HLA Restriction Element Research Articles

Unbiased, High-Throughput Identification of T Cell Epitopes by ELISPOT.

Recent systematic immune monitoring efforts suggest that, in humans, epitope recognition by T cells is far more complex than has been assumed based on minimalistic murine models. The increased complexity is due to the higher number of HLA loci in humans, the typical heterozygosity for these loci in the outbred population, and the high number of peptides that each HLA restriction element can bind with an affinity that suffices for antigen presentation. The sizable array of potential epitopes on any given antigen is due to each individual's unique HLA allele makeup. Of this individualized potential epitope space, chance events occurring in the course of the T cell response determine which epitopes induce dominant T cell expansions. Establishing the actually-engaged T cell repertoire in each human subject, including the individualized peptides targeted, therefore requires the systematic testing of all peptides that constitute the potential epitope space in that person. The goal of comprehensive, high-throughput epitope mapping can be readily established by the methods described in this chapter.

Determining if T cell antigens are naturally processed and presented on HLA class I molecules

BackgroundDetermining T cell responses to naturally processed and presented antigens is a critical immune correlate to determine efficacy of an investigational immunotherapeutic in clinical trials. In most cases, minimal epitopes and HLA restriction elements are unknown.ResultsHere, we detail the experimental use of ex vivo expanded autologous B cells as antigen presenting cells to overcome the limitation of unknown HLA restriction, and the use of electroporated full length mRNA encoding full length parental proteins to ensure that any observed T cell responses are specific for antigens that are naturally processed and presented. ConclusionsThis technique can serve as useful experimental approach to determine the induction or enhancement of specific responses to naturally processed and presented antigens on HLA class I molecules in peripheral blood or tumor infiltrating T cells.

TVGN-489: A novel approach for the production of SARS-CoV-2-specific cytotoxic T lymphocytes to treat COVID-19

Abstract SARS-COV-2 (COVID-19) has resulted in over 2 million deaths. While vaccination is expected to decrease mortality, there remains a need for curative therapies for active infections. Uncertainties regarding the duration of post-vaccination immunity and the rapidity of mutational evolution by this virus suggest that it is unwise to rely on preventative measures alone. Dysregulation of endogenous cellular immunity has been implicated in the failure to control COVID-19 infections suggesting that allogeneic T cell therapy using nature’s curative approach to viral infections could successfully be applied to vulnerable patients. Allogeneic virus-specific cytotoxic T lymphocytes (CTLs) have a long track record of safety and efficacy in treating viral infections occurring after allogeneic stem cell transplantation. We hypothesize that this approach can be applied to the treatment of Covid-19. Covid-19 specific CTLs were produced by priming and enriching T cells from convalescent patients using COVID-19 peptides predicted or demonstrated to bind to specific HLA alleles. Utilizing the globally common HLA-A*02:01 allele as the restriction element, 7 peptides from 4 COVID-19 gene/ORF products were identified and used as a pool for serial restimulation and enrichment on a peptide pulsed, HLA-A*02:01 antigen presenting cell monolayer. This preclinical effort produced CTLs of high purity (>95% CD3+/CD8+, >60% positive by tetramer staining) and potency (60% lysis of peptide pulsed targets at an effector to target ratio of 3:1) in sufficient quantities for clinical application. Trials using these TVGN-489 CTLs and those produced with other SARS-COV-2 peptides and HLA restriction elements will hopefully be launched shortly.

Multi-HLA class II tetramer analyses of citrulline-reactive T cells and early treatment response in rheumatoid arthritis

BackgroundHLA class II tetramers can be used for ex vivo enumeration and phenotypic characterisation of antigen-specific CD4+ T cells. They are increasingly applied in settings like allergy, vaccination and autoimmune diseases. Rheumatoid arthritis (RA) is a chronic autoimmune disorder for which many autoantigens have been described.ResultsUsing multi-parameter flow cytometry, we developed a multi-HLA class II tetramer approach to simultaneously study several antigen specificities in RA patient samples. We focused on previously described citrullinated HLA-DRB1*04:01-restricted T cell epitopes from α-enolase, fibrinogen-β, vimentin as well as cartilage intermediate layer protein (CILP). First, we examined inter-assay variability and the sensitivity of the assay in peripheral blood from healthy donors (n = 7). Next, we confirmed the robustness and sensitivity in a cohort of RA patients with repeat blood draws (n = 14). We then applied our method in two different settings. We assessed lymphoid tissue from seropositive arthralgia (n = 5) and early RA patients (n = 5) and could demonstrate autoreactive T cells in individuals at risk of developing RA. Lastly, we studied peripheral blood from early RA patients (n = 10) and found that the group of patients achieving minimum disease activity (DAS28 < 2.6) at 6 months follow-up displayed a decrease in the frequency of citrulline-specific T cells.ConclusionsOur study demonstrates the development of a sensitive tetramer panel allowing simultaneous characterisation of antigen-specific T cells in ex vivo patient samples including RA ‘at risk’ subjects. This multi-tetramer approach can be useful for longitudinal immune-monitoring in any disease with known HLA-restriction element and several candidate antigens.

