Alloreactivity of Virus Specific T-Cells.

Abstract Graft versus host disease (GVHD) in allogeneic stem cell transplantation (SCT) and graft rejection is caused by alloreactive T-cells. Alloreactivity can be exerted by naïve as well as by memory T-cells. Persistent latent viral infections, like those with herpes viruses, have a profound impact on the repertoire of memory T-cells. This implies that virus specific memory T-cells are also potentially alloreactive. Previously it has been shown that virus specific T-cell clones can cross react against allo-HLA. We investigated the frequency of alloreactivity mediated by virus specific T-cells. Mixed lymphocyte reactions, previously used to determine precursor frequencies of alloreactive T-cells, give an underestimation of the total frequency of alloreactive T-cells, due to limited number of allo-HLA alleles tested in this system. Therefore, in this study multiple CD8+ virus specific T-cells lines and clones were tested for alloreactivity against almost all frequent HLA class I and II alleles. From different healthy individuals we derived CD8+ virus specific T-cell lines, specific for Epstein Barr virus (EBV), Cytomegalovirus (CMV), Varicella Zoster virus (VZV) and Influenza virus (Flu) which were restricted to different HLA molecules. The generation of the T-cell lines and clones was performed by bulk sorting and single cell sorting, based on staining with viral peptide/MHC complex specific tetramers. The viral specificity of the expanded lines and clones was confirmed by tetramer staining and cytotoxicity and cytokine production assays. Polyclonality of the T-cell lines and monoclonality of the T-cell clones was confirmed by TCR Vβ analysis. Next, the T-cell lines and clones were screened for alloreactivity by testing against a panel of 29 different EBV transformed LCLs, together covering almost all frequent HLA class I and II molecules. 90% of tested virus specific T-cell lines and 40% of virus specific T-cell clones were found to be alloreactive, recognizing at least one of the allo-HLA alleles. For several lines and clones the specific recognized allo-HLA molecule was further identified using a panel of HLA typed target cells in combination with HLA specific blocking antibodies. Additionally, single HLA antigen expressing cell lines were used as target cells. Thus far we found EBV EBNA3A specific, HLA-A3 restricted T-cell clones to recognize HLA-A31. A CMV pp50 specific, HLA-A1 restricted T-cell line recognized HLA-A68. One VZV IE62 specific, HLA-A2 restricted clone showed recognition of HLA-B57, while another clone with the same specificity but with a different TCR Vβ recognized HLA-B55. An EBV BMLF specific, HLA-A2 restricted T-cell line showed recognition of HLA-A11. Finally an EBV BRLF specific, HLA-A3 restricted clone recognized HLA-A2. Our results show that a high percentage of virus specific T-cells can exert alloreactivity against allo- HLA molecules. Previously it was assumed that virus specific T-cells are not alloreactive against foreign HLA, allowing safe application of virus specific T-cell lines derived from HLA disparate donors in patients without the risk of inducing GVHD. Our data indicate that applying virus specific T-cell lines over HLA barriers does give a significant risk of GVHD and suggest that lines should be tested for alloreactivity against patient specific HLA alleles prior to application. A substantial part of the memory T-cell pool consists of virus specific T-cells, which are dominated by a limited repertoire of virus specific T-cell clones, present in high frequencies. Thus, virus specific T-cells recognizing allo-HLA alleles may also play an essential role in graft rejection.

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A Novel Strategy for Generation of Human Tumor-Specific T Cell Clones for Adoptive Transfer.

Blood ◽

10.1182/blood.v108.11.3713.3713 ◽

2006 ◽

Vol 108 (11) ◽

pp. 3713-3713

Author(s):

