ways (60). For instance, interaction

of complement opsonized HIV-‐1 with CR1 on erythrocytes might facilitate the spread of

opsonized HIV-‐1 to the liver and spleen where HIV-‐1 can be transferred to target cells

(104). CR2 is involved in trapping HIV-‐1 in the centers in the lymphoid organs by binding

complement and immune complex opsonized HIV-‐1 to FDCs. In fact, CR2 is the main HIV-‐

1 binding receptor on FDCs in vivo, no involvement of CR1 or CR4 (105). HIV-‐1 opsonized

with complement and/or immune complex binds to the surface of the FDCs and can stay

trapped there for months without infecting the FDCs (106). During this time the trapped

virions are highly infectious for CD4+ T cells even in the presence of neutralizing

antibodies (107).

Virions opsonized by complement fragments and immune complexes mark them for

uptake by phagocytosis and destruction. Phagocytes like DCs and macrophages

internalize the opsonized virus mainly via FcRs or CRs. The presence of iC3b on the viral

surface leads to the interaction with CR3 and CR4 and several studies have shown a

highly increased HIV-‐1 infection in cells expressing these CRs (73). For instance, DCs

infected with HIV opsonized with complement and anti HIV-‐IgG had a 10-‐fold increased

infection compared to cells infected with free virions (108). Of note, viral replication

increased in latently infected monocytes following stimulation of CR3 (109). A twofold

increase in HIV-‐1 infection was seen in an epithelial cells line when infected with seminal

fluid opsonized virions compared to free virions and this enhanced infection was due to

CR3 engagement (110). We have previously shown in our group that complement

opsonized virions are more efficiently internalized via receptor mediated endocytosis

than free viral particles (111).

28

Dendritic cells

16

4. DENDRITIC CELLS

For a long time immunology research focused on lymphocytes and antigens but a major

player essential for immunity was missing. However in 1972 a third party, the dendritic

cell (DC) was discovered by Ralph Steinman and Zanvil Cohn at the Rockefeller

University, USA (112).

Ralph Steinman passed away 2011 three days before he was announced as one of the

laureates for this years Nobel Prize in Medicine due to his life’s work on DCs (113). This

antigen presenting cell (APC) was found to be an initiator and modulator of the adaptive

immune response (114). The fist DC subtype was visualized already 1868 in the skin by

Paul Langerhans and named Langerhans cells (LCs) (115), but the characterization of

other DC subtypes did not start until the finding by Ralph Steinman 38 years ago. We

know today that DCs are specialized APCs, crucial for our protection against pathogens,

bridging both the innate and the adaptive immunity (116).

4.1 The role of dendritic cells in immunity DCs are specialized APCs capable of priming naïve T cells, which leads to induction

memory responses. These cells are essential in the defense against pathogens and

throughout the whole body they are forming a tight network with their long arms, i.e.

dendrites that sense pathogens. DCs are continuously produced from hematopoietic stem

cells and distributed from the bone marrow to all organs in the body, such as mucosa

tissues, skin, liver, lung, heart, and blood making them well positioned to quickly

encounter invading pathogens (117).

The DCs exist in three different differentiation stages including precursor, immature, and

mature. Precursor myeloid DCs (MDCs) migrate to the tissues attracted by the chemokine

and cytokine gradients and in the tissue the DCs remain in an immature state (118, 119).

Immature MDCs are highly endocytic but less potent immune stimulators than mature

MDCs (117). After antigen exposure the immature DCs become activated and undergo a

maturation process. This process guides the DCs during the migration to the lymph nodes

where large numbers of naïve T cells and resting memory T cells continuously circulate

through. The maturation of the DCs increases their immunostimulatory capacity by

upregulating of an array of receptors resulting in high surface expression of MHC class I

and II-‐ peptide complexes, costimulatory molecules CD40, CD86, and CD80 (120),

29

Dendritic cells

17

adhesion molecules CD44 (121), and α6β1integrin (121), and receptors for T cell

activation such as CD48 and CD58 (122) all which benefit their interaction with and

activation of T cells. At the same time the antigen capture activity is downregulated (120).

The maturation also results in a change in the chemokine receptor expression with a

decreased expression of, e.g. CC-‐chemokines CCR5 and CXCR4, and upregulation of CCR7

(119). In the lymph node paracortex area, the mature DCs present antigen peptides in the

context of MHC class I and II molecules to naïve T cells. T cells with a TCR specificity that

recognize the peptide-‐MHC complex presented by the DCs and are activated and an

immune response is triggered (114). However, only mature DCs can elicit an immune

response resulting in long lasting immunologic memory (116). Even in the absence of

inflammation and maturation signals, a small number of DCs migrate to the lymph nodes

carrying self antigens and induce and maintain tolerance rather than inducing immune

activation (118). In addition, priming of immune responses by the wrong DC subset can

also lead to suboptimal and even silencing of the immune response (122).

PDCs are the most potent responders to viral infection by production of type I IFNs,

which alerts and sets the body in an antiviral state. The MDC subsets can also produce

type I IFNs, but not at the same high levels as PDCs (123). In addition, MDC subsets and

PDCs differ in which TLRs they express, indicating that they sense different sets of danger

molecules and their response to a specific pathogen differs. These overlapping and

distinct functions of the DC subsets have in all probability evolved in order to achieve an

optimal sensing and immune defense against different pathogens.

4.2 Dendritic cell lineages and subsets 4.2.1 Plasmacytoid dendritic cells (PDCs)

There are two major lineages of DCs, MDCs and PDCs. PDCs are of lymphoid origin and

found mainly in blood, cerebrospinal fluid, and lymphoid tissues (118). This subtype of

DCs lack the classical myeloid markers and are called “linage negative” and express high

levels of CD123, and CD62L (119, 124), but they are also known to express receptors

including BDCA-‐4 (CD304), CD45RA, CD4, CCR5, TLR7, and TLR9. PDCs are the most

potent inducers of type I IFNs and represent a key effector cell in the innate response

against pathogens, especially viral infections (125-‐127). In addition, PDCs activate NK

cells and are important APCs that activate T cell responses (128).

30

Dendritic cells

18

4.2.2 Myeloid dendritic cells (MDCs)

MDCs are considered to be the “classical” population of DCs and consist of several

different subtypes, including blood MDCs, LCs and tissue interstitial MDCs (129). The

majority of DCs in the body are of myeloid origin and express the myeloid markers

CD11c, and CD33 and lack markers like CD14, CD19, CD20, and CD56 (130). Immature

MDCs have a high capacity to capture antigens via receptor mediated endocytosis,

macropinocytosis, and phagocytosis and mature MDCs have very potent T cell activating

capacity. The LCs are located in the epidermis, the mucosal epithelium of the male and

female genital tracts and rectum (131). They express surface markers such as HLA-‐DR,

CD11c, CD1a, CD4, CCR5, Langerin (CD207), and E-‐Cadherin, (132), with the C-‐type lectin

Langerin being a unique marker for LCs. LCs were initially identified by the presence of

Birbeck granules, “tennis-‐racket” shaped granules in the cytoplasm (133). It was recently

discovered that Langerin is crucial for the formation of the Birbeck granules and that this

organelle is a part of the endosomal recycling pathway (134). LCs, as all DCs, capture

antigens and migrate to the lymph nodes and mount specific immune responses (118).

The antigen capture and migration leads to maturation, which changes the receptor

expression with upregulation of MHC class I and II and costimulatory molecules and

downregulation of Langerin and E-‐Cadherin. LCs express only one C-‐type lectin, i.e.

Langerin, and are negative for other C-‐type lectins such as DC-‐SIGN, DCIR, DEC-‐205, and

MMR, commonly found on other subtypes of MDCs (135).

Blood MDCs constitute only a small percentage (~0.5%) of mononuclear cells in blood

and are characterized by the expression of HLA-‐DR, CD11c, CD11b, BDCA1 (CD301),

CD45RO, and DC-‐SIGN. They also express an array of TLRs including TLR 3, 4, 5, 6, and 8

(136). Blood MDCs are believed to migrate from the blood out to infected or inflamed

tissues (117). However not much is known about the differentiation and migration of

blood MDCs into tissue MDCs and some studies have shown that blood MDCs are destined

for direct migration to lymph nodes or thymus (117). Another subtype of MDCs is

interstitial MDCs and these cells are found in e.g. the submucosa, heart, liver, and kidneys.

However, unlike LCs, interstitial MDCs do not express Langerin and lack Birbeck granules

and have 10 times higher capacity for antigen uptake than LCs (137).

31

Dendritic cells

19

4.2.3 Monocyte derived dendritic cells (MDDCs)

In vitro model MDCs, monocyte derived DCs (MDDCs), are DCs propagated from

stimulation of blood monocyte precursors with IL-‐4 and GM-‐CSF for 5-‐7 days. This model

of interstitial tissue MDCs is widely used in experiments since blood MDCs, LCs and MDCs

derived from human skin or mucosa explants are difficult to isolate in high numbers. The

immature MDDCs express markers such as CD11b, CD11c, MHC class I and II molecules,

CD1a, DC-‐SIGN, MMR, and low levels of CD80 and CD86 (117, 138). These immature

MDDCs have similar characteristics to tissue MDCs, such as immature dermal MDCs and

intestinal MDCs (138). In response to different stimuli such as TLR3 ligand poly I:C or

TLR4 ligand LPS, immature MDDCs differentiate into mature cells (111). The use of

MDDCs have provided many important insights in the interaction of HIV-‐1 with DCs,

however it is important to remember that MDDCs do not fully resemble the MDC subsets

interacting with HIV-‐1 in vivo.

4.3 HIV-‐1 capture by dendritic cells DCs are important in the pathogenesis of HIV-‐1 but also in the generation of a specific

immune response against HIV-‐1 (139). After transmission through the mucosal

epithelium DCs are among the first potential targets for HIV-‐1 (140). The virus hijack the

DCs to be transported to the lymph nodes and HIV-‐1 utilize several receptors expressed

by DCs for binding and uptake. Except for the infection receptors CD4 and coreceptors

(CCR5 or CXCR4), HIV-‐1 can interact with CRs, FcRs, integrins, syndecan-‐3, and C-‐type

lectin receptors on the DCs (Fig. 7). However, C-‐type lectins including DC-‐SIGN, MMR,

DEC-‐205, DCIR, and Langerin are the main attachment receptors for HIV-‐1 on dermal and

mucosal DCs and this family of receptors facilitates endocytosis of glycolsylated antigens

(139). Uptake by C-‐type lectins may be a natural route for degradation of HIV-‐1, but viral

usage of different C-‐type lectins guides virions to different routes inside the DCs (119). It

has been shown for MMR that ligand binding to this receptor can route the ligand to a

recycling early endosome (141). However, this seems to depend on the type of ligand that

binds to MMR, for instance, MMR-‐bovine serum albumin enters the early recycling

endosome and avoids degradation by recycling back to the plasma membrane (142).

However, the binding of ligands rich in oligomannose residues like gp120 routes to

endosomal uptake and trafficking to late endosome (143). DC-‐SIGN is shown to be a

major receptor for HIV-‐1 gp120 and HIV-‐1 binding to DC-‐SIGN mediates internalization

32

Dendritic cells

20

into a nonlysosomal compartment (144). In addition, DC-‐SIGN facilitates HIV-‐1 transfer

from MDDCs to CD4+ T cells (145). Moreover, Langerin has a great capacity to bind large

amounts of HIV-‐1 gp120 and probably play an important role in the transmission of HIV-‐

1 through the epithelium in the genital mucosa (146). Free and complement opsonized

virions interact both with C-‐type lectins and integrins and free HIV-‐1 binds to β1-‐integrin

and complement opsonized HIV-‐1 to β7-‐integrin (111). Integrins are one of the major

families of cell adhesion receptors and they all contain one α-‐chain and one β-‐chain. It has

previously been shown that HIV-‐1 binds to α4β7 integrin expressed on CD4+ T cells. The

region of HIV-‐1 gp120 interacting with α4β7 has been mapped to the V2 loop (147, 148).

The integrins CR3 and CR4 are involved in the binding, uptake, and HIV-‐1 infection of

target cells (60, 111).

DCs express relatively small amounts of the infection receptors, CD4 and coreceptors

CCR5/CXCR4, but both R5 and X4 tropic HIV-‐1 can productively infect DCs. However, this

infection is 10-‐100 times lower compared to CD4+T cells (149-‐151). In in vitro cultures it

is clear that only a fraction of the DCs become productively infected and that immature

DCs are more susceptible to the HIV-‐1 infection than mature DCs (152). Some of the

reasons for the moderate infection in DCs may include the low expression of CD4 and

coreceptors, degradation of internalized virions, expression of restriction factors, and the

low metabolic activity in these cells.

A direct productive HIV-‐1 infection of the DCs is called cis infection. DC mediated HIV-‐1

trans infection is a distinct mechanism where virions are internalized into nonacidic

compartments and transported to the contact zone between the DC and a CD4+ T cell

without directly infecting the DC. At this contact zone, i.e. the virological synapse, whole

HIV-‐1 virions are transferred from the DC to the CD4+ T cell. DC mediated trans infection

of T cells is the most efficient way to establish viral replication in CD4+ T cells (153, 154).

DC-‐SIGN has been shown to enhance both the infection of DCs and the transfer of HIV-‐1

through the virological synapse (155-‐157).

33

Dendritic cells

21

Figure 7. Interaction of free and complement opsonized HIV-‐1 with immature and mature DCs.

HIV-‐1 interacts with many receptors expressed by the DCs. Except for the main receptors utilized by HIV-‐

1 for infection, CD4 and CCR5/CXCR4, HIV-‐1 interacts with several receptor families, e.g. complement

receptors (CR), Fc-‐receptors (FcRs), integrins, syndecan-‐3, and C-‐type lectins. The expression levels of

these receptors are dependent on the subtype and maturation status of the DC. In addition, HIV-‐1 utilizes

different sets of receptor depending on if the virions are free, opsonized with complement, immune

complexed, or covered in both complement and antibodies.

4.4 Intrinsic antiretroviral factors Approximately 8% of the human genome is comprised of endogenous retroviral

elements. Over the past years it has become apparent that the human genome

counteracts this by the development of antiviral proteins referred to as restriction

factors (158). DCs, but also monocytes and to a lesser extent macrophages, utilize

different mechanisms to restrict retroviral infection, such as the production of

restriction factors including APOBEC3G, TRIM5α, tetherin, and SAMHD-‐1 (159).

APOBEC3G was first identified as a cellular factor making the host able to resist HIV-‐1

infection when the virus was lacking the vif (viral infectivity factor) gene (160).

APOBEC3G targets the negative sense ssDNA generated during HIV-‐1 reverse

transcription and catalyzes the hydrolysis of cytosines (C) to uridines (U). The editing of

C to U leads to substitutions of guanine (G) to adenine (A) in the positive sense strand

leading to a noninfectious virion. However, the Vif protein counteracts APOBEC3G by

tagging it for ubiquitylation and proteasomal degradation and the intracellular levels of

APOBEC3G are reduced (161). For this reason APOBEC3G is not incorporated into

progeny virions. Of note, APOBEC3F also inhibits HIV-‐1 in a Vif dependent manner (161-‐

164).

34

Dendritic cells

22

TRIM5α interferes with the viral coat when HIV-‐1 enters the cytoplasm, causing

premature uncoating (159, 165). However, exactly how TRIM5α blocks HIV-‐1 infection

is not known. The restriction factor tetherin (BEST2 or CD317) inhibits the release of

enveloped viral particles from infected cells. Tetherin has been shown to restrict the

release of a wide range of viruses including filoviruses, arenaviruses, rhabdoviruses, and

all retroviruses tested so far. The HIV-‐1 protein Vpu binds to tetherin and counteracts its

antiviral function (166, 167). SAMHD-‐1 was recently identified as an antiretroviral

protein and for HIV-‐2 this is counteracted by the viral protein Vpx (50, 168).