Negative selection of human T cells recognizing a naturally-expressed tissue-restricted antigen in the human thymus

During T cell development in mice, thymic negative selection deletes cells with the potential to recognize and react to self-antigens. In human T cell-dependent autoimmune diseases such as Type 1 diabetes, multiple sclerosis, and rheumatoid arthritis, T cells reactive to autoantigens are thought to escape negative selection, traffic to the periphery and attack self-tissues. However, physiological thymic negative selection of autoreactive human T cells has not been previously studied. We now describe a human T-cell receptor-transgenic humanized mouse model that permits the study of autoreactive T-cell development in a human thymus. Our studies demonstrate that thymocytes expressing the autoreactive Clone 5 TCR, which recognizes insulin B:9–23 presented by HLA-DQ8, are efficiently negatively selected at the double and single positive stage in human immune systems derived from HLA-DQ8+ HSCs. In the absence of hematopoietic expression of the HLA restriction element, negative selection of Clone 5 is less efficient and restricted to the single positive stage. To our knowledge, these data provide the first demonstration of negative selection of human T cells recognizing a naturally-expressed tissue-restricted antigen. Intrathymic antigen presenting cells are required to delete less mature thymocytes, while presentation by medullary thymic epithelial cells may be sufficient to delete more mature single positive cells. These observations set the stage for investigation of putative defects in negative selection in human autoimmune diseases.

CMV-Specific T Cells Restricted By Shared and Donor, but Not By Host HLA Molecules Reconstitute in the First 180 Days after Allogeneic HSCT and Protect from CMV Reactivation: Results of a Prospective Observational Study