Seung-Tae Lee ◽

Shujuan Liu ◽

Pariya Sukhumalchandra ◽

Jeffrey Molldrem ◽

Patrick Hwu ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Tumor Cells ◽

Cell Lines ◽

Cd4 T Cells ◽

Cd4 T Cell ◽

Autologous Tumor ◽

T Cell Clones ◽

Cell Clones ◽

T Cell Lines

Abstract Adoptive T-cell therapy using donor lymphocyte infusions is a promising approach for treating hematological malignancies. But, efficacy is limited by the induction of graft-versus-host disease. Transfer of tumor-specific T-cell clones could enhance the graft-versus-tumor effect and eliminate graft-versus-host disease. However, isolating antigen-specific T-cell clones by the traditional limiting dilution approach is a time-consuming and laborious process. Here, we describe a novel strategy for rapidly cloning tumor-specific T cells. Lymphoma-specific T-cell lines were generated from two follicular lymphoma patients by repeated in vitro stimulation of lymphocytes isolated from tumor or blood with autologous soluble CD40 ligand-activated tumor cells. After four in vitro stimulations at 10-day intervals in the presence of IL-2 and IL-15, T-cell lines were found to be predominantly CD4+ T cells and produced significant amounts of TNF-a, GM-CSF, and IFN-γ in response to autologous tumor cells. The tumor reactivity was MHC class II restricted suggesting that it was mediated by CD4+ T cells. Staining with a TCR Vb antibody panel, a set of monoclonal antibodies against 24 human TCR Vb families, revealed that certain Vb families were overrepresented in each CD4+ T-cell line. In patient 1, 51% of CD4+ T cells were Vb1 positive, and in patient 2, 27% of CD4+ T cells were Vb8 positive. To clone lymphoma-specific T cells, CD4+ T-cell lines were labeled with CFSE and stimulated with autologous tumor cells. After 9 days of in vitro expansion in the presence of IL-2 and IL-15, CD4+ T-cell lines were stained with an anti-human CD4-APC monoclonal antibody and an anti-human TCR Vb-PE monoclonal antibody for each CD4+ T-cell line. Proliferating Vb1 cells from patient 1 and Vb8 cells from patient 2 were identified by their reduction in CFSE staining, and CD4+TCRV b +CFSEdim cells were sorted by flow cytometer. Monoclonality of the sorted cells was confirmed by PCR using a panel of optimized primers specific for 24 TCR Vb families, by TCR Vb spectratype analysis, and finally, by sequencing the TCR Vb gene used by each T-cell clone. Sorted tumor-specific T-cell clones could be expanded to large numbers using a 14-day rapid expansion protocol with allofeeder PBMCs, and confirmed to retain specificity against autologous tumor cells in a cytokine induction assay. This approach was also successfully used to isolate melanoma-specific CD8+ T-cell clones from two patients. We conclude that this approach is highly reproducible, rapid, and efficient for generating antigen-specific T-cell clones for adoptive T-cell therapy against human malignancies in the autologous or allogeneic setting.

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HLA-DPB1 Mismatching Results in the Generation of a Full Repertoire of HLA-DPB1 Specific T Cell Responses Showing Immunogenicity of All HLA-DPB1 Alleles

Blood ◽

10.1182/blood.v112.11.3504.3504 ◽

2008 ◽

Vol 112 (11) ◽

pp. 3504-3504

Author(s):

Caroline E. Rutten ◽

Simone A.P. van Luxemburg-Heijs ◽

Edith D. van der Meijden ◽

Marieke Griffioen ◽

Roelof Willemze ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cell Lines ◽

