Erratum: A library of cancer testis specific T cell receptors for T cell receptor gene therapy.

[This corrects the article DOI: 10.1016/j.omto.2022.11.007.].

PRAME and CTCFL-reactive TCRs for the treatment of ovarian cancer.

Recurrent disease emerges in the majority of patients with ovarian cancer (OVCA). Adoptive T-cell therapies with T-cell receptors (TCRs) targeting tumor-associated antigens (TAAs) are considered promising solutions for less-immunogenic 'cold' ovarian tumors. In order to treat a broader patient population, more TCRs targeting peptides derived from different TAAs binding in various HLA class I molecules are essential. By performing a differential gene expression analysis using mRNA-seq datasets, PRAME, CTCFL and CLDN6 were selected as strictly tumor-specific TAAs, with high expression in ovarian cancer and at least 20-fold lower expression in all healthy tissues of risk. In primary OVCA patient samples and cell lines we confirmed expression and identified naturally expressed TAA-derived peptides in the HLA class I ligandome. Subsequently, high-avidity T-cell clones recognizing these peptides were isolated from the allo-HLA T-cell repertoire of healthy individuals. Three PRAME TCRs and one CTCFL TCR of the most promising T-cell clones were sequenced, and transferred to CD8+ T cells. The PRAME TCR-T cells demonstrated potent and specific antitumor reactivity in vitro and in vivo. The CTCFL TCR-T cells efficiently recognized primary patient-derived OVCA cells, and OVCA cell lines treated with demethylating agent 5-aza-2'-deoxycytidine (DAC). The identified PRAME and CTCFL TCRs are promising candidates for the treatment of patients with ovarian cancer, and are an essential addition to the currently used HLA-A*02:01 restricted PRAME TCRs. Our selection of differentially expressed genes, naturally expressed TAA peptides and potent TCRs can improve and broaden the use of T-cell therapies for patients with ovarian cancer or other PRAME or CTCFL expressing cancers.

Broadly applicable TCR-based therapy for multiple myeloma targeting the immunoglobulin J chain.

BACKGROUND: The immunoglobulin J chain (Jchain) is highly expressed in the majority of multiple myeloma (MM), and Jchain-derived peptides presented in HLA molecules may be suitable antigens for T-cell therapy of MM. METHODS: Using immunopeptidomics, we identified Jchain-derived epitopes presented by MM cells, and pHLA tetramer technology was used to isolate Jchain-specific T-cell clones. RESULTS: We identified T cells specific for Jchain peptides presented in HLA-A1, -A24, -A3, and -A11 that recognized and lysed JCHAIN-positive MM cells. TCRs of the most promising T-cell clones were sequenced, cloned into retroviral vectors, and transferred to CD8 T cells. Jchain TCR T cells recognized target cells when JCHAIN and the appropriate HLA restriction alleles were expressed, while JCHAIN or HLA-negative cells, including healthy subsets, were not recognized. Patient-derived JCHAIN-positive MM samples were also lysed by Jchain TCR T cells. In a preclinical in vivo model for established MM, Jchain-A1, -A24, -A3, and -A11 TCR T cells strongly eradicated MM cells, which resulted in 100-fold lower tumor burden in Jchain TCR versus control-treated mice. CONCLUSIONS: We identified TCRs targeting Jchain-derived peptides presented in four common HLA alleles. All four TCRs demonstrated potent preclinical anti-myeloma activity, encouraging further preclinical testing and ultimately clinical development.

A library of cancer testis specific T cell receptors for T cell receptor gene therapy.