Introduction: Cytomegalovirus (CMV) reactivation and disease are important risk factors after allogeneic hematopoietic stem cell transplantation (allo-HSCT), and strongly affect morbidity and mortality after transplant. CMV-specific T cell reconstitution controls CMV reactivation and protects against serious adverse events but a protective level of CMV-specific T cell response or standardized method for its monitoring have not been yet determined. Methods: We designed a prospective, single-center observational study to assess if the kinetic and quality of CMV specific T-cell reconstitution impact the incidence and severity of CMV reactivations. We have enrolled 84 consecutive patients affected by hematological malignancies receiving allo-HSCT followed by Cyclophosphamide and Rapamycin between December 2017 and February 2019. Here we report preliminary data on the first 61 patients. Patients received allo-HSCT from family (siblings=10; HLA haploidentical=24), unrelated HLA-matched (n= 24) donors or cord blood (CB, n=3). The CMV serostatus of host (H) and donor (D) pairs was: H+/D+(n=40, 65%), H+/D-(n=20, 33%) and H-/D+ (n=1, 2%); H-/D-(7% of the overall transplanted population at our center) were excluded. CMV DNAemia was assessed weekly in whole blood (WB). Absolute numbers of polyclonal and CMV-specific T cells were quantified by flow cytometry using Troucount™Tubes (BD) and Dextramer®CMV-Kit (Immudex), respectively, in the graft and fresh WB at days -7, +30, +45, +60, +90, +120, +150, +180 and +360. Dextramer CMV kit includes reagents for the identification of CMV-specific lymphocytes restricted for several HLA class I molecules: A*01:01/*02:01/*03:01/*24:02 and B*07:02/*08:01/*35:01. These alleles allowed the longitudinal evaluation of 54 out of 61 (89%) patients. Results: At a median follow-up of 226 days post-HSCT, 31 (57%) patients experienced a CMV-related clinically relevant event (CRE, median +63 days), including 8 patients (15%) with CMV disease (median +59 days). Univariate analyses showed that the incidence of CMV clinically-relevant reactivation (CRE) was influenced by H/D CMV serostatus (0.90 in H+/D- versus 0.44 in H+/D+pairs, p=0.015) and by previous acute Graft-versus-Host Disease (aGvHD) requiring systemic immunosuppression (0.82 in aGvHD grade II-IV versus 0.52 in aGvHD grade 0-I, p=0.051). The disease status at transplant, the donor type (HLA-matched versus HLA-haploidentical/CB donors), donor's or host's age did not significantly affect the probability to develop CRE. For each time-point, we compared the absolute number of CMV-specific lymphocytes in patients experiencing or not a subsequent CRE. Our data demonstrate that higher levels of CMV-specific CD8+T cells in the donor apheresis and at +45 days after allo-HSCT are associated with reduced risk of subsequent CRE (median CMV-specific CD8+cells/kg in the apheresis=5x103in CRE-positive patients (CRE+) and 5x105in CRE-negative patients (CRE-), p=0.012; median CMV-specific CD8+at +45 days=0.14 cells/μL in CRE+and 1.21 cells/μL in CRE-, p=0.034). Furthermore, patients with any Dextramer positivity at +45 days displayed a lower incidence of CRE compared with subjects who were negative (CRE probability: 0.5 vs 1.0, p=0.003). Conversely, the absolute number of neither polyclonal CD3+CD8+T lymphocytes nor total CD3+T cells correlate with subsequent CRE. Taking advantage of the HLA mismatched-HSCT setting, we then dissected CMV-specific T-cell response according to HLA restriction elements (H/D=shared n=45, D-restricted n=14, H-restricted n=11). In H+/D+pairs, we observed a fast and similar kinetic of reconstitution of CMV-specific lymphocytes restricted by H/D and D HLAs. Conversely, in H+/D-pairs, we detected only CMV-specific CD8+lymphocytes restricted for H/D haplotypes. Host-restricted cells remained undetectable for the first 180 days after HSCT. Conclusion: Early after allo-HSCT and in the donor apheresis, the level of CMV-specific CD8+T cells measured by Dextramer staining differs in patients experiencing or not subsequent CRE. Furthermore, our findings indicate that CMV reactivations can prime H/D-restricted T cells presumably educated in the donor thymus; conversely, D- and H-restricted donor-derived lymphocytes have not yet undergone neither cross-priming nor thymic education respectively. Disclosures Brix: Immudex: Employment. Bonini:Kite/Gilead: Consultancy; Intellia Therapeutics: Consultancy; Intellia Therapeutics: Research Funding; Novartis: Consultancy; GSK: Consultancy; Allogene: Consultancy; Molmed: Consultancy; TxCell: Consultancy; -: Patents &amp; Royalties: Adoptive T cell therapy field.

Key Features Relevant to Select Antigens and TCR From the MHC-Mismatched Repertoire to Treat Cancer.

Adoptive transfer of T cells transgenic for tumor-reactive T-cell receptors (TCR) is an attractive immunotherapeutic approach. However, clinical translation is so far limited due to challenges in the identification of suitable target antigens as well as TCRs that are concurrent safe and efficient. Definition of key characteristics relevant for effective and specific tumor rejection is essential to improve current TCR-based adoptive T-cell immunotherapies. We here characterized in-depth two TCRs derived from the human leukocyte antigen (HLA)-mismatched allogeneic repertoire targeting two different myeloperoxidase (MPO)-derived peptides presented by the same HLA-restriction element side by side comprising state of the art biochemical and cellular in vitro, in vivo, and in silico experiments. In vitro experiments reveal comparable functional avidities, off-rates, and cytotoxic activities for both TCRs. However, we observed differences especially with respect to cytokine secretion and cross-reactivity as well as in vivo activity. Biochemical and in silico analyses demonstrate different binding qualities of MPO-peptides to the HLA-complex determining TCR qualities. We conclude from our biochemical and in silico analyses of peptide-HLA-binding that rigid and high-affinity binding of peptides is one of the most important factors for isolation of TCRs with high specificity and tumor rejection capacity from the MHC-mismatched repertoire. Based on our results, we developed a workflow for selection of such TCRs with high potency and safety profile suitable for clinical translation.

Rapid Construction of Antitumor T-cell Receptor Vectors from Frozen Tumors for Engineered T-cell Therapy.

