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International Immunology Advance Access originally published online on February 28, 2006
International Immunology 2006 18(4):591-601; doi:10.1093/intimm/dxh401
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© The Japanese Society for Immunology. 2006. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org

T cell re-targeting to EBV antigens following TCR gene transfer: CD28-containing receptors mediate enhanced antigen-specific IFN{gamma} production

Niels Schaft1,7, Birgit Lankiewicz1, Joost Drexhage1, Cor Berrevoets1, Denis J. Moss2, Victor Levitsky3, Marc Bonneville4, Steven P. Lee5, Andrew J. McMichael6, Jan-Willem Gratama1, Reinier L. H. Bolhuis1,8, Ralph Willemsen1 and Reno Debets1

1 Laboratory of Tumor Immunology, Department of Medical Oncology, Erasmus MC-Daniel den Hoed Cancer Center, Groene Hilledijk 301, 3075 EA Rotterdam, the Netherlands
2 Tumor Immunology Laboratory, Division of Infectious Diseases and Immunology, Queensland Institute of Medical Research, University of Queensland, Brisbane, Australia
3 Microbiology and Tumorbiology Center, Karolinska Institutet, Stockholm, Sweden
4 INSERM U463, Institut de Biologie, Nantes, France
5 Cancer Research UK, Institute for Cancer Studies, University of Birmingham, Birmingham, UK
6 MRC Human Immunology Unit, Weatherall Institute of Molecular Medicine, The John Radcliffe, Oxford, UK
7 Present address: Department of Dermatology, University Hospital Erlangen, Erlangen, Germany
8 Present address: Archifact, Meerewijck 79, 2451 XC Leimuiden, the Netherlands

Correspondence to: R. Debets; E-mail: j.debets{at}erasmusmc.nl


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
EBV is associated with a broad range of malignancies. Adoptive immunotherapy of these tumors with EBV-specific CTL proved useful. We generated a panel of primary human T cells specific to various EBV antigens (i.e. Epstein–Barr nuclear antigen 3A, 3B and BamHI-M leftward reading frame) via transfer of modified TCR genes that are either coupled to CD3{zeta} or Fc({varepsilon})RI{gamma}. TCR-transduced T cells from 20–60% of donors (total number of 25) demonstrated specific lysis of EBV peptide-loaded target cells, whereas lymphoblastoid cell lines expressing native EBV antigens were not killed by any of the EBV-specific T cell populations. This non-responsiveness, confirmed at the level of nuclear factor of activated T cells activation, is not due to receptor configuration since identical receptor formats specific for melanoma antigens successfully re-targeted T cells to native melanoma cells. In an effort to generate a more potent receptor, we introduced a CD28 domain into one of the EBV-specific TCR. This TCR did not affect the cytotoxic response of re-targeted T cells, but dramatically enhanced antigen-specific IFN{gamma} production. We therefore conclude that these novel CD28-containing EBV-specific TCRs provide a basis for further development of TCR gene transfer to treat EBV-induced diseases.

Keywords: cytotoxicity, gene therapy, signal transduction, T cell co-stimulation, tumor immunity


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
EBV is a gamma-1 B-lymphotropic herpesvirus found in all human populations, with a prevalence of over 90% in adults, and it typically persists in its natural hosts for life. B cells constitute the long-term reservoir for the virus, whilst the oropharyngeal epithelium is the site for periodical virus replication. Important to the immune control of EBV are EBV-specific CTLs. In healthy individuals, EBV-specific CTL recognize MHC class I-restricted latent and lytic viral antigens, and are thought to prevent virus spreading (1, 2). Only a small subset of at least 11 viral genes is expressed in latently infected B cells and recognized by EBV-specific CTL. Latent viral gene products can be divided into three groups: (i) the EBV-encoded RNAs (EBERs) 1 and 2; (ii) the EBV nuclear antigens Epstein–Barr nuclear antigen (EBNA) 1, 2, 3A, 3B, 3C and EBNA-LP and (iii) the latent membrane proteins (LMP)-1, 2A and 2B [reviewed in (3, 4)]. EBV is associated with a broad range of malignancies of mostly hematopoietic or epithelial origin, which can be divided into the following types of latencies based on EBV gene expression patterns: type I latency expresses the EBER and EBNA-1 genes; type II latency expresses the EBER, EBNA-1 and the LMP genes; and type III latency expresses the EBER, all EBNA and LMP genes. These types of expression patterns are unique to various forms of tumors, such as (immunosuppression related) lymphomas in transplant recipients and patients with AIDS, gastric carcinomas and the Reed-Sternberg cells in Hodgkin's disease (5–11).

Adoptive immunotherapy of EBV-positive tumors in allogeneic bone marrow transplant recipients with HLA-restricted, EBV-specific T cells has proven effective. This immunotherapy, when performed with non-selected lymphocytes from EBV-seropositive bone marrow donors, is associated with graft-versus-host disease (GVHD) (12, 13). However, EBV-specific CTL generated in vitro by stimulation with autologous EBV-transformed B-lymphoblast cell lines (B-LCL) have clear antiviral effects without causing GVHD (14, 15). Clinical responses were observed when patients with EBV-positive post-transplant lymphoproliferative disease were treated with either autologous or partly HLA-matched allogeneic CTL (15, 16). Unfortunately, identification, isolation and expansion of tumor-specific T cells are laborious and time consuming and have not been successful in all patients (17).

Alternatively, autologous T cells can be genetically programmed to express MHC-restricted antigen-specific receptors. Primary human T cells can be re-targeted to antigen-expressing cells by transfer of human TCR{alpha}/ß genes. Successfully targeted MHC class I-restricted antigens range from melanoma cancer/testis antigens [i.e. MAGE-1/HLA-A1 (18, 19)], melanocyte differentiation antigens [i.e. MART-1/HLA-A2 (20) or gp100/HLA-A2 (21)] to viral antigens [i.e. HIV gag/HLA-A3 (22), HIV pol/HLA-B35 (23) or EBV LMP2/HLA-A2 (24)]. These re-targeted human T cells were shown not only to bind the relevant peptide/MHC ligand but also to produce cytokines and kill target cells upon antigen-specific stimulation.

The therapeutic use of full-length TCR genes, however, may face problems. The formation of newly formed TCR{alpha}/ß heterodimers comprising both introduced and endogenous TCR chains may induce unknown and possibly dangerous specificities, and constitute a theoretical side effect of TCR gene transfer (18, 22, 25). In addition, the introduction of TCR{alpha}/ß transgenes into T cells may result in a low and unstable surface expression of TCR{alpha} protein (26). Moreover, TCR{alpha}/ß ‘mispairing’ as described above dilutes the expression of the desired TCR{alpha}/ß heterodimers. Indeed, we recently observed that only a fraction of T cells transduced with TCR{alpha} and ß genes and expressing the introduced TCRß chain is able to bind the corresponding peptide/MHC tetramer (21), suggesting that the introduced and surface-expressed TCRß chain pairs with the endogenous TCR{alpha} chain. We have pioneered the use of modified two-chain (tc) and single-chain (sc) TCR molecules as an alternative to full-length TCR, and which addresses the above-mentioned issues (27). Please note that the term-modified TCR is chosen deliberately to distinguish these receptors from chimeric TCRs, a term mostly covering classical (non-MHC restricted) antibody-based receptors that are used for gene transfer. The modified tcTCR-based receptors, that for instance comprise CD3 signaling molecules, i.e. CD3{zeta}, result in exclusive pairing between the introduced TCR{alpha} and ß chains, and ‘rescue’ the surface expression of the introduced TCR{alpha} chain.