To increase the number of cancer patients that can be treated with T cell receptor (TCR) gene therapy, we aimed to identify a set of high-affinity cancer-specific TCRs targeting different melanoma-associated antigens (MAGEs). In this study, peptides derived from MAGE genes with tumor-specific expression pattern were identified by human leukocyte antigen (HLA) peptidomics. Next, peptide-HLA tetramers were generated, and used to sort MAGE-specific CD8+ T cell clones from the allogeneic (allo) HLA repertoire of healthy donors. To evaluate the clinical potential, most potent TCRs were sequenced, transferred into peripheral blood-derived CD8+ T cells, and tested for antitumor efficacy. In total we identified, seven MAGE-specific TCRs that effectively target MAGE-A1, MAGE-A3, MAGE-A6, and MAGE-A9 in the context of HLA-A∗01:01, -A∗02:01, -A∗03:01, -B∗07:02, -B∗35:01, or -C∗07:02. TCR gene transfer into CD8⁺ T cells resulted in efficient reactivity against a variety of different tumor types, while no cross-reactivity was detected. In addition, major in vivo antitumor effects of MAGE-A1 specific TCR engineered CD8⁺ T cells were observed in the orthotopic xenograft model for established multiple myeloma. The identification of seven MAGE-specific TCRs expands the pool of cancer patients eligible for TCR gene therapy and increases possibilities for personalized TCR gene therapy.

WT1-specific TCRs directed against newly identified peptides install antitumor reactivity against acute myeloid leukemia and ovarian carcinoma.

BACKGROUND: Transcription factor Wilms' tumor gene 1 (WT1) is an ideal tumor target based on its expression in a wide range of tumors, low-level expression in normal tissues and promoting role in cancer progression. In clinical trials, WT1 is targeted using peptide-based or dendritic cell-based vaccines and T-cell receptor (TCR)-based therapies. Antitumor reactivities were reported, but T-cell reactivity is hampered by self-tolerance to WT1 and limited number of WT1 peptides, which were thus far selected based on HLA peptide binding algorithms. METHODS: In this study, we have overcome both limitations by searching in the allogeneic T-cell repertoire of healthy donors for high-avidity WT1-specific T cells, specific for WT1 peptides derived from the HLA class I associated ligandome of primary leukemia and ovarian carcinoma samples. RESULTS: Using broad panels of malignant cells and healthy cell subsets, T-cell clones were selected that demonstrated potent and specific anti-WT1 T-cell reactivity against five of the eight newly identified WT1 peptides. Notably, T-cell clones for WT1 peptides previously used in clinical trials lacked reactivity against tumor cells, suggesting limited processing and presentation of these peptides. The TCR sequences of four T-cell clones were analyzed and TCR gene transfer into CD8+ T cells installed antitumor reactivity against WT1-expressing solid tumor cell lines, primary acute myeloid leukemia (AML) blasts, and ovarian carcinoma patient samples. CONCLUSIONS: Our approach resulted in a set of naturally expressed WT1 peptides and four TCRs that are promising candidates for TCR gene transfer strategies in patients with WT1-expressing tumors, including AML and ovarian carcinoma.

Cutting Edge: Unconventional CD8+ T Cell Recognition of a Naturally Occurring HLA-A*02:01-Restricted 20mer Epitope.

Unconventional HLA class I-restricted CD8+ T cell epitopes, longer than 10 aa, have been implicated to play a role in human immunity against viruses and cancer. T cell recognition of long peptides, centrally bulging from the HLA cleft, has been described previously. Alternatively, long peptides can contain a linear HLA-bound core peptide, with a N- or C-terminal peptide "tail" extending from the HLA peptide binding groove. The role of such a peptide "tail" in CD8+ T cell recognition remains unclear. In this study, we identified a 20mer peptide (FLPTPEELGLLGPPRPQVLA [FLP]) derived from the IL-27R subunit α gene restricted to HLA-A*02:01, for which we solved the crystal structure and demonstrated a long C-terminal "tail" extension. FLP-specific T cell clones demonstrated various recognition modes, some T cells recognized the FLP core peptide, while for other T cells the peptide tail was essential for recognition. These results demonstrate a crucial role for a C-terminal peptide tail in immunogenicity.

A broad and systematic approach to identify B cell malignancy-targeting TCRs for multi-antigen-based T cell therapy.

