UTY-specific TCR-transfer generates potential graft-versus-leukaemia effector T cells

In recent years donor lymphocyte infusions (DLIs) have gained

acceptance as the most successful approach to the treatment of

relapsed lymphoid malignancies (Barrett, 2003; Sureda &

Schmitz, 2003; Kolb et al, 2004). The key therapeutic mech-

anism of the DLI is an immune response of donor

T lymphocytes against leukaemic cells of a patient. This

beneficial graft-versus-leukaemia (GVL) effect is accompanied

by the T cell reactivity towards healthy tissues that frequently

causes graft-versus-host disease (GVHD). Although GVL and

GVHD are closely related, numerous observations have been

made that suggest a distinct antigenic basis of these two DLI

effects (Jiang et al, 1997; Michalek et al, 2003). If true, this may

permit the development of strategies that augment GVL

without exacerbation of GVHD. Obviously, this would signi-

ficantly improve the clinical results of DLI therapy. Therefore,

the identification of antigens that can induce T cell responses

that specifically target leukaemic cells is a major goal of

tumour immunologists. The adoptive transfer of T cells that

are genetically engineered to react against such leukaemia-

specific antigens is a new and promising treatment modality

(Stanislawski et al, 2001).

In a human leucocyte antigen (HLA)-identical setting, T cell

responses are directed against minor histocompatibility anti-

gens (mHags) – products of polymorphic proteins, which are

different between a patient and a donor. H–Y antigens

represent a separate class of mHags. These antigens are

encoded by genes located on the male Y chromosome and are

therefore absent in females. Recent observations have estab-

lished that male recipients of female haematopoietic stem cells

have the lowest risk of relapse in comparison with other

patients and that this phenomenon is independent of the

concomitant GVHD (Randolph et al, 2004). Presumably, this

selective GVL effect is mediated by T cells recognizing certain

H–Y antigens on leukaemic cells. Although no H–Y genes with

a haematopoietic-restricted expression have been found

(Skaletsky et al, 2003), the preferential immune recognition

Roman Ivanov,1 Samantha Hol,1 Tineke

Aarts,1 Anton Hagenbeek,1 Elisabeth H.

Slager2 and Saskia Ebeling1

1Jordan Laboratory for Haemato-Oncology,

Department of Haematology, University Medical

Centre Utrecht, Heidelberglaan 100, Utrecht, the

Netherlands, and 2Department of Haematology,

Leiden University Medical Centre, PO Box 9600,

Leiden, the Netherlands

Received 26 December 2004; accepted for

publication 21 February 2005

Correspondence: Saskia Ebeling, PhD,

Department of Haematology, University

Medical Centre Utrecht Heidelberglaan 100,

3584 CX Utrecht, the Netherlands.

E-mail: s.ebeling@azu.nl

Summary

Immunotherapeutic approaches that target antigens that are differentially

recognized on haematopoietic and non-haematopoietic cells may specifically

enhance the graft-versus-leukaemia (GVL) effect of donor lymphocyte

infusion. In this study, we have characterized a new HLA-B*5201-restricted

epitope of the UTY gene. Unusually, presentation of this epitope was

restricted to lymphoblasts. As a result, a T cell clone specific to this epitope

recognized normal and malignant male B and T lymphoblasts, while showing

little reactivity towards male HLA-B*5201+ fibroblasts. Transfer of its T cell

receptor (TCR) into donor T cells led to the generation of large numbers of

T cells, which acquired the specificity of the original clone, its avidity and the

differential pattern of reactivity towards lymphoblasts and fibroblasts.

Remarkably, the specific response of TCR-transferred T cells was

significantly higher than that of the original clone. This is the first

demonstration of the possibility to preserve the specific pattern of a T cell

response to a differentially expressed antigen after TCR-transfer and to

augment the amplitude of this response concomitantly. These results indicate

that it may be feasible to enhance the GVL effect of donor lymphocyte

infusions in lymphoproliferative malignancies by the transfer of TCRs specific

to epitopes that are differentially recognized on lymphoblasts.

Keywords: H–Y antigens, T cell receptor-transfer, graft-versus-leukaemia,

immunotherapy, donor lymphocyte infusion.

research paper

doi:10.1111/j.1365-2141.2005.05461.x ª 2005 Blackwell Publishing Ltd, British Journal of Haematology, 129, 392–402

of leukaemic cells may be caused by overexpression or altered

processing of some H–Y antigens.

In an attempt to reveal the nature of the GVL response in

sex-mismatched settings, we stimulated female T cells with

haematopoietic cells from a male patient. A number of T cell

clones were generated that recognized Epstein–Barr virus

(EBV)-transformed B cells (EBV-LCL) of patient, but not

donor, origin, while showing no significant reactivity towards

resting haematopoietic cells and fibroblasts. Detailed analysis

of the specificity of one of these clones led to the identification

of a new cytotoxic T lymphocyte (CTL) epitope of the UTY

gene as a target of an HLA-B*5201-restricted lymphoblast-

specific CTL response. Notably, HLA-B8- and B60-restricted

epitopes of the UTY gene have been reported previously (Vogt

et al, 2000; Warren et al, 2000) and the leucoblast-specific

recognition of HLA-B8-restricted epitope has been demon-

strated (Warren et al, 2000). The results of this study indicate

that the new epitope of UTY can be considered as a leukaemia-

associated antigen because of its predominant presentation on

normal and malignant lymphoblasts. Furthermore, the feasi-

bility to generate large numbers of lymphoblast-specific CTL

through retroviral transfer of the UTY-specific T cell receptor

(TCR) was shown.