T cells genetically engineered with tumor antigen-specific T-cell receptor (TCR) genes have demonstrated therapeutic potential in patients with solid tumors. In order to achieve broader application, an efficient method to identify TCR genes for an array of tumor antigens and HLA restriction elements is required. Here, we have developed a method to construct a TCR-expression library from specimens, including frozen tumor biopsies, that contain antigen-specific T cells. TCR-expressing cassettes were constructed and cloned in a retroviral plasmid vector within 24 hours by unbiased PCR amplification of TCR α and β chain variable regions assembled with TCR constant regions. The method was validated by constructing TCR-expressing vectors from tumor antigen-specific T-cell clones and functionally assessing TCR gene-transduced T cells. We applied this method to frozen ovarian tumor specimens that were infiltrated by tumor antigen-specific T cells. The tumor-derived TCR libraries were expressed in peripheral T cells from healthy volunteers and screened for tumor antigen-specific TCR pairs with the use of an MHC/peptide tetramer reagent. Tumor antigen-specific TCR-expressing transgenes were recovered from isolated tetramer-positive T cells. Peripheral T cells that were engineered with library-derived TCR gene showed potent therapeutic antitumor effect in a tumor xenograft model. Our method can efficiently and rapidly provide tumor-specific TCR-expressing viral vectors for the manufacture of therapeutic and personalized antitumor T-cell products. Cancer Immunol Res; 6(5); 594-604. ©2018 AACR.

Abstract 4977: Isolation and characterization of a PRAME-specific TCR with high avidity, potent antitumor efficacy and a favorable preclinical safety profile

Abstract Some of the challenges regarding adoptive T cell transfer as an immunotherapeutic approach for the treatment of cancer patients include the initial induction, isolation and later characterization of suitable TCR candidates. In addition, the selection of appropriate target antigens is important and complex as such antigens need to meet some critical demands. Published data as well as in depth in silico analyses revealed the tumor associated antigen PRAME a suitable candidate. Classified as cancer/testis antigen, PRAME is highly expressed in tumors with only limited or no expression in normal tissues, other than testis. Here we describe the induction of PRAME-specific, HLA-A*02:01-restricted CD8-positive T cells by using an in vitro priming approach. Mature dendritic cells transfected with ivtRNA encoding full-length PRAME were cocultured with autologous CD8-enriched PBL from a healthy, HLA-A*02:01-positive blood donor. Initially primed T cells were expanded for 2 weeks and T cells bearing TCRs specific for PRAME-derived epitopes were identified by multimer staining and FACS analysis. Single cells were sorted into 96 well plates and after an expansion period of another 2 weeks, growing clones were screened for specificity. TCR sequences of specific clones were identified by using NGS and reconstructed in a retroviral vector system enabling stable transduction of effector cells. The resulting TCR-modified T cells were thoroughly analyzed and characterized using a dedicated set of assays to evaluate multiple parameters concerning safety and efficacy. This characterization revealed TCR T4.8-1-29 to be highly specific for a PRAME-derived epitope presented on HLA-A*02:01. Only epitope-positive target cells expressing the appropriate HLA-restriction element induced cytokine secretion by T4.8-1-29 expressing effector cells, leading to recognition as well as specific lysis of these target cells. Recognition and lysis, not just of peptide-loaded T2 cells, but also of PRAME-transfected APC, transfected tumor cells as well as endogenously PRAME-positive tumor cells was mediated by this TCR. T4.8-1-29-expressing effectors show a high natural avidity for the target epitope without the need of further manipulation, e.g. functional efficacy enhancement by affinity maturation. In addition, the toxicity assessment using various in vitro and in silico tools revealed a favorable preclinical safety profile for this TCR. In summary, by using an in vitro priming approach utilizing cells from healthy donors, it was possible to generate a TCR recognizing a PRAME-derived epitope with high natural avidity and specificity. The TCR shows a favorable safety profile, demonstrating that this approach is useful to generate TCRs potentially qualifying for evaluation in clinical trials. Citation Format: Manon Weis, Carina Wehner, Christian Ellinger, Susanne Wilde, Dolores J. Schendel. Isolation and characterization of a PRAME-specific TCR with high avidity, potent antitumor efficacy and a favorable preclinical safety profile [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4977. doi:10.1158/1538-7445.AM2017-4977