Here we studied whether the modified TCR approach is applicable to re-target primary human T cells to EBV antigen-expressing target cells. Such an approach would allow for an ‘off-the-shelf’ treatment regimen based on the ex vivo transduction and expansion of patient T cells with genes encoding for class I MHC-restricted and EBV-specific TCR.


   Methods
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 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Cells and reagents
Peripheral blood lymphocytes from healthy donors were isolated by centrifugation through Ficoll–Isopaque (density = 1.077 g cm–3; Pharmacia Biotech, Uppsala, Sweden). Transduced primary human T cells, the EBV-specific CTL clones BK289 [specific for the HLA-A*1101-presented EBNA-3B peptide IVTDFSVIK (IVT/A11) (28, 29)], A4.5 [specific for the HLA-A*0201-presented BamHI-M leftward reading frame (BMLF-1) peptide GLCTLVAML (GLC/A2) (30)] and CF3 [specific for the HLA-B*0802-presented EBNA-3A peptide FLRGRAYGL (FLR/B8) (31)] and the melanoma-specific CTL clone 82/30 (specific for the HLA-A*0101-presented melanoma peptide EADPTGHSY (EAD/A1)) were all cultured with RPMI 1640 medium supplemented with 25 mM Hepes, 200 nM L-glutamine, 10% human serum, 360 IU ml–1 rIL-2 (Proleukin, Chiron, Amsterdam, The Netherlands) and the antibiotics streptomycin (100 µg ml–1) and penicillin (100 U ml–1). Cells were stimulated every 2 weeks with a mixture of irradiated allogeneic feeder cells, as described elsewhere (32). The EBV-transformed B-lymphoblast cell lines BSM (GLCneg/HLA-A2pos), CJO (IVTpos/HLA-A11pos), HAR (FLRneg/HLA-B8pos), APD (EADneg/HLA-A1pos) (all four target cells kindly provided by M. Giphart, Leiden, the Netherlands), RL, CFpuy (both FLRpos/HLA-B8pos), BK (IVTpos/HLA-A11pos) and the Jurkat T cell clone E6.1 were cultured with RPMI 1640 medium supplemented with 200 nM L-glutamine, 10% bovine calf serum (BCS: Hyclone, Logan, UT, USA) and antibiotics. The human amphotropic packaging cell line Phoenix, the transporter associated with antigen processing-deficient TxB cell hybrid T2 cells, HLA-A11-transfected T2 cells (T2-A11) and the melanoma cell lines G43 (EADpos/HLA-A1pos) and FM3 (YLEpos/HLA-A2pos) were grown in DMEM (Gibco BRL, Paisley, Scotland, UK) supplemented with 10% BCS and antibiotics. The mAbs used in this study comprised anti-TCRVß8 mAb, anti-TCRVß22 mAb, PE-conjugated anti-TCRVß22 mAb, PE-conjugated anti-TCRVß4 mAb (all from Beckman-Coulter, Marseille, France), mouse Ig (mIg; Jackson Immuno Research Laboratories, West Grove, PA, USA) and anti-MHC class I mAb (clone W6/32, Sera-Lab, Crawley Down, UK). Other reagents used in this study were RetroNectin (human fibronectin fragments CH-296: Takara Shuzo Co. Ltd, Otsu, Japan); FLR/HLA-B*0802 monomers; streptavidin–PE (Becton Dickinson Biosciences, San Jose, CA, USA); the EBV-encoded peptides GLCTLVAML, IVTDFSVIK and FLRGRAYGL, the gp100 peptide YLEPGPVTA, and the MAGE-1 peptide EADPTGHSY (all peptides from J. Drijfhout, Leiden, the Netherlands); phorbol myristate acetate (PMA) (Sigma, St Louis, MO, USA) and ionomycin (Calbiochem, La Jolla, CA, USA).

Modification of EBV-specific TCR genes
Modified TCRs specific for EBV were constructed as follows. TCR{alpha} and ß DNA was obtained by PCR using template cDNA from the CTL clones BK289 (which expresses the TCR genes V{alpha}1s1b/J{alpha}45, Vß22s1/Jß2s1), A4.5 (V{alpha}15s1/J{alpha}23, Vß4s1/Jß1s4) and CF3 (V{alpha}2s1/J{alpha}9s14, Vß8s6/Jß1s2). Two-chain TCR{alpha}ß linked to CD3{zeta} (tcTCR{alpha}ß:{zeta}) specific for EBNA-3B or BMLF-1 were constructed by linking the extracellular domains of the TCR {alpha} and ß chain to the CD3{zeta} molecule (i.e. V{alpha}C{alpha}{zeta} and VßCß{zeta}) [as described in (18)]. A single-chain TCR{alpha}ß linked to Fc({varepsilon})RI{gamma} (scTCR{alpha}ß:{gamma}) specific for EBNA-3A was constructed by coupling the TCR V{alpha} and VßCß domains, interspersed by a flexible linker, to a few amino acids of the constant domain of the {kappa} light chain, the transmembrane domain of the CD4 molecule and the signaling domain of Fc({varepsilon})RI{gamma} (i.e. V{alpha}VßCßC{kappa}CD4{gamma}) [as described in (33)]. tcTCR{alpha}ß:{zeta} specific for the melanoma antigens MAGE-1 (18) or gp100 (34) were used as control receptors. Additionally, we generated EBNA-3B-specific TCR{alpha} and ß chains containing CD28 (transmembrane and intracellular domains: aa 153–220 according to gi4557430) and CD3{zeta} (intracellular domain: aa 50–162 according to gi4557430). The CD28-CD3{zeta} DNA (kindly provided by H. Abken, Cologne, Germany) was re-amplified and put downstream of the variable and constant domains of the TCR DNA via BamHI and XhoI sites. Specific primer sequences to amplify the TCR and other domains will be given upon request. The modified TCR genes were subsequently cloned into the retroviral vector pBullet, as described elsewhere (18).

Retroviral gene transduction of TCR into human T cells
Primary human T cells of healthy donors were depleted for cells endogenously expressing TCRVß22 and Vß4 by using anti-TCRVß22 and anti-TCRVß4 mAbs and goat anti-mouse Ig-labeled microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) or magnetic beads (Dynal, Oslo, Norway). Following depletion, T cells were pre-activated with anti-CD3 mAb prior to retroviral transductions. The activated T cells as well as Jurkat T cells were transduced with pBullet retroviral vectors containing the TCR genes of interest. Supernatant containing retroviral vector was produced by the packaging cell line Phoenix. The transduction procedure used was optimized for human T cells and described by Lamers et al. (35). In short, 24-well culture plates were coated with RetroNectin and pre-treated with retroviral particles by centrifugation. Next, 106 human T cells were centrifuged in fresh retroviral supernatant, and cultured for 4–5 h at 37°C/5% CO2. T cells were allowed to recover in normal T cell medium overnight prior to a second transduction cycle, after which cells were harvested and transferred to T25 culture flasks. After sufficient numbers were obtained, TCR-transduced T cells were analyzed for receptor expression by flow cytometry.