Materials and methods

Cell culture

The generation and culture of the YKII.39 T cell clone were

performed according to the previously described protocol

(Ebeling et al, 2003). Activation and expansion of B cells were

carried out using the 293-CD40-sCD40L system (Ivanov et al,

2005a). Briefly, B cells were expanded from peripheral blood

mononuclear cells (PBMC) by incubation with 1Æ2 · 104

irradiated (75 Gy) 293 cells expressing both membrane-bound

and soluble CD40L in the presence of 10 ng/ml interleukin

(IL)-10 (Peprotech, London, UK) and 200 U/ml IL-4 (Pepro-

tech). B and T cells were cultured in Roswell Park Memorial

Institute (RPMI) 1640 medium (Invitrogen, Carlsbad, CA,

USA) supplemented with 10% heat-inactivated pooled human

AB serum, 100 U/ml penicillin (Gibco, Gaithersburg, MD,

USA), 100 lg/ml streptomycin (Gibco) and 5 · 10)5 mol/l

b-mercaptoethanol (Merck, Haarlem, the Netherlands) (here-

after referred to as PSb). B cells were harvested every 3–4 d and

plated again at 6 · 104 B cells per well in a 24-well plate (Nunc,

Rochester, NY, USA), with addition of irradiated 293-CD40-

sCD40L cells, IL-4 and IL-10, as described above. Fibroblasts,

the amphotropic Phoenix packaging cell line and 293-EBNA-

B7 cell line were cultured in Dulbecco’s modified Eagles

medium (DMEM) (Invitrogen), supplemented with 10% heat-

inactivated fetal calf serum (FCS) (Integro BV, Leuvenheim,

the Netherlands) and PSb. All other cell lines were cultured in

RPMI 1640 medium (Invitrogen) with 10% FCS and PSb. Insome cases, cell lines were cultured in presence of 5 ng/ml

interferon-c (IFN-c; Peprotech) and 200 U/ml tumour

necrosis factor-a (TNF-a; R & D Systems, Minneapolis, MN,

USA) for 2 d before the test.

Cloning of HLA-B molecules and transduction of cell lines

RNA was isolated from the EBV-LCL of patient origin (EBVp)

with Trizol reagent (Invitrogen). Reverse transcription polym-

erase chain reaction (RT-PCR) was performed with HLA-B-

specific primers 5¢-GGGGTCGACATGCGGGTCAC-3¢ and

5¢-GGGGCGGCCGCTCAAGCTGTGAGAGA-3¢. The PCR

product was purified with the Qiagen Gel Extraction kit

(Qiagen, Hilden, Germany) and digested with Sal I and Not I

restriction endonucleases (New England Biolabs, Beverly, MA,

USA). The resulting fragment was subcloned in the pEGFP-N1

vector (Clontech, Palo Alto, CA, USA) and sequenced with the

BigDye Terminator v3Æ1 Cycle Sequencing Kit (Applied

Biosystems, Foster City, CA, USA). Subsequently, HLA-

B*3508 and HLA-B*5201 were cloned separately into the

retroviral pMX vector (Onishi et al, 1996) upstream of the

internal ribosomal entry sequence (IRES) and either the nerve

growth factor receptor (NGFR) or enhanced green fluorescent

protein (EGFP) marker genes.

Resulting vectors were transfected into the amphotropic

Phoenix packaging cell line with the calcium-phosphate preci-

pitationmethod (Qiagen). Viral supernatants were harvested on

the second and third days after transfection. 293-EBNA-B7 cells

were incubated for 24 h with the 1:3 diluted viral supernatant in

the presence of 6 lg/ml polybrene (Sigma-Aldrich, Steinheim,

Germany). On the third day after transduction, NGFR-positive

cells were purified by means of the MiniMACSTM separation

method (Myltenyi Biotech, Bergisch Gladbach, Germany) using

anti-NGFR antibody as described elsewhere (Weijtens et al,

2002). Fibroblasts were transduced in the same way. EGFP-

positive fibroblasts were sorted by fluorescence-activated cell

sorting (FACS). Phytohaemagglutinin (PHA) blasts and EBV-

LCLs were transduced in untreated flasks (Becton Dickinson,

Franklin Lakes, NJ, USA) coated with 12Æ5 lg/ml retronectin

(Takara, Otsu, Shiga, Japan). Cells were added at a concentra-

tion of 1 · 106 cells/ml in the culture medium, supplemented

with 300 IU/ml IL-2 (Proleukin, Chiron, Amsterdam, the

Netherlands) in case of PHA blasts. Fresh viral supernatant

was added on the second day. On the third day cells were

harvested and resuspended in freshmediumwith addition of the

corresponding cytokines. Two days after harvesting the fraction

of transduced cells was determined by flow cytometry.

Construction of mini-genes

The plasmid containing the full-length UTY sequence was used

as a template for PCR with a common sense primer

5¢-GAAGGTACCATGAAATCCTGCGCAGTG-3¢ and one of

antisense primers containing a TAA stop codon in frame with

the main open reading frame: minigene 1–334 – 5¢-TA-AGCGGCCGCTTAAGCATCCATAGGCTG-3¢; minigene 1–408 –

5¢-TAAGCGGCCGCTTATGAAAGACTCTGGCC-3¢; minigene

Generation of GVL effector T cells through TCR-transfer

ª 2005 Blackwell Publishing Ltd, British Journal of Haematology, 129, 392–402 393

1–413 – 5¢-TAAGCGGCCGCTTACTGTACTGGATGATG-3¢;minigene 1–426 – 5¢-TAAGCGGCCGCTTACTGTAATTTCT-GTGG-3¢; minigene 1–443 – 5¢-TAAGCGGCCGCTTACTTC-TGTGCTGGATT-3¢; minigene 1–453 – 5¢-TAAGCGGCCGC-TTAAAACTGACTTTCTAA-3¢; minigene 1–468 – 5¢-TAAGC-GGCCGCTTATCGTACCTGAGCAAC-3¢; minigene 1–528 –

5¢-TAAGCGGCCGCTTAACAAGGAATACAGCC-3¢; minigene

1–596 – 5¢-TAAGCGGCCGCTTACTGACTTTTATGAAG-3¢;minigene 1–667 – 5¢-TAAGCGGCCGCTTAGCTCTCTTT-GGTAAA-3¢. These PCR products were digested with KpnI

and NotI and cloned into the pCEP4 vector. The resulting

constructs contained truncated UTY genes lacking different

parts of the carboxyl terminus of UTY.