35

Antigen presentation by dendritic cells

23

5. ANTIGEN PRESENTATION BY DENDRITIC CELLS

5.1 Overview DCs are specialized for antigen presentation and T cell activation and can capture and

present a broad variety of different classes of antigens in the body, e.g. soluble and

particulate antigens, pathogens, and dying cells. Most exogenously acquired antigens are

processed in acidic endosomal/lysosomal vesicles and presented on MHC class II

molecules and activate CD4+ T cells (169). DCs that have been exposed to viruses and

bacteria can have antigens derived from the pathogens in their cytosol, either due to

that they are productively infected or that pathogenic antigens have been delivered to

this site. These cytosolic antigens are degraded and almost exclusively loaded on MHC

class I molecules in the ER by a process called direct or classical endogenous MHC class I

presentation. Furthermore, exogenous antigens can also be presented on MHC class I

molecules and this process is called cross presentation or cross priming and was

discovered in 1976 by Michael J Bevan (170) in mouse APCs and 1998 by Nina Bhardwaj

in human DCs (171). Several antigens have been reported to be cross presented and

include proteins, immune complexes, intracellular bacteria, parasites, and infected dying

cells (172, 173). The antigens are in part proteolysed in the endosomal compartment

and presented either by direct loading on MHC class I molecules in recycling endosomes

or by active translocation out to the cytosol (174-‐178). In the cytosol the

proteins/antigens are further processed by the proteasome and either guided back into

the endosomal compartment for loading on MHC class I molecules or transported into

ER via transporter associated with antigen processing (TAP) and loaded onto MHC class

I molecules. When the peptides are loaded on MHC class I molecules, these complexes

are transported to the cell surface where they can be presented to CD8+ T cells (174-‐

178). In the case for HIV-‐1, DCs can efficiently cross present antigens from HIV-‐1

infected dying cells leading to efficient CD8+ T cell activation (179). A third way of

presenting antigens in the context of MHC class I molecules is called cross dressing.

Cross-‐dressing is a phenomenon where a peptide-‐MHC class I complex is transferred

from the cell surface of an infected APC to an uninfected APC that can present this

complex (180). Cross dressing occurs in viral infections and activates the memory CD8+

T cell population (180).

36


24

The DC is the only APC with the ability prime naïve T cells. An activated naïve T cell

rapidly undergoes up to 15 cell divisions and these cells develop into effector T cells that

migrate to and enter specific tissues where they can destroy infected cells and clear the

infection (181). In addition, some of the primed naïve T cells develop into central

memory T cells that persist long term and can be reactivated when the body encounters

the antigen a second time (181).

5.2 MHC class I restricted antigen presentation Endogenous HIV-‐1 antigen presentation on MHC class I molecules require fusion of the

viral gp120 with the infection receptors CD4 and coreceptors (CCR5/CXCR4), i.e. viral

access to the host cell cytosol. The viral proteins are processed in the cytosol by the

proteasome and guided into the ER for MHC class I molecule loading by TAP1/2 (182,

183). In early 1970, ubiquitin was recognized as a molecules that covalently marks

intracellular proteins for transport and degradation by the 26S proteasome, a multi

enzymatic complex, and is therefore known as the ubiquitin-‐proteasome system (UBS)

(184) (Fig. 8). The UBS plays a major role in the MHC class I restricted processing of

cytosolic proteins and ER targeted proteins, including mutant, damaged, partially

unfolded, or miss folded proteins (185). Some of the viral proteins released in the

cytosol are ubiquitinated and ubiquitins are progressively added and form a chain, a

process catalyzed by three enzymes E1, E2, and E3. The ubiquitin is activated by E1,

conveyed to E2 ubiquitin carrier proteins, and subsequently transferred by E3 ubiquitin

ligase to a substrate protein. This ubiquitinated protein is transported, recognized, and

degraded by the 26S proteasome (186). Over time, several proteins similar to ubiquitin

have been identified, i.e. ubiquitin like proteins (UBLs). UBLs are divided into two

groups, ubiquitin domain proteins (UDP) and ubiquitin-‐like modifiers (ULM) (187-‐189),

some involved in escorting polyubiquitinated proteins to the 26S proteasome and others

function in an ubiquitin like manner (190). The binding of UBLs can affect different

biological events, such the enzymatic activity, half life of a protein, facilitate or inhibit

the binding of the protein to another molecule (191). ISG15 was the first identified UBL

and it tags proteins and interacts with the proteasome system (191, 192). For instance,

inhibition of the proteasome increases the levels of proteins conjugated with ISG15 and

overexpression of ISG15 decreases polyubiquitinilation of proteins, indicating that

ISG15 antagonizes the activity of the ubiquitin-‐proteasome system (193-‐195). HIV-‐1

37


25

replication, assembly, and release of virions are inhibited by ISG15 (196, 197). NEDD8 is

another UBL and conjugation of this UBL to proteins can lead to their degradation via

the proteasome. This is mediated by the adaptor protein NEDD8 ultimate buster-‐1

(NUB1) (198, 199). A portion of the viral proteins entering a cell is ubiquitinated and

tagged for proteasomal degradation, whereas misfolded HIV-‐1 proteins are degraded in

the 20S proteasome in an ubiquitin independent manner (200).

However, many viruses modulate the ubiquitin-‐proteasome pathway thereby

modulating cellular signaling and antiviral responses (201, 202). HIV-‐1 Vif modulates

this pathway by ubiquitination of APOBEC3G, which helps viral fitness. Vif itself is

monoubiquitinated, which helps to recruit this protein to the site of viral assembly.

Ubiquitination of Gag by an Ubiquitin ligase is also an important step for the assembly of

HIV-‐1 proteins (20, 203-‐206).

The proteasome is involved in many degenerative and biological processes in the cytosol

including removing ubiquitinated and misfolded proteins (200). Several kinds of

proteasomes exist in the cell, 20S, 26S proteasome, and immunoproteasome. The 20S

proteasome contains multiple peptidase activities and is shaped as a barrel, composed

of four rings, each with seven β1, β2, and β5 subunits (207). The 26S proteasome

recognizes polyubiquitinated proteins (208) and consists of a 20S subunit and a 19S

regulatory complex, which degrades the ubiquitinated proteins (200). 20S subunit also

associates with the 11S regulatory complex and this opens a channel through the

complex and this proteasome complex degrades non-‐ubiquitinated short peptides. In

the presence of IFN-‐γ the homologous subunits β1i (LMP2), β2i (MECL1), and β5i

(LMP7) are incorporated in the 26S proteasome and 19S subunit is replaced with the

11S subunit, giving rise to the immunoproteasome (200, 209, 210). The

immunoproteasome has an increased proteolytic activity favoring the production of

peptides for MHC class I presentation (209). Immature MDDCs express equal amounts of

the 20S proteasomes and the immunoproteasomes, while mature MDDCs contain only

immunoproteasomes (211). The proteasomal cleavage generates the N-‐terminus of the

peptides presented by MHC class I molecules, whereas the amino terminus of the

proteins and peptides can be further edited by cytosolic aminopeptidases (210). For

instance, the cytosolic aminopeptidase tripeptidyl peptidase II is essential for the

generation of the immunodominant HIV-‐1 Nef MHC class I epitope in DCs (212).

38


26

Peptides generated in the cytosol by proteasome/immunoproteasome and

aminopeptidase proteolysis are transported to TAP, which translocates the peptides

into the lumen of ER (213). In the ER, the peptides can be further trimmed by ER

aminopeptidases (ERAP) to produce 8-‐10 amino acid long peptides that fit into the MHC

class I molecule peptide binding grove (213).

Newly synthesized MHC class I proteins, i.e. MHC class I heavy chain and β2

microglobulin (β2m), are transported to assemble in the ER lumen and remain there

until they bind peptides. This process is strictly controlled to ensure the highest possible

efficiency of the antigen presentation (214). For instance, the folding and assembly of

the MHC class I heavy chain is controlled by the molecular chaperones including BiP,

calnexin, calreticulin, and ERp57. The chaperones also play an important role in

stabilizing the empty MHC class I molecule. The MHC class I peptide loading complex is

composed of MHC class I molecule, TAP1/2, calreticulin, tapasin, and ERp57 (215). The

association of MHC class I molecule with the TAP-‐tapasin-‐calreticulin complexes leads to

the release of calnexin, but the MHC class I molecule is released first when binding of a

peptide with fitness for its peptide grove has occurred. The fully assembled peptide

loaded MHC class I molecule is transported from the ER, through the Golgi apparatus,

and via vesicles out to the cell surface (Fig. 9).

39


27

Figure 8. Transportation and degradation of ubiquitinated proteins by the 26S proteasome.

When a substrate protein becomes polyubiquitinated by a chain of at least four ubiquitins (Ub), it can bind

to an adaptor protein, containing binding domains for both the polyubiquitin and the proteasome. In

addition, the polyubiquitinated protein can also bind directly to intrinsic Ub binding sites in the 19S

regulatory complex of the 26S proteasome. Why some substrates must be escorted to the proteasome by

an adaptor protein and others can associate directly with polyubiquitin-‐binding subunits in the

proteasome is not fully understood. Binding of the substrate protein to the proteasome is followed by

protein unfolding by the half-‐dozen ATPases encircling the pore of the proteasome catalytic core, removal

of the polyubiquitin chain by proteasome-‐associated deubiquitylation enzymes (DUBs), and translocation

of the unfolded protein into the central proteolytic chamber, where it is cleaved into short peptides (216).

5.3 MHC class II restricted antigen presentation Before HIV-‐1 derived antigens can be presented by the DCs on MHC class II molecules

they are processed in the endosomal/lysosomal compartment (217). Virions are

internalized by DCs via macropinocytosis, receptor mediated endocytosis, and/or

40


28

clathrin mediated endocytosis (218, 219). The endocytic compartments become

gradually more and more acid and in the lysosomes the pH is 4.5-‐5.0. Protein antigens

are processed in endosomal compartments by endosomal proteases, e.g. Cathepsin B, C,

D, and E, and hydrolases and this degradation in the acidic endosomal compartments is

necessary for MHC class II presentation (220). The degradation of antigens in the

endosomal compartments includes reduction of disulfide bounds by IFN-‐γ inducible

thiol reductase (GILT), and proteolysis by an array of proteases, e.g. Cathepsin B and D,

and aminopeptidases (221). These enzymes function best in an acidic environment and

an optimal rate of peptide production is achieved at pH 4.5-‐5.0. The pH optimum does

not only depend on the peptide, but also on properties of the whole peptide-‐MHC class II

complex (221).

The MHC class II molecule is synthesized in the ER and the invariant chain (Ii) prevents

peptides from binding to its peptide binding grove. When assembled, the MHC class II

molecules are transported through the Golgi and directed in to the endocytic route by

the help of the Ii. Before the MHC class II molecule can bind a peptide, the Ii must be

removed and this is achieved by a stepwise proteolytic degradation of Ii by the

endosomal protease Cathepsin S, which is found in a very high concentration in the late

endosomes, e.g. MHC class II compartments (MIIC) (222). The proteolysis of Ii leaves

only the one part of the Ii attached to the binding grove, i.e. CLIP. Antigenic peptides

compete with CLIP for the binding site on MHC class II molecules and peptide binding

can only occur when CLIP is removed. HLA-‐DM retains the MHC class II molecule in the

MIIC and assists in the process of removing CLIP from the peptide binding groove. First

when a peptide has bound to the groove of MHC class II molecule can it migrate up to

the cell surface and present the antigenic peptide to the CD4+ T cells (221) (Fig. 9).

41


29

Figure 9. MHC class I and II antigen processing and presentaton pathways by HIV-‐1.

HIV-‐1 can be internalized by endocytosis and processed in the MHC class II restricted pathway. The

internalized antigens traffic via early endosomes to late endosomes. This pathway is dependent on a

gradual acidification for proteolytoc activity and endosomal hydrolases for the removal of the invariant

chain and loading on antigen peptides on MHC class II molecules. The peptide-‐MHC class II complex is

transported to the surface and the antigen peptide is presented to CD4+ T cells (223). HIV-‐1 can enter the

endogenous processing pathway by fusion following interaction of HIV-‐1 gp120 with CD4 and coreceptor.

The viral proteins are processed in the cytosol by the proteasome and guided into the ER for MHC class I

molecule loading by TAP (182, 183). In the ER, the MHC class I molecules are assembled with the help

molecular chaperones that stabilize them when they are assembled. The peptides transported into the ER

are loaded on the stabilized MHC class I molecules and the complexes transported to the cell surface for

presentation to CD8+ T cells (221).

endosome

ER

MHC I

golgi

lateendosome

tapasin

ERp57 calreticulin

TAP 1/2

proteasome

HIVcell membrane

cytososol

cytosol

cell membrane

cathepsinMHC II

TCR

CD8 T cell

MHC I+HIV peptide

TCR

CD4 T cell

MHC II+HIV peptide

42

Mucosal immunity and HIV

30

6. MUCOSAL IMMUNITY AND HIV

The innate immunity of the mucosa is the first line of defense against invading

pathogens. The mucosa in the female genital tract has a unique architecture to provide

protection against pathogenic microbes. However, the epithelial layer does not only

serve as a passive barrier with epithelial integrity and low pH, the mucosa innate

immunity also has active defense mechanisms and secretes mucus, defensins, whey

acidic proteins, type 1 IFNs, secretory leukocyte protease inhibitors (SLPIs), and

complement proteins (224). Defensins are produced by the epithelial cells and inhibit

HIV-‐1 replication in vivo (225) and whey acidic proteins have anti-‐microbial activities.

SLPIs can reduce viral transmission through the mucosal epithelium and they are

produced by epithelial cells upon sensing pathogens through TLRs (226). The

microenvironment in the female genital tract is influenced by female reproductive

hormones and these hormones have diverse effects on the HIV-‐1 infection. For instance,

progesterone increases the susceptibility to HIV-‐1 infection, whereas estradiol has a

preventive effect (227-‐230).

6.1 Transfer of HIV-‐1 through the female genital mucosa

The typical mode of HIV-‐1 transmission is by heterosexual intercourse and the mucosa

is the first site of interaction between the virus and the host. Mucosal tissues are

characterized as either type I or type II. Type I mucosa consists of a single layer

epithelium covering the intestine, lungs, endocervix, and uterus. Physiologically type I

mucosa serves as an area of respiration, absorption, and exchange. Type II mucosa

consists of a stratified epithelium, covering vagina and ectocervix, and this is a

protective barrier. In general, DCs residing in type I mucosa have a regulatory function

and maintain the balance between tolerance and inflammation while DCs in type II

mucosa protect the host against pathogens through the induction of inflammatory

responses (231, 232). The most common site of HIV-‐1 infection the female genital tract is

unknown (7). However, HIV-‐1 can cross both the stratified epithelium of vagina and

ectocervix and the single layer epithelium of endocervix and the transformation zone.

Penetration through the mucosal epithelium occurs fast, within 30-‐60 minutes after

exposure the virus have reached the submucosa (233). The stratified epithelium

provides the better mechanical protection than the single layer epithelium. However,

43


31

the greater surface area of vagina and ectocervix provides a more potential target (7).

The transformation zone may be especially susceptible to HIV-‐1 infection since this area

is enriched with CD4+ target cells (7). HIV-‐1 can penetrate the mucosal epithelium in

several different modes; it can penetrate down between the epithelial cells and this may

give HIV-‐1 an opportunity to come in contact with and infect LCs and CD4+ T cells (234).

However, a recent study concluded that productive HIV-‐1 infection of vaginal LCs is

absent or minimal but that they transmit infectious virions to CD4+ T cells (235). It is

also likely that virus can enter the submucosa by a mechanism called transcytosis.

Transcytosis is a nondegenerative process and the virus is transported in vesicles from

the apical side of apolarized epithelial cell in the ectocervical columnar epithelium to the

baselateral side without infecting the cell itself (236, 237). In addition, if there is a break

in the epithelium, virions get direct access to the submucosa and the target cells located

at this site. LCs in the epithelium can pick up HIV-‐1 and migrate to the lymph nodes, and

have therefore been proposed to be the first target cells for HIV-‐1 (145) (Fig. 10). Of

note, genital mucosa CD4+ T cells, macrophages, and DCs have also been shown to be

targeted by HIV-‐1 upon the first encounter (238). Our study showed that HIV-‐1

opsonized with complement fragments gave rise to an increased infection in the DCs and

infection decreased infection in the CD4 + T cells (239).