Hela Cells ◽

Cd4 T Cells ◽

Cross Reactivity ◽

T Cell Clones ◽

Cell Clones ◽

T Cell Lines

Abstract In unrelated donor hematopoietic stem cell transplantation (URD-SCT) patients are preferably transplanted with stem cells from a fully HLA matched donor, usually defined as identical for HLA-class I, -DR and -DQ. Since HLA-DPB1 is often not taken into consideration in donor selection, 80–90% of URD-SCTs are mismatched for HLA-DPB1. The role of HLA-DPB1 as transplantation antigen has been unclear, since clinical reports on the impact of matching for HLA-DPB1 on transplant outcome showed conflicting results. HLA-DPB1 mismatching has been associated with an increased risk of graft versus host disease (GVHD). However, we recently demonstrated that HLA-DPB1 specific T cells can mediate a potent graft versus leukemia effect without inducing GVHD. It has been suggested that the controversial effects of matching for HLA-DPB1 in URD-SCT could partly be explained by the assumption that not all HLA-DPB1 differences are immunogenic. This theory was based on the cross-reactive recognition of two HLA-DPB1* 09 specific T cell clones that recognized other HLA-DPB1 alleles sharing amino acids (aa) in position 8–11 of HLA-DPB1 (Zino et al, blood 2004). It was hypothesized that there would be no induction of T cell responses between individuals expressing HLA-DPB1 molecules sharing this aa sequence. This was translated into a classification of permissive and non-permissive HLA-DPB1 mismatches in order to allow a broader donor selection. To investigate whether cross-reactive recognition of other HLA-DPB1 molecules by our previously generated HLA-DPB1*02 or *03 specific CD4+ T cell clones depended on the presence of specific aa sequences we tested recognition of a panel of 14 EBV-LCL expressing 9 different HLA-DPB1 molecules. All HLA-DPB1*02 as well as all *03 specific T cell clones showed cross-reactivity with other HLA-DPB1 alleles and each T cell clone exhibited its own pattern of cross-reactivity. Two HLA-DPB1*0201 specific T cell clones with different TCR-Vβ showed also recognition of EBV-LCL expressing HLA-DPB1*1001 and *1701 or HLA-DPB1*1001, *0901 and *1601 respectively. Five HLA-DPB1*03 reactive T cells clones with different TCR-Vβ showed differential cross-recognition of EBV-LCL expressing HLA-DPB1*0101, *0601, *1101, *1301 and *1401. To identify immunogenic differences the aa sequences of the HLA-DPB1 molecules recognized by the various T cell clones were compared. The HLA-DPB1 molecules recognized by the HLA-DPB1*02 specific T cell clones shared an aa substitution at position 69 compared to the responder cell. However, HLA-DPB1*0601,*0901 and *1901 with the same substitution were not recognized by both T cell clones. This phenomenon was also observed for the HLA-DPB1*03 specific T cell clones, indicating that the cross-reactive recognition of HLA-DPB1 could not be predicted by aa sequences. Next, we analyzed the immunogenicity of various HLA-DPB1 alleles in different stimulator/responder combinations to verify the classification of permissive and non-permissive mismatches. We developed a model to generate allo-HLA-DP responses by transducing HLA-class II negative HELA cells with various HLA-DP molecules and used these cells to stimulate purified CD4+ T cells from HLA-DPB1 homozygous donors. HELA cells transduced with HLA-DPB1*0101, *0201, *0301, *0401, *0402, *0501, *0601, *0901, *1101, *1301, *1401 or *1701 were used as stimulator cells. Responder CD4+ T cells were typed HLA-DPB1* 0201, *0301, *0401 or *0402. 14 days after stimulation, CD4+ T cells were tested for recognition of the stimulator cells and of HELA cells transduced with the responder HLA-DPB1 molecule as a negative control. For these 4 responders, stimulation with 12 different HLA-DP transduced HELA cell lines resulted in specific IFN-γ production in response to the stimulator cells in 47 out of 48 stimulations. 28 CD4+ T cell lines also showed cross-reactive recognition of HELA cells transduced with at least one other HLA-DPB1 molecule. In conclusion, we showed that cross-reactive recognition of various HLA-DPB1 molecules by HLA-DPB1 specific T cells is a common observation. However, we demonstrated that cross-reactivity between HLA-DPB1 molecules by allo-HLA-DPB1 specific T cells does not exclude the generation of immune response between individuals expressing these HLA-DPB1 molecules. By generating multiple allo-HLA-DP specific T cell lines, we showed that all HLA-DPB1 mismatch combinations are immunogenic.

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Alloreactive cloned T cell lines. I. Interactions between cloned amplifier and cytolytic T cell lines.

Journal of Experimental Medicine ◽

10.1084/jem.151.4.876 ◽

1980 ◽

Vol 151 (4) ◽

pp. 876-895 ◽

Cited By ~ 163

Author(s):

A L Glasebrook ◽

F W Fitch

Keyword(s):

T Cell ◽

Cell Lines ◽

Cytolytic Activity ◽

Surface Proteins ◽

Target Cells ◽

Spleen Cells ◽

T Cell Clones ◽

Cell Clones ◽

T Cell Lines ◽

Cytolytic Cell

Several T cell clones have been derived by limiting dilution of secondary mixed leukocyte culture cells stimulated by H-2- and M locus (Mls)-disparate spleen cells. When examined for the expression of cytolytic activity and the ability to proliferate, these cell clones can be classified into two major categories. One type of cell is noncytolytic; when cultured with irradiated spleen cells, such clones proliferate in response to Mls determinants. Some, but not all, of these clones express Lyt-1 alloantigens. The other type of cell is cytolytic; these clones do not proliferate when cultured with irradiated allogeneic spleen cells unless supernatant fluid (SF) is added. These cytolytic clones express Lyt-2 alloantigens. Some cytolytic clones are specific for H-2Kd and others for H-2Dd alloantigens. Still other cytolytic cell clones exhibit cross-reactive lysis of different H-2-bearing tumor and Con A blast target cells. Noncytolytic T cell clones, when stimulated by Mls antigens, were examined for their ability to promote proliferation of cytolytic T cell clones. All of the noncytolytic cell clones tested were able to promote proliferation of cytolytic cell clones with the concomitant expression of cytolytic activity directed toward the original stimulating alloantigen (H-2d). Amplification of cytolytic activity was dependent upon stimulation of the noncytolytic amplifier T cell clones by Mls antigens. Specific alloantigen (signal 1), however, was not required for proliferation of the cytolytic cell clones; the amplifying signal (signal 2), delivered by the amplifier cell clones, was sufficient alone to promote proliferation of the cytolytic cell clones. Whereas proliferation of the amplifier cells was radiosensitive, the generation of the soluble amplifying signal was radioresistant. Amplification of cytolytic activity was observed when either amplifier cells were physically separated from responding cytolytic cells in Marbrook cultures or when cytolytic cells were cultured with SF collected from amplifier cell cultures. The amplifying factors were neither antigen specific nor strain specific and could be produced by Lyt-1- cells. The availability of cloned T cell lines that retain specific biologic function offers unique opportunities to characterize cell surface proteins and cell-cell interactions.