CAR T cell therapy has shown great promise for the treatment of B cell malignancies. However, antigen-negative escape variants often cause disease relapse, necessitating the development of multi-antigen-targeting approaches. We propose that a T cell receptor (TCR)-based strategy would increase the number of potential antigenic targets, as peptides from both intracellular and extracellular proteins can be recognized. Here, we aimed to isolate a broad range of promising TCRs targeting multiple antigens for treatment of B cell malignancies. As a first step, 28 target genes for B cell malignancies were selected based on gene expression profiles. Twenty target peptides presented in human leukocyte antigen (HLA)-A∗01:01, -A∗24:02, -B∗08:01, or -B∗35:01 were identified from the immunopeptidome of B cell malignancies and used to form peptide-HLA (pHLA)-tetramers for T cell isolation. Target-peptide-specific CD8 T cells were isolated from HLA-mismatched healthy donors and subjected to a stringent stepwise selection procedure to ensure potency and eliminate cross-reactivity. In total, five T cell clones specific for FCRL5 in HLA-A∗01:01, VPREB3 in HLA-A∗24:02, and BOB1 in HLA-B∗35:01 recognized B cell malignancies. For all three specificities, TCR gene transfer into CD8 T cells resulted in cytokine production and efficient killing of multiple B cell malignancies. In conclusion, using this systematic approach we successfully identified three promising TCRs for T cell therapy against B cell malignancies.

Healthy cells functionally present TAP-independent SSR1 peptides: implications for selection of clinically relevant antigens.

Tumors with an impaired transporter associated with antigen processing (TAP) present several endoplasmic reticulum-derived self-antigens on HLA class I (HLA-I) which are absent on healthy cells. Selection of such TAP-independent antigens for T cell-based immunotherapy should include analysis of their expression on healthy cells to prevent therapy-induced adverse toxicities. However, it is unknown how the absence of clinically relevant antigens on healthy cells needs to be validated. Here, we monitored TAP-independent antigen presentation on various healthy cells after establishing a T cell tool recognizing a TAP-independent signal sequence receptor 1-derived antigen. We found that most but not all healthy cells present this antigen under normal and inflammatory conditions, indicating that TAP-independent antigen presentation is a variable phenomenon. Our data emphasize the necessity of extensive testing of a wide variety of healthy cell types to define clinically relevant TAP-independent antigens that can be safely targeted by immunotherapy.

Mutated nucleophosmin 1 as immunotherapy target in acute myeloid leukemia.

The most frequent subtype of acute myeloid leukemia (AML) is defined by mutations in the nucleophosmin 1 (NPM1) gene. Mutated NPM1 (ΔNPM1) is an attractive target for immunotherapy, since it is an essential driver gene and 4 bp frameshift insertions occur in the same hotspot in 30%-35% of AMLs, resulting in a C-terminal alternative reading frame of 11 aa. By searching the HLA class I ligandome of primary AMLs, we identified multiple ΔNPM1-derived peptides. For one of these peptides, HLA-A*02:01-binding CLAVEEVSL, we searched for specific T cells in healthy individuals using peptide-HLA tetramers. Tetramer-positive CD8+ T cells were isolated and analyzed for reactivity against primary AMLs. From one clone with superior antitumor reactivity, we isolated the T cell receptor (TCR) and demonstrated specific recognition and lysis of HLA-A*02:01-positive ΔNPM1 AML after retroviral transfer to CD8+ and CD4+ T cells. Antitumor efficacy of TCR-transduced T cells was confirmed in immunodeficient mice engrafted with a human AML cell line expressing ΔNPM1. In conclusion, the data show that ΔNPM1-derived peptides are presented on AML and that CLAVEEVSL is a neoantigen that can be efficiently targeted on AML by ΔNPM1 TCR gene transfer. Immunotherapy targeting ΔNPM1 may therefore contribute to treatment of AML.

PRAME and HLA Class I expression patterns make synovial sarcoma a suitable target for PRAME specific T-cell receptor gene therapy.

Synovial sarcoma expresses multiple cancer testis antigens that could potentially be targeted by T-cell receptor (TCR) gene therapy. In this study we investigated whether PRAME-TCR-gene therapy could be an effective treatment for synovial sarcoma by investigating the potential of PRAME-specific T-cells to recognize sarcoma cells and by evaluating the expression patterns of PRAME and HLA class I (HLA-I) in synovial sarcoma tumor samples. All PRAME expressing sarcoma cell lines, including 2 primary synovial sarcoma cell cultures (passage < 3), were efficiently recognized by PRAME-specific T-cells. mRNA FISH demonstrated that PRAME was expressed in all synovial sarcoma samples, mostly in an homogeneous pattern. Immunohistochemistry demonstrated low HLA-I baseline expression in synovial sarcoma, but its expression was elevated in specific areas of the tumors, especially in biphasic components of biphasic synovial sarcoma. In 5/11 biphasic synovial sarcoma patients and in 1/17 monophasic synovial sarcoma patients, elevated HLA-I on tumor cells was correlated with infiltration of T-cells in these specific areas. In conclusion, low-baseline expression of HLA-I in synovial sarcoma is elevated in biphasic areas and in areas with densely infiltrating T-cells, which, in combination with homogeneous and high PRAME expression, makes synovial sarcoma potentially a suitable candidate for PRAME-specific TCR-gene therapy.