Flow cytometric analysis of retrovirally transduced T cells
TCR-transduced T cells were analyzed for transgene expression by flow cytometry using PE-conjugated anti-TCR mAb (Vß22 or Vß4) or FLR–HLA-B8 tetramer complexes. Soluble FLR/HLA-B8 tetramers were made by incubating streptavidin–PE and biotinylated FLR/HLA-B8 monomers at a 1:4 molar ratio for 1 h at 4°C. For immunostaining, 0.25–0.5 x 106 transduced T cells were incubated with the mAbs or tetramers on ice for 30 min, washed, fixed (1% PMA) and analyzed on a flow cytometer (Becton Dickinson). TCR-transduced T cells were subsequently enriched via anti-TCRVß mAbs or FLR/HLA-B8 tetramer and anti-PE MACS MicroBeads (Miltenyi Biotec) according to the manufacturer's instructions. In short, 10 x 106 cells were labeled with PE-conjugated antibody or tetramer for 30 min at 4°C, washed and re-suspended in 160 µl of degassed PBS containing 1% BSA and 2 mM EDTA (wash buffer). Magnetic anti-PE MicroBeads were added to the cells in a final volume of 200 µl, after which cells were incubated for 15 min at 4°C, washed and again re-suspended in wash buffer. Next, cells were loaded onto a pre-washed miniMACS column (MACS High Gradient Magnetic Separation Column MS, Miltenyi Biotec). PE-bound cells were washed several times and carefully collected in wash buffer by applying a plunger to the column. T cells enriched for the introduced TCR were expanded on feeder plates prior to experiments.

Cytotoxicity assay
Cytotoxic activity of CTL clones and TCR-transduced primary human T cells versus indicated target cells was assayed in a standard 6-h 51Cr-release assay (36). CTL clones and TCR-transduced T cells were co-cultivated with either peptide-pulsed target cells or target cells expressing the native antigen of interest. Peptide loading was performed by the addition of the peptide (10 µM final) to the target cells prior to incubation with effector T cells. Specific blocking of cytolytic activity was studied by adding anti-MHC class I mAb or mIg (both at 10 µg ml–1 final) to the target cells 15 min prior to co-cultivation with effector T cells.

Nuclear factor of activated T cells reporter gene assay
Nuclear factor of activated T cells (NFAT) reporter gene assays were performed as described elsewhere (34). In short, exponentially growing TCR-transduced Jurkat T cells (5 x 106) were transiently transfected by electroporation with 5 µg of both the NFAT–luciferase (Stratagene, La Jolla, CA, USA) and ß-galactosidase constructs. Twenty hours post-transfection, Jurkat T cells were transferred to round-bottom 96-well tissue culture-treated plates (Costar, Corning, NY, USA) at 2 x 105 cells per well and were stimulated for 6 h with anti-TCR mAbs, peptide–MHC complexes or target cells (at 105 cells per well) in RPMI 1640 medium supplemented with 1% BCS at 37°C/5% CO2. Cells were subsequently lysed and luciferase and ß-galactosidase activities were determined. Luciferase activities were normalized on the basis of ß-galactosidase activities and expressed relative to a non-stimulated condition (i.e. medium only: set to 1.0). Antibodies used to stimulate TCR-transduced Jurkat T cells comprise anti-TCRVß22, Vß8 mAb or mIg, pre-coated in 96-well plates at 0.1 µg per well. For stimulations with FLR–HLA-B8 complexes, biotinylated BSA (at 1 µg ml–1 final) was pre-coated in 96-well plates (overnight at 4°C), after which wells were washed with PBS and streptavidin (10 µg ml–1) was bound to the biotin for 45 min at room temperature. Plates were washed again and biotinylated FLR–HLA-B8 complexes (at indicated concentrations) were added to wells and incubated for 30 min at room temperature. Finally, for stimulations with target cells, CJO, T2-A11 cells and CFpuy cells were peptide loaded for 30 min at 37°C/5% CO2 with 10 µM of the relevant peptide prior to their use in NFAT reporter gene assays. As a positive control for NFAT activation, cells were stimulated with 10 ng ml–1 PMA and 1 µM ionomycin for 6 h at 37°C/5% CO2.

IFN{gamma} and tumor necrosis factor {alpha} ELISA
To quantify secreted IFN{gamma} and tumor necrosis factor {alpha} (TNF{alpha}) by TCR-transduced human T cells after antigen-specific stimulation, 6 x 104 T cells were cultured in the presence of 3 x 104 EBV-expressing target cells with or without additional peptide for 18 h. As a positive control, transduced T cells were stimulated with PMA and ionomycin. Supernatants were harvested and levels of IFN{gamma} and TNF{alpha} were measured by standard ELISA according to the manufacturer's instructions (Central Laboratory for Blood Transfusions, Amsterdam, the Netherlands).


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
Expression of EBV-specific TCR on transduced T cells
Primary human T cells and the human Jurkat T cell line E6.1 were retrovirally transduced with modified TCRs derived from various EBV-specific CTL clones. Transduced T cells were enriched for receptor-positive cells to obtain similarly high expression levels of the introduced TCR on both cell types. Surface expression levels of the TCR transgenes on primary human T cells were 88% [mean fluorescence intensity (MFI) on a scale of 1–104 relative linear channels, 440], 65% (MFI, 312) and 67% (MFI, 315) for the tcTCR{alpha}ß:{zeta} specific for EBNA-3B and BMLF-1 and the scTCR{alpha}ß:{gamma} specific for EBNA-3A, respectively (Fig. 1A). Expression levels on Jurkat T cells were 55% (MFI, 450) and 59% (MFI, 290) for the tcTCR{alpha}ß:{zeta} specific for EBNA-3B and the scTCR{alpha}ß:{gamma} specific for EBNA-3A, respectively (Fig. 1B). Jurkat T cells were co-transduced with the human CD8{alpha} gene (expression level 100%; MFI, 590). The frequency of CD8-positive T cells within the transduced primary human T cells was higher than 50% (data not shown). Mock-transduced T cells did not bind anti-TCRVß mAbs or tetramers specific for the introduced TCR (Fig. 1).


Figure 1
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  		Fig. 1. Cell-surface expression of EBV-specific modified TCR on transduced human T cells. Primary human T cells (A) or Jurkat T cells (B) transduced with modified TCR genes and enriched for receptor-positive cells were labeled with PE-conjugated anti-TCRVß mAbs or tetramers, and analyzed by flow cytometry. Mock-transduced human T cells (dotted lines) served as negative controls. Marker M1 was set in the corresponding histogram of mock-transduced T cells at a 5% expression level, and percentages given reflect the fraction of stained TCR transductants relative to M1. For each TCR construct results of one representative donor are shown.