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Allo-HLA reactivity of virus-specific memory T cells is common

Blood ◽

10.1182/blood-2009-07-234906 ◽

2010 ◽

Vol 115 (15) ◽

pp. 3146-3157 ◽

Cited By ~ 207

Author(s):

Avital L. Amir ◽

Lloyd J. A. D'Orsogna ◽

Dave L. Roelen ◽

Marleen M. van Loenen ◽

Renate S. Hagedoorn ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Memory T Cells ◽

Cross Reactivity ◽

Target Cells ◽

T Cell Clones ◽

Specific Memory ◽

Cell Clones ◽

The Cross ◽

Hla Molecules

Abstract Graft-versus-host disease and graft rejection are major complications of allogeneic HLA-mismatched stem cell transplantation or organ transplantation that are caused by alloreactive T cells. Because a range of acute viral infections have been linked to initiating these complications, we hypothesized that the cross-reactive potential of virus-specific memory T cells to allogeneic (allo) HLA molecules may be able to mediate these complications. To analyze the allo-HLA reactivity, T cells specific for Epstein-Barr virus, cytomegalovirus, varicella zoster virus, and influenza virus were tested against a panel of HLA-typed target cells, and target cells transduced with single HLA molecules. Eighty percent of T-cell lines and 45% of virus-specific T-cell clones were shown to cross-react against allo-HLA molecules. The cross-reactivity of the CD8 and CD4 T-cell clones was directed primarily against HLA class I and II, respectively. However, a restricted number of CD8 T cells exhibited cross-reactivity to HLA class II. T-cell receptor (TCR) gene transfer confirmed that allo-HLA reactivity and virus specificity were mediated via the same TCR. These results demonstrate that a substantial proportion of virus-specific T cells exert allo-HLA reactivity, which may have important clinical implications in transplantation settings as well as adoptive transfer of third-party virus-specific T cells.

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Allogeneic T-cell clones able to selectively destroy Philadelphia chromosome-bearing (Ph1+) human leukemia lines can also recognize Ph1- cells from the same patient

Blood ◽

10.1182/blood.v83.11.3390.bloodjournal83113390 ◽

1994 ◽

Vol 83 (11) ◽

pp. 3390-3402 ◽

Cited By ~ 1

Author(s):

KR Oettel ◽

OH Wesly ◽

MR Albertini ◽

JA Hank ◽

O Iliopolis ◽

...

Keyword(s):

T Cell ◽

Cell Lines ◽

In Vitro Model ◽

Target Cells ◽

Human Leukemia ◽

T Cell Clones ◽

Cell Clones ◽

Vitro Model ◽

T Cell Lines

Immunocompetent cells in bone marrow allografts have been associated with a graft-versus-leukemia (GVL) effect. To further characterize effector mechanisms that may be involved in this GVL phenomenon, we have previously established an in vitro model to identify allogeneic T- cell clones that selectively mediate cytotoxicity against a patient's leukemic cells, but not against nonleukemic lymphocytes from the same patient. We have modified this in vitro model to test whether the Ph1 chromosome and the P210 fusion protein it controls have a detectable role in leukemia-specific recognition by allogeneic T-cell clones. In this report, T-cell lines reactive with allogeneic Ph1 chromosome- bearing (Ph1+) chronic myeloid leukemia (CML) cell lines were derived and selected to be minimally reactive with Ph1 negative (Ph1-) lymphoid lines from the same patient. However, after prolonged culture, these same T-cell lines also mediated significant destruction of the Ph1- target cells from the same patients. These T-cell lines specifically recognized cells from the allogeneic CML patient to which they were sensitized, and were not contaminated by an outgrowth of natural killer cells. Furthermore, subclones could be derived from these T-cell lines, and some of these subclones again showed selective killing of the allogeneic Ph1+ leukemia cell lines, and not of the Ph1- cell line from the same patient. Analyses of T-cell receptor (TCR) genes showed the alloreactive T-cell lines and the Ph1+ selective subclones derived from them to be of the same clonal origin. This suggests that the same T cells reacting with antigens expressed on the nonleukemic Ph1- targets can at times selectively and preferentially kill the allogeneic Ph1+ cells. As the same TCR that recognizes Ph1+ cells also can recognize the Ph1- targets, it appears that the Ph1+ chromosome does not play a detectable role in recognition by these allogeneic T-cell clones. This in vitro observation may provide a model for evaluating the relationship between GVL and graft-versus-host disease effects.