Abstract B074: Tumor-reactive CD4+ T cells in metastatic gastrointestinal cancer refractory to chemotherapy

Abstract Purpose: To evaluate whether patients with metastatic gastrointestinal adenocarcinomas refractory to chemotherapy harbor tumor-reactive CD4+ T cells. Experimental Design: Expansion of CD4+ tumor-infiltrating lymphocytes (TIL) and cancer cell lines was performed from gastrointestinal cancer metastases in 5 patients for the study of antitumor immune recognition. Retroviral transduction of genes encoding the class II, major histocompatibility complex, transactivator (CIITA) was used to induce the expression of major histocompatibility complex (MHC) class II in tumor cell lines. Recognition of autologous tumor cell lines by TIL was evaluated by up-regulation of 4-1BB and OX40 by flow cytometry, and/or secretion of IFNg by ELISA. Results: TIL were expanded from metastases, and new tumor cell lines were generated in 5 patients. Retroviral transduction of CIITA in tumor cell lines effectively induced expression of MHC class II in &amp;gt;80% of cells. Autologous tumor recognition was found in CD4+ TIL from 2 of these 5 patients. In a patient with gastric cancer liver metastases, tumor-reactive CD4+OX40+ TIL were cell-sorted from a TIL cell line. These cells up-regulated OX40 in the presence of all 4 autologous cancer cell lines albeit at different levels, but they did not produce IFNg. This recognition was specifically abolished by pan-MHC class II blocking antibodies. CD4+ TIL clones have been isolated and are being further characterised. Interestingly, tumor-reactive CD8+ TIL were previously identified and characterized in this patient. In a second patient with colon cancer abdominal wall metastases, tumor-reactive CD4+OX40+ TIL were cell-sorted from a TIL cell line. These cells were reactive to all 4 autologous cancer cell lines as they up-regulated OX40 and secreted IFNg. Recognition was blocked by anti-HLA-DR blocking antibodies. CD4+ TIL clones have been isolated and are being further characterized. In the near future, we expect to identify the TCR and HLA restriction element for both patients. We will also determine if CD4+ TIL clones have cross-reactivity against allogeneic HLA-matched gastrointestinal tumor cell lines. Conclusions: This study provides a basis for the development of immunotherapy for patients with advanced gastrointestinal malignancies by first establishing the presence of naturally occurring tumor-reactive CD4+ TIL at the molecular level. Citation Format: Sandy Pelletier, Simon Turcotte. Tumor-reactive CD4+ T cells in metastatic gastrointestinal cancer refractory to chemotherapy. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B074.

Individual cultures of melanoma tumor infiltrating lymphocytes possess distinct patterns of reactivity with mutated antigens identified by whole exome sequencing of autologous tumors

The adoptive transfer of autologous tumor infiltrating lymphocytes (TIL) can mediate the regression of tumors in up to 70% of patients with metastatic melanoma. These clinical trials have involved infusion of either bulk T cells generated by culturing cells from enzyme digests of fresh tumors in interleukin-2 (IL-2), or of T cells derived from small tumor fragments cultured in IL-2, that were subsequently expanded using the anti-CD3 antibody, OKT3, in the presence of irradiated feeder cells derived from peripheral blood. Individual fragment cultures may contain a more diverse repertoire of antigen reactivities, which may be advantageous in treating heterogeneous tumor populations. The diversity of antigen recognition by individual TIL fragment cultures has not, to date, been directly evaluated. The current study was carried out to evaluate antigen reactivity of individual fragment TIL cultures from patient 2369, who experienced a complete response to autologous TIL administration. Previous results indicated that the TIL that were administered to patient 2369 predominantly recognized two unique mutated tumor antigens. In the present study, fragment cultures were evaluated for their ability to recognize mutated candidate antigens identified by exomic sequencing encoded within tandem minigene constructs (TMGs) using recently described methods. The screening of twelve individual fragment cultures of the fresh tumor, two tumor digest cultures, and the bulk population of infused T cells for their ability to recognize TMGs encoding 162 mutated gene products resulted in the identification of two additional mutated antigens that were recognized in the context of multiple HLA restriction elements. Individual cultures recognized between zero to four of the mutated targets, and these antigens were recognized to varying degrees by individual cultures. Future studies will be directed towards evaluating TIL from additional patients for their ability to recognize mutated antigens expressed by autologous tumors in an attempt to generate cells that are more effective at mediating long-term tumor regressions.