In the submucosa and lymph nodes, the DCs transfer the virus to CD4+ T cells in a trans-‐

infectious mode (240, 241) and this DC-‐T cell spread highly amplifies the CD4+ T cell

infection. The productive HIV-‐1 infection following mucosal transmission is usually the

expansion from a single founder virus (242, 243). 3-‐4 days postinfection, small founder

populations can be detected in the transformation zone or the endocervix (244). The

small founder population must be sustained at a basic reproductive rate to give rise to

systemic HIV-‐1 infection. The founder populations consist of CD4+ T cells and around

90% of these cells are resting CD4+ T cells. The virions in the founder population have a

small diversity until the host develops an adaptive immune response, which drives the

virus to great diversity (6). We showed that blocking the integrins α4 and β7

significantly reduced the establishment of founder populations in cervical tissue (239).

DCs exposed to HIV-‐1 at the portal of entry migrate to the lymph nodes, within 18-‐24h

after exposure the DCs reach the lymphoid tissue, which is before the local infection has

expanded to the lymph nodes (233). In the lymph nodes the DCs activate the CD4+ and

CD8+ T cells and mount a specific immune response directed against HIV-‐1 (Fig. 10).

44


32

However, the adaptive immune response develops late too prevent the systemic spread

of HIV-‐1 and the massive destruction of CD4+T cells in the mucosa and can in most

individuals only partially control the infection.

Figure 10. Interaction of HIV-‐1 in the female genital tract-‐ entry, infection and delivery to lymph

nodes. Endocervix is composed of a single layer columnar epithelium while ectocervix and vagina include

a stratified epithelium. HIV-‐1 can penetrate the epithelium in several different modes and CCR5 tropic

HIV-‐1 is preferentially transmitted through the epithelium and has an advantage in establishing an

infection in the female genital tract. After crossing the cervicovaginal barrier, macrophages, DCs, and T

cells become infected. DCs transfer HIV-‐1 to T cells in DC-‐T cell conjugates and the virus is then spread to

the lymph nodes. DCs pick up HIV-‐1 and migrate to the lymph nodes where they present HIV-‐1 antigens

on MHC class I and II molecules. This activates both CD4 and CD8+ T cells, mounting a HIV-‐1 specific

immune response (6, 245, 246)

45

Aims of thesis

33

7. AIMS OF THIS THESIS

The majority of HIV-‐1 infections are acquired by mucosal exposure and the DCs are one of

the first cells targeted by HIV-‐1. The general aims of this thesis were to study the infection

of DCs and T cells in the cervical mucosa and to investigate how free and opsonized HIV-‐1

interact with human MDDCs. Little is known about the cellular mechanisms leading to

antigen presentation of HIV-‐1 and wether opsonized HIV-‐1 has a different fate inside the

DCs compared to free HIV-‐1.

The specific objectives were:

Paper I: To study the different steps leading to MHC class I and II restricted antigen

presentation by immature and mature DCs of HIV-‐1 antigens derived from whole

noninfectious and infectious HIV-‐1 particles.

Paper II: To study the binding and uptake of free and opsonized HIV-‐1 by immature and

mature MDDCs and establish the receptors involved in binding and internalization of

virions.

Paper III: To assess how opsonization of HIV-‐1 affects the processing pathways,

degradation, and storage of HIV-‐1 by immature and mature DCs and how this affects MHC

class I and II antigen presentation.

Paper IV: To study the effects opsonization of HIV-‐1 has on infection of cervical mucosa

using a human cervical tissue explant model and identification of cellular receptors

involved in the HIV-‐1 infection of immune cells and establishment of founder populations.

46

Methods

34

8. METHODS

8.1 Propagation of monocyte derived DCs Peripheral blood mononuclear cells (PBMCs) were isolated from Buffy coats or

leukapheresis after Ficoll-‐Hypaque density gradient centrifugation. DC progenitors, i.e.

CD14+ monocytes, were differentiated into immature DCs by the growth factor GM-‐CSF

and cytokine IL-‐4. Maturation of immature DCs was induced by exposing these cells to Toll-‐

like receptor ligands such as dsRNA (poly-‐I:C) or LPS. The immunophenotype of immature

and mature DCs was assessed by analyzing the surface expression of CD14 and CD83 using

flow cytometry.

Figure 11. Propagation of human monocyte derived dendritic cells (MDDCs) from PBMCs.

To differentiate the CD14+ precursor cell into immature DCs, the cells were stimulated with

GM-‐CSF and IL-‐4. To induce maturation were the DCs exposed to dsRNA (poly I:C).

8.2 Virus propagation and opsonization HIV-‐1BaL/SUPT1-‐CCR5 CL.30 was produced using chronically-‐infected cultures of

ACVP/BCP cell line. Virus was purified by continuous flow centrifugation and sucrose

density-‐gradient fractions were collected and virus containing fractions pooled and virus

pelleted. The virus pellet was resuspended and aliquots frozen in liquid N2 vapor. All

virus preparations were assayed for infectivity. Non-‐infectious HIV-‐1BaL and HIV-‐1MN

virions were prepared by chemical inactivation with 2,2’-‐dithiodipyridine (Aldrithiol-‐2,

AT-‐2); AT-‐2 inactivation eliminates infectivity by covalent modification of internal virion

proteins but preserves conformationally and functionally intact envelope glycoproteins

on the virion surface (247). GFP HIV-‐1BaL was propagated by transfecting a 239 T cell line

with HIV-‐1BaL and GFP-‐VPR plasmids. The virus were harvested after

47

Methods

35

36h and concentrated by ultracentrifugarion before determing the p24 concentration by

ELISA.

Opsonization of HIV-‐1 was performed using human serum (HS) or seminal plasma (SP)

from healthy volunteers. Different constellations of HIV-‐1; free HIV-‐1 (F-‐HIV),

complement opsonized HIV-‐1 (C-‐HIV), HIV-‐specific and unspecific IgG opsonized (IgG-‐

HIV), or a combination of both complement and IgG (C-‐IgG-‐HIV) was used to mimic the in

vivo situation. C-‐HIV was obtained by incubation of HIV-‐1 with HS or SP in Veronal buffer

(248). For C-‐IgG-‐HIV, HIV-‐specific and unspecific IgG were added besides the HS in

Veronal buffer, whereas IgG-‐HIV was obtained by adding the HIV-‐specific and unspecific

IgG. F-‐HIV was treated with media alone. As a control for the complement opsonization

was heat inactivated HS was used to opsonize HIV-‐1 as heat inactivation abolishes the

complement activation.

8.3 ELISPOT assays Immature dendritic cells (IDCs) or mature dendritic cells (MDCs) were exposed to different

binding and uptake inhibitors for 30 min at 37°C. IDCs and MDCs, were exposed to free

HIV-‐1BaL (F-‐HIV), complement opsonized HIV-‐1, antibody opsonized HIV-‐1 (IgG-‐HIV) or

HIV-‐1 opsonized with a combination of complement and antibody. The samples were

incubated over night at 37°C and unbound virus were removed before coculturing MDCs

and IDCs with HIV-‐specific CD4+ (HLA-‐A*DRβ04+) or CD8+ T cell (HLA-‐A*0201+) clones in

precoated 96 well IFN-‐γ ELISPOT plates or in 96-‐well plates. After the overnight coculture

the ELISPOT plates were washed and developed and the detection of IFN-‐γ spots was

performed as described previously (249).

8.4 Quantification using Real time PCR Real-‐time PCR is a method to analyze gene expression and has an advantage compared to

more traditional PCR with a greater sensitivity, reduced time per analysis. Compared to

traditional PCR which only measures the final phase of the reaction, Real time PCR

measures the kinetics of the reaction and therefore allows the detection of amplification

of DNA in real time. The method involves several steps including isolation of RNA and

digestion of genomic DNA. Using the RNA Easy Mini kit, RNA was prepared Quantitative

PCR was performed with Fast SYBER Green Master Mix on 7900 Fast Real-‐Time PCR

system with 7900 system SDS 2.3 Software. SYBER Green binds dsDNA with high

48

Methods

36

specificity and the fluorescence emission of the DNA-‐dye complex will be measured, and

the intensity of the fluorescence emission is in direct proportion to the amount of DNA

being produced. In our experiments the final results were analyzed using the ΔΔCt

equation.

Figure 12. Real time PCR technology.

In contrary to conventional PCR, real time PCR includes detection steps combined with the

amplification. First the primer and the polymerase bind to the target nucleic acid (cDNA) to

make a complementary strand. To visualize the target nucleic acid specific oligonucleotide

probe, linked to a fluorophore dye(SYBR green), hybridizes with the amplified dsDNA and

emits light when exposed to blue light (488 nm).In every cycle the optical module of the

real-‐time PCR system measures the fluorescence signal, and the associated software plots a

graph of the fluorescence intensity versus the number of cycles.

8.5 Preparation of cervical tissue samples Cervical tissue was received from women undergoing partial or full hysterectomy at the

Gynecology Clinic in Linköping, Sweden and women with conditions not involving the

cervix were chosen to be involved in this study. The tissues were kept on ice and

processed in the lab within 30 minutes after resection and the epithelial layer and lamina

propria were separated from the underlying stroma using a surgical scissor.

Cervical tissues with a size of 3mm2 or 8mm2 were placed in a cell culturing plate and pre

incubated for 30 min at 37°C with mock or different inhibitors.

Tissue samples (8mm2) were challenged with HIV-‐1or incubated with GFP-‐HIV-‐1

(3mm2), spinoculated, cultured and washed. The tissue were then transferred to 6-‐well

plates and cultured at 37ºC. After 3-‐6 days the emigrating cervical cells were collected

and stained for acquisition by flow cytometry.

49

Methods

37

8.6 Flow Cytometry On day 3, 5, or 6 cells migrating from the cervical tissue were harvested and subsequently

stained using the following anti-‐human antibodies; CD4-‐APC (BD Biosciences), CD3-‐

PerCP (BD Biosciences), and CD1a-‐PE (BD Biosciences). The cells were incubated for 30

min at 4°C and than fixed in 4% PFA and permeabilized in 0.2% Saponin. For detection of

HIV-‐1 the cells were incubated with the anti-‐HIV-‐1 mAb (KC57, clone FH190-‐1-‐1) and the

corresponding isotype control (BD PharMingen, San Diego, CA) for 45 min at room

temperature. The stained emigrating cells were assessed by four color flow cytometry

using FACS Calibur. The acquired data was analyzed using FLowJo (Treestar, Ashland, OR,

USA).

Figure 13. The samples were analyzed using fluorescence activated cell sorting (FACS).

Cells migrating from cervical tissue were labeled with different fluorochromes and directed into a

hydrodynamically focused single stream. As the cells pass through the laser the fluorochromes will emit light.

The emitted light is then transferred to detectors that convert the emitted light into signals that can be

processed by computers.

50

Methods

38

8.7 Immunofluorescence and confocal microscopy Uninfected cervical tissues or tissues frozen down 2h or 5 days post-‐infection were cryo

sectioned. Tissue sections or slides from cytospin of a single cell suspension using IDCs

and MDCs were fixed in 4% PFA, quenched, and stained with primary antibodies and

incubated over night at 4°C, washed 3 times and stained with a secondary mAb

(Rhodamine Red-‐X conjugated) for 1h at room temperature (RT). The sections were then

washed and mounted using mounting medium for fluorescence containing DAPI (Vector

Laboratories, Burlingame, CA). The samples were analyzed by a LSM 510 META confocal

microscope (Carl Zeiss AB, Stockholm, Sweden) using the LSM 510 software.

8.8 Immunohistochemistry (IHC) Infected and uninfected cervical tissue (4% PFA fixed), cultured for 5-‐6 days were

embedded in paraffin and cut in 5μm sections. The sections were rehydrated and

endogenous peroxidase was eliminated by incubating in H2O2 for 10 min. To prevent

nonspecific binding the sections were quenched in 1% bovine serum albumin for 10 min.

The sections were immunostained with anti-‐HIV-‐1 mAb overnight, washed and incubated

with a biotinylated secondary rabbit-‐anti-‐mouse Ab (DAKO), followed by streptavidin-‐

biotin-‐peroxidase complex (HRP). HRP was detected by development in TRIS-‐buffer

containing diaminobenzidine tetrahydrochloride (DAB) and 10μl 30% H2O2. The sections

were counterstained with methyl green solution containing 1% Methyl-‐green.

51

Methods

39

Figure 14. General method of Immunohistochemisty.

A primary antibody recognizes an antigen and a secondary biotinylated antibody binds the

primary antibody. Subsequently a complex consisting of avidin, biotin and HRP binds to the

biotin attached to the secondary antibody. The peroxidase is developed by diaminobenzidine

(DAB) and a brown colorimetric end product is developed.

8.9 Statistical analysis The statistical analysis was performed using GraphPad Prism 5 (GraphPad Software, La

Jolla, CA). Results were tested for statistical significance using a two-‐sided paired t-‐test or

non-‐parametric Mann-‐Whitney test. A p-‐value of less than 0.05 was considered

statistically significant. N denotes the number of times each experiment was replicated, in

all figures.

52

Results and discussion

40

9. RESULTS AND DISCUSSION

Most HIV-‐1 infections are transmitted through the mucosal epithelium and activate the

complement system immediately. Therefore, both free and opsonized virions are present in

the initiation and throughout the infection. The altered surface of the virus changes how

the virus interacts with immune cells. For this reason is it important to take both free and

opsonized HIV-‐1 particles into consideration when studying this chronic disease. One of the

first cells that comes in contact with HIV-‐1 is the DC but the virus hijacks this cell for

transportation to the lymph nodes (250). In the lymphoid organs, the virions are

transferred from the DCs to the CD4+ T cells, the foremost target cell for HIV-‐1 infection

(251). However, at the same time in the lymph the DCs present antigens derived from the

captured HIV-‐1 and activate HIV-‐1 specific CD8+ and CD4+ T cells. We have studied the

initial infection of mucosa and the events leading to DCs uptake, processing, and activation

of HIV-‐1 specific T cells. The increased knowledge of how HIV-‐1 interacts with the DCs,

mucosal immunity, and initiation of HIV-‐1 infection is essential and should be the most

promising approach to take when considering how to design HIV-‐1 vaccines.

9.1 Paper I 9.1.1 Background

The primary function of the immune system is to protect the individual against pathogens,

such as viruses (252). The DCs are the most efficient APCs, linking the innate and the

adaptive immune system. In HIV-‐1 pathogenesis, DCs play a central role throughout the

infection (152). DCs pick up antigens, process, and present them on MHC class I and MHC

class II molecules. In general, peptides originating from the cytosolic or nuclear proteins

are processed by the proteasome, a multi catalytic protein complex. This is followed by a

transport of peptides via TAP1/2 into ER, loading onto MHC class I molecules, and

presentation at the cell surface and activation of CD8+ T cells. Activation of the CD4+ T cells

is dependent on endocytosis of exogenous antigens, degradation in the acidified endocytic

compartments, and peptide loading onto MHC class II molecules. DCs express many

different receptors on the surface and the array of receptors utilized by the antigen for

binding and uptake can determine processing route and the fate of the antigen. In this

paper we have studied the pathways utilized in immature or mature MDDCs for MHC class I

and II presentation of antigens derived from whole virions.

53


41

9.1.2 Principal findings

We examined whether C-‐type lectins were involved in the uptake of virions leading to

processing and antigen presentation in the context of MHC class I and MHC class II

molecules by DCs. When the use of C-‐type lectins by HIV-‐1 was inhibited, the level of MHC

class I presentation of HIV-‐1 derived antigens by immature and mature DCs decreased

leading to a reduced CD8+ T cell activation. Furthermore, blocking the use of MMR, DC-‐

SIGN, or DEC-‐205 with antibodies did not affect the level of MHC class I and II antigen

presentation. Active receptor mediated endocytosis and viral proteolysis in late endosomal

compartments was required for optimal MHC class II presentation of HIV-‐1 derived

antigens by both immature and mature DCs. Viral gp120 CD4 binding and subsequent

fusion was required for MHC class I presentation of HIV-‐1 derived antigens by DCs. The

role of endosomal acidification was examined and we found significantly increased MHC

class I presentation while MHC class II presentation was significantly decreased. This

indicated that MHC class II presentation was dependent on endosomal acidification. In the

case for MHC class I presentation, the elevated levels were due to the prolonged ability of

virions localized in the endosomes to bind and fuse with CD4/coreceptor located inside

these compartments. After viral fusion the virions were subsequently degraded by the

proteasome, transported via TAP1/2 into ER, and HIV-‐1 derived peptides were loaded on

newly synthesized MHC class I molecules.