Transfection of the 293 cell line

293-EBNA-B7-B*5201 cells, plated in flat-bottom 96-well

plates (4 · 104 cells per well) 24 h in advance, were transfected

in duplicates with 1Æ2 ll Lipofectamine (Invitrogen) and

100 ng of the plasmid DNA. After 24 h, the culture medium

was substituted for 200 ll of RPMI 1640 with 10% HS and

5 · 103 T cells of the YKII.39 clone per well. After another 24 h

culture supernatant was harvested and IFN-c production was

measured by enzyme-linked immunosorbent assay (ELISA).

Peptide recognition assays

Candidate peptides were synthesized by solid phase peptide

synthesis and characterized by mass spectrometry (Pepscan

Systems, Lelystad, the Netherlands). Cells were washed twice

with RPMI 1640 medium supplemented with 2% FCS and

incubated overnight in serum-free RPMI 1640 with candidate

peptides at 10)5 mol/l concentration, if not mentioned oth-

erwise. The following day peptide-loaded cells were tested for

their ability to induce IFN-c production by YKII.39.

Chromium release assay

The chromium release assay was performed according to the

previously described protocol (Ebeling et al, 2003) with minor

modifications. Briefly, 2500 labelled target cells per well were

seeded in triplicate in 96-well round-bottomed plates (Costar,

Cambridge, MA, USA) in 200 ll of RPMI 1640 medium

supplemented with 5% FCS. Thereafter, target cells were

incubated for 4 h at 37�C with T cells at different effector to

target cell ratios. Supernatants were collected using the

Supernatant Harvesting System (Molecular Devices Corpora-

tion, Sunnyvale, CA, USA) and radioactivity was measured

with a Cobra autogamma betaplate reader (Packard, Gronin-

gen, the Netherlands).

IFN-c production assay

3 · 104 target cells were co-cultured with 3 · 104 T cells in

96-well round-bottomed plates in triplicates. After 24 h the

supernatant was harvested and IFN-c concentration was

measured by the PeliPair human IFN-c ELISA reagent set

(CLB, Amsterdam, the Netherlands), according to the manu-

facturer’s instructions.

Flow cytometric determination of cell surface markers

Flow cytometry was performed on a Calibur flow cytometer

(BD Biosciences, San Jose, CA, USA). Monoclonal antibodies

used for the flow cytometry were purchased from BD

Biosciences, except 20Æ4 anti-NGFR antibody (culture super-

natant) and goat-anti-mouse immunoglobulin (Ig) phyco-

erythrin (PE)-conjugated antibody (SBA, Birmingham, AL,

USA). Data analysis was performed using CellQuest software

(BD Biosciences).

Real time RT-PCR

RNA was isolated from cell pellets or homogenized foreskins

with Trizol reagent (Invitrogen). cDNA was prepared with the

First Strand cDNA Synthesis Kit for RT-PCR (AMV) (Roche

Diagnostics, GmbH, Mannheim, Germany) using a random

primer. Amplification reactions were performed in a 25 llfinal volume with 1X SybrGreen PCR Master Mix (Applied

Biosystems) and 300 nmol/l primers designed to specifically

amplify UTY (forward primer 5¢-TCTACAGAATGGTTCT-GATAACTGGAA-3¢, reverse primer 5¢-GGTGTCAAACA-CAACGAATAAACTTG-3¢). Amplification and detection

were performed with an ABI Prism 7700 sequence detection

system (ABI/PE, Foster City, CA, USA) under the following

conditions: 10 min at 95�C, 45 cycles of 15 s at 95�C and

1 min at 60�C. Fluorescence spectra were recorded during the

elongation phase of each PCR cycle. The Sequence Detection

Software (SDS v1.7) of the ABI-Prism 7700 was used to

generate the amplification curves for each reaction. To

differentiate specific amplicons from nonspecific products, a

DNA association curve was generated after each reaction with

the ABI Prism 7700 Sequence detection system (Applied

Biosystems). After denaturation, the temperature was

decreased from 95�C to 25�C in 1�C steps with holds for

20 s. SybrGreen fluorescence was measured during each step

and the first derivative of the fluorescence was plotted as a

function of the temperature. The cycle threshold (Ct) value

represented the refraction cycle number at which a positive

amplification reaction was measured and was set at 10 times

the standard deviation (SD) of the mean baseline emission

calculated for PCR cycles 3–15. For comparison of Ct values of

different samples correction for the amount of input material

was performed using control PCR reactions for the porpho-

bilinogen deaminase.

Cloning of the YKII.39 TCR

Usage of variable regions of TCRa (AV) and TCRb (BV)

chains in YKII.39 was analysed by RT-PCR using forward

R. Ivanov et al

394 ª 2005 Blackwell Publishing Ltd, British Journal of Haematology, 129, 392–402

primers specific for different AV and BV gene families; an

oligonucleotide specific for either Ca or Cb was used as a

reverse primer (Arden et al, 1995). Full-length YKII.39 TCRa(AV9-2*02�AJ42�AC) was amplified with primers 5¢-TTTGGATCCGCCCACCATGAACTATTCTC-3¢ and 5¢-CCCGCGGCCGCCCTCAGCTGGACCACAGC-3¢, then cloned into Bam-

HI and NotI sites of the pMX-mTCRa-IRES-EGFP vector.