Preclinical Strategies to Identify Off-Target Toxicity of High-Affinity TCRs.

Adoptive transfer of T cells engineered with a cancer-specific T cell receptor (TCR) has demonstrated clinical benefit. However, the risk for off-target toxicity of TCRs remains a concern. Here, we examined the cross-reactive profile of T cell clone (7B5) with a high functional sensitivity for the hematopoietic-restricted minor histocompatibility antigen HA-2 in the context of HLA-A*02:01. HA-2pos Epstein-Barr virus-transformed B lymphoblastic cell lines (EBV-LCLs) and primary acute myeloid leukemia samples, but not hematopoietic HA-2neg samples, are effectively recognized. However, we found unexpected off-target recognition of human fibroblasts and keratinocytes not expressing the HA-2 antigen. To uncover the origin of this off-target recognition, we performed an alanine scanning approach, identifying six out of nine positions to be important for peptide recognition. This indicates a low risk for broad cross-reactivity. However, using a combinatorial peptide library scanning approach, we identified a CDH13-derived peptide activating the 7B5 T cell clone. This was confirmed by recognition of CDH13-transduced EBV-LCLs and cell subsets endogenously expressing CDH13, such as proximal tubular epithelial cells. As such, we recommend the use of a combinatorial peptide library scan followed by screening against additional cell subsets to validate TCR specificity and detect off-target toxicity due to cross-reactivity directed against unrelated peptides before selecting candidate TCRs for clinical testing.

PRAME as a Potential Target for Immunotherapy in Metastatic Uveal Melanoma.

Importance: Uveal melanoma (UM) is an intraocular primary malignant neoplasm that often gives rise to metastatic disease for which there are no effective therapies. A substantial proportion of UMs express the cancer-testis antigen PRAME (preferentially expressed antigen in melanoma), which can potentially be targeted by adoptive T-cell therapy. Objective: To determine whether there may be a rationale for PRAME-directed T-cell therapy for metastatic UM. Design, Setting, and Participants: An experimental study using a retrospective cohort of 64 patients with UM (median follow-up, 62 months) was conducted from January 8, 2015, to November 20, 2016, at the Leiden University Medical Center. Clinical, histopathologic, and genetic parameters were compared between 64 PRAME-positive and PRAME-negative UMs. HLA class I restricted, PRAME-specific T cells were stimulated with UM cell lines to measure their antigen-specific reactivity against these cell lines, which were analyzed for PRAME expression by real-time quantitative polymerase chain reaction. Uveal melanoma metastases from 16 unrelated patients were assessed for PRAME expression by messenger RNA fluorescence in situ hybridization and for HLA class I expression by immunofluorescence staining. Main Outcomes and Measures: Interferon γ production for antigen-specific reactivity and detection of PRAME and HLA class I expression in primary and metastatic UM. Results: Of the 64 patients in the study (31 women and 33 men; mean [SD] age at the time of enucleation, 60.6 [15.6] years), PRAME expression was negative in 35 primary UMs and positive in 29 primary UMs. Positive PRAME expression was associated with a high largest basal diameter (15.0 vs 12.0 mm; P = .005), ciliary body involvement (59% vs 26%; P = .008), and amplification of chromosome 8q (66% vs 23%; P = .002). PRAME-specific T cells reacted against 4 of 7 UM cell lines, demonstrating that T-cell reactivity correlated with PRAME expression. Metastatic UM samples were positive for PRAME messenger RNA in 11 of 16 patients and for HLA class I in 10 of 16 patients, with 8 of 16 patients demonstrating coexpression of both PRAME and HLA class I. Conclusions and Relevance: PRAME is expressed in many primary and metastatic UMs, and about half of the metastatic UMs coexpress PRAME and HLA class I. The finding that PRAME-specific T cells in this study reacted against PRAME-positive UM cell lines suggests a potential role for PRAME-directed immunotherapy for selected patients with metastatic UM.