 
Peptide-loaded target cells are specifically lysed by primary human T cells transduced with EBV-specific TCR
Human T cells positive for EBV-specific TCR were first tested for their cytolytic reactivity versus target cells loaded with EBV-peptides. For pulsing experiments with peptides derived from the latent viral proteins EBNA-3A and B, we chose target cells that do not natively present the corresponding EBV-epitope, i.e. the B-LCL HAR (HLA-B8 positive) and T2-A11 cells, respectively. For loading experiments with the lytic cycle protein-derived BMLF-1 peptide, we used the B-LCL BSM and T2 cells (both HLA-A2 positive) as target cells. Primary human T cells from 5 out of 9 (56%), 2 out of 6 (33%) and 2 out of 10 (20%) different donors transduced with the tcTCR{alpha}ß:{zeta} specific for EBNA-3B or BMLF-1 and the scTCR{alpha}ß:{gamma} specific for EBNA-3A, respectively, specifically lysed target cells that were loaded with 10 µM of the corresponding peptide. Target cells without peptide were not lysed (Table 1 and data not shown). A representative example of a cytotoxic response of TCR-transduced primary human T cells is shown in Fig. 2A (left panel). Primary human T cells of the same donor transduced with the MAGE-1/HLA-A1-specific tcTCR{alpha}ß:{zeta} (18), which served as a positive control, lysed MAGE-1 peptide-pulsed target cells (Fig. 2B). Peptide-pulsed target cells were also co-cultivated with the corresponding parental CTL clones (EBV-as well as MAGE-1-specific CTLs) and in all cases target cells pulsed with the relevant peptide were specifically lysed, whereas target cells without peptide were not lysed (see Fig. 2A and B, right panels). Mock-transduced T cells were not able to lyse any of the target cells.


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  		Table 1. Re-targeting of primary human T cells with modified TCR specific for EBV

 

Figure 2
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  		Fig. 2. Primary human T cells transduced with EBV-specific TCR specifically lyse peptide-loaded target cells. Human T cells transduced with the tcTCR{alpha}ß:{zeta} specific for EBNA-3B (A) or MAGE-1 (B) were tested in a 6-h 51Cr-release assay. The following target cells were used: the IVTneg/A11pos T2-A11 cell line (A) or the EADneg/A1pos B-LCL APD (B) in the absence (open symbols) or presence (closed symbols) of the relevant peptide. Target cells were pre-incubated with peptide for 30 min at 37°C at a final concentration of 10 µM. The effector cell to target cell ratio is indicated in the figure. Representative examples of cytotoxic responses of TCR-transduced primary human T cells derived from one donor are shown. Antigen specificity was confirmed by blocking experiments with anti-MHC class I mAb or control mIg (10 µg ml–1 final) which was added to the target cells 30 min prior to co-cultivation with T cells (data not shown). The EBNA-3B- and MAGE-1-specific parental CTL clones were used as positive controls. Mock-transduced primary human T cells were not cytolytic toward the mentioned targets.

 
Primary human T cells transduced with EBV-specific TCR do not lyse target cells that express endogenously processed antigen
Next, T cells positive for EBV-specific TCR were tested for their cytolytic reactivity versus target cells that express natively processed antigen. tcTCR{alpha}ß:{zeta}pos primary human T cells specific for EBNA-3B from nine different donors were co-cultivated with the native IVTpos/A11pos EBV-transformed B-LCL BK (Table 1). Surprisingly, only exogenously peptide-loaded BK cells were lysed by the tcTCR{alpha}ß:{zeta}pos T cells, whereas BK cells without exogenously added peptide were not lysed (Fig. 3A). Also the IVTpos/A11pos EBV-transformed B-LCL CJO was only lysed by tcTCR{alpha}ß:{zeta}pos T cells after addition of IVT-peptide (data not shown). T cells positive for scTCR{alpha}ß:{gamma} specific for EBNA-3A from 10 different donors were tested for cytolytic reactivity versus the FLRpos/B8pos EBV-transformed B-LCL target cells RL or CFpuy (Table 1). Again, only peptide-loaded target cells were lysed by scTCR{alpha}ß:{gamma}pos T cells, whereas target cells without exogenous peptide were not lysed. Since there is no cell line available that natively expresses the epitope recognized by the BMLF-1-specific TCR, the tcTCR{alpha}ß:{zeta}pos T cells specific for this EBV-epitope have only been tested versus peptide-loaded target cells. Even at extremely high effector to target cell ratios (81:1), T cells positive for EBV-specific TCR were not able to lyse native targets (see Fig. 3A). In contrast, primary human T cells of the same donor transduced with the MAGE-1/A1-specific control TCR did lyse target cells that expressed the natively processed MAGE-1 epitope without addition of exogenous peptide (Fig. 3B). The parental CTL clones, including the EBV-specific ones, were also able to lyse target cells that express natively processed antigen (Fig. 3A and B). Again, mock-transduced T cells were not able to lyse any of the target cells tested.


Figure 3
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  		Fig. 3. Native target cells are not lysed by primary human T cells transduced with EBV-specific TCR. TCR-transduced human T cells with specificities for EBNA-3B (A) or MAGE-1 (B) were tested in a 6-h 51Cr-release assay with the following target cells: the IVTpos/A11pos B-LCL BK in the absence (open squares) or presence (closed squares) of (additional) IVT-peptide, the IVTneg/A11pos T2-A11 cell line (open diamonds), the EADpos/A1pos melanoma cell line G43 (open squares) or the EADneg/A1pos B-LCL APD (open diamonds). Target cells were pre-incubated with peptide for 30 min at 37°C at a final concentration of 10 µM. The effector cell to target cell ratio is indicated in the figure. Data of one representative donor are shown. Antigen specificity was confirmed by blocking experiments as described in the legend to Fig. 2 (data not shown). The parental CTL clones were used as positive controls. Mock-transduced primary human T cells were not cytolytic toward the above-mentioned target cells.

 
Receptor-specific activation of NFAT via EBV-specific TCR in Jurkat T cells
The observed non-responsiveness of EBV-specific TCRpos T cells, i.e. cytolytic responses versus peptide-loaded target cells by T cells from only 20 to 60% of the donors and no cytolytic responses versus native targets by T cells from any of the donors, was followed up by NFAT reporter gene assays in Jurkat T cells. Receptor-mediated activation of NFAT in EBV-specific TCRpos Jurkat T cells was analyzed by stimulation with anti-TCRVß mAbs, peptide–MHC complexes or B-LCL with or without prior loading with the relevant peptides. Receptor-specific activation of NFAT was seen in tcTCR{alpha}ß:{zeta}pos and scTCR{alpha}ß:{gamma}pos Jurkat T cells specific for EBNA-3B and EBNA-3A, respectively, following stimulation with anti-TCRVß mAb or peptide–MHC complexes (Fig. 4A). However, co-cultivation of tcTCR{alpha}ß:{zeta}pos Jurkat T cells specific for EBNA-3B with CJO B-LCL and T2-A11 stimulator cells, with or without prior loading with the IVT-peptide, or co-cultivation of scTCR{alpha}ß:{gamma}pos Jurkat T cells specific for EBNA-3A with CFpuy B-LCL loaded with or without the FLR-peptide, did not result in NFAT activation (Fig. 4A and data not shown). In contrast, we did demonstrate receptor-specific activation of NFAT in Jurkat T cells transduced with a melanoma gp100/A2-specific tcTCR{alpha}ß:{zeta} in response to not only anti-TCRVß mAb but also T2 cells pulsed with the gp100 peptide as well as native gp100pos/A2pos FM3 melanoma cells (Fig. 4B). Non-transduced Jurkat T cells only showed NFAT activation upon stimulation with anti-TCRVß8 mAb (specific for the endogenous TCRß of Jurkat T cells), but not with anti-TCRVß22 mAb, anti-TCRVß14 mAb, FLR/HLA-B8 peptide–MHC complexes or target cells.