Next, EGFP marker gene in the pMX-TCRa-IRES-EGFPconstruct was substituted with the NGFR marker gene by

excising EGFP with NcoI and SalI and sequential cloning of

NGFR fragment 26–844 into NcoI and SalI sites and then NGFR

fragment 1–25 into the NcoI site. YKII.39 TCRb type could not

be found in previous experiments; therefore it was amplified

with a degenerate primer 5¢-CCCGTCGACATGGGYHSCD-

GBCTCCTMTG-3¢, where Y ¼ C + T, H ¼ A + T + C,

S ¼ C + G, D ¼ A + T + G, B ¼ T + C + G, M ¼ A + C,

and a primer 5¢-TTTGCGGCCGCTCAGAAATCCTTTCTC-3¢.The PCR product was cloned into Sal I and Not I sites of

pEGFP-N1 vector (Clontech) and sequenced with the BigDye

Terminator v3Æ1 Cycle Sequencing Kit (Applied Biosystems).

Reaction mixtures were analysed on an ABI Prism 3100 genetic

analyzer (Applied Biosystems). Sequencing of the insert

revealed BV5-5*02�BD2*01�BJ2-1�BC2 type of TCRb. ThenPCR was performed with primers 5¢-TTTCCGCGGCCAC-CATGGGCC CTGGGCTC-3¢ and 5¢-CCGTCGACCTAGCCT-CTGGAATCCTTTCTCTTGACC-3¢, the PCR product was

blunted and cloned into blunt BamHI and SalI sites of

pMX-mTCRa-IRES-EGFP vector. The sequence of all cloned

TCR genes was verified.

Results

YKII.39 recognizes an HLA-B*5201-restricted minorhistocompatibility antigen expressed on proliferatinglymphocytes

The YKII T cell line was generated through repetitive

stimulation of T cells of a female donor with chronic myeloid

leukaemia (CML) cells and, later, EBV-LCL of her HLA-

identical brother. The CD8+ T cell clone YKII.39, generated in

a limiting dilution culture of YKII, produced IFN-c in

response to stimulation with EBV-LCL of patient (EBVp)

and not donor (EBVd) origin (Fig 1A). When YKII.39

reactivity towards different subsets of the patient’s primary

leucocytes was tested, comparable reactivity was observed

towards PHA-activated T cell blasts and CD40-activated B

cells. In contrast, much lower reactivity was observed towards

CML cells, normal CD14+ cells and non-stimulated primary T

and B cells (Fig 1B). This suggested that YKII.39 is specific for

a differentially expressed mHag.

YKII.39 reactivity towards EBVp was completely blocked by

antibodies against HLA Class I, HLA-B & C molecules and

CD8, but not with antibodies against HLA Class II or CD4

(Fig 1C). These results indicated that YKII.39 reacted in a

CD8-dependent manner towards an antigen presented on

either HLA-B or HLA-C. To identify the exact restriction

element of YKII.39 we transduced a random selection of 19

EBV-LCL cell lines, derived from members of two CEPH

Fig 1. YKII.39 recognizes a minor histocompatibility antigen, which is

mainly expressed on proliferating lymphocytes and presented on either

HLA-B or HLA-C. IFN-c production by YKII.39 in response to sti-

mulation with different cell types for 24 h was measured by an IFN-cELISA. Average measurements for triplicates and their standard devia-

tions are shown. (A) YKII.39 recognizes patient (EBVp) but not donor

EBV-LCL (EBVd). (B) YKII.39 reacts mainly towards activated

lymphocytesmainly. Data from independent experiments are presented.

Absolute numbers of IFN-c production differed in individual experi-

ments; therefore the IFN-c production by YKII.39 in response to sti-

mulation with different cell types is depicted in the graph as percentages

from the reactivity towards EBVp in the same experiment. (C) IFN-cproduction by YKII.39 in response to stimulation with EBVp for 24 h

was blocked by antibodies against HLA Class I, HLA-B and C molecules

and CD8, but not with antibodies against HLA Class II or CD4.

Generation of GVL effector T cells through TCR-transfer

ª 2005 Blackwell Publishing Ltd, British Journal of Haematology, 129, 392–402 395

families, with retroviruses encoding either of the two HLA-B

alleles expressed by the patient – HLA-B*3508 and HLA-

B*5201. None of the mock-transduced EBV-LCLs or EBV-

LCLs transduced with B*3508 were recognized by YKII.39,

while 10 of 19 EBV-LCLs transduced with B*5201 induced

IFN-c production by YKII.39 (data not shown). Remarkably,

all recognized EBV-LCLs were derived from male donors,

demonstrating that YKII.39 recognized an H-Y antigen

presented on HLA-B*5201.

As shown in Fig 1B, YKII.39 responded to stimulation not

only with EBV-LCL, but also with normal B and T lympho-

blasts of the patient. To confirm the ability of YKII.39 to

recognize proliferating male lymphocytes other than EBV-

LCLs, we tested YKII.39 reactivity towards B*5201-positive B

and T cell blasts of three male individuals. Due to the low

frequency of the B*5201 allele in Caucasians, we had to

perform B*5201 transductions of EBV-LCLs and PHA blasts of

three B*5201-negative male donors. Transduction efficiency,

measured by FACS analysis of the NGFR marker gene

expression, was 18Æ3 ± 2Æ9% and 42Æ7 ± 14Æ7 for EBV-LCL

and PHA blasts, respectively. A sharp increase of reactivity

towards both EBV-LCLs and PHA blasts was observed upon

transduction with B*5201 (Fig 2A).