9.1.3 Discussion/Conclusion

This study clearly showed that MHC class I presentation of HIV-‐1 derived antigens followed

the classical pathway. In addition, the viral gp120 binding to CD4/coreceptor and fusion

followed by proteasomal degradation and transport via TAP1/2 was essential for MHC

class I presentation of the HIV-‐1 derived gag SL9 epitope to CD8+T cells. Of note, the level

of MHC class I presentation was highly elevated when acidification of endosomal

compartments was inhibited. This clearly indicated that viral fusion and access to cytosol

can occur in the endosomal compartment when the virions are rescued from degradation

by acid dependent proteases. A large fraction of the CD4 and coreceptors are located in the

endosomal compartments and not at the plasma membrane of the DC, providing a source of

binding and fusion receptors in these compartments. It has been proven that the HIV-‐1

infection is initiated in the endosomal compartments and not at the cell surface (253). C-‐

type lectin receptors were involved in the uptake leading to MHC

54


42

class I presentation but is far from the only receptor family used by HIV-‐1. Most likely there

are multiple receptors involved in the uptake and trafficking of viral particles leading to

MHC class I and II antigen presentation. In addition, how the virus has been propagated

and the HIV-‐1 strain might affect the viral interaction with host cells including the specific

array of receptor used for the initial cell binding. The MHC class II restricted antigen

presentation of HIV-‐1 derived antigens by DCs was strongly dependent on endosomal

acidification and transport from early to late endosomal compartments where the MHC

class II peptide loading occurs. This process is independent on viral binding and fusion via

CD4 and coreceptor (176, 183, 218).

9.2 Paper II 9.2.1 Background

The DCs play a central role in the establishment of and throughout the HIV-‐1 infection. The

initial interaction and uptake of HIV-‐1 involves the viral binding to an array of cell surface

receptors. DCs express many different surface receptors, including C-‐type lectins, integrins,

FcRs, CD4, and chemokine receptors. Receptors differ in their ability and affinity to interact

with and to endocytose and phagocytose antigens. For instance, cross linking of FcRs and

CRs is required for efficient endocytosis of antigens (254). Consequently, the array of

receptors used by virions influences the rate of internalization and where the HIV-‐1 ends

up inside the cell. The interaction between DC receptors and HIV-‐1 might be influenced by

the state of the virions, i.e. if they are free, opsonized or immune complexed (255). In

addition, the binding of the HIV-‐1 to infection receptors gp120 to CD4 and CCR5/CXCR4

expressed on DCs can give rise in a direct productive infection, i.e. cis infection. At the same

time, binding and uptake of HIV-‐1 by other receptors such as the C-‐type lectin DC-‐SIGN

leads to internalization into the endocytic compartments where the virions remain

infectious and can be transferred by the DC to CD4+ T cells, i.e. trans infection. Taken

together, it is clear that the initial interactions between HIV-‐1 and DCs influence the uptake

and handling of the virions but also the effect the virions exert on the DCs.


Several integrins were involved in the binding and uptake of F-‐HIV and C-‐HIV. Both C-‐HIV

and F-‐HIV utilized the two chains of CR3, however distinctive for C-‐HIV was the use of β1

integrin and for F-‐HIV the use of β7 integrin. We showed in this study that C-‐HIV was

55


43

internalized more efficiently by the endocytic receptors, C-‐type lectins and integrins, into

both immature and mature DCs compared to free HIV-‐1 despite the fact that similar

receptor repertoire was used and the same amount of C-‐HIV and F-‐HIV bound to the DC

surface. This is probably due to higher levels of cross-‐linking of endocytic receptors due to

the complement fragments and anybodies disposed on the viral surface. Blocking C-‐type

lectins, integrins, and CD4 more or less abolished both F-‐HIV and C-‐HIV binding to DC cell

surface at 4°C, when the endocytic machinery is inactive.

9.2.3 Discussion/ Conclusion

This study showed that even when HIV-‐1 was opsonized with complement fragments, the

virus still interacted with the same receptor families as free virions, i.e. CD4, C-‐type lectins,

and integrins. Blocking viral binding to CD4 together with the C-‐type lectins and integrins

to the DC surface nearly abolished the ability of HIV-‐1 to bind, indicating that we identified

most of the receptor families used by HIV-‐1 for binding to MDDCs. Although opsonization

of the virus did not affect the amount of virus binding to the surface of immature and

mature DCs, they both internalized significantly more C-‐HIV. This indicated that C-‐HIV was

more efficiently taken up by DC than F-‐HIV and this depended on the complement

fragments and antibodies deposited on the viral surfaces, which gives higher levels of

cross-‐linking of endocytic receptors leading to a more efficient receptor mediated

endocytosis. This provides distinct intracellular handling of C-‐HIV leading to both

increased infection and altered activation of HIV specific immune responses.

9.3 Paper III 9.3.1 Background

DCs are essential for initiating and regulating T cell activation and they play a central role

in the pathogenesis of HIV-‐1. Immature DC pick up and proteolyticly process HIV-‐1. This

initiates activation of the DCs leading to their migration to the afferent lymph nodes. The

DCs that reach the lymph nodes will be in a mature state and present HIV-‐1 derived

antigens to CD8+ and CD4+ T cells. The complement system is an important part of the

innate defense against viral infections and is potently activated by HIV-‐1. However, like

other retroviruses HIV-‐1 acquire proteins from the host cell’s plasma membrane during the

process of budding that have the ability to protect the virions from complement lysis (91).

Instead, the virions become covered with complement fragments, which may change how

56


44

the virus is taken up, processed, and presented in the context of MHC class I and II

molecules by immature and mature DCs. In addition, the HIV-‐1 might exert detrimental

effects on the DCs antigen presenting machinery. For instance, it has been shown that the

protease activity is affected in immature DCs after exposure to HIV-‐1 (220).


In this study we investigated the effects opsonized HIV-‐1 had on the uptake and processing

pathways in mature and immature DCs, leading to antigen presentation and T cell

activation. The results showed that C-‐HIV and C-‐IgG-‐HIV significantly enhanced MHC class I

presentation by immature and mature DCs compared to F-‐HIV. Of note, C-‐HIV did not affect

the MHC class II presentation for immature DCs, whereas the effect on mature DCs

resembled the profile seen for MHC class I presentation with enhanced presentation for the

opsonized virions. CR3 was involved in guiding the free and complement opsonized virions

to both MHC class I and MHC class II presentation pathways as inhibition of this receptor

decreased the presentation. In contrast, blocking of the β7-‐integrin on immature and

mature DCs resulted in a significantly enhanced MHC class I and II presentation for both F-‐

HIV and C-‐HIV. We also examined if C-‐type lectin receptors intersected the antigen

presentation pathway and concluded that binding of F-‐HIV or C-‐HIV to C-‐type lectins

promoted the delivery of antigen for MHC class I presentation in immature DCs but not in

mature DCs. However, when F-‐HIV or C-‐HIV used the specific C-‐type lectin receptors MMR

or DEC-‐205, decreased the amount of antigen accessible for MHC class I presentation by

immature DCs. C-‐HIV had a slower degradation rate in DCs than F-‐HIV. In addition,

neutralization of pH in endosomes did not affect DC MHC class I presentation of antigens

derived from C-‐HIV, while it had a strong enhancing effect on F-‐HIV, indicating that C-‐HIV

was processed differently by the DCs than F-‐HIV. In addition, the exposure to C-‐HIV

decreased the protease activity in both immature and matured DCs, whereas F-‐HIV

increased this activity. In the case for the proteolysis in the cytosol, C-‐HIV enhanced the

proteasome proteolytic activity, whereas F-‐HIV decreased its activity. Quantitative

proteomics and PCR showed that several proteins and genes involved in degradation and

regulation of the proteasome were affected in immature DCs after exposure to either F-‐HIV

or C-‐HIV.

57


45


This study takes our previous findings further by investigating in detail the processing

pathway and machinery in immature and mature DCs leading to MHC class I and II

presentation of HIV antigens derived from whole free or opsonized HIV-‐1. The elevated

uptake of C-‐HIV lead to enhanced MHC class I presentation in DCs. Neutralization of the

endosomal compartments proved that the free and opsonized virions were handled

differently inside the DCs and this gave the free virions greater opportunity to enter the

cytosol and the MHC class I restricted pathway, whereas it did not affect the C-‐HIV. This

indicated that C-‐HIV ended up in endosomal compartments with a higher pH than F-‐HIV. In

addition, C-‐HIV had a slower degradation process, probably due to the decreased protease

activity this source of HIV-‐1 induced in DCs. The guiding to special more protective

endosomal compartments and a slower degradation process might help the virus to enter

the cytosol and the MHC class I restricted antigen presentation pathway.

HIV-‐1 interacts with many receptors expressed on the surface on the DC and uptake by

these receptors can lead to different pathways inside the cell. Clearly the use of β7-‐integrin

and MMR guide viral particles to pathways is less involved in MHC class I and II

presentation, whereas CR3 enhances the delivery of virions to the presentation pathways.

In addition, when virions are unable to bind β7-‐integrin or MMR, other receptors might

take over that induce a more efficient uptake and guiding of the HIV-‐1 to MHC class I and II

presentation.

The majority of all HIV-‐1 particles is taken up by the endosomal route and a part of these

virions binds and fuses and accesses the cytoplasm where the viral proteins are tagged for

destruction by the ubiquitin-‐proteasome pathway. The exposure of DCs to C-‐HIV enhanced

the proteasomal activity in both immature and mature DCs, which could play a role in the

enhanced MHC class I presentation. Both F-‐HIV and C-‐HIV had a different expression

profile of proteins and genes affecting the regulation and degradation of the proteasome,

which also could affect MHC class I presentation. Taken together, the initial interactions

between HIV-‐1 and DCs highly influence the uptake, processing, and antigen presentation

of virions and also the direct effects virions have on the DCs.

9.4 Paper IV 9.4.1 Background

58


46

The female genital tract is the major portal of entry for HIV-‐1 and at this mucosal site, DCs

and CD4+ T cells are considered early targets for HIV-‐1 (256, 257). It is still not completely

clear how HIV-‐I penetrate the epithelial layer but evidence from the SIV studies in

macaques and a few in vitro studies using mucosal explants, indicate that the LCs localized

in ectocervix and vagina pick up virions and transfer them to the submucosa (7). In

addition, if there is a break in the epithelium, the virus gets direct access to the submucosal

tissue and DCs and CD4+ T cells located at this site (7). In the submucosa can DCs that have

captured HIV-‐1 can transmit the virions to nearby CD4+ T cells. The DC-‐CD4+ T cells

conjugates drastically facilitate the viral infection of the CD4+ T cells. After 3-‐4 days of

virus inoculation, small founder populations of infected cells emerge and they first expand

locally in CD4+ T cells and then spread the infection to the draining lymphoid tissues and

give rise to a systemic infection. HIV-‐1 exposed mucosal DCs/LCs also migrate to the lymph

nodes and spread the virus to CD4+ T cells. DCs/LCs and CD4+ T cells localized in the

female genital tract might interact differently with free and opsonized HIV-‐1 and therefore

these different groups are examined in this study of the initial HIV-‐1 interaction and

infection of mucosa using human cervical explants.


We found in this study that C-‐HIV significantly increased the infection in DCs emigrating

from cervical mucosal tissue compared to F-‐HIV. In contrast, the infection in emigrating

CD4+ T cells was decreased when the tissue was challenged with C-‐HIV. In addition, the

level of C-‐HIV infection was higher in mucosal DCs in women younger than 50 years of age

compared to women older than 50, even if this infection in both age categories was higher

than for F-‐HIV. We studied several receptors known to interact with HIV-‐1 and detected

that cervical mucosa tissue explants and emigrating DCs and T cells expressed C-‐type

lectins, DC-‐SIGN, MMR, and DEC-‐205, and integrins β1, β2, β7, α4, and αM. We next studied

the involvement of these receptors in HIV-‐1 infection. Blocking the viral use of C-‐type

lectins decreased the infection in migrating DCs but not in CD4+ T cells. The decreased

infection in the CD4+ T cells is probably due to the lack of complement receptors on the

surface of CD4+ T cells and possibly also because the CR fragments on the surface of HIV-‐1

left gp120 poorly accessible for binding to other receptors expressed on the CD4+ T cell.

Inhibition of αM/β2 integrins (CD11b/CD18) decreased HIV-‐1 infection of emigrating DCs

and T cells, with the highest effect for C-‐HIV. In addition, the blocking of the α4-‐integrin,

59


47

β1-‐integrin, and β7-‐integrin decreased the HIV-‐1 infection in both DCs and T cells and the

level of inhibition of HIV-‐1 infection was more prominent in DCs than the T cells. Of note,

we detected viral founder populations in the cervical tissue explants challenged with HIV-‐1

and when α4-‐integrin or β7-‐integrin was blocked the establishment of these populations

was gone from more than 70% of the tissues examined.


The initial events of the HIV-‐1 infection in the genitals are poorly characterized and little is

known about the factors influencing initiation of HIV-‐1 replication in the cervical mucosa.

The cervical mucosa immune cells expressed an array of receptors known to affect the HIV-‐

1 infection. In this study we show that complement opsonization of virions gave a higher

infection in the emigrating DCs than free virions. This is probably due to the C-‐HIV use of

the CR3 expressed by mucosal DCs. Of note, infection of DCs was reduced even for F-‐HIV,

when CR3 was blocked, although not to the same degree as C-‐HIV, and this can be

explained by the expression of a CR3 ligand, i.e. ICAM-‐1 in the envelope the virions. In

addition, the HIV-‐1 envelope protein gp41 shares four regions of homology with the

complement protein C3, another ligand for CR3. The decrease in infection of T cells with,

which are CR3 negative for C-‐HIV is possibly due to that the deposition of inactivated C3

fragments on the surface of HIV-‐1 sterically interferes with viral attachment of HIV-‐1 to the

T cell surface and masks some viral gp120 epitopes so they cant bind CD4 and/or

coreceptor (60). Blocking CR3 significantly reduced the infection in both emigrating DCs

and CD4+ T cells using C-‐HIV. Of note, blocking integrins had the best effect regarding

infection of DCs, T cells and preventing establishment of founder populations. Therefore,

integrins should be taken in consideration when developing microbicides since the

blocking of integrins seems to reduce the infection in thereby also the spread of HIV-‐1.

60

Conclusion and future directions

48

10. CONCLUSION AND FUTURE DIRECTIONS

10.1 Complement activation-‐ Good or Evil in HIV-‐1 Infection? HIV-‐1 immediately activates the complement system, which is a part of our innate defense,

but will the complement system really do us any good in this setting? Is complement good

or evil in HIV-‐1 infection?