Figure 4
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  		Fig. 4. EBV-specific TCRs mediate NFAT activation following strong TCR stimuli but not following co-cultivation with antigen-positive target cells. Jurkat T cells transduced with the tcTCR{alpha}ß:{zeta} specific for EBNA-3B, scTCR{alpha}ß:{gamma} specific for EBNA-3A (A: black and white bars, respectively) or tcTCR{alpha}ß:{zeta} specific for gp100 (B: black bars) were transfected with 5 µg of both the NFAT reporter and ß-galactosidase constructs. TCR-transduced Jurkat T cells were stimulated for 6 h with anti-TCRVß mAb, control mIg (both at 0.1 µg per well final), peptide–MHC complexes (20 µg ml–1 final) or target cells. T2 target cells, the EBNA-3Bpos CJO and the EBNA-3Apos CFpuy B-LCLs were pre-incubated with peptide for 30 min at 37°C at a final concentration of 1 µM. FM3 cells (gp100pos/HLA-A2pos) were pre-incubated O/N with cytokines, and co-cultivation of these melanoma cells with TCR-transduced Jurkat T cells was performed in the presence of anti-CD28 mAb (34). Luciferase activities were determined in cell lysates, normalized for ß-galactosidase activities, and expressed relative to medium only (tcTCR{alpha}ß:{zeta} specific for EBNA-3B: Relative Light Units (RLU) = 0.09; scTCR{alpha}ß:{gamma} specific for EBNA-3A: RLU = 0.05 and tcTCR{alpha}ß:{zeta} specific for gp100: RLU = 0.02, which are all set to 1.0). Results of one (out of three) representative experiment are shown.

 
Incorporation of CD28 into EBV-specific TCR allows re-targeted T cells to respond to EBV antigen-expressing target cells
In an effort to improve genetic re-targeting of T cells to EBV antigen-expressing cells, we extended the observations obtained with CD28-containing antibody-based receptors (37) to TCR and generated EBNA-3B-specific TCR containing CD3{zeta} preceded by CD28 [two-chain TCR{alpha}ß linked to CD28 and CD3{zeta} (tcTCR{alpha}ß:28{zeta})]. T cells expressing either tcTCR{alpha}ß:{zeta} or tcTCR{alpha}ß:28{zeta} were tested for their cytotoxic responses as well as their ability to produce IFN{gamma} and TNF{alpha} following co-culture with EBV antigen-positive B-LCL that were or were not pre-loaded with additional IVT-peptide. The inclusion of CD28 into TCR did not affect the cytotoxic responses of re-targeted T cells (Fig. 5), whereas it dramatically increased the antigen-specific IFN{gamma} production (Fig. 6). The antigen-specific production of TNF{alpha} was low and not affected by the inclusion of CD28.


Figure 5
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  		Fig. 5. The inclusion of a CD28 domain into EBV-specific TCR does not affect the cytotoxic responsiveness of re-targeted T cells. Human T cells transduced with the tcTCR{alpha}ß:{zeta} or tcTCR{alpha}ß:28{zeta} specific for EBNA-3B (A: first donor, B: second donor) were tested in a 6-h 51Cr-release assay with the IVTpos/A11pos cell line CJO as target cells in the absence (open symbols) or presence (closed symbols) of (additional) IVT-peptide. Target cells were pre-incubated with peptide for 30 min at 37°C at a final concentration of 10 µM. The effector cell to target cell ratio is indicated in the figure. The parental CTL clones were used as positive controls. Mock-transduced primary human T cells were not cytolytic toward the mentioned targets and the TCR-transduced T cells were not cytolytic toward an IVTneg target cell line (i.e. T2-A11).

 

Figure 6
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  		Fig. 6. CD28-containing EBV-specific TCRs mediate a significantly increased production of IFN{gamma} following stimulation with EBV antigen-expressing LCL. Human T cells transduced with the tcTCR{alpha}ß:{zeta} (white bars) or tcTCR{alpha}ß:28{zeta} (black bars) specific for EBNA-3B were co-cultivated for 18 h with the IVTpos/A11pos target cell line CJO in the absence [without (w/o) peptide] or presence (with (w/) IVT-peptide) of (additional) IVT-peptide, and IFN{gamma} production was measured. Effector cell to target cell ratio is 2:1. Target cells were pre-incubated with peptide for 30 min at 37°C at a final concentration of 10 µM. Data of one representative donor are shown out of two different donors (with one donor tested in two separate experiments and the other donor tested in a single experiment). In all stimulation experiments, the same antigen-positive target cell line CJO, with or without addition of exogenous IVT-peptide, and antigen-negative target cell line T2-A11 cell line were used. The parental CTL clones were used as positive controls. Mock-transduced primary human T cells were not responsive toward the above-mentioned target cells. T cells transduced with the tcTCR{alpha}ß:{zeta} or tcTCR{alpha}ß:28{zeta} specific for EBNA-3B produced 23 and 179 pg ml–1 IFN{gamma} in response to T2-A11 cells, respectively. PMA/ionomycin stimulations resulted in IFN{gamma} production levels of >5000 pg ml–1.

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
This paper reports on the functional characterization of EBV-specific modified TCR that are considered promising tools to treat EBV-induced diseases following gene transfer into human T cells. We generated a panel of TCRs with specificities to various EBV antigens such as EBNA-3A, 3B and BMLF-1. Our analysis of the antigen reactivity and specificity of TCR-transduced human T cells demonstrated that EBV-specific TCR can mediate a cytolytic response versus peptide-loaded B-LCL but not native EBVpos target cells. Table 1 shows that 20 to about 60% of donors of which the T cells express the introduced TCR specifically lyse EBV-peptide-loaded target cells (see also Fig. 2). This MHC-restricted killing of target cells showed a clear dependence on the antigen specificity and type of modified TCR used, with the EBNA-3B-specific tcTCR{alpha}ß:{zeta} being the most effective TCR. The non-responsiveness toward B-LCL that express endogenously processed antigen (Table 1 and Fig. 3) was somewhat expected for T cells transduced with TCR specific for BMLF-1 since only a minor fraction of B-LCLs express EBV lytic proteins. However, EBNA-3A and B antigens are expressed by numerous B-LCLs, yet appear unable to initiate a cytotoxic response by T cells transduced with the relevant TCR. In fact, prolonging the duration of the cytotoxicity assay (from 6 up to 16 h) or measuring cytokine production (i.e. IFN{gamma} or TNF{alpha}) hardly resulted in detectable antigen-specific T cell responses versus antigen-positive target cells. Gene transfer or perhaps donor-related aspects as possible explanations for the absence of cytolysis of native targets by EBV-specific TCRpos T cells are highly unlikely since a melanoma-specific TCR{alpha}ß with an identical format, when introduced into T cells from the same donor, is clearly able to mediate the expected response versus native target cells (Fig. 3B).

The Jurkat T cell-based NFAT reporter gene assay provides a fast and sensitive method to functionally validate TCRs (34), and was used to monitor signaling events downstream of EBV-specific TCRs. NFAT activation data confirmed the cytotoxicity findings. Jurkat T cells expressing TCRs specific either for EBNA-3B or 3A activated NFAT after stimulation with anti-TCRVß mAb and peptide–MHC complexes, but not following co-cultivation with antigen-positive target cells (Fig. 4A). This is in contrast to melanoma-specific and similarly modified TCR{alpha}ß expressed on the same Jurkat T cell line, which did induce NFAT activation after stimulation with peptide-pulsed target cells as well as native antigen-positive melanoma cells (Fig. 4B). These data demonstrate that EBV-specific TCRs are functional and are, at least following a strong TCR stimulus, able to initiate a signal transduction cascade ultimately leading to activation of NFAT. The ability of these EBV-specific modified TCR to initiate a signal was confirmed in primary human T cells in which CD3{zeta} phosphorylation was observed in response to strong TCR stimuli (data not shown).