In a separate experiment, the NGFR+ fraction of male

fibroblasts transduced with B*5201 induced only a very weak

YKII.39 response (2Æ8% of IFN-c production on EBVp)

(Fig 2B). To increase the immunogenicity of B*5201-positive

fibroblasts, they were pre-treated with IFN-c and TNF-a,which increased HLA Class I expression approximately fivefold

(Fig 2C). A twofold increase in the YKII.39 reactivity was

observed as a result, although it still constituted a negligible

fraction of the IFN-c production towards EBVp (5Æ9%,

Fig 2B). Taken together, these data indicated that YKII.39

reacted towards a B*5201-restricted mHag specifically

expressed by male lymphoblasts.

Identification of a novel HLA-B*5201-restricted UTYepitope

In order to identify the molecular nature of the antigen

recognized by YKII.39, we investigated the YKII.39 response to

a panel of H-Y antigens. First, EBVd were transduced with the

retroviral LZRS vector, encoding one of five H–Y antigens

(RPS4Y, SMCY, EIF1AY, DBY and TB4Y) followed by the

internal ribosomal entry sequence (IRES) and the EGFP

marker gene (Vogt et al, 2000). EGFP-positive EBVd were

sorted by FACS and tested for their ability to induce IFN-cproduction by YKII.39. None of the cell lines was recognized

(data not shown). Then a panel of H–Y antigens consisting of

the UTY, ZFY and DFFRY genes cloned into the pCR3Æ1plasmid, and the TBL1Y, CYorf15A, CYorf15B genes cloned

into the pCEP4 vector, were transfected into 293-EBNA-B7-

B*5201 cells. Only transfection with pCR3Æ1-UTY led to the

recognition of transfected 293 cells by YKII.39 (408 pg/ml

IFN-c versus no detectable IFN-c production on 293 cells

transfected with other constructs). UTY transcript variant 1

(GenBank accession number NM_182660), encoded by the

pCR3Æ1-UTY, consists of 1079 amino acids and has multiple

Fig 2. YKII.39 recognizes male B*5201-transduced EBV-LCLs and

PHA blasts but not fibroblasts. (A) An IFN-c ELISA was used to

measure IFN-c production by YKII.39 in response to stimulation for

24 h with male EBV-LCLs and PHA blasts transduced with B*5201.

Average measurements for three patients and their standard deviations

are shown. IFN-c production in response to stimulation with the

corresponding cell types of the original B5201+ patient is shown for

comparison. (B) IFN-c production by YKII.39 in response to 24 h

stimulation with EBVp, B*5201+ male fibroblasts and the same fibro-

blasts pretreated with 5 ng/ml IFN-c and 100 U/ml TNF-a for 48 h

(fibroblasts + cyt). Average data of triplicate measurements and their

standard deviations are shown. (C) HLA class I expression on fibro-

blasts was measured by FACS analysis. HLA class I expression on non-

treated fibroblasts and fibroblasts incubated with 5 ng/ml IFN-c and

100 U/ml TNF-a for 48 h is shown by empty contours with continuous

and dotted borders, respectively. The filled contour represents non-

stained fibroblasts. Mean fluorescence intensities (MFI) are indicated.

R. Ivanov et al

396 ª 2005 Blackwell Publishing Ltd, British Journal of Haematology, 129, 392–402

amino acid differences with its female analogue (protein

divergence 13Æ6%) (Skaletsky et al, 2003).

YKII.39 recognizes the MQQMRHKEV peptide of UTY

To identify the epitope of UTY recognized by YKII.39, we cloned

10 truncated UTY genes, which had different parts of the

3¢ sequence missing. Only minigenes containing a sequence

coding for amino acids 454–468 induced YKII.39 activation

upon their transfection into 293-EBNA-B7-B*5201 cells

(Fig 3A). The male and female isoforms differed in this region

at three amino acid positions; moreover, two amino acids of

UTX are absent in UTY. Ten overlapping non-apeptides

spanning this region were synthesized (Fig 3B). EBVd were

loaded with these candidate peptides and tested for their ability

to induce IFN-c production by YKII.39. EBVd loaded with

peptides LMQQMRHKE, MQQMRHKEV and QQMRHKEVA

were recognized by YKII.39, while no significant reactivity was

observed towards non-treated EBVd or EBVd loaded with other

peptides (Fig 3C). Next, the response of YKII.39 to EBVd loaded

with decreasing concentrations of these three peptides was

measured. We found that peptide MQQMRHKEV was recog-

nized much more efficiently at low concentrations; a half-

maximal lysis was observed at a peptide concentration of

10)8 mol/l (Fig 3D).

Increased generation of MQQMRHKEV in normal andmalignant lymphoblasts determines their susceptibility tolysis by YKII.39

The level of UTY expression in a selection of haematopoietic

and non-haematopoietic cell types was studied to analyse

whether the differential reactivity of YKII.39 towards various

cell subsets correlates with differential levels of UTY expres-

sion. As shown in Fig 4A, relative UTY mRNA levels,

normalized for the amount of porphobilinogen deaminase

transcripts, were very high in bone marrow samples contain-

ing >80% leukaemic blasts. The UTY mRNA content was

significantly higher in leukaemic cells than in bone marrow

stromal cells and homogenized foreskins (25Æ8 ± 10Æ7 vs.