To answer the title of my thesis one must remember that HIV-‐1 has developed escape

mechanisms that protect it from complement destruction. The complement system is

generally a dangerous weapon against microorganisms and is strongly activated during a

HIV-‐1 infection, but instead of getting lysed the virions are opsonized with complement

fragments. Usually, when pathogens are opsonized they are eliminated and cleared from

the system by binding to phagocytes expressing complement receptors (CR) (64). However,

for HIV-‐1, the opsonization seems to be rather an advantage, even though a minor fraction

of virions are destroyed by the complement system (60). The advantages of opsonization

for HIV-‐1 include the ability to interact with cells expressing CRs, e.g. erythrocytes

expressing CR1, or FDCs expressing CR2 and CR3, B cells expressing CR1, and DCs

expressing CR1, CR3, and CR4, the elevated infectivity (64), and transfer of virions from

DCs to target cells (258). C-‐HIV binding to FDCs on one hand activates B cells and

production of antibodies, but on the other hand the deposition of C-‐HIV on FDCs is the far

largest reservoir of HIV-‐1 in an infected individual (64). C3a and C5a are factors produced

by the activated complement cascade, these anaphylatoxins attract DCs and other APCs to

the site of infection, which then can then be exploited by HIV-‐1 (60). Results from our

group have shown that opsonized virions are more efficiently internalized via receptor

mediated endocytosis by DCs compared to free HIV-‐1 (259). In vivo, DCs capture and

internalize C-‐HIV and migrate to the lymph nodes where they subsequently transfer

infectious virions to the CD4+ T cells. Our studies demonstrated that C-‐HIV significantly

enhanced immature and mature DCs MHC class I presentation of HIV derived antigens by

DCs. An increased MHC class I presentation and activation of CD8+ T cells is indeed good

for the host. However, during the priming of the naïve T cells HIV-‐1 will be transferred

through the infectious synapse and infect the newly activated cells. An increased MHC class

I presentation implies that a larger amount of C-‐HIV gains access to the cytosol compared

to F-‐HIV, which can lead to a higher infection of the DCs. A higher infection was indeed seen

in cervical mucosal DCs using C-‐HIV compared to F-‐HIV. C-‐HIV slightly decreased the direct

61

Conclusion and future directions

49

infection of CD4+ T cells. Then again, transfer of complement opsonized virions from DCs

to CD4+ T cells creates a much higher infection (218, 260, 261), probably due to the DC

induced activation of the CD4+ T cells. To answer the question I made in the beginning; the

virus infects the host and the initial immune system immediately strikes back. However, in

the long run the virus seem to be the winner of the battle and complement activation does

more harm than good in HIV-‐1 infection.

Figure 15. The battle between complement opsonized HIV-‐1 and the immune system.

The complement system plays a role in clearing HIV-‐1. However, at the same time complement opsonized

HIV-‐1 facilitates the DC spread of the virus, increases the level of infection of DCs, and functions as a viral

reservoir on FDCs in the lymph nodes. In summary, it seems the balance will be tipped in favor ofHIV-‐1.

10.2 Future challenges The results from our studies show the importance of studying both free and complement

opsonized HIV-‐1. Complement opsonized HIV-‐1 exists in every compartment in the body

and the C3 fragments deposited on the viral surface may alter the interaction with the

immune cells. Therefore, the effect complement opsonized HIV-‐1 has on the ability of the

DCs to function as potent antigen presenting cells and the mechanism behind the increased

infection induced by opsonized virions should be taken in consideration for future studies.

The finding that blocking of CR3, α4 and β7 integrins inhibited HIV-‐1 infection of both

immune cells and cervical mucosa tissues would be of a great interest to continue to

explore to identify potent inhibitors of these receptors that can be used as microbicides or

vaccines.

62

Populärvetenskaplig sammanfattning

50

11. POPULÄRVETENSKAPLIG SAMMANFATTNING

Humant immunbrist virus-‐1 (HIV-‐1) identifierades av två forskare, Luc Montagnier och

Françoise Barré Sinoussie på Pasteurinstitutet i Paris. HIV är en infektion som angriper

kroppens immunförsvar och långsamt bryter ner det och orsakar en immunbristsjukdom

som 1982 fick namnet förvärvat immunbrist syndrom (AIDS). AIDS uppträder först när

kroppens immunförsvar blivit så försvagat att vi inte längre kan försvara oss emot andra

virus och bakterieinfektioner. Fram till idag har HIV-‐1 orsakat 25 miljoner dödsfall i

världen vilket gör den här infektionen till en av de värsta epidemierna som drabbat

mänskligheten under det här årtiondet. Det finns idag inget botemedel eller vaccin

emot HIV-‐1 men det finns bromsmediciner som kan kontrollera virusinfektionen. De är

dock inte tillgängliga för alla, har höga priser och ger många gånger biverkningar. Det finns

idag ett akut behov av ett HIV vaccin eller microbicider för att hindra spridningen av HIV

och stoppa epidemin.

Majoriteten av HIV infektionerna sker via samlag och när viruset kommer in i kroppen

kommer det omedelbart att aktivera en del av vårt immunförsvar som heter

komplementsystemet. Komplementsystemets funktion är bland annat att försvara oss emot

inkräktare som bakterier och virus. HIV-‐1 har emellertid utvecklat försvar som gör att

komplementsystemet inte kommer att förgöra viruset, istället kommer virusytan att täckas

av komplement proteinfragment vilket kallas opsonisering. Den förändrade ytan kommer

viruset istället använda till sin fördel genom hela infektionsförloppet. När HIV-‐1 tagit sig in

i kroppen angriper det celler viktiga för ett fungerade immunförsvar, dvs.

immunceller. HIV-‐1 tas upp av en specifik immuncell som kallas dendritiska cellen (DC)

som transporterar viruset vidare från slemhinnan, dvs. platsen för sexuellsmitta, till

speciella körtlar, kallade lymfkörtlar där viruset överförs till ytterligare en immuncell dvs.

T hjälparceller. T hjälparcellerna är de celler i kroppen som HIV-‐1 framför allt infekterar

och tar död på. DC är viktiga både i vårt naturliga och förvärvade immunförsvar och vid en

infektion verkar de lokalt bland annat genom att attrahera andra immunceller till platsen

för infektion. Deras viktigaste uppgift och special funktion är att plocka upp t.ex. virus eller

bakterier från sin omgivning och bryta ned dem i småbitar, så kallade antigen peptider.

Dessa peptider visas upp för både T hälparceller och T mördarceller (en immuncell som

kan ta död på infekterade celler) och leder till aktivering av ett specifikt immunförsvar som

kan bekämpa det virus eller bakterie som angripit kroppen.

63


51

Vi har studerat mekanismer involverade i DC upptag och nedbrytning av fritt HIV-‐1 som

leder till uppvisade av antigenpeptider och aktivering av T hälparceller och T mördarceller.

Uppvisandet av HIV-‐1 antigenpeptider för aktivering av T mördarceller krävde att viruset

band och togs upp av DC och levererades till cellens insida (cytosol) och denna aktivering

blev effektivare när viruset fick längre tid på sig att förflytta sig till cellens cytosol.

Aktivering av T hjälparceller krävde att viruset togs upp i specialavdelningar så kallade

endosomer och att de hade en sur miljö så att viruset kunde brytas ned.

HIV-‐1 kan binda till olika proteiner som finns på immuncellernas yta och vilka proteiner

det binder kan bero på om viruset är fritt eller täckt med komplement proteinfragment. Vi

studerade inbindningen och upptaget av fritt och opsoniserat HIV-‐1 till DC och om det

fanns skillnader i receptoranvändning. Komplement opsoniserat HIV-‐1 togs upp effektivare

av DC antagligen pga. av effektivare upptag via komplementreceptorer. Trots den ökade

mängden opsoniserat virus som togs upp av DC var det inga stora skillnader när det gäller

receptoranvändning.

Nästa studie undersökte hur fritt och opsoniserat virus hanterades och bröts ned inne i den

DC samt hur antigenpeptider från virusen presenterades för att starta ett immunförsvar

mot HIV. Vi hittade att DC som presenterade opsoniserat virus aktiverade mer T

mördarceller än de som presenterade fritt virus. Vid jämförelse mellan fritt och opsoniserat

HIV kunde vi också se intressanta skillnader som indikerade att när viruset var maskerat

med komplement fragment så processades det annorlunda än fritt HIV. Dessutom så hade

fritt och komplement opsoniserat HIV olika effekt på DC maskineri som ansvarar för

nedbrytning av antigen så som virus. Våra resultat visar också att olika receptorer

transporterar HIV-‐1 till olika avdelningar inne i DC, vilket påverkar aktiveringen av

immunsystemet.

I den sista studien undersökte vi hur fritt och opsonizerat HIV-‐1 påverkar immunceller i

livmoderhalsslemhinnan eftersom HIV-‐1 vanligen överförs och smittar denna slemhinna

vid samlag. Dessutom var syftet att se om det gick att stoppa infektionen genom att

blockera olika receptorer som finns på cellerna och i vävnaden. I vår studie fann vi bland

annat att blockering av integriner drastiskt minskade infektionen i både DC och CD4+ T

celler, samt uppkomsten av infektion i de kluster av T celler ”founder populationer” som

normalt initierar att denna infektion kan sprida sig i hela kroppen

64


52

För att kunna ta fram HIV vaccin och microbicider är det viktigt att förstå hur HIV påverkar

DCs och hur viruset processas och presenteras av DC och att förstå hur infektionen av

slemhinnor så som den i livmoderhalsen går till.

65

Acknowledgements

53

12. ACKNOWLEDGEMENTS

This work could not have been completed without the help from others.

First I want to acknowledge and thank my supervisor Professor Marie Larsson for her

guidance and mentorship during my training experience. The support and enthusiasm from

my mentor have encouraged me though my projects and the scientific freedom,

constructive criticism and many discussions have contributed a lot towards my

understanding and personal development, this are invaluable tools for my future carrier.

To my co-‐supervisor Professor Jorma Hinkula thank you for helping me whenever you

could and always taking time to discuss science with enthusiasm, I have always felt like I

can come to you with questions.

I also want to thank my co-‐supervisor Professor Karl-‐Erik Magnusson.

A special thanks to my colleagues and friends in my lab group. Rada Ellegård, I was so

impressed by you when you first became my student. After a while I also learned that you

were quit a computer expert and a talent when it comes to making illustrative pictures.

Thank you for always having time, your help in the lab and the pictures in this thesis. Also

want to thank you for all the discussions and your intelligent inputs regarding dendritic

cells and science in general. Karlhans Che you mean a lot to me and you have always been

a big support, if I ever needed to talk about work or anything else you took your time plus

you almost always had candy for me in your office. Sundaram Muthu thanks for all the

discussions we had, I enjoy talking science with you and thank you for helping me keeping

me updated on the latest big new news in the field of HIV-‐1.

I also want to acknowledge Professor Lennart Svensson’s group we go way back and we

share the passion for virology. Lennart Svensson thank you for always having time for me,

all the advices I have got, and sharing your experience with me. Caroline Jönsson you keep

track of absolutely everything and you have a lot of experience, thank you for all the help

I’ve got during my PhD period but also for being a wonderful coworker and friend. I also

want to thank Johan Nordgren, Malin Vildevall, Elin Kindberg, Beatrice Karlsson,

Marie Hagbom, Sumit Sharma, and Claudia Istrate you have all helped me in different

ways and made me look forward to come to come to work every day.

66

Acknowledgements

54

Lena Svensson we have known each other since I first came to Linköping and you have

always been supporting and helpful. I enjoyed working together with you but also spending

time outside work. Thank you for everything!

Lotta Lenner and Pia Druid thank you for always being so helpful!

I want to thank all my coworkers in Professor Stephan Thor’s group. I’m glad you guys

moved from Valla to us here at floor 13 so that we could get some action up here!

Especially I want to acknowledge Daniel Karlsson for discussing immunofluorescence

staining, confocal etc and for being a good friend. Magnus Baumgaurt for supporting my

research in his own way, I can’t thank you enough! I also want to thank you for “saving” my

computers a couple of times, discussing experiments, making me laugh so much and being

the person you are. Annika Starkenberg for always making me laugh and being the happy

person you are.

I want to thank Tina Falkeborn, Josefine Åberg, Camilla and Johanna for being nice

coworkers and all the discussions over a cup of coffee.

Thanks to Amanda Nordigården for always being so happy and positive! In addition, you

know you inspire me with your classy clothes and a sophisticated life style!

I wish you all good luck with your PhD and in the future!

Thanks to Robert Blomgran for being who you are and good luck with your research in

the future!

Thanks to all of my colleagues at floor 13, “Labbettan”!

I want to acknowledge our collaborators at the National Laboratory for HIV Immunology,

Winnipeg, Canada. A special thanks to Dr. Adam Burgener for making me better in

English, many interesting discussions and thank you so much for analyzing our samples it

have resulted in great and valuable data.

I also want to acknowledge our collaborators at the division of Gynecology and especially

Preben Kjölhede.

67

Acknowledgements

55

I want to acknowledge Café Cellskapet for their great food and the nice people who work

there, Rebecka, Jessicka and Kristina.

I wish to express my sincere gratitude to the my opponent Associate professor Barbra L.

Shacklett and review committee; Professor Kristina Broliden, Associate professor

Maria Jenmalm and Professor Anders Rosén. I’m sure you will give me a hard time at my

defense and I look forward to interesting discussions.

Finally I want to acknowledge my family.

My mother Annicka Dennerqvist who supported me a lot by babysitting Isabelle when I

was working. To my father Miran Dennerqvist who always have been interested and

supportive of my carrier. To my wonderful sister Caroline Dennerqvist who lives in USA.

Thank you for making such a beautiful cover to my thesis!

To my extended family Tjomsland in Norway, thank you for your concern!

To my wonderful friend Jenny Hällsten, who always support and encourage me, you

should know a lot about viruses by now! You are making me run when I feel like I don’t

have the strength to stand. I want you to know that I consider myself very lucky to have

you as a friend!

To my children Isabelle Tjomsland and Alexander Tjomsland for being a source of

happiness in my life!

A very special thanks to my loving husband, coworker and office-‐mate Vegard Tjomsland.

You have encouraged me through all the ups and downs in science. Being in the same group

for five years, married for seven years and before that class-‐mate as undergraduates, what

can I say, no one understands me like you do! We share the same passion for science and

you have motivated me during hard times and you have been the most important support

in my life! Remember, amor vincit omnia…

68

Acknowledgements

56

All good things come to an end but I will take with me everything I learned and the experience

I got and I won’t forget the people who helped me!

69

Acknowledgements

57

13. REFERENCES

1. A. Vahlne, A historical reflection on the discovery of human retroviruses. Retrovirology 6, 40 (2009).

2. U. R. o. t. G. A. Epidemic. 3. T. Hirbod, K. Broliden, Mucosal immune responses in the genital tract of HIV-‐1-‐exposed

uninfected women. Journal of internal medicine 262, 44 (Jul, 2007). 4. J. K. Leyenaar, HIV/AIDS and Africa's orphan crisis. Paediatrics & child health 10, 259

(May, 2005). 5. UN AIDS data on sub-‐Saharan Africa. Joint United Nations Program on HIV/AIDS. Journal

of the International Association of Physicians in AIDS Care 2, 54 (May, 1996). 6. A. T. Haase, Targeting early infection to prevent HIV-‐1 mucosal transmission. Nature

464, 217 (Mar 11, 2010). 7. F. Hladik, M. J. McElrath, Setting the stage: host invasion by HIV. Nature reviews.

Immunology 8, 447 (Jun, 2008). 8. S. P. Buchbinder et al., Efficacy assessment of a cell-‐mediated immunity HIV-‐1 vaccine

(the Step Study): a double-‐blind, randomised, placebo-‐controlled, test-‐of-‐concept trial. Lancet 372, 1881 (Nov 29, 2008).

9. E. Check, Scientists rethink approach to HIV gels. Nature 446, 12 (Mar 1, 2007). 10. M. Altfeld, T. M. Allen, Hitting HIV where it hurts: an alternative approach to HIV vaccine

design. Trends in immunology 27, 504 (Nov, 2006). 11. P. D. Kwong, R. Wyatt, Q. J. Sattentau, J. Sodroski, W. A. Hendrickson, Oligomeric

modeling and electrostatic analysis of the gp120 envelope glycoprotein of human immunodeficiency virus. Journal of virology 74, 1961 (Feb, 2000).

12. H. R. Gelderblom, M. Ozel, G. Pauli, Morphogenesis and morphology of HIV. Structure-‐function relations. Archives of virology 106, 1 (1989).

13. S. Sierra, B. Kupfer, R. Kaiser, Basics of the virology of HIV-‐1 and its replication. Journal of clinical virology : the official publication of the Pan American Society for Clinical Virology 34, 233 (Dec, 2005).

14. S. L. McGovern, E. Caselli, N. Grigorieff, B. K. Shoichet, A common mechanism underlying promiscuous inhibitors from virtual and high-‐throughput screening. Journal of medicinal chemistry 45, 1712 (Apr 11, 2002).

15. D. C. Chan, D. Fass, J. M. Berger, P. S. Kim, Core structure of gp41 from the HIV envelope glycoprotein. Cell 89, 263 (Apr 18, 1997).