TCR-based therapy for multiple myeloma and other B-cell malignancies targeting intracellular transcription factor BOB1.

Immunotherapy for hematological malignancies or solid tumors by administration of monoclonal antibodies or T cells engineered to express chimeric antigen receptors or T-cell receptors (TCRs) has demonstrated clinical efficacy. However, antigen-loss tumor escape variants and the absence of currently targeted antigens on several malignancies hamper the widespread application of immunotherapy. We have isolated a TCR targeting a peptide of the intracellular B cell-specific transcription factor BOB1 presented in the context of HLA-B*07:02. TCR gene transfer installed BOB1 specificity and reactivity onto recipient T cells. TCR-transduced T cells efficiently lysed primary B-cell leukemia, mantle cell lymphoma, and multiple myeloma in vitro. We also observed recognition and lysis of healthy BOB1-expressing B cells. In addition, strong BOB1-specific proliferation could be demonstrated for TCR-modified T cells upon antigen encounter. Furthermore, clear in vivo antitumor reactivity was observed of BOB1-specific TCR-engineered T cells in a xenograft mouse model of established multiple myeloma. Absence of reactivity toward a broad panel of BOB1- but HLA-B*07:02+ nonhematopoietic and hematopoietic cells indicated no off-target toxicity. Therefore, administration of BOB1-specific TCR-engineered T cells may provide novel cellular treatment options to patients with B-cell malignancies, including multiple myeloma.

Generation of CD20-specific TCRs for TCR gene therapy of CD20low B-cell malignancies insusceptible to CD20-targeting antibodies.

Immunotherapy of B-cell leukemia and lymphoma with CD20-targeting monoclonal antibodies (mAbs) has demonstrated clinical efficacy. However, the emergence of unresponsive disease due to low or absent cell surface CD20 urges the need to develop additional strategies. In contrast to mAbs, T-cells via their T-cell receptor (TCR) can recognize not only extracellular but also intracellular antigens in the context of HLA molecules. We hypothesized that T-cells equipped with high affinity CD20-targeting TCRs would be able to recognize B-cell malignancies even in the absence of extracellular CD20. We isolated CD8+ T-cell clones binding to peptide-MHC-tetramers composed of HLA-A*02:01 and CD20-derived peptide SLFLGILSV (CD20SLF) from HLA-A*02:01neg healthy individuals to overcome tolerance towards self-antigens such as CD20. High avidity T-cell clones were identified that readily recognized and lysed primary HLA-A2pos B-cell leukemia and lymphoma in the absence of reactivity against CD20-negative but HLA-A2pos healthy hematopoietic and nonhematopoietic cells. The T-cell clone with highest avidity efficiently lysed malignant cell-lines that had insufficient extracellular CD20 to be targeted by CD20 mAbs. Transfer of this TCR installed potent CD20-specificity onto recipient T-cells and led to lysis of CD20low malignant cell-lines. Moreover, our approach facilitates the generation of an off-the-shelf TCR library with broad applicability by targeting various HLA alleles. Using the same methodology, we isolated a T-cell clone that efficiently lysed primary HLA-B*07:02pos B-cell malignancies by targeting another CD20-derived peptide. TCR gene transfer of high affinity CD20-specific TCRs can be a valuable addition to current treatment options for patients suffering from CD20low B-cell malignancies.

A CD22-reactive TCR from the T-cell allorepertoire for the treatment of acute lymphoblastic leukemia by TCR gene transfer.