2Æ5 ± 2Æ2, P < 0Æ001). Surprisingly, non-stimulated CD3+ and

CD20+ lymphocytes isolated from healthy donors had much

higher levels of UTY mRNA expression than EBV-LCLs and

PHA blasts (35Æ2 ± 12Æ4 vs. 2Æ9 ± 2Æ6, P < 0Æ0001). In fact,

the level of UTY expression in lymphoblasts was not

significantly different from that in non-haematopoietic cells

(P ¼ 0Æ78).These findings suggest that overexpression of UTY is not the

cause of the lymphoblast-specific reactivity of YKII.39. Instead,

this differential pattern of reactivity is caused either by a more

efficient generation of the UTY epitope by lymphoblasts or by

Fig 3. YKII.39 recognizes the MQQMRHKEV peptide of the UTY gene. (A) IFN-c production by YKII.39 in response to 24 h stimulation with 293-

EBNA-B7-B*5201 cells transfected with UTY mini-genes was measured by an IFN-c ELISA. Average measurements for triplicates and their standard

deviations are shown. YKII.39 recognized only cells transfected with minigenes containing amino acids 453–468. (B) The region of UTY between

amino acids 453 and 468 contains three different amino acid residues compared with UTX (indicated with bold italics) and two amino acid deletions

(indicated by hyphens). Ten candidate non-apeptides, which were tested in the next experiment, are shown. (C) Reactivity of YKII.39 towards EBVd

loaded with candidate peptides was determined in an IFN-c ELISA. Three peptides [underlined in (B)], were found to be recognized by YKII.39. (D)

Lytic activity of YKII.39 towards EBVd loaded with increasing concentrations of peptides LMQQMRHKE, MQQMRHKEV and QQMRHKEVA was

tested in a 51Cr release assay at an effector:target ratio of 10:1.

Generation of GVL effector T cells through TCR-transfer

ª 2005 Blackwell Publishing Ltd, British Journal of Haematology, 129, 392–402 397

an enhanced ability of these cells to trigger T cell activation due

to a higher expression of co-stimulatory and adhesion

molecules. In order to distinguish between these two possibil-

ities, we investigated YKII.39-reactivity towards B*5201-pos-

itive female fibroblasts, and resting and activated T cells loaded

with increasing concentrations of MQQMRHKEV. In the

absence of significant lytic activity towards resting T cells and

fibroblasts of male origin (Fig 4B), loading of female cells with

the antigenic peptide led to efficient lysis of all tested cell types

(Fig 4C,D). Upregulation of HLA class I expression on target

cells through treatment with IFN-c and TNF-a moderately

increased lysis, but was not essential for recognition. Together,

these data demonstrate that the lack of recognition of male

fibroblasts and resting T cells by YKII.39 is a result of highly

inefficient generation of MQQMRHKEV in these cells.

To prove susceptibility of tumour cells to lysis by YKII.39,

we investigated its reactivity towards malignant lymphoblasts.

Due to the lack of B*5201-positive samples of leukaemic cells,

we could not test the lytic activity of YKII.39 towards primary

leukaemic cells. Therefore, leukaemic cell lines were trans-

duced with the pMX-B*5201/NGFR vector and NGFR-positive

cells were immunomagnetically sorted. Specific lytic activity

was observed in EBV-LCL (92%), the Burkitt’s lymphoma cell

line Raji (54%), ALL YT cells (56%) and the cytokine-treated

myeloma cell line RPMI 8226 (49%) at Effector:Target ratios

of 30:1 (data not shown).

Retroviral transfer of the YKII.39 TCR into naı̈ve donorT cells generates vast numbers of highly effectivelymphoblast-specific CTL

Next, the immunotherapeutical potential of UTY-targeting

through TCR-transfer was evaluated. T cells of the female

donor were co-transduced with two retroviral vectors enco-

ding the a- and b-chains of the YKII.39 TCR (TCRYKII.39),

respectively (Fig 5A,B). Then TCRa/NGFR-positive T cells

were immunomagnetically purified to obtain a cell population

with 57% cells positive for both a- and b-chains of TCRYKII.39

(Fig 5C,D). Unlike mock-transduced cells, TCRYKII.39-trans-

duced donor T cells displayed profound reactivity to EBVp

(Fig 5E). The reactivity to EBVd was much lower, at a level

slightly above background. This may be caused by the presence

Fig 4. Enhanced MQQMRHKEV generation rather than UTY overexpression determines susceptibility to lysis by YKII.39. (A) Real-time RT-PCR

was performed with UTY-specific primers. Resulting Ct values were corrected for the different amount of input material by concomitant real-time

RT-PCR for porphobilinogen deaminase. The sample with the lowest corrected Ct value was considered to have one arbitrary unit of the UTYmRNA.

The fold difference between this sample and other samples was calculated; the UTY mRNA content expressed in arbitrary units is shown. Three

different samples of each category were tested. Average results for three samples and their standard deviations are shown. (B) YKII.39 exerted lytic

activity in a 51Cr release assay on male B*5201-positive PHA blasts (solid squares) but neither on resting cells (solid triangles) of the same patient nor

on B*5201-transduced male fibroblasts (solid circles). IFN-c and TNF-a treatment of target cells slightly increased lysis of fibroblasts (open circles)

and did not influence lysis of resting T cells (data not shown). (C) Lysis of MQQMRHKEV-loaded B*5201-positive female T cells by YKII.39 was

tested in a 51Cr release assay at an effector:target ratio 10:1. PHA blasts are indicated with solid squares, resting T cells – with solid circles, cytokine-

treated resting T cells – with open circles. (D) YKII.39 lytic activity on EGFP-positive fraction of B*5201-tranduced female fibroblasts loaded with

MQQMRHKEV was measured in a 51Cr release assay at effector:target ratio 10:1. Solid circles represent fibroblasts not treated with cytokines, open

circles – cytokine-treated fibroblasts. Mock-transduced female fibroblasts (solid triangles) were tested as a negative control.

R. Ivanov et al

398 ª 2005 Blackwell Publishing Ltd, British Journal of Haematology, 129, 392–402

of EBV-specific T cells in the polyclonal population of

TCRYKII.39-transduced cells. The EBVp-specific reactivity was

completely confined to the CD8+ fraction, confirming the

necessity of co-receptor engagement for T cell activation via

TCRYKII.39, which was found in blocking experiments.