Tumor-Reactive CD8+ T Cells in Metastatic Gastrointestinal Cancer Refractory to Chemotherapy

To evaluate whether patients with metastatic gastrointestinal adenocarcinomas refractory to chemotherapy harbor tumor-reactive cytotoxic T cells. Expansion of CD8(+) tumor-infiltrating lymphocytes (TIL) and cancer cell lines was attempted from gastrointestinal cancer metastases in 16 consecutive patients for the study of antitumor immune recognition. Retroviral transduction of genes encoding T-cell receptors (TCR) was used to define HLA-restriction elements and specific reactivity. TIL were expanded from metastases in all patients, and new tumor cell lines were generated in 5 patients. Autologous tumor recognition without cross-reactivity against allogeneic HLA-matched gastrointestinal tumors was found in CD8(+) TIL from 3 of these 5 patients. In a patient with gastric cancer liver metastases, the repertoire of CD8(+) TIL was dominated by cytolytic sister clones reactive to 2 out of 4 autologous cancer cell lines restricted by HLA-C*0701. From the same patient, a rare CD8(+) TIL clone with a distinct TCR recognized all four cancer cell lines restricted by HLA-B*4901. In a patient with bile duct cancer, two distinct antitumor cytolytic clones were isolated from a highly polyclonal CD8(+) TIL repertoire. TCRs isolated from these clones recognized epitopes restricted by HLA-A*0201. In a third patient, CD8(+) TIL reactivity was progressively lost against an autologous colon cancer cell line that displayed loss of HLA haplotype. This study provides a basis for the development of immunotherapy for patients with advanced gastrointestinal malignancies by first establishing the presence of naturally occurring tumor-reactive CD8(+) TIL at the molecular level.

Identification of a Novel UTY‐Encoded Minor Histocompatibility Antigen

Minor histocompatibility antigens (mHags) encoded by the Y-chromosome (H-Y-mHags) are known to play a pivotal role in allogeneic haematopoietic cell transplantation (HCT) involving female donors and male recipients. We present a new H-Y-mHag, YYNAFHWAI (UTY(139-147)), encoded by the UTY gene and presented by HLA-A*24:02. Briefly, short peptide stretches encompassing multiple putative H-Y-mHags were designed using a bioinformatics predictor of peptide-HLA binding, NetMHCpan. These peptides were used to screen for peptide-specific HLA-restricted T cell responses in peripheral blood mononuclear cells obtained post-HCT from male recipients of female donor grafts. In one of these recipients, a CD8+ T cell response was observed against a peptide stretch encoded by the UTY gene. Another bioinformatics tool, HLArestrictor, was used to identify the optimal peptide and HLA-restriction element. Using peptide/HLA tetramers, the specificity of the CD8+ T cell response was successfully validated as being HLA-A*24:02-restricted and directed against the male UTY(139-147) peptide. Functional analysis of these T cells demonstrated male UTY(139-147) peptide-specific cytokine secretion (IFNγ, TNFα and MIP-1β) and cytotoxic degranulation (CD107a). In contrast, no responses were seen when the T cells were stimulated with patient tumour cells alone. CD8+ T cells specific for this new H-Y-mHag were found in three of five HLA-A*24:02-positive male recipients of female donor HCT grafts available for this study.

A CD8+ T-Cell Clone Directed Against AML Blasts that Recognizes a Tumor Specific Antigen Expressed Also by Solid Tumors