We postulate the following explanations for the observed non-responsiveness of EBV-specific TCR-transduced T cells, or perhaps virus-specific TCR-transduced T cells in general.

First, the non-responsiveness may be inherent to a sub-optimal balance between TCR density and antigen density. We have shown earlier that such a functional balance exists between receptor density on the one hand and tumor-antigen density on the other (25). Interestingly, the expression level of the introduced EBV-specific TCR was about 19% lower in T cells from donors lacking a cytotoxic T cell response against peptide-loaded target cells relative to T cells from donors showing a peptide response (25 TCR-transduced T cell populations tested in 48 experiments, Student's t-test: P < 0.006). In addition, intra-donor comparisons of T cells from responding donors showed that responses were only observed when T cells expressed a sufficiently high level of TCR (data not shown). These observations are in line with a study on T cells transduced with an HIV-specific TCR, and suggest that a high receptor density on transduced T cells compensates for low antigen density on target cells (22). We speculate that the density of EBV antigen on in vitro cultured B-LCL target cells is too low to functionally interact with a bulk culture of human T cells transduced with EBV-specific TCR, whereas it is sufficiently high to trigger lysis by the original and highly differentiated EBV-specific CTL clones from which the DNA sequences coding for the TCR variable domains had been derived. Next to TCR expression, the surface expression of accessory and adhesion molecules such as CD2, CD3, CD11a/CD18, co-stimulatory molecules such as CD27 and CD28 as well as homing molecules such as CD62L also affect T cell responses in vitro (38, 39). Differences in the expression levels of such T cell markers, especially in the context of low antigen expression, may further contribute to the observed EBV-specific T cell non-responsiveness.

Second, gene transduction and culture conditions may also adversely affect the reactivity of EBV-specific T cells. Transduction of primary human T cells with retrovirus produced by other packaging cells (i.e. 293T or PG13) had no effect on the observed non-responsiveness. It is of interest, however, to note that EBV-specific T cells are sensitive to activation-induced cell death during the first days of culture (such as after anti-CD3 mAb T cell pre-activation), and do not fully differentiate into terminal effector T cells (CD45RA+, CD27–, CCR7–) during subsequent culture (40). Cultured T cells, expressing either an endogenous or introduced EBV-specific TCR, may therefore show a hampered response to antigen-positive target cells. Choosing alternative recipient cells for TCR transduction, such as antigen-experienced virus- or allo-specific T cells (41–43), may benefit EBV-specific T cell re-targeting. To this end, we have tried a small panel of different CTL clones and cell lines (CTL cell lines were kindly provided by D. H. Crawford, Edinburgh, UK) but experienced sub-optimal TCR gene transfer efficiencies when compared with primary human T cells (data not shown). Highly differentiated T cells may be more resistant to gene transfer than primary T cells, which possibly impacts gene transfer of TCR more than that of scFv (41) because of the inherent low ligand-binding affinity of TCR when compared with scFv. The use of antigen-experienced primary T cells, pre-sorted from PBMC with virus-specific tetramers, is reported to allow functional TCR gene transfer (43) and may represent an attractive strategy to improve EBV-specific TCR gene transfer.

Third, the receptor's ligand-binding affinity and/or signaling capacity may simply be sub-optimal to mediate T cell responses toward EBV antigen-positive target cells. Studies on gene transfer of full-length (non-modified) TCR{alpha} and ß chains specific for various viral antigens suggest that the success of TCR gene transfer is determined by the specific TCR used. For example and in line with our data, genetic introduction of TCR specific for EBV and HIV antigens has demonstrated clear T cell responses to peptide-loaded target cells but no responses to native antigen-positive cells other than virally super-infected cells (22, 24) or at most low responses to virus-infected target cells (23), whereas gene transfer of TCR specific for Human Papillomavirus and Cytomegalovirus antigens has demonstrated clear T cell responses to either peptide-loaded target cells or virus-expressing target cells (44, 45). Properties inherent to the specific TCRs, such as ligand-binding affinity and/or signaling capacity, may have contributed to the observed differences. In this respect, the incorporation of a CD28 domain in classical (non-MHC restricted) antibody-based receptors has already proven its surplus value for T cell re-targeting. scFv receptors harboring both CD28 (transmembrane plus intracellular domains) and either the Fc({varepsilon})RI{gamma} or CD3{zeta} chain (intracellular domains only) are clearly superior in their capacity to proliferate and mediate production of Th1-type cytokines (but not cytotoxicity) when compared with identical receptors lacking CD28 (37, 46). Primary mouse CD8+ T cells transduced with a CD28-containing scFv had a greater capacity to proliferate, secrete IFN{gamma} and inhibit established tumor growth and metastases in mice bearing tumors when compared with T cells transduced with an scFv lacking CD28 (46). In fact, it is currently believed that antigen-specific IFN{gamma} secretion may represent a better in vitro measure than cytotoxicity to predict antitumor efficacy of T cells in vivo. Also primary human T cells expressing a CD28-containing scFv exerted significant and durable antitumor activity in SCID mice with a transplanted human tumor (47). In the present study, we extended these observations to human TCR, and generated an EBNA-3B-specific tcTCR{alpha}ß:28{zeta}. This CD28-containing TCR did not result in enhanced cytotoxic responses toward antigen-positive target cells (Fig. 5), but dramatically increased receptor-mediated IFN{gamma} (but not TNF{alpha}) production (Fig. 6). The cytokine production appeared maximal in response to EBNA-3B-positive targets (CJO, Fig. 6; BK, not shown) and was not increased further due to peptide loading. This is the first report showing the functional relevance of human T cells genetically re-targeted with CD28-containing TCR, which, at least in this setting, confirms in vitro findings with CD28-containing antibody-based receptors (37).

Taken together, this paper shows that the success of genetic re-targeting of primary human T cells, as a therapeutic tool to treat cancers or viral diseases, depends on the chosen target antigen and type of receptor used, and works well for melanoma-specific receptors but is limited for EBV-specific receptors. The inclusion of a CD28 co-stimulatory domain provides a receptor that mediates enhanced EBV antigen-specific responses (i.e. IFN{gamma} production) and represents a strategy that can be further developed to treat EBV-induced diseases with re-targeted T cells.


   Acknowledgements
 
We thank T. Boon and P. Coulie for providing the 82/30 CTL clone and G43 melanoma cell line, M. Giphart for the BSM, CJO, HAR, and APD B-LCLs and J. W. Drijfhout for peptide synthesis. We further thank Ms. S. Langeveld for performing the necessary ELISA. This work was supported by the Dutch Cancer Society (DDHK 1996-1253).