16. E. Fanales-‐Belasio, M. Raimondo, B. Suligoi, S. Butto, HIV virology and pathogenetic mechanisms of infection: a brief overview. Annali dell'Istituto superiore di sanita 46, 5 (2010).

17. D. C. Chan, P. S. Kim, HIV entry and its inhibition. Cell 93, 681 (May 29, 1998). 18. R. Wyatt, J. Sodroski, The HIV-‐1 envelope glycoproteins: fusogens, antigens, and

immunogens. Science 280, 1884 (Jun 19, 1998). 19. N. Arhel, Revisiting HIV-‐1 uncoating. Retrovirology 7, 96 (2010). 20. A. M. Sheehy, N. C. Gaddis, J. D. Choi, M. H. Malim, Isolation of a human gene that inhibits

HIV-‐1 infection and is suppressed by the viral Vif protein. Nature 418, 646 (Aug 8, 2002). 21. Y. Wu, HIV-‐1 gene expression: lessons from provirus and non-‐integrated DNA.

Retrovirology 1, 13 (2004). 22. Y. Wu, J. W. Marsh, Selective transcription and modulation of resting T cell activity by

preintegrated HIV DNA. Science 293, 1503 (Aug 24, 2001). 23. M. D. Miller, C. M. Farnet, F. D. Bushman, Human immunodeficiency virus type 1

preintegration complexes: studies of organization and composition. Journal of virology 71, 5382 (Jul, 1997).

24. M. Adams et al., Cellular latency in human immunodeficiency virus-‐infected individuals with high CD4 levels can be detected by the presence of promoter-‐proximal transcripts.

70

Acknowledgements

58

Proceedings of the National Academy of Sciences of the United States of America 91, 3862 (Apr 26, 1994).

25. V. W. Pollard, M. H. Malim, The HIV-‐1 Rev protein. Annual review of microbiology 52, 491 (1998).

26. W. C. Greene, B. M. Peterlin, Charting HIV's remarkable voyage through the cell: Basic science as a passport to future therapy. Nature medicine 8, 673 (Jul, 2002).

27. C. A. Lundquist, M. Tobiume, J. Zhou, D. Unutmaz, C. Aiken, Nef-‐mediated downregulation of CD4 enhances human immunodeficiency virus type 1 replication in primary T lymphocytes. Journal of virology 76, 4625 (May, 2002).

28. P. Stumptner-‐Cuvelette et al., HIV-‐1 Nef impairs MHC class II antigen presentation and surface expression. Proceedings of the National Academy of Sciences of the United States of America 98, 12144 (Oct 9, 2001).

29. M. Geyer, O. T. Fackler, B. M. Peterlin, Structure-‐-‐function relationships in HIV-‐1 Nef. EMBO reports 2, 580 (Jul, 2001).

30. J. F. Roeth, K. L. Collins, Human immunodeficiency virus type 1 Nef: adapting to intracellular trafficking pathways. Microbiology and molecular biology reviews : MMBR 70, 548 (Jun, 2006).

31. R. L. Willey, F. Maldarelli, M. A. Martin, K. Strebel, Human immunodeficiency virus type 1 Vpu protein induces rapid degradation of CD4. Journal of virology 66, 7193 (Dec, 1992).

32. Y. H. Zheng, N. Lovsin, B. M. Peterlin, Newly identified host factors modulate HIV replication. Immunology letters 97, 225 (Mar 15, 2005).

33. P. M. Pitha, Innate Antiviral Response: Role in HIV-‐1 Infection. Viruses 3, 1179 (Jul, 2011).

34. S. Mehandru et al., Primary HIV-‐1 infection is associated with preferential depletion of CD4+ T lymphocytes from effector sites in the gastrointestinal tract. The Journal of experimental medicine 200, 761 (Sep 20, 2004).

35. J. J. Mattapallil et al., Massive infection and loss of memory CD4+ T cells in multiple tissues during acute SIV infection. Nature 434, 1093 (Apr 28, 2005).

36. T. Lehner, Y. Wang, T. Whittall, T. Seidl, Innate immunity and HIV-‐1 infection. Advances in dental research 23, 19 (Apr, 2011).

37. T. Lehner et al., The emerging role of innate immunity in protection against HIV-‐1 infection. Vaccine 26, 2997 (Jun 6, 2008).

38. J. A. Levy, The importance of the innate immune system in controlling HIV infection and disease. Trends in immunology 22, 312 (Jun, 2001).

39. R. M. Steinman et al., The interaction of immunodeficiency viruses with dendritic cells. Current topics in microbiology and immunology 276, 1 (2003).

40. T. Taniguchi, K. Ogasawara, A. Takaoka, N. Tanaka, IRF family of transcription factors as regulators of host defense. Annual review of immunology 19, 623 (2001).

41. B. J. Barnes, P. A. Moore, P. M. Pitha, Virus-‐specific activation of a novel interferon regulatory factor, IRF-‐5, results in the induction of distinct interferon alpha genes. The Journal of biological chemistry 276, 23382 (Jun 29, 2001).

42. A. Iwasaki, R. Medzhitov, Toll-‐like receptor control of the adaptive immune responses. Nature immunology 5, 987 (Oct, 2004).

43. R. N. Harty, P. M. Pitha, A. Okumura, Antiviral activity of innate immune protein ISG15. Journal of innate immunity 1, 397 (Aug, 2009).

44. S. Akira, TLR signaling. Current topics in microbiology and immunology 311, 1 (2006). 45. S. S. Diebold, T. Kaisho, H. Hemmi, S. Akira, C. Reis e Sousa, Innate antiviral responses by

means of TLR7-‐mediated recognition of single-‐stranded RNA. Science 303, 1529 (Mar 5, 2004).

46. A. Lepelley et al., Innate sensing of HIV-‐infected cells. PLoS pathogens 7, e1001284 (Feb, 2011).

47. Y. L. Chiu et al., Cellular APOBEC3G restricts HIV-‐1 infection in resting CD4+ T cells. Nature 435, 108 (May 5, 2005).

71

Acknowledgements

59

48. E. E. Nakayama, T. Shioda, Anti-‐retroviral activity of TRIM5 alpha. Reviews in medical virology 20, 77 (Mar, 2010).

49. S. J. Neil, T. Zang, P. D. Bieniasz, Tetherin inhibits retrovirus release and is antagonized by HIV-‐1 Vpu. Nature 451, 425 (Jan 24, 2008).

50. N. Laguette et al., SAMHD1 is the dendritic-‐ and myeloid-‐cell-‐specific HIV-‐1 restriction factor counteracted by Vpx. Nature 474, 654 (Jun 30, 2011).

51. J. L. Croxford, S. Gasser, Damage control: how HIV survives the editor APOBEC3G. Nature immunology 12, 925 (2011).

52. P. Marrack, J. Kappler, T. Mitchell, Type I interferons keep activated T cells alive. The Journal of experimental medicine 189, 521 (Feb 1, 1999).

53. S. S. Cho et al., Activation of STAT4 by IL-‐12 and IFN-‐alpha: evidence for the involvement of ligand-‐induced tyrosine and serine phosphorylation. J Immunol 157, 4781 (Dec 1, 1996).

54. T. Lehner et al., Protective mucosal immunity elicited by targeted iliac lymph node immunization with a subunit SIV envelope and core vaccine in macaques. Nature medicine 2, 767 (Jul, 1996).

55. N. Yan, A. D. Regalado-‐Magdos, B. Stiggelbout, M. A. Lee-‐Kirsch, J. Lieberman, The cytosolic exonuclease TREX1 inhibits the innate immune response to human immunodeficiency virus type 1. Nature immunology 11, 1005 (Nov, 2010).

56. D. T. Fearon, R. M. Locksley, The instructive role of innate immunity in the acquired immune response. Science 272, 50 (Apr 5, 1996).

57. T. Fujita, Y. Endo, M. Nonaka, Primitive complement system-‐-‐recognition and activation. Molecular immunology 41, 103 (Jun, 2004).

58. B. P. Morgan, P. Gasque, Extrahepatic complement biosynthesis: where, when and why? Clinical and experimental immunology 107, 1 (Jan, 1997).

59. C. Speth et al., Human immunodeficiency virus type 1 induces expression of complement factors in human astrocytes. Journal of virology 75, 2604 (Mar, 2001).

60. G. Huber, Z. Banki, S. Lengauer, H. Stoiber, Emerging role for complement in HIV infection. Current opinion in HIV and AIDS 6, 419 (Sep, 2011).

61. M. Bennett, T. Leanderson, Was it there all the time? Scandinavian journal of immunology 57, 499 (Jun, 2003).

62. D. Ricklin, G. Hajishengallis, K. Yang, J. D. Lambris, Complement: a key system for immune surveillance and homeostasis. Nature immunology 11, 785 (Sep, 2010).

63. C. B. Chen, R. Wallis, Two mechanisms for mannose-‐binding protein modulation of the activity of its associated serine proteases. The Journal of biological chemistry 279, 26058 (Jun 18, 2004).

64. H. Stoiber, Z. Banki, D. Wilflingseder, M. P. Dierich, Complement-‐HIV interactions during all steps of viral pathogenesis. Vaccine 26, 3046 (Jun 6, 2008).

65. H. U. Lutz, E. Jelezarova, Complement amplification revisited. Molecular immunology 43, 2 (Jan, 2006).

66. P. J. Lachmann, The amplification loop of the complement pathways. Advances in immunology 104, 115 (2009).

67. P. F. Zipfel, C. Skerka, Complement regulators and inhibitory proteins. Nature reviews. Immunology 9, 729 (Oct, 2009).

68. D. D. Kim, W. C. Song, Membrane complement regulatory proteins. Clinical immunology 118, 127 (Feb-‐Mar, 2006).

69. A. Rochowiak, Z. I. Niemir, [The role of CR1 complement receptor in pathology]. Polski merkuriusz lekarski : organ Polskiego Towarzystwa Lekarskiego 28, 84 (Jan, 2010).

70. M. S. Diamond, J. Garcia-‐Aguilar, J. K. Bickford, A. L. Corbi, T. A. Springer, The I domain is a major recognition site on the leukocyte integrin Mac-‐1 (CD11b/CD18) for four distinct adhesion ligands. The Journal of cell biology 120, 1031 (Feb, 1993).

71. S. D. Wright et al., Identification of the C3bi receptor of human monocytes and macrophages by using monoclonal antibodies. Proceedings of the National Academy of Sciences of the United States of America 80, 5699 (Sep, 1983).

72

Acknowledgements

60

72. H. V. Nielsen, J. P. Christensen, E. C. Andersson, O. Marker, A. R. Thomsen, Expression of

type 3 complement receptor on activated CD8+ T cells facilitates homing to inflammatory sites. J Immunol 153, 2021 (Sep 1, 1994).

73. H. Stoiber, M. Pruenster, C. G. Ammann, M. P. Dierich, Complement-‐opsonized HIV: the free rider on its way to infection. Molecular immunology 42, 153 (Feb, 2005).

74. L. Kacani et al., Dendritic cells transmit human immunodeficiency virus type 1 to monocytes and monocyte-‐derived macrophages. Journal of virology 72, 6671 (Aug, 1998).

75. M. Suresh et al., Complement component 3 is required for optimal expansion of CD8 T cells during a systemic viral infection. J Immunol 170, 788 (Jan 15, 2003).

76. B. L. Sullivan et al., Susceptibility of HIV-‐1 plasma virus to complement-‐mediated lysis. Evidence for a role in clearance of virus in vivo. J Immunol 157, 1791 (Aug 15, 1996).

77. X. Ji, H. Gewurz, G. T. Spear, Mannose binding lectin (MBL) and HIV. Molecular immunology 42, 145 (Feb, 2005).

78. U. Kishore et al., Modular organization of the carboxyl-‐terminal, globular head region of human C1q A, B, and C chains. J Immunol 171, 812 (Jul 15, 2003).

79. H. Stoiber, L. Kacani, C. Speth, R. Wurzner, M. P. Dierich, The supportive role of complement in HIV pathogenesis. Immunological reviews 180, 168 (Apr, 2001).

80. Y. Xu et al., A novel approach to inhibit HIV-‐1 infection and enhance lysis of HIV by a targeted activator of complement. Virology journal 6, 123 (2009).

81. S. Dopper et al., Mechanism(s) promoting HIV-‐1 infection of primary unstimulated T lymphocytes in autologous B cell/T cell co-‐cultures. European journal of immunology 33, 2098 (Aug, 2003).

82. Z. Banki et al., Complement dependent trapping of infectious HIV in human lymphoid tissues. AIDS 19, 481 (Mar 25, 2005).

83. D. C. Montefiori et al., Complement control proteins, CD46, CD55, and CD59, as common surface constituents of human and simian immunodeficiency viruses and possible targets for vaccine protection. Virology 205, 82 (Nov 15, 1994).

84. H. Stoiber, C. Pinter, A. G. Siccardi, A. Clivio, M. P. Dierich, Efficient destruction of human immunodeficiency virus in human serum by inhibiting the protective action of complement factor H and decay accelerating factor (DAF, CD55). The Journal of experimental medicine 183, 307 (Jan 1, 1996).

85. D. Tortorella, B. E. Gewurz, M. H. Furman, D. J. Schust, H. L. Ploegh, Viral subversion of the immune system. Annual review of immunology 18, 861 (2000).

86. Z. Banki, H. Stoiber, M. P. Dierich, HIV and human complement: inefficient virolysis and effective adherence. Immunology letters 97, 209 (Mar 15, 2005).

87. P. D. Kwong et al., Structure of an HIV gp120 envelope glycoprotein in complex with the CD4 receptor and a neutralizing human antibody. Nature 393, 648 (Jun 18, 1998).

88. M. Scherl et al., Targeting human immunodeficiency virus type 1 with antibodies derived from patients with connective tissue disease. Lupus 15, 865 (2006).

89. B. L. Sullivan, D. M. Takefman, G. T. Spear, Complement can neutralize HIV-‐1 plasma virus by a C5-‐independent mechanism. Virology 248, 173 (Sep 1, 1998).

90. B. Falkensammer et al., Role of complement and antibodies in controlling infection with pathogenic simian immunodeficiency virus (SIV) in macaques vaccinated with replication-‐deficient viral vectors. Retrovirology 6, 60 (2009).

91. I. Frank et al., Acquisition of host cell-‐surface-‐derived molecules by HIV-‐1. AIDS 10, 1611 (Dec, 1996).

92. M. Pruenster et al., C-‐type lectin-‐independent interaction of complement opsonized HIV with monocyte-‐derived dendritic cells. European journal of immunology 35, 2691 (Sep, 2005).

93. H. Stoiber, M. Pruenster, C. G. Ammann, M. P. Dierich, Complement-‐opsonized HIV: the free rider on its way to infection. Mol Immunol 42, 153 (Feb, 2005).

73

Acknowledgements

61

94. P. Tacnet-‐Delorme et al., In vitro analysis of complement-‐dependent HIV-‐1 cell infection

using a model system. J Immunol 162, 4088 (Apr 1, 1999). 95. V. Boyer, C. Desgranges, M. A. Trabaud, E. Fischer, M. D. Kazatchkine, Complement

mediates human immunodeficiency virus type 1 infection of a human T cell line in a CD4-‐ and antibody-‐independent fashion. The Journal of experimental medicine 173, 1151 (May 1, 1991).

96. Z. Bajtay, L. Kacani, A. Erdei, M. P. Dierich, HIV-‐1 induces human monocyte-‐derived macrophages to produce C3 and to fix C3 on their surface. Journal of leukocyte biology 63, 463 (Apr, 1998).

97. C. F. Ebenbichler et al., Human immunodeficiency virus type 1 activates the classical pathway of complement by direct C1 binding through specific sites in the transmembrane glycoprotein gp41. The Journal of experimental medicine 174, 1417 (Dec 1, 1991).

98. N. M. Thielens, P. Tacnet-‐Delorme, G. J. Arlaud, Interaction of C1q and mannan-‐binding lectin with viruses. Immunobiology 205, 563 (Sep, 2002).

99. P. K. Datta, J. Rappaport, HIV and complement: hijacking an immune defense. Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie 60, 561 (Nov, 2006).