CD22 is currently evaluated as a target-antigen for the treatment of B-cell malignancies using chimeric antigen receptor (CAR)-engineered T-cells or monoclonal antibodies (mAbs). CAR- and mAbs-based immunotherapies have been successfully applied targeting other antigens, however, occurrence of refractory disease to these interventions urges the identification of additional strategies. Here, we identified a TCR recognizing the CD22-derived peptide RPFPPHIQL (CD22RPF) presented in human leukocyte antigen (HLA)-B*07:02. To overcome tolerance to self-antigens such as CD22, we exploited the immunogenicity of allogeneic HLA. CD22RPF-specific T-cell clone 9D4 was isolated from a healthy HLA-B*07:02neg individual, efficiently produced cytokines upon stimulation with primary acute lymphoblastic leukemia and healthy B-cells, but did not react towards healthy hematopoietic and nonhematopoietic cell subsets, including dendritic cells (DCs) and macrophages expressing low levels of CD22. Gene transfer of TCR-9D4 installed potent CD22-specificity onto recipient CD8+ T-cells that recognized and lysed primary B-cell leukemia. TCR-transduced T-cells spared healthy CD22neg hematopoietic cell subsets but weakly lysed CD22low-expressing DCs and macrophages. CD22-specific TCR-engineered T-cells could form an additional immunotherapeutic strategy with a complementary role to CAR- and antibody-based interventions in the treatment of B-cell malignancies. However, CD22 expression on non-B-cells may limit the attractiveness of CD22 as target-antigen in cellular immunotherapy.

Natural killer cells facilitate PRAME-specific T-cell reactivity against neuroblastoma.

Neuroblastoma is the most common solid tumor in children with an estimated 5-year progression free survival of 20-40% in stage 4 disease. Neuroblastoma actively avoids recognition by natural killer (NK) cells and cytotoxic T lymphocytes (CTLs). Although immunotherapy has gained traction for neuroblastoma treatment, these immune escape mechanisms restrain clinical results. Therefore, we aimed to improve neuroblastoma immunogenicity to further the development of antigen-specific immunotherapy against neuroblastoma. We found that neuroblastoma cells significantly increase surface expression of MHC I upon exposure to active NK cells which thereby readily sensitize neuroblastoma cells for recognition by CTLs. We show that oncoprotein PRAME serves as an immunodominant antigen for neuroblastoma as NK-modulated neuroblastoma cells are recognized by PRAMESLLQHLIGL/A2-specific CTL clones. Furthermore, NK cells induce MHC I upregulation in neuroblastoma through contact-dependent secretion of IFNγ. Our results demonstrate remarkable plasticity in the peptide/MHC I surface expression of neuroblastoma cells, which is reversed when neuroblastoma cells experience innate immune attack by sensitized NK cells. These findings support the exploration of NK cells as adjuvant therapy to enforce neuroblastoma-specific CTL responses.

Therapeutic targeting of the BCR-associated protein CD79b in a TCR-based approach is hampered by aberrant expression of CD79b.

Immunotherapy of B-cell malignancies using CD19-targeted chimeric antigen receptor-transduced T cells or CD20-targeted therapeutic monoclonal antibodies has shown clinical efficacy. However, refractory disease and the emergence of antigen-loss tumor escape variants after treatment demonstrate the need to target additional antigens. Here we aimed to target the B-cell receptor-associated protein CD79b by a T-cell receptor (TCR)-based approach. Because thymic selection depletes high-avidity T cells recognizing CD79b-derived peptides presented in self-HLA molecules, we aimed to isolate T cells recognizing these peptides presented in allogeneic HLA. Peptide-HLA tetramers composed of CD79b peptides bound to either HLA-A2 or HLA-B7 were used to isolate T-cell clones from HLA-A*0201 and B*0702-negative individuals. For 3 distinct T-cell clones, CD79b specificity was confirmed through CD79b gene transduction and CD79b-specific shRNA knockdown. The CD79b-specific T-cell clones were highly reactive against CD79b-expressing primary B-cell malignancies, whereas no recognition of nonhematopoietic cells was observed. Although lacking CD79b-cell surface expression, intermediate reactivity toward monocytes, hematopoietic progenitor cells, and T-cells was observed. Quantitative reverse transcriptase polymerase chain reaction revealed low CD79b gene expression in these cell types. Therefore, aberrant gene expression must be taken into consideration when selecting common, apparently lineage-specific self-antigens as targets for TCR-based immunotherapies.

HLA monomers as a tool to monitor indirect allorecognition.