Remarkably, the amount of IFN-c produced by TCRYKII.39-

transduced T cells was significantly higher than that of YKII.39

(5455 ± 1286 pg/ml vs. 721 ± 134 pg/ml, P ¼ 0Æ003).In a separate experiment, we tested the reactivity of

TCRYKII.39-transduced donor cells in response to allogeneic

fibroblasts transduced with B*5201. YKII.39 had demonstrated

very low reactivity towards these fibroblasts, as shown in

Fig 2B. TCRYKII.39-transduced T cells produced some IFN-c in

response to stimulation with B*5201-positive fibroblasts,

although it was not significantly different from the reactivity

towards B*5201-negative fibroblasts (Fig 5F). Therefore, the

observed response was mediated by alloreactive T cells in the

absence of a significant B*5201-restricted UTY-specific

response. The differential pattern of recognition mediated

through TCRYKII.39 is hence preserved in TCRYKII.39-trans-

duced cells. This finding suggests that YKII.39 and TCRYKII.39-

transduced T cells require a similar amount of peptide

Fig 5. Donor T cells transduced with YKII.39 TCR preserve the specificity, the sensitivity and the differential pattern of recognition of the original

clone. Donor T cells were co-transduced with vectors pMX-TCRaYKII.39/NGFR and pMX-TCRbYKII.39. Staining with antibodies against TCRVb5Æ3and NGFR was performed on mock-transduced (A), transduced (B), transduced and immunomagnetically purified (C) donor T cells. (D) The

TCRbYKII.39 expression on TCR-transduced cells was measured by FACS analysis after staining with PE-conjugated BV5Æ3-specific antibody. Empty

contours represent staining of the original YKII.39 clone. For the Mean Fluorescence Intensity (MFI) estimation only BV5Æ3-positive TCR-transducedcells were gated (marker M1) to enable valid comparison with the original clone. The MFI of YKII.39 was 57Æ0 and the MFI of TCRYKII.39-transduced

cells was 40Æ2. (E) CD4+ and CD8+ fractions of TCR-transduced donor T cells were obtained by immunomagnetic depletion of CD8+ and CD4+ cells,

respectively. Reactivity of YKII.39 and different fractions of TCRYKII.39-transduced T cells to EBVp and EBVd was tested in an IFN-c ELISA. Average

results for triplicates and their standard deviations are shown in the figure. (F) Reactivity of CD8+ TCRYKII.39-transduced T cells towards non-

transduced versus B*5201-transduced fibroblasts, either pretreated with 5 ng/ml IFN-c and 200 U/ml TNF-a or not, was tested in an IFN-c ELISA.

IFN-c production in response to stimulation with EBVp and EBVd is shown for comparison. (G) Reactivity of YKII.39 and TCRYKII.39-transduced

T cells to EBVd loaded with increasing amounts of MQQMRHKEV was compared in a 51Cr release assay at the effector:target ratio of 10:1.

Generation of GVL effector T cells through TCR-transfer

ª 2005 Blackwell Publishing Ltd, British Journal of Haematology, 129, 392–402 399

presented on the target cell surface for activation. Indeed, we

found that both the original T cell clone and T cells transduced

with TCRYKII.39 reacted to EBVd loaded with the

MQQMRHKEV peptide at the minimal concentration of

10)9 mol/l (Fig 5G).

Discussion

H–Y antigens were initially identified as targets of immune

responses leading to the rejection of a male graft in female

patients (Scott et al, 1997). The increased severity of GVHD in

female to male SCT is associated with responses to H–Y

antigens (Goulmy et al, 1996). Recently, it was shown that

anti-H–Y responses also contribute to the GVL effect, which is

independent from the parallel GVHD reactivity (Randolph

et al, 2004). This important observation indicates that certain

H–Y antigens should be able to elicit responses that target

leukaemic cells in the absence of significant collateral damage.

However, all H–Y antigens characterized so far as targets of

T cell responses are ubiquitously expressed (Wang et al, 1995;

Pierce et al, 1999; Vogt et al, 2000, 2002). Moreover, mapping

of the Y chromosome did not reveal any antigens with a

haematopoietic-restricted expression (Skaletsky et al, 2003).

This indicates that either overexpression, post-transcriptional

regulation or altered processing of some H–Y antigens leads to

a differential pattern of recognition of their epitopes on

leukaemic cells, resulting in GVL reactions.

We have recently found evidence to support this notion. The

male isoform of the ribosomal protein S4 (RPS4Y) induces an

HLA-B*5201-restricted CTL response that specifically targets

proliferating B and T cells (Ivanov et al, 2005b). Despite the

constantly high levels of RPS4 mRNA in different cell types, a

significantly larger amount of RPS4 protein was found in

malignant and normal lymphoblasts. This observation pro-

vides a proof of principle that an immune response to a

ubiquitously expressed antigen may result in a differential

recognition of leukaemic cells. A second H–Y antigen to

corroborate this notion is UTY. Warren et al (2000) demon-

strated a significant cell-type specificity of presentation of UTY

by cultured human cells. In that study an increased level of

UTY expression and/or processing in haematopoietic cells was

thought to play a role in the preferential recognition of UTY

on blast cells.

In the present study, a new HLA-B*5201-restricted epitope

of UTY was identified. UTY encodes a tetratricopeptide repeat

protein of unknown function. Tetratricopeptide repeats were

reported to facilitate protein–protein interactions and the

assembly into high-order complexes (Blatch & Lassle, 1999).