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Chlamydia trachomatis-Specific Human CD8+ T Cells Show Two Patterns of Antigen Recognition

Infection and Immunity ◽

10.1128/iai.72.8.4357-4367.2004 ◽

2004 ◽

Vol 72 (8) ◽

pp. 4357-4367 ◽

Cited By ~ 8

Author(s):

Malgosia K. Matyszak ◽

J. S. Hill Gaston

Keyword(s):

T Cells ◽

T Cell ◽

Chlamydia Trachomatis ◽

Cell Lines ◽

Class I ◽

T Cell Clones ◽

Cell Clones ◽

Infected Cells ◽

Ifn Γ ◽

T Cell Lines

ABSTRACT Chlamydia trachomatis is an intracellular gram-negative bacteria which causes several clinically important diseases. T-cell-mediated immunity and the production of gamma interferon (IFN-γ) are known to be essential for the clearance of the bacteria in vivo. Here we have investigated CD8+-T-cell responses to C. trachomatis in patients with previous episodes of chlamydia infection. To isolate C. trachomatis-specific CD8+-T-cell lines, dendritic cells (DC) were infected with C. trachomatis and cocultured with purified CD8+ T cells to generate C. trachomatis-specific CD8+-T-cell lines which were then cloned. Two patterns of recognition of C. trachomatis-infected cells by CD8+-T-cell clones were identified. In the first, C. trachomatis antigens were recognized in association with classical class I HLA antigens, and responses were inhibited by class I HLA-specific monoclonal antibodies. The second set of clones was unrestricted by classical HLA class I, and further studies showed that CD1 molecules were also not the restriction element for those clones. Both types of clones produced IFN-γ in response to C. trachomatis and were able to lyse C. trachomatis-infected target cells. However, unrestricted clones recognized C. trachomatis-infected cells at much earlier time points postinfection than HLA-restricted clones. Coculture of C. trachomatis-infected DC with the C. trachomatis-specific clones induced DC activation and a rapid enhancement of interleukin-12 (IL-12) production. Early production of IL-12 during C. trachomatis infection, facilitated by unrestricted CD8+-T-cell clones, may be important in ensuring a subsequent Th1 T-cell-mediated response by classical major histocompatibility complex-restricted CD4+ and CD8+ T cells.

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Allogeneic T-cell clones able to selectively destroy Philadelphia chromosome-bearing (Ph1+) human leukemia lines can also recognize Ph1- cells from the same patient

Blood ◽

10.1182/blood.v83.11.3390.3390 ◽

1994 ◽

Vol 83 (11) ◽

pp. 3390-3402 ◽

Cited By ~ 37

Author(s):

KR Oettel ◽

OH Wesly ◽

MR Albertini ◽

JA Hank ◽

O Iliopolis ◽

...

Keyword(s):