   Abbreviations
 
BCS   bovine calf serum
BMLF-1   BamHI-M leftward reading frame
EBER   EBV-encoded RNA
EBNA   Epstein–Barr nuclear antigen
GVHD   graft-versus-host disease
LCL   lymphoblast cell line
LMP   latent membrane proteins
MFI   mean fluorescence intensity
mIg   mouse Ig
NFAT   nuclear factor of activated T cells
PMA   phorbol myristate acetate
RLU   relative light units
sc   single chain
scTCR{alpha}ß:{gamma}   single-chain TCR{alpha}ß linked to Fc({varepsilon})RI{gamma}
T2-A11   HLA-A11-transfected T2 cells
tc   two chain
tcTCR{alpha}ß:{zeta}   two-chain TCR{alpha}ß linked to CD3{zeta}
tcTCR{alpha}ß:28{zeta}   two-chain TCR{alpha}ß linked to CD28 and CD3{zeta}
TNF{alpha}   tumor necrosis factor {alpha}

   Notes
 
Transmitting editor: L. Lanier

Received 16 April 2005, accepted 23 January 2006.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

  1. Rickinson, A. and Kieff, E. 1996. Epstein-Barr virus. In Fields, B. N., Knipe, D. M., Howley, P. M., Chanock, R. M., Melnick, J. L., Monath, T. P., Roizman, B., and Strauss, S. E., eds, Fields Virology, p. 2397. Lippincott-Raven, Philadelphia.
  2. Rickinson, A. B., Moss, D. J., Wallace, L. E. et al. 1981. Long-term T-cell-mediated immunity to Epstein-Barr virus. Cancer Res. 41:4216.[Free Full Text]
  3. Cohen, J. I. 2000. Epstein-Barr virus infection. N. Engl. J. Med. 343:481.[Free Full Text]
  4. Kieff, E. 1996. Epstein-Barr virus and its replication. In Fields, B. N., Knipe, D. M., Howley, P. M., Chanock, R. M., Melnick, J. L., Monath, T. P., Roizman, B., and Strauss, S. E., eds, Fields Virology, p. 2343. Lippincott-Raven, Philadelphia.
  5. Brooks, L., Yao, Q. Y., Rickinson, A. B. and Young, L. S. 1992. Epstein-Barr virus latent gene transcription in nasopharyngeal carcinoma cells: coexpression of EBNA1, LMP1, and LMP2 transcripts. J. Virol. 66:2689.[Abstract/Free Full Text]
  6. Deacon, E. M., Pallesen, G., Niedobitek, G. et al. 1993. Epstein-Barr virus and Hodgkin's disease: transcriptional analysis of virus latency in the malignant cells. J. Exp. Med. 177:339.[Abstract/Free Full Text]
  7. Hitt, M. M., Allday, M. J., Hara, T. et al. 1989. EBV gene expression in an NPC-related tumour. EMBO J. 8:2639.[Web of Science][Medline]
  8. Imai, S., Koizumi, S., Sugiura, M. et al. 1994. Gastric carcinoma: monoclonal epithelial malignant cells expressing Epstein-Barr virus latent infection protein. Proc. Natl. Acad. Sci. USA 91:9131.[Abstract/Free Full Text]
  9. Rowe, M., Rowe, D. T., Gregory, C. D. et al. 1987. Differences in B cell growth phenotype reflect novel patterns of Epstein-Barr virus latent gene expression in Burkitt's lymphoma cells. EMBO J. 6:2743.[Web of Science][Medline]
  10. Thomas, J. A., Allday, M. J. and Crawford, D. H. 1991. Epstein-Barr virus-associated lymphoproliferative disorders in immunocompromised individuals. Adv. Cancer Res. 57:329.[Web of Science][Medline]
  11. Young, L., Alfieri, C., Hennessy, K. et al. 1989. Expression of Epstein-Barr virus transformation-associated genes in tissues of patients with EBV lymphoproliferative disease. N. Engl. J. Med. 321:1080.[Abstract]
  12. Heslop, H. E., Brenner, M. K. and Rooney, C. M. 1994. Donor T cells to treat EBV-associated lymphoma. N. Engl. J. Med. 331:679.[Free Full Text]
  13. Papadopoulos, E. B., Ladanyi, M., Emanuel, D. et al. 1994. Infusions of donor leukocytes to treat Epstein-Barr virus-associated lymphoproliferative disorders after allogeneic bone marrow transplantation. N. Engl. J. Med. 330:1185.[Abstract/Free Full Text]
  14. Comoli, P., Labirio, M., Basso, S. et al. 2002. Infusion of autologous Epstein-Barr virus (EBV)-specific cytotoxic T cells for prevention of EBV-related lymphoproliferative disorder in solid organ transplant recipients with evidence of active virus replication. Blood 99:2592.[Abstract/Free Full Text]
  15. Khanna, R., Bell, S., Sherritt, M. et al. 1999. Activation and adoptive transfer of Epstein-Barr virus-specific cytotoxic T cells in solid organ transplant patients with posttransplant lymphoproliferative disease. Proc. Natl. Acad. Sci. USA 96:10391.[Abstract/Free Full Text]
  16. Haque, T., Wilkie, G. M., Taylor, C. et al. 2002. Treatment of Epstein-Barr-virus-positive post-transplantation lymphoproliferative disease with partly HLA-matched allogeneic cytotoxic T cells. Lancet 360:436.[CrossRef][Web of Science][Medline]
  17. Bolhuis, R. L., Willemsen R. A. and Gratama, J. W. 2000. Clinical applications of redirected cytotoxicity. In Sitkovsky, M. V. and Henkart, P. A., eds, Cytotoxic cells, p. 423. Lippincott-Raven, Philadelphia.
  18. Willemsen, R. A., Weijtens, M. E., Ronteltap, C. et al. 2000. Grafting primary human T lymphocytes with cancer-specific chimeric single chain and two chain TCR. Gene Ther. 7:1369.[CrossRef][Web of Science][Medline]
  19. Willemsen, R. A., Debets, R., Hart, E., Hoogenboom, H. R., Bolhuis, R. L. and Chames, P. 2001. A phage display selected fab fragment with MHC class I-restricted specificity for MAGE-A1 allows for retargeting of primary human T lymphocytes. Gene Ther. 8:1601.[CrossRef][Web of Science][Medline]
  20. Clay, T. M., Custer, M. C., Sachs, J., Hwu, P., Rosenberg, S. A. and Nishimura, M. I. 1999. Efficient transfer of a tumor antigen-reactive TCR to human peripheral blood lymphocytes confers anti-tumor reactivity. J. Immunol. 163:507.[Abstract/Free Full Text]
  21. Schaft, N., Willemsen, R. A., de Vries, J. et al. 2003. Peptide fine specificity of anti-glycoprotein 100 CTL is preserved following transfer of engineered TCR alpha beta genes into primary human T lymphocytes. J. Immunol. 170:2186.[Abstract/Free Full Text]
  22. Cooper, L. J., Kalos, M., Lewinsohn, D. A., Riddell, S. R. and Greenberg, P. D. 2000. Transfer of specificity for human immunodeficiency virus type 1 into primary human T lymphocytes by introduction of T-cell receptor genes. J. Virol. 74:8207.[Abstract/Free Full Text]
  23. Ueno, T., Fujiwara, M., Tomiyama, H., Onodera, M. and Takiguchi, M. 2004. Reconstitution of anti-HIV effector functions of primary human CD8 T lymphocytes by transfer of HIV-specific alphabeta TCR genes. Eur. J. Immunol. 34:3379.[CrossRef][Web of Science][Medline]