100. C. Pinter, A. G. Siccardi, R. Longhi, A. Clivio, Direct interaction of complement factor H with the C1 domain of HIV type 1 glycoprotein 120. AIDS research and human retroviruses 11, 577 (May, 1995).

101. C. Pinter, A. G. Siccardi, L. Lopalco, R. Longhi, A. Clivio, HIV glycoprotein 41 and complement factor H interact with each other and share functional as well as antigenic homology. AIDS research and human retroviruses 11, 971 (Aug, 1995).

102. H. Stoiber, R. Schneider, J. Janatova, M. P. Dierich, Human complement proteins C3b, C4b, factor H and properdin react with specific sites in gp120 and gp41, the envelope proteins of HIV-‐1. Immunobiology 193, 98 (Jun, 1995).

103. J. Alsenz, J. D. Lambris, T. F. Schulz, M. P. Dierich, Localization of the complement-‐component-‐C3b-‐binding site and the cofactor activity for factor I in the 38kDa tryptic fragment of factor H. The Biochemical journal 224, 389 (Dec 1, 1984).

104. C. Hess et al., Association of a pool of HIV-‐1 with erythrocytes in vivo: a cohort study. Lancet 359, 2230 (Jun 29, 2002).

105. L. Kacani et al., Detachment of human immunodeficiency virus type 1 from germinal centers by blocking complement receptor type 2. Journal of virology 74, 7997 (Sep, 2000).

106. R. Tsunoda, K. Hashimoto, M. Baba, S. Shigeta, N. Sugai, Follicular dendritic cells in vitro are not susceptible to infection by HIV-‐1. AIDS 10, 595 (Jun, 1996).

107. J. Schmitz et al., Follicular dendritic cells retain HIV-‐1 particles on their plasma membrane, but are not productively infected in asymptomatic patients with follicular hyperplasia. J Immunol 153, 1352 (Aug 1, 1994).

108. Z. Bajtay, C. Speth, A. Erdei, M. P. Dierich, Cutting edge: productive HIV-‐1 infection of dendritic cells via complement receptor type 3 (CR3, CD11b/CD18). J Immunol 173, 4775 (Oct 15, 2004).

109. N. Thieblemont et al., Triggering of complement receptors CR1 (CD35) and CR3 (CD11b/CD18) induces nuclear translocation of NF-‐kappa B (p50/p65) in human monocytes and enhances viral replication in HIV-‐infected monocytic cells. J Immunol 155, 4861 (Nov 15, 1995).

110. H. Bouhlal et al., Opsonization of HIV-‐1 by semen complement enhances infection of human epithelial cells. J Immunol 169, 3301 (Sep 15, 2002).

111. V. Tjomsland et al., Complement Opsonization of HIV-‐1 Enhances the Uptake by Dendritic Cells and Involves the Endocytic Lectin and Integrin Receptor Families. PloS one 6, e23542 (2011).

74

Acknowledgements

62

112. R. M. Steinman, Z. A. Cohn, Identification of a novel cell type in peripheral lymphoid organs of mice. I. Morphology, quantitation, tissue distribution. The Journal of experimental medicine 137, 1142 (May 1, 1973).

113. J. Travis, Nobel Prize in physiology or medicine. Immunology prize overshadowed by untimely death of awardee. Science 334, 31 (Oct 7, 2011).

114. R. M. Steinman, Dendritic cells: understanding immunogenicity. European journal of immunology 37 Suppl 1, S53 (Nov, 2007).

115. K. Tamaki, G. Stingl, S. I. Katz, The origin of Langerhans cells. The Journal of investigative dermatology 74, 309 (May, 1980).

116. J. Banchereau, R. M. Steinman, Dendritic cells and the control of immunity. Nature 392, 245 (Mar 19, 1998).

117. H. Donaghy, J. Wilkinson, A. L. Cunningham, HIV interactions with dendritic cells: has our focus been too narrow? Journal of leukocyte biology 80, 1001 (Nov, 2006).

118. L. L. Cavanagh, U. H. Von Andrian, Travellers in many guises: the origins and destinations of dendritic cells. Immunology and cell biology 80, 448 (Oct, 2002).

119. S. Turville, J. Wilkinson, P. Cameron, J. Dable, A. L. Cunningham, The role of dendritic cell C-‐type lectin receptors in HIV pathogenesis. Journal of leukocyte biology 74, 710 (Nov, 2003).

120. I. Mellman, R. M. Steinman, Dendritic cells: specialized and regulated antigen processing machines. Cell 106, 255 (Aug 10, 2001).

121. J. M. Weiss et al., An essential role for CD44 variant isoforms in epidermal Langerhans cell and blood dendritic cell function. The Journal of cell biology 137, 1137 (Jun 2, 1997).

122. J. Banchereau et al., Immunobiology of dendritic cells. Annual review of immunology 18, 767 (2000).

123. S. J. Gibson et al., Plasmacytoid dendritic cells produce cytokines and mature in response to the TLR7 agonists, imiquimod and resiquimod. Cellular immunology 218, 74 (Jul-‐Aug, 2002).

124. A. Dzionek et al., Plasmacytoid dendritic cells: from specific surface markers to specific cellular functions. Human immunology 63, 1133 (Dec, 2002).

125. N. Teleshova, I. Frank, M. Pope, Immunodeficiency virus exploitation of dendritic cells in the early steps of infection. Journal of leukocyte biology 74, 683 (Nov, 2003).

126. M. Cella et al., Plasmacytoid monocytes migrate to inflamed lymph nodes and produce large amounts of type I interferon. Nature medicine 5, 919 (Aug, 1999).

127. F. P. Siegal et al., The nature of the principal type 1 interferon-‐producing cells in human blood. Science 284, 1835 (Jun 11, 1999).

128. M. Lambotin, S. Raghuraman, F. Stoll-‐Keller, T. F. Baumert, H. Barth, A look behind closed doors: interaction of persistent viruses with dendritic cells. Nature reviews. Microbiology 8, 350 (May, 2010).

129. D. N. Hart, Dendritic cells: unique leukocyte populations which control the primary immune response. Blood 90, 3245 (Nov 1, 1997).

130. K. Shortman, Y. J. Liu, Mouse and human dendritic cell subtypes. Nature reviews. Immunology 2, 151 (Mar, 2002).

131. M. A. de Jong, T. B. Geijtenbeek, Langerhans cells in innate defense against pathogens. Trends in immunology 31, 452 (Dec, 2010).

132. T. Kawamura et al., R5 HIV productively infects Langerhans cells, and infection levels are regulated by compound CCR5 polymorphisms. Proceedings of the National Academy of Sciences of the United States of America 100, 8401 (Jul 8, 2003).

133. K. Wolff, The fine structure of the Langerhans cell granule. The Journal of cell biology 35, 468 (Nov, 1967).

134. R. McDermott et al., Reproduction of Langerin/CD207 traffic and Birbeck granule formation in a human cell line model. The Journal of investigative dermatology 123, 72 (Jul, 2004).

75

Acknowledgements

63

135. T. Kawamura et al., Candidate microbicides block HIV-‐1 infection of human immature Langerhans cells within epithelial tissue explants. The Journal of experimental medicine 192, 1491 (Nov 20, 2000).

136. N. Kadowaki et al., Subsets of human dendritic cell precursors express different toll-‐like receptors and respond to different microbial antigens. The Journal of experimental medicine 194, 863 (Sep 17, 2001).

137. C. Caux et al., CD34+ hematopoietic progenitors from human cord blood differentiate along two independent dendritic cell pathways in response to granulocyte-‐macrophage colony-‐stimulating factor plus tumor necrosis factor alpha: II. Functional analysis. Blood 90, 1458 (Aug 15, 1997).

138. T. B. Geijtenbeek et al., Identification of DC-‐SIGN, a novel dendritic cell-‐specific ICAM-‐3 receptor that supports primary immune responses. Cell 100, 575 (Mar 3, 2000).

139. L. Wu, V. N. KewalRamani, Dendritic-‐cell interactions with HIV: infection and viral dissemination. Nature reviews. Immunology 6, 859 (Nov, 2006).

140. V. Holl et al., Efficient inhibition of HIV-‐1 replication in human immature monocyte-‐derived dendritic cells by purified anti-‐HIV-‐1 IgG without induction of maturation. Blood 107, 4466 (Jun 1, 2006).

141. A. J. Engering et al., The mannose receptor functions as a high capacity and broad specificity antigen receptor in human dendritic cells. European journal of immunology 27, 2417 (Sep, 1997).

142. M. C. Tan et al., Mannose receptor-‐mediated uptake of antigens strongly enhances HLA class II-‐restricted antigen presentation by cultured dendritic cells. European journal of immunology 27, 2426 (Sep, 1997).

143. T. I. Prigozy et al., The mannose receptor delivers lipoglycan antigens to endosomes for presentation to T cells by CD1b molecules. Immunity 6, 187 (Feb, 1997).

144. A. Engering, T. B. Geijtenbeek, Y. van Kooyk, Immune escape through C-‐type lectins on dendritic cells. Trends in immunology 23, 480 (Oct, 2002).

145. T. B. Geijtenbeek et al., DC-‐SIGN, a dendritic cell-‐specific HIV-‐1-‐binding protein that enhances trans-‐infection of T cells. Cell 100, 587 (Mar 3, 2000).

146. S. G. Turville et al., Diversity of receptors binding HIV on dendritic cell subsets. Nature immunology 3, 975 (Oct, 2002).

147. Q. Sattentau, HIV's gut feeling. Nature immunology 9, 225 (Mar, 2008). 148. J. Arthos et al., HIV-‐1 envelope protein binds to and signals through integrin

alpha4beta7, the gut mucosal homing receptor for peripheral T cells. Nature immunology 9, 301 (Mar, 2008).

149. A. Granelli-‐Piperno et al., Efficient interaction of HIV-‐1 with purified dendritic cells via multiple chemokine coreceptors. The Journal of experimental medicine 184, 2433 (Dec 1, 1996).

150. S. Ayehunie et al., Human immunodeficiency virus-‐1 entry into purified blood dendritic cells through CC and CXC chemokine coreceptors. Blood 90, 1379 (Aug 15, 1997).

151. I. M. Klagge, S. Schneider-‐Schaulies, Virus interactions with dendritic cells. The Journal of general virology 80 ( Pt 4), 823 (Apr, 1999).

152. V. Piguet, R. M. Steinman, The interaction of HIV with dendritic cells: outcomes and pathways. Trends in immunology 28, 503 (Nov, 2007).

153. C. Dong, A. M. Janas, J. H. Wang, W. J. Olson, L. Wu, Characterization of human immunodeficiency virus type 1 replication in immature and mature dendritic cells reveals dissociable cis-‐ and trans-‐infection. Journal of virology 81, 11352 (Oct, 2007).

154. J. H. Wang, A. M. Janas, W. J. Olson, L. Wu, Functionally distinct transmission of human immunodeficiency virus type 1 mediated by immature and mature dendritic cells. Journal of virology 81, 8933 (Sep, 2007).

155. A. Engering, S. J. Van Vliet, T. B. Geijtenbeek, Y. Van Kooyk, Subset of DC-‐SIGN(+) dendritic cells in human blood transmits HIV-‐1 to T lymphocytes. Blood 100, 1780 (Sep 1, 2002).

76

Acknowledgements

64

156. B. Lee et al., cis Expression of DC-‐SIGN allows for more efficient entry of human and simian immunodeficiency viruses via CD4 and a coreceptor. Journal of virology 75, 12028 (Dec, 2001).

157. D. S. Kwon, G. Gregorio, N. Bitton, W. A. Hendrickson, D. R. Littman, DC-‐SIGN-‐mediated internalization of HIV is required for trans-‐enhancement of T cell infection. Immunity 16, 135 (Jan, 2002).

158. D. SenGupta et al., Strong human endogenous retrovirus-‐specific T cell responses are associated with control of HIV-‐1 in chronic infection. Journal of virology 85, 6977 (Jul, 2011).

159. M. Stremlau et al., Specific recognition and accelerated uncoating of retroviral capsids by the TRIM5alpha restriction factor. Proceedings of the National Academy of Sciences of the United States of America 103, 5514 (Apr 4, 2006).

160. K. L. Martin, M. Johnson, R. T. D'Aquila, APOBEC3G complexes decrease human immunodeficiency virus type 1 production. Journal of virology 85, 9314 (Sep, 2011).

161. Y. H. Zheng et al., Human APOBEC3F is another host factor that blocks human immunodeficiency virus type 1 replication. Journal of virology 78, 6073 (Jun, 2004).

162. K. N. Bishop et al., Cytidine deamination of retroviral DNA by diverse APOBEC proteins. Current biology : CB 14, 1392 (Aug 10, 2004).

163. M. T. Liddament, W. L. Brown, A. J. Schumacher, R. S. Harris, APOBEC3F properties and hypermutation preferences indicate activity against HIV-‐1 in vivo. Current biology : CB 14, 1385 (Aug 10, 2004).

164. H. L. Wiegand, B. P. Doehle, H. P. Bogerd, B. R. Cullen, A second human antiretroviral factor, APOBEC3F, is suppressed by the HIV-‐1 and HIV-‐2 Vif proteins. The EMBO journal 23, 2451 (Jun 16, 2004).

165. D. Wolf, S. P. Goff, Host restriction factors blocking retroviral replication. Annual review of genetics 42, 143 (2008).

166. J. Martin-‐Serrano, S. J. Neil, Host factors involved in retroviral budding and release. Nature reviews. Microbiology 9, 519 (Jul, 2011).

167. D. T. Evans, R. Serra-‐Moreno, R. K. Singh, J. C. Guatelli, BST-‐2/tetherin: a new component of the innate immune response to enveloped viruses. Trends in microbiology 18, 388 (Sep, 2010).

168. E. S. Lim, M. Emerman, HIV: Going for the watchman. Nature 474, 587 (Jun 30, 2011). 169. L. J. Young et al., Dendritic cell preactivation impairs MHC class II presentation of

vaccines and endogenous viral antigens. Proceedings of the National Academy of Sciences of the United States of America 104, 17753 (Nov 6, 2007).

170. M. J. Bevan, Cross-‐priming. Nature immunology 7, 363 (Apr, 2006). 171. M. L. Albert, B. Sauter, N. Bhardwaj, Dendritic cells acquire antigen from apoptotic cells

and induce class I-‐restricted CTLs. Nature 392, 86 (Mar 5, 1998). 172. M. Larsson, J. F. Fonteneau, N. Bhardwaj, Dendritic cells resurrect antigens from dead

cells. Trends in immunology 22, 141 (Mar, 2001). 173. W. R. Heath et al., Cross-‐presentation, dendritic cell subsets, and the generation of

immunity to cellular antigens. Immunological reviews 199, 9 (Jun, 2004). 174. A. Y. Huang et al., Role of bone marrow-‐derived cells in presenting MHC class I-‐restricted

tumor antigens. Science 264, 961 (May 13, 1994). 175. M. J. Bevan, Cross-‐priming for a secondary cytotoxic response to minor H antigens with

H-‐2 congenic cells which do not cross-‐react in the cytotoxic assay. The Journal of experimental medicine 143, 1283 (May 1, 1976).

176. F. Buseyne et al., MHC-‐I-‐restricted presentation of HIV-‐1 virion antigens without viral replication. Nature medicine 7, 344 (Mar, 2001).

177. P. Guermonprez, S. Amigorena, Pathways for antigen cross presentation. Springer Semin Immunopathol 26, 257 (Jan, 2005).

178. M. Gromme et al., Recycling MHC class I molecules and endosomal peptide loading. Proceedings of the National Academy of Sciences of the United States of America 96, 10326 (Aug 31, 1999).

77

Acknowledgements

65

179. M. Larsson et al., Activation of HIV-‐1 specific CD4 and CD8 T cells by human dendritic cells: roles for cross-‐presentation and non-‐infectious HIV-‐1 virus. AIDS 16, 1319 (Jul 5, 2002).

180. L. M. Wakim, M. J. Bevan, Cross-‐dressed dendritic cells drive memory CD8+ T-‐cell activation after viral infection. Nature 471, 629 (Mar 31, 2011).

181. C. Stemberger et al., A single naive CD8+ T cell precursor can develop into diverse effector and memory subsets. Immunity 27, 985 (Dec, 2007).