AbstractAbstract 4289The aim of this study was to generate cytotoxic T-lymphocytes (CTL) clones directed against AML cells and to identify new immunogenic antigens with the following properties: i) to be overexpressed by leukemic cells and not by normal tissues; ii) to be shared among different AML subtypes; iii) to play a role in leukemic growth/survival; iv) to be expressed by leukemic stem cells.To this end, we loaded normal dendritic cells (DC) from a healthy donor with apoptotic bodies from primary AML cells and used loaded DC to stimulate autologous lymphocytes (i.e. lymphocytes from the donor). Donor was selected to be partially matched with the leukemic patient for MHC-class I, in that he shared two MHC-class I alleles at the HLA-supertype level (HLA-B7 and HLA-B44). With this strategy, a CD8+ T-cell line was generated that recognized both loaded DC and AML cells used for loading, but not DC loaded with normal cells derived from the patient (PHA-blasts). This CTL line was cloned by limiting dilutions and 180 clones were screened by IFN-g elispot against 2 different AML samples that were expressing both HLA-B7 and HLA-B44; one additional mismatched AML sample was used as negative control. Two clones were selected that recognized HLA-B7+/HLA-B44+ AML cells but not the negative control (namely, clone 31D3 and 8E12). To determine the HLA-restriction element and to verify shared antigen expression among AML subtypes, we tested both clones against a panel of 18 HLA-typed primary AML samples. We found that clone 31D3 was restricted by HLA-B7 and clone 8E12 by HLA-B44. Each clone recognized 5/5 HLA-matched AML samples of different subtype. In addition, the clones did not recognize both resting and activated normal myeloid, nor lymphoid and CD34+ cells expressing the proper HLA-restriction allele. In addition to elispot activity, both clones showed killing of AML cells in a CFSE-based cytotoxic assay. To confirm the lack of reactivity against normal tissues, we analyzed the activity of both clones against HLA matched or mismatched fibroblasts and we found that clone 8E12 displayed a low reactivity activity against normal matched fibroblasts, while clone 31D3 was not reactive.We then focused the analysis on clone 31D3 and tested whether its activity was restricted to leukemia or also directed against a panel of HLA-B7 positive (N=3) or negative (N=5) solid tumors. Interestingly, we found that the clone 31D3 could recognize one colon carcinoma and one melanoma cell line expressing the HLA-B7 supertype. In particular, the melanoma cell line (G4-mel) was HLA-B35+ (HLA-B35 belongs to the HLA-B7 supertype) and was recognized at very high levels. The availability of a tumor cell line expressing adequate amounts of antigen will make the generation of a cDNA library for antigen identification more feasible than with primary leukemic cells. Finally, to further confirm that HLA-B35 is the proper MHC restriction element and to determine to what extent the antigen is shared among different tumors, we transduced 8 tumor cell lines, 3 normal B-lymphoblastoid and 1 fibroblast cell line with the HLA-B35 allele. Clone 31D3 efficiently recognized 6/8 HLA-B35 transduced tumor cell lines (1 AML, 2 melanomas, 2 colon and 1 breast carcinoma) but none of the control cell lines.In conclusion, clone 31D3 is restricted by the HLA-B7 supertype, it recognizes an antigen that is expressed by leukemic cells and not by normal tissues and, most importantly, is shared among tumors of different histology. Since the vast majority of malignant cells tested (both primary cells and in vitro established cell lines) were targeted by this clone, the putative antigen might be a protein playing an important role in tumor growth or survival. We are now testing the activity of the clone against purified leukemic stem cells and attempting to identify this shared tumor antigen. Disclosures:No relevant conflicts of interest to declare.

HLArestrictor—a tool for patient-specific predictions of HLA restriction elements and optimal epitopes within peptides

Traditionally, T cell epitope discovery requires considerable amounts of tedious, slow, and costly experimental work. During the last decade, prediction tools have emerged as essential tools allowing researchers to select a manageable list of epitope candidates to test from a larger peptide, protein, or even proteome. However, no current tools address the complexity caused by the highly polymorphic nature of the restricting HLA molecules, which effectively individualizes T cell responses. To fill this gap, we here present an easy-to-use prediction tool named HLArestrictor ( http://www.cbs.dtu.dk/services/HLArestrictor ), which is based on the highly versatile and accurate NetMHCpan predictor, which here has been optimized for the identification of both the MHC restriction element and the corresponding minimal epitope of a T cell response in a given individual. As input, it requires high-resolution (i.e., 4-digit) HLA typing of the individual. HLArestrictor then predicts all 8-11mer peptide binders within one or more larger peptides and provides an overview of the predicted HLA restrictions and minimal epitopes. The method was tested on a large dataset of HIV IFNγ ELIspot peptide responses and was shown to identify HLA restrictions and minimal epitopes for about 90% of the positive peptide/patient pairs while rejecting more than 95% of the negative peptide-HLA pairs. Furthermore, for 18 peptide/HLA tetramer validated responses, HLArestrictor in all cases predicted both the HLA restriction element and minimal epitope. Thus, HLArestrictor should be a valuable tool in any T cell epitope discovery process aimed at identifying new epitopes from infectious diseases and other disease models.