T Cell ◽

Cell Lines ◽

In Vitro Model ◽

Target Cells ◽

Human Leukemia ◽

T Cell Clones ◽

Cell Clones ◽

Vitro Model ◽

T Cell Lines

Abstract Immunocompetent cells in bone marrow allografts have been associated with a graft-versus-leukemia (GVL) effect. To further characterize effector mechanisms that may be involved in this GVL phenomenon, we have previously established an in vitro model to identify allogeneic T- cell clones that selectively mediate cytotoxicity against a patient's leukemic cells, but not against nonleukemic lymphocytes from the same patient. We have modified this in vitro model to test whether the Ph1 chromosome and the P210 fusion protein it controls have a detectable role in leukemia-specific recognition by allogeneic T-cell clones. In this report, T-cell lines reactive with allogeneic Ph1 chromosome- bearing (Ph1+) chronic myeloid leukemia (CML) cell lines were derived and selected to be minimally reactive with Ph1 negative (Ph1-) lymphoid lines from the same patient. However, after prolonged culture, these same T-cell lines also mediated significant destruction of the Ph1- target cells from the same patients. These T-cell lines specifically recognized cells from the allogeneic CML patient to which they were sensitized, and were not contaminated by an outgrowth of natural killer cells. Furthermore, subclones could be derived from these T-cell lines, and some of these subclones again showed selective killing of the allogeneic Ph1+ leukemia cell lines, and not of the Ph1- cell line from the same patient. Analyses of T-cell receptor (TCR) genes showed the alloreactive T-cell lines and the Ph1+ selective subclones derived from them to be of the same clonal origin. This suggests that the same T cells reacting with antigens expressed on the nonleukemic Ph1- targets can at times selectively and preferentially kill the allogeneic Ph1+ cells. As the same TCR that recognizes Ph1+ cells also can recognize the Ph1- targets, it appears that the Ph1+ chromosome does not play a detectable role in recognition by these allogeneic T-cell clones. This in vitro observation may provide a model for evaluating the relationship between GVL and graft-versus-host disease effects.

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EBV-Specific Cytotoxic T Lymphocytes by LMP2a- and EBNA3a-Specific T Cell Receptor Gene Transfer for Adoptive Therapy in EBV-Associated Hodgkin’s Disease and Post-Transplant Lymphoma.

Blood ◽

10.1182/blood.v110.11.2334.2334 ◽

2007 ◽

Vol 110 (11) ◽

pp. 2334-2334

Author(s):

Tuan D. Nguyen ◽

Haike Gelfort ◽

Kathrin Sebelin-Wulf ◽

Oliver Schmetzer ◽

Wolfgang Uckert ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cell Lines ◽

Adoptive Transfer ◽

Adoptive Therapy ◽

T Cell Clones ◽

Cell Clones ◽

Human T Cells ◽

Ifn Γ ◽

T Cell Lines

Abstract Adoptive transfer of polyclonal EBV-specific T cell lines has been used as prophylaxis and therapy in patients with EBV-associated malignancies. However, this strategy is time-consuming and demands difficult generation of lymphoblastoid cell lines (LCLs) and corresponding T cells for each individual patient. We applied an alternative strategy to confer T cell immunity against EBV-antigens by isolating EBV antigen-specific T cell receptors (TCRs) for transduction of primary human T cells for adoptive therapy. Previously, we have demonstrated the feasibility of using peptide-pulsed dendritic cells (DC) for generating high-affinity EBV antigen-specific T cell lines and T cell clones. Based on this strategy, T cell clones directed against LMP2a and EBNA3a were generated and functionally analyzed. Monospecificity was demonstrated by homogeneous double staining with CD8 and appropriate tetramers. High avidity of T cell clones (< 0.01 μM) was shown by peptide titration in an ELISPOT assay for IFN-γ secretion. In addition, the cytokine secretion profiles of some of the T cell clones were tested by cytokine bead array assay. High secretion levels of IFN-γ, IL-2 as well as TNF-α after stimulation with the EBNA3a- or LMP2a-peptide were shown for the corresponding T cell clones. Potent TCRs from one LMP2a-specific, HLA-A2-restricted and one EBNA3a-specific, HLA-B8-restricted T cell clone were isolated and cloned into the retroviral vector MP71. Transduction efficiency of TCR-deficient T cell lines was > 40% (TCR-LMP2a) and > 30% (TCR-EBNA3a) as measured by tetramer staining. Both TCR-LMP2a- and TCR-EBNA3a-redirected T cell lines were functional as indicated by NFAT-mediated luciferase expression upon TCR-MHC-peptide ligation. Primary human T cells were successfully transduced with TCR-LMP2a (∼ 12% tetramer-positive) and TCR-EBNA3a (∼ 3% tetramer-positive). Importantly, both TCRs conferred similar cytolytic activity against EBV-transformed B cell lines. Our data support the development of TCR-transduced T cells for adoptive transfer in EBV-associated malignancies, including Hodgkin′ s disease and nasopharyngeal carcinoma in which only subdominant EBV antigens are expressed. The feasibility and the therapeutic potential of TCR-transduced T cells for adoptive transfer have already been shown in a clinical phase I trial in patients with metastatic melanoma. We believe that redirecting human PBLs is a rapid and efficient tool toward adoptive transfer in EBV-associated malignancies.

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