  24. Orentas, R. J., Roskopf, S. J., Nolan, G. P. and Nishimura, M. I. 2001. Retroviral transduction of a T cell receptor specific for an Epstein-Barr virus-encoded peptide. Clin. Immunol. 98:220.[CrossRef][Web of Science][Medline]
  25. Weijtens, M. E., Hart, E. H. and Bolhuis, R. L. 2000. Functional balance between T cell chimeric receptor density and tumor associated antigen density: CTL mediated cytolysis and lymphokine production. Gene Ther. 7:35.[CrossRef][Web of Science][Medline]
  26. Chung, S., Wucherpfennig, K. W., Friedman, S. M., Hafler, D. A. and Strominger, J. L. 1994. Functional three-domain single-chain T-cell receptors. Proc. Natl. Acad. Sci. USA 91:12654.[Abstract/Free Full Text]
  27. Debets, R., Willemsen, R. and Bolhuis, R. 2002. Adoptive transfer of T-cell immunity: gene transfer with MHC-restricted receptors. Trends Immunol. 23:435.[CrossRef][Web of Science][Medline]
  28. Campos-Lima, P. O., Levitsky, V., Imreh, M. P., Gavioli, R. and Masucci, M. G. 1997. Epitope-dependent selection of highly restricted or diverse T cell receptor repertoires in response to persistent infection by Epstein-Barr virus. J. Exp. Med. 186:83.[Abstract/Free Full Text]
  29. Levitsky, V., Campos-Lima, P. O., Frisan, T. and Masucci, M. G. 1998. The clonal composition of a peptide-specific oligoclonal CTL repertoire selected in response to persistent EBV infection is stable over time. J. Immunol. 161:594.[Abstract/Free Full Text]
  30. Lim, A., Trautmann, L., Peyrat, M. A. et al. 2000. Frequent contribution of T cell clonotypes with public TCR features to the chronic response against a dominant EBV-derived epitope: application to direct detection of their molecular imprint on the human peripheral T cell repertoire. J. Immunol. 165:2001.[Abstract/Free Full Text]
  31. Burrows, S. R., Silins, S. L., Moss, D. J., Khanna, R., Misko, I. S. and Argaet, V. P. 1995. T cell receptor repertoire for a viral epitope in humans is diversified by tolerance to a background major histocompatibility complex antigen. J. Exp. Med. 182:1703.[Abstract/Free Full Text]
  32. Van de Griend, R. J., Van Krimpen, B. A., Bol, S. J., Thompson, A. and Bolhuis, R. L. 1984. Rapid expansion of human cytotoxic T cell clones: growth promotion by a heat-labile serum component and by various types of feeder cells. J. Immunol. Methods 66:285.[CrossRef][Web of Science][Medline]
  33. Weijtens, M. E., Willemsen, R. A., Hart, E. H. and Bolhuis, R. L. 1998. A retroviral vector system ‘STITCH’ in combination with an optimized single chain antibody chimeric receptor gene structure allows efficient gene transduction and expression in human T lymphocytes. Gene Ther. 5:1195.[CrossRef][Web of Science][Medline]
  34. Schaft, N., Lankiewicz, B., Gratama, J. W., Bolhuis, R. L. and Debets, R. 2003. Flexible and sensitive method to functionally validate tumor-specific receptors via activation of NFAT. J. Immunol. Methods 280:13.[CrossRef][Web of Science][Medline]
  35. Lamers, C. H., Willemsen, R. A., Luider, B. A., Debets, R. and Bolhuis, R. L. 2002. Protocol for gene transduction and expansion of human T lymphocytes for clinical immunogene therapy of cancer. Cancer Gene Ther. 9:613.[CrossRef][Web of Science][Medline]
  36. Lamers, H. J., Gratama, J. W., van Putten, W. L., Stoter, G. and Bolhuis, R. L. 1991. Exogenous interleukin 2 recruits in vitro lymphokine-activated killer activity by in vivo activated lymphocytes. Cancer Res. 51:2324.[Abstract/Free Full Text]
  37. Hombach, A., Wieczarkowiecz, A., Marquardt, T. et al. 2001. Tumor-specific T cell activation by recombinant immunoreceptors: CD3 zeta signaling and CD28 costimulation are simultaneously required for efficient IL-2 secretion and can be integrated into one combined CD28/CD3 zeta signaling receptor molecule. J. Immunol. 167:6123.[Abstract/Free Full Text]
  38. Weijtens, M. E., Willemsen, R. A., Van Krimpen, B. A. and Bolhuis, R. L. 1998. Chimeric scFv/gamma receptor-mediated T-cell lysis of tumor cells is coregulated by adhesion and accessory molecules. Int. J. Cancer 77:181.[CrossRef][Web of Science][Medline]
  39. Gattinoni, L., Klebanoff, C. A., Palmer, D. C. et al. 2005. Acquisition of full effector function in vitro paradoxically impairs the in vivo antitumor efficacy of adoptively transferred CD8+ T cells. J. Clin. Invest. 115:1616.[CrossRef][Web of Science][Medline]
  40. Sauce, D., Rufer, N., Mercier, P. et al. 2003. Retrovirus-mediated gene transfer in polyclonal T cells results in lower apoptosis and enhanced ex vivo cell expansion of CMV-reactive CD8 T cells as compared with EBV-reactive CD8 T cells. Blood 102:1241.[Abstract/Free Full Text]
  41. Rossig, C., Bollard, C. M., Nuchtern, J. G., Rooney, C. M. and Brenner, M. K. 2002. Epstein-Barr virus-specific human T lymphocytes expressing antitumor chimeric T-cell receptors: potential for improved immunotherapy. Blood 99:2009.[Abstract/Free Full Text]
  42. Kershaw, M. H., Westwood, J. A. and Hwu, P. 2002. Dual-specific T cells combine proliferation and antitumor activity. Nat. Biotechnol. 20:1221.[CrossRef][Web of Science][Medline]
  43. Heemskerk, M. H., Hoogeboom, M., Hagedoorn, R., Kester, M. G., Willemze, R. and Falkenburg, J. H. 2004. Reprogramming of virus-specific T cells into leukemia-reactive T cells using T cell receptor gene transfer. J. Exp. Med. 199:885.[Abstract/Free Full Text]
  44. Scholten, K. B., Schreurs, M. W., Ruizendaal, J. J. et al. 2005. Preservation and redirection of HPV16E7-specific T cell receptors for immunotherapy of cervical cancer. Clin. Immunol. 114:119.[CrossRef][Web of Science][Medline]
  45. Van der Veken, L. T., Hoogeboom, M., Van Loenen, M. M., Hagedoorn, R., Falkenburg, J. H. F. and Heemskerk, M. H. M. 2004. Generation of {gamma}{delta} T cells with redirected antigen specificity for immunotherapy. (Abstract). Immunobiology 209:370.
  46. Haynes, N. M., Trapani, J. A., Teng, M. W. et al. 2002. Single-chain antigen recognition receptors that costimulate potent rejection of established experimental tumors. Blood 100:3155.[Abstract/Free Full Text]
  47. Pinthus, J. H., Waks, T., Kaufman-Francis, K. et al. 2003. Immuno-gene therapy of established prostate tumors using chimeric receptor-redirected human lymphocytes. Cancer Res. 63:2470.[Abstract/Free Full Text]

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