182. R. L. Sabado et al., Pathways utilized by dendritic cells for binding, uptake, processing and presentation of antigens derived from HIV-‐1. European journal of immunology 37, 1752 (Jul, 2007).

183. A. Moris et al., DC-‐SIGN promotes exogenous MHC-‐I-‐restricted HIV-‐1 antigen presentation. Blood 103, 2648 (Apr 1, 2004).

184. A. Ciechanover, Proteolysis: from the lysosome to ubiquitin and the proteasome. Nature reviews. Molecular cell biology 6, 79 (Jan, 2005).

185. L. Huang, J. M. Marvin, N. Tatsis, L. C. Eisenlohr, Selective role of ubiquitin in MHC class I antigen presentation. J Immunol 186, 1904 (Feb 15, 2011).

186. D. Voges, P. Zwickl, W. Baumeister, The 26S proteasome: a molecular machine designed for controlled proteolysis. Annual review of biochemistry 68, 1015 (1999).

187. O. Kerscher, R. Felberbaum, M. Hochstrasser, Modification of proteins by ubiquitin and ubiquitin-‐like proteins. Annual review of cell and developmental biology 22, 159 (2006).

188. K. J. Walters, A. M. Goh, Q. Wang, G. Wagner, P. M. Howley, Ubiquitin family proteins and their relationship to the proteasome: a structural perspective. Biochimica et biophysica acta 1695, 73 (Nov 29, 2004).

189. R. L. Welchman, C. Gordon, R. J. Mayer, Ubiquitin and ubiquitin-‐like proteins as multifunctional signals. Nature reviews. Molecular cell biology 6, 599 (Aug, 2005).

190. S. Elsasser et al., Proteasome subunit Rpn1 binds ubiquitin-‐like protein domains. Nature cell biology 4, 725 (Sep, 2002).

191. J. Herrmann, L. O. Lerman, A. Lerman, Ubiquitin and ubiquitin-‐like proteins in protein regulation. Circulation research 100, 1276 (May 11, 2007).

192. D. Zhang, D. E. Zhang, Interferon-‐stimulated gene 15 and the protein ISGylation system. Journal of interferon & cytokine research : the official journal of the International Society for Interferon and Cytokine Research 31, 119 (Jan, 2011).

193. J. A. Hamerman et al., Serpin 2a is induced in activated macrophages and conjugates to a ubiquitin homolog. J Immunol 168, 2415 (Mar 1, 2002).

194. M. P. Malakhov et al., High-‐throughput immunoblotting. Ubiquitiin-‐like protein ISG15 modifies key regulators of signal transduction. The Journal of biological chemistry 278, 16608 (May 9, 2003).

195. M. Liu, X. L. Li, B. A. Hassel, Proteasomes modulate conjugation to the ubiquitin-‐like protein, ISG15. The Journal of biological chemistry 278, 1594 (Jan 17, 2003).

196. A. Okumura, G. Lu, I. Pitha-‐Rowe, P. M. Pitha, Innate antiviral response targets HIV-‐1 release by the induction of ubiquitin-‐like protein ISG15. Proceedings of the National Academy of Sciences of the United States of America 103, 1440 (Jan 31, 2006).

197. B. Skaug, Z. J. Chen, Emerging role of ISG15 in antiviral immunity. Cell 143, 187 (Oct 15, 2010).

198. T. Tanaka, H. Kawashima, E. T. Yeh, T. Kamitani, Regulation of the NEDD8 conjugation system by a splicing variant, NUB1L. The Journal of biological chemistry 278, 32905 (Aug 29, 2003).

199. K. Tanji, T. Tanaka, T. Kamitani, Interaction of NUB1 with the proteasome subunit S5a. Biochemical and biophysical research communications 337, 116 (Nov 11, 2005).

200. O. Schwartz, V. Marechal, B. Friguet, F. Arenzana-‐Seisdedos, J. M. Heard, Antiviral activity of the proteasome on incoming human immunodeficiency virus type 1. Journal of virology 72, 3845 (May, 1998).

78

Acknowledgements

66

201. C. Boutell, S. Sadis, R. D. Everett, Herpes simplex virus type 1 immediate-‐early protein ICP0 and is isolated RING finger domain act as ubiquitin E3 ligases in vitro. Journal of virology 76, 841 (Jan, 2002).

202. Y. Yu, S. E. Wang, G. S. Hayward, The KSHV immediate-‐early transcription factor RTA encodes ubiquitin E3 ligase activity that targets IRF7 for proteosome-‐mediated degradation. Immunity 22, 59 (Jan, 2005).

203. X. Yu et al., Induction of APOBEC3G ubiquitination and degradation by an HIV-‐1 Vif-‐Cul5-‐SCF complex. Science 302, 1056 (Nov 7, 2003).

204. H. Zhang et al., The cytidine deaminase CEM15 induces hypermutation in newly synthesized HIV-‐1 DNA. Nature 424, 94 (Jul 3, 2003).

205. S. Dussart et al., The Vif protein of human immunodeficiency virus type 1 is posttranslationally modified by ubiquitin. Biochemical and biophysical research communications 315, 66 (Feb 27, 2004).

206. V. M. Vogt, Ubiquitin in retrovirus assembly: actor or bystander? Proceedings of the National Academy of Sciences of the United States of America 97, 12945 (Nov 21, 2000).

207. B. Guillaume et al., Two abundant proteasome subtypes that uniquely process some antigens presented by HLA class I molecules. Proceedings of the National Academy of Sciences of the United States of America 107, 18599 (Oct 26, 2010).

208. S. Elsasser, D. Chandler-‐Militello, B. Muller, J. Hanna, D. Finley, Rad23 and Rpn10 serve as alternative ubiquitin receptors for the proteasome. The Journal of biological chemistry 279, 26817 (Jun 25, 2004).

209. B. J. Van den Eynde, S. Morel, Differential processing of class-‐I-‐restricted epitopes by the standard proteasome and the immunoproteasome. Current opinion in immunology 13, 147 (Apr, 2001).

210. P. M. Kloetzel, F. Ossendorp, Proteasome and peptidase function in MHC-‐class-‐I-‐mediated antigen presentation. Current opinion in immunology 16, 76 (Feb, 2004).

211. A. Macagno et al., Dendritic cells up-‐regulate immunoproteasomes and the proteasome regulator PA28 during maturation. European journal of immunology 29, 4037 (Dec, 1999).

212. U. Seifert et al., An essential role for tripeptidyl peptidase in the generation of an MHC class I epitope. Nature immunology 4, 375 (Apr, 2003).

213. D. H. Margulies, Antigen-‐processing and presentation pathways select antigenic HIV peptides in the fight against viral evolution. Nature immunology 10, 566 (Jun, 2009).

214. E. S. Song, Y. Yang, M. R. Jackson, P. A. Peterson, In vivo regulation of the assembly and intracellular transport of class I major histocompatibility complex molecules. The Journal of biological chemistry 269, 7024 (Mar 4, 1994).

215. L. Y. Hwang, P. T. Lieu, P. A. Peterson, Y. Yang, Functional regulation of immunoproteasomes and transporter associated with antigen processing. Immunologic research 24, 245 (2001).

216. T. Ravid, M. Hochstrasser, Diversity of degradation signals in the ubiquitin-‐proteasome system. Nature reviews. Molecular cell biology 9, 679 (Sep, 2008).

217. C. Muratori et al., DC contact with HIV-‐1-‐infected cells leads to high levels of Env-‐mediated virion endocytosis coupled with enhanced HIV-‐1 Ag presentation. European journal of immunology 39, 404 (Feb, 2009).

218. A. Moris et al., Dendritic cells and HIV-‐specific CD4+ T cells: HIV antigen presentation, T-‐cell activation, and viral transfer. Blood 108, 1643 (Sep 1, 2006).

219. I. Frank et al., Infectious and whole inactivated simian immunodeficiency viruses interact similarly with primate dendritic cells (DCs): differential intracellular fate of virions in mature and immature DCs. Journal of virology 76, 2936 (Mar, 2002).

220. A. N. Harman et al., HIV-‐1-‐infected dendritic cells show 2 phases of gene expression changes, with lysosomal enzyme activity decreased during the second phase. Blood 114, 85 (Jul 2, 2009).

79

Acknowledgements

67

221. T. Zavasnik-‐Bergant, B. Turk, Cysteine proteases: destruction ability versus immunomodulation capacity in immune cells. Biological chemistry 388, 1141 (Nov, 2007).

222. J. A. Villadangos et al., MHC class II expression is regulated in dendritic cells independently of invariant chain degradation. Immunity 14, 739 (Jun, 2001).

223. O. J. Landsverk, O. Bakke, T. F. Gregers, MHC II and the endocytic pathway: regulation by invariant chain. Scandinavian journal of immunology 70, 184 (Sep, 2009).

224. P. Borrow, R. J. Shattock, A. Vyakarnam, Innate immunity against HIV: a priority target for HIV prevention research. Retrovirology 7, 84 (2010).

225. H. Nakashima, N. Yamamoto, M. Masuda, N. Fujii, Defensins inhibit HIV replication in vitro. AIDS 7, 1129 (Aug, 1993).

226. C. R. Wira, J. V. Fahey, The innate immune system: gatekeeper to the female reproductive tract. Immunology 111, 13 (Jan, 2004).

227. C. R. Wira et al., Sex hormone regulation of innate immunity in the female reproductive tract: the role of epithelial cells in balancing reproductive potential with protection against sexually transmitted pathogens. American journal of reproductive immunology 63, 544 (Jun, 2010).

228. S. M. Smith et al., Topical estrogen protects against SIV vaginal transmission without evidence of systemic effect. AIDS 18, 1637 (Aug 20, 2004).

229. S. M. Smith, G. B. Baskin, P. A. Marx, Estrogen protects against vaginal transmission of simian immunodeficiency virus. The Journal of infectious diseases 182, 708 (Sep, 2000).

230. E. M. Stringer et al., A randomized trial of the intrauterine contraceptive device vs hormonal contraception in women who are infected with the human immunodeficiency virus. American journal of obstetrics and gynecology 197, 144 e1 (Aug, 2007).

231. A. Iwasaki, Mucosal dendritic cells. Annual review of immunology 25, 381 (2007). 232. A. C. Soloff, S. M. Barratt-‐Boyes, Enemy at the gates: dendritic cells and immunity to

mucosal pathogens. Cell research 20, 872 (Aug, 2010). 233. J. Hu, M. B. Gardner, C. J. Miller, Simian immunodeficiency virus rapidly penetrates the

cervicovaginal mucosa after intravaginal inoculation and infects intraepithelial dendritic cells. Journal of virology 74, 6087 (Jul, 2000).

234. K. M. Fahrbach, S. M. Barry, M. R. Anderson, T. J. Hope, Enhanced cellular responses and environmental sampling within inner foreskin explants: implications for the foreskin's role in HIV transmission. Mucosal immunology 3, 410 (Jul, 2010).

235. L. Ballweber et al., Vaginal Langerhans cells non-‐productively transporting HIV-‐1 mediate infection of T cells. Journal of virology, (Oct 5, 2011).

236. D. M. Phillips, X. Tan, M. E. Perotti, V. R. Zacharopoulos, Mechanism of monocyte-‐macrophage-‐mediated transmission of HIV. AIDS research and human retroviruses 14 Suppl 1, S67 (Apr, 1998).

237. R. Shen et al., GP41-‐specific antibody blocks cell-‐free HIV type 1 transcytosis through human rectal mucosa and model colonic epithelium. J Immunol 184, 3648 (Apr 1, 2010).

238. F. Hladik et al., Initial events in establishing vaginal entry and infection by human immunodeficiency virus type-‐1. Immunity 26, 257 (Feb, 2007).

239. V. Tjomsland et al., Blocking of integrins significantly inhibits HIV-‐1 infection of human cervical mucosa immune cells and development of founder populations. Submitted, (2011).

240. S. G. Turville et al., Immunodeficiency virus uptake, turnover, and 2-‐phase transfer in human dendritic cells. Blood 103, 2170 (Mar 15, 2004).

241. V. Piguet, Q. Sattentau, Dangerous liaisons at the virological synapse. The Journal of clinical investigation 114, 605 (Sep, 2004).

242. M. Sagar et al., Selection of HIV variants with signature genotypic characteristics during heterosexual transmission. The Journal of infectious diseases 199, 580 (Feb 15, 2009).

243. B. F. Keele et al., Identification and characterization of transmitted and early founder virus envelopes in primary HIV-‐1 infection. Proceedings of the National Academy of Sciences of the United States of America 105, 7552 (May 27, 2008).

80

Acknowledgements

68

244. M. K. Norvell, G. I. Benrubi, R. J. Thompson, Investigation of microtrauma after sexual intercourse. The Journal of reproductive medicine 29, 269 (Apr, 1984).

245. P. J. Klasse, R. Shattock, J. P. Moore, Antiretroviral drug-‐based microbicides to prevent HIV-‐1 sexual transmission. Annual review of medicine 59, 455 (2008).

246. M. Pope, A. T. Haase, Transmission, acute HIV-‐1 infection and the quest for strategies to prevent infection. Nature medicine 9, 847 (Jul, 2003).

247. J. L. Rossio et al., Inactivation of human immunodeficiency virus type 1 infectivity with preservation of conformational and functional integrity of virion surface proteins. Journal of virology 72, 7992 (Oct, 1998).

248. H. Bouhlal et al., Opsonization of HIV-‐1 by semen complement enhances infection of human epithelial cells. J Immunol 169, 3301 (Sep 15, 2002).

249. M. Subklewe et al., Presentation of epstein-‐barr virus latency antigens to CD8(+), interferon-‐gamma-‐secreting, T lymphocytes. European journal of immunology 29, 3995 (Dec, 1999).

250. M. Larsson, HIV-‐1 and the hijacking of dendritic cells: a tug of war. Springer Semin Immunopathol 26, 309 (Jan, 2005).

251. D. C. Douek et al., HIV preferentially infects HIV-‐specific CD4+ T cells. Nature 417, 95 (May 2, 2002).

252. M. Gromme, J. Neefjes, Antigen degradation or presentation by MHC class I molecules via classical and non-‐classical pathways. Molecular immunology 39, 181 (Oct, 2002).

253. K. Miyauchi, Y. Kim, O. Latinovic, V. Morozov, G. B. Melikyan, HIV enters cells via endocytosis and dynamin-‐dependent fusion with endosomes. Cell 137, 433 (May 1, 2009).

254. L. A. van Es, M. R. Daha, Factors influencing the endocytosis of immune complexes. Adv Nephrol Necker Hosp 13, 341 (1984).

255. D. Wilflingseder et al., IgG opsonization of HIV impedes provirus formation in and infection of dendritic cells and subsequent long-‐term transfer to T cells. J Immunol 178, 7840 (Jun 15, 2007).

256. M. Firoz Mian, A. A. Ashkar, Induction of innate immune responses in the female genital tract: friend or foe of HIV-‐1 infection? American journal of reproductive immunology 65, 344 (Mar, 2011).

257. L. Wu, Biology of HIV mucosal transmission. Current opinion in HIV and AIDS 3, 534 (Sep, 2008).

258. H. Bouhlal et al., Opsonization of HIV with complement enhances infection of dendritic cells and viral transfer to CD4 T cells in a CR3 and DC-‐SIGN-‐dependent manner. J Immunol 178, 1086 (Jan 15, 2007).

259. V. Tjomsland et al., Complement opsonization of HIV-‐1 enhances the uptake by dendritic cells and involves the endocytic lectin and integrin receptor families. PLoS ONE 6, e23542 (2011).

260. Z. Zhang et al., Sexual transmission and propagation of SIV and HIV in resting and activated CD4+ T cells. Science 286, 1353 (Nov 12, 1999).

261. A. C. Saphire, M. D. Bobardt, Z. Zhang, G. David, P. A. Gallay, Syndecans serve as attachment receptors for human immunodeficiency virus type 1 on macrophages. Journal of virology 75, 9187 (Oct, 2001).

81

Complement activation-‐ good or evil in HIV-‐1 infection?

Documents

Transcript of Complement activation-‐ good or evil in HIV-‐1 infection?