As a result of the alternative splicing, three transcripts are

generated of the UTY gene, resulting in UTY proteins with

different carboxyl termini. The sequence encoding the

MQQMRHKEV peptide recognized by YKII.39 can be found

in all three isoforms. This peptide, as well as its female

analogue, has a Valine residue in the ninth position that can

serve as an anchor residue for binding to B*5201, according to

published data (Rammensee et al, 1995). However, compared

with its female analogue, it has three amino acid substitutions

and two amino acid deletions, making the existence of a female

counterpart peptide and its recognition by YKII.39 rather

improbable.

Like the previously reported HLA-B8-restricted UTY-speci-

fic CTL, YKII.39 displayed a differential pattern of recognition

(Warren et al, 2000). Only B and T lymphoblasts were

recognized, while resting haematopoietic cells and fibroblasts

did not induce activation of YKII.39. Quantitative analysis of

UTY expression showed that, although the level of UTYmRNA

in lymphoblasts was higher than in skin or bone marrow

stromal cells, it was lower than in non-recognized resting

haematopoietic cells. Thus YKII.39-reactivity is not correlated

to UTY expression levels. Furthermore, elevated expression of

co-stimulatory and adhesion molecules on lymphoblasts is not

the cause of their specific recognition either, since loading of

fibroblasts and resting T cells with the antigenic peptide

induced their lysis by YKII.39 at levels similar to, or higher

than, those of PHA blasts. Thus, provided that sufficient

amounts of the MQQMRHKEV peptide are presented, resting

cells are equally susceptible to YKII.39 as blast cells. These data

suggest that inefficient generation of MQQMRHKEV in

resting cells is the cause of their YKII.39-resistance. Immuno-

proteasome involvement in the generation of this epitope does

not explain lymphoblast recognition, because neither fibro-

blasts treated with IFN-c, nor CML cells differentiated into

dendritic cell-like cells, were recognized. However, other

factors involved in antigen-processing may be responsible for

this phenomenon, such as the lymphoblast-restricted activity

of aminopeptidases that participate in peptide-trimming

downstream of the proteasome (Rock et al, 2004). Alternat-

ively, considering the similar recognition pattern of another

UTY epitope (Warren et al, 2000), post-transcriptional regu-

lation of expression of the UTY protein or its turnover may be

responsible for the accumulation of the UTY protein in

lymphoblasts.

Retroviral transfer of the TCR of the original clone into large

numbers of donor T cells provides an attractive solution to this

problem (Schumacher, 2002). However, tissue-specific recog-

nition of ubiquitously expressed antigens depends on a subtle

balance between the avidity of antigen-specific T cells and the

level of the antigen presented by target cells. Primary T cells of

the HLA-identical donor transduced with TCRYKII.39 consist of

T lymphocytes with different functional properties. More

importantly, they have variable levels of expression of the

exogenous TCR, which is shown to have an impact on T cell

reactivity (Heemskerk et al, 2004). Nevertheless, we demon-

strated the preservation of the avidity and the recognition

pattern of YKII.39 in donor T cells transduced with TCRYKII.39.

Both TCRYKII.39-transduced T cells and YKII.39 demonstrated

the same efficient recognition of lymphoblasts in the absence

of significant recognition of fibroblasts, which represent

principal substrates of GVL and GVHD reactions, respectively.

Although the preservation of the fine peptide specificity after

R. Ivanov et al

400 ª 2005 Blackwell Publishing Ltd, British Journal of Haematology, 129, 392–402

TCR-transfer was reported previously (Schaft et al, 2003;

Rubinstein et al, 2003), this report represents the first

description of the preserved differential pattern of recognition

of a ubiquitously expressed tumour-associated antigen after

transfer of a human TCR into peripheral blood lymphocytes.

In other studies, either antigens with a very restricted pattern

of expression were targeted (Clay et al, 1999; Morgan et al,

2003; Heemskerk et al, 2004) or a murine TCR was used (Liu

et al, 2000; Stanislawski et al, 2001).

Differentially recognized H–Y antigens represent a promis-

ing target for immunotherapy. Unlike many mHags that are

present in a small proportion of the patient population, H–Y

antigens are shared by all male patients. A universally attractive

H–Y epitope has to be presented on an HLA allele that is

frequent in the population. HLA-B*5201 is present in 34% of

Japanese and 27% of Chinese individuals (Imanishi et al,

1992), although it is relatively rare (2–6%) in Caucasians. For

the latter patients, differentially processed UTY epitopes

presented on more frequent HLA alleles need to be identified

to make the clinical application of UTY-targeting immuno-

therapies more feasible. The significant divergence of UTY and

its female counterpart UTX ensures an abundance of potential

T cell epitopes in UTY.

In conclusion, we have identified a new epitope of the UTY

antigen that is able to elicit a male lymphoblast-specific CTL

response because of its superior generation in proliferating B

and T cells. Transfer of the anti-UTY TCR into donor T cells

resulted in generation of large numbers of lymphoblast-specific

effector cells. To our knowledge, this is the first demonstration

that a differential pattern of recognition of a ubiquitously

expressed antigen is preserved in a heterogeneous population

of TCR-transduced cells. Our findings support the feasibility of

immunogenetherapy of lymphoproliferative diseases by gen-

etically engineered T cells redirected towards the patient’s

lymphoblasts. Use of T cells transduced with the UTY-specific

TCR may be especially beneficial in case of DLI for male

patients transplanted previously with haematopoietic stem

cells of a female sibling.

Acknowledgements

The authors are grateful to Dr Pierre G. Coulie and Dr Benoit

van den Eynde (Institute of Cellular Pathology, Universite de

Louvain, Brussels, Belgium) for the 293-EBNA-B7 cell line;

Dr Ton N.M. Schumacher (Netherlands Cancer Institute,

Amsterdam, the Netherlands) for the pMX vector; Dr Garry

Nolan (Stanford University, Palo Alto, CA, USA) for the

amphotrophic packaging cell line Phoenix; M. Otten and

F. Melman for excellent technical assistance.

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