BACKGROUND: Recognition of donor antigens can occur through two separate pathways: the direct pathway (non-self HLA on donor cells) and the indirect pathway (self-restricted presentation of donor derived peptides on recipient cells). Indirect allorecognition is important in the development of humoral rejection; therefore, there is an increasing interest in the monitoring of indirect alloreactive T-cells. We have used an in vitro model to determine the optimal requirements for indirect presentation and assessed the risk for semidirect presentation in this system. METHODS: HLA-typed monocyte-derived dendritic cells (moDCs) were incubated with cellular fragments or necrotic cells and incubated with either indirect or direct alloreactive T-cell clones. T-cell reactivity was measured through proliferation or cytokine secretion. HLA-typed moDC, monocytes, or PBMCs were incubated with HLA class I monomers, in combination with either direct/indirect T-cell clones. RESULTS: Although both were efficiently taken up, alloreactivity was limited to the semi-direct pathway, as measured by allospecific CD4 (indirect) and CD8 T-cell clones (direct) when cells were used. In contrast, HLA-A2 monomers were not only efficiently taken up but also processed and presented by HLA-typed moDC, monocytes, and PBMCs. Activation was shown by a dose-dependent induction of IFN-γ production and proliferation by the CD4 T-cell clone. Antigen presentation was most efficient when the monomers were cultured for longer periods (24-48 hr) in the presence of the T-cells. Using this method, no reactivity was observed by the CD8 T-cell clone, confirming no semidirect alloreactivity. CONCLUSION: We have developed a system that could be used to monitor indirect alloreactive T-cells.

Allo-HLA-reactive T cells inducing graft-versus-host disease are single peptide specific.

T-cell alloreactivity directed against non-self-HLA molecules has been assumed to be less peptide specific than conventional T-cell reactivity. A large variation in degree of peptide specificity has previously been reported, including single peptide specificity, polyspecificity, and peptide degeneracy. Peptide polyspecificity was illustrated using synthetic peptide-loaded target cells, but in the absence of confirmation against endogenously processed peptides this may represent low-avidity T-cell reactivity. Peptide degeneracy was concluded based on recognition of Ag-processing defective cells. In addition, because most investigated alloreactive T cells were in vitro activated and expanded, the previously determined specificities may have not been representative for alloreactivity in vivo. To study the biologically relevant peptide specificity and avidity of alloreactivity, we investigated the degree of peptide specificity of 50 different allo-HLA-reactive T-cell clones which were activated and expanded in vivo during GVHD. All but one of the alloreactive T-cell clones, including those reactive against Ag-processing defective T2 cells, recognized a single peptide allo-HLA complex, unique for each clone. Down-regulation of the expression of the recognized Ags using silencing shRNAs confirmed single peptide specificity. Based on these results, we conclude that biologically relevant alloreactivity selected during in vivo immune response is peptide specific.

PRAME-specific Allo-HLA-restricted T cells with potent antitumor reactivity useful for therapeutic T-cell receptor gene transfer.

PURPOSE: In human leukocyte antigen (HLA)-matched stem cell transplantation (SCT), it has been shown that beneficial immune response mediating graft-versus-tumor (GVT) responses can be separated from graft-versus-host disease (GVHD) immune responses. In this study, we investigated whether it would be possible to dissect the beneficial immune response of allo-HLA-reactive T cells with potent antitumor reactivity from GVHD-inducing T cells present in the detrimental immune response after HLA-mismatched SCT. EXPERIMENTAL DESIGN: The presence of specific tumor-reactive T cells in the allo-HLA repertoire was analyzed at the time of severe GVHD after HLA-mismatched SCT, using tetramers composed of different tumor-associated antigens (TAA). RESULTS: High-avidity allo-HLA-restricted T cells specific for the TAA preferentially expressed antigen on melanomas (PRAME) were identified that exerted highly single-peptide-specific reactivity. The T cells recognized multiple different tumor cell lines and leukemic cells, whereas no reactivity against a large panel of nonmalignant cells was observed. These T cells, however, also exerted low reactivity against mature dendritic cells (DC) and kidney epithelial cells, which was shown to be because of low PRAME expression. CONCLUSIONS: On the basis of potential beneficial specificity and high reactivity, the T-cell receptors of these PRAME-specific T cells may be effective tools for adoptive T-cell therapy. Clinical studies have to determine the significance of the reactivity observed against mature DCs and kidney epithelial cells.