High-Throughput Characterization of 10 New Minor Histocompatibility Antigens by Whole Genome Association Scanning

Patients with malignant diseases can be effectively treated with allogeneic hematopoietic stem cell transplantation (allo-SCT). Polymorphic peptides presented in HLA molecules, the so-called minor histocompatibility antigens (MiHA), play a crucial role in antitumor immunity as targets for alloreactive donor T cells. Identification of multiple MiHAs is essential to understand and manipulate the development of clinical responses after allo-SCT. In this study, CD8+ T-cell clones were isolated from leukemia patients who entered complete remission after allo-SCT, and MiHA-specific T-cell clones were efficiently selected for analysis of recognition of a panel of EBV-transformed B cells positive for the HLA restriction elements of the selected T-cell clones. One million single nucleotide polymorphisms (SNP) were determined in the panel cell lines and investigated for matching with the T-cell recognition data by whole genome association scanning (WGAs). Significant association with 12 genomic regions was found, and detailed analysis of genes located within these genomic regions revealed SNP disparities encoding polymorphic peptides in 10 cases. Differential recognition of patient-type, but not donor-type, peptides validated the identification of these MiHAs. Using tetramers, distinct populations of MiHA-specific CD8+ T cells were detected, demonstrating that our WGAs strategy allows high-throughput discovery of relevant targets in antitumor immunity after allo-SCT.

High Avidity HLA-A2-Restricted CD8+ T Cells against the Wilms Tumor Protein (WT1) Can Be Isolated Only from HLA-A2 Negative Donors Not Subjected to HLA-A2-Mediated Thymic Deletion

The anti-tumor effect of donor lymphocyte infusions (DLI) after HLA-matched allogeneic stem cell transplantation (allo-SCT) can be mediated by donor T cells recognizing minor histocompatibility antigens (mHag) in the context of self HLA on the malignant cells of the recipient. However, T cells recognizing self antigens (Ag) that are overexpressed in malignant cells like the Wilms tumor protein (WT1) have also been proposed to contribute to the anti-tumor reactivity both after allo- and autologous SCT. WT1 is expressed in various types of cancer, including hematological malignancies at higher levels compared to their non-malignant counterparts. Different groups have isolated CD8+ HLA-A2 restricted WT1-specific T cells after HLA-matched transplantation and/or WT1 vaccination. Although these T cells stain with HLA-A2/WT1 peptide tetrameric complexes and are capable of recognizing peptide loaded HLA-A2+ target cells, recognition of primary leukemic cells could hardly be demonstrated. Stauss et al demonstrated recognition of endogenously processed Ag by WT1 specific T cells. However, these CD8 T cells were isolated in a situation where responder and stimulator cells were mismatched for HLA-A2. Based on these data we hypothesized that high avidity HLA-A2 restricted WT1 specific T cells may be deleted during thymic selection in HLA-A2+ donors. As a result, it is likely that only low avidity HLA-A2 restricted WT1 specific CD8+ T cells can be isolated from HLA-A2 positive donors, whereas it might be possible to isolate high avidity HLA-A2 restricted WT1 specific T cells from HLA-A2 negative donors due to the absence of the HLA restriction element in the thymus irrespective of the local WT1 expression. To test this hypothesis, we induced immune responses against the HLA-A2 binding WT1-derived peptide RMFPNAPYL using either HLA-A2+ or A2- donor T cells as responder cells. In case of HLA-A2+ donors (n=3) we stimulated CD45RO depleted donor T cells with autologous monocyte-derived antigen-presenting cells (APCs) loaded with 1E-6M WT1 peptide in the presence of 5ng/mL IL-7. At day 10 we either enriched tetramer-positive cells using immunomagnetic beads (MACS) or restimulated the responses with peptide-loaded autologous PBMC. At day 20 tetramer positive cells were isolated in bulk cultures and single cell/well by flowcytometric cell sorting. When using HLA-A2 negative donor T cells as responder cells, we used HLA-A2+ stimulator cells from an unrelated donor matched for all other class I alleles. The similar protocol was used for the induction of the immune response. At day 10 CD4+ T cells were depleted and the WT1 specific T cells were either enriched using tetramers and MACS or restimulated with peptide loaded HLA-A2+ PBMC. Using these approaches we were capable of isolating CD8+ T cell clones staining brightly with the A2/WT1 tetramer and expressing different T cell receptors (TCRs). HLA-A2+ WT1 specific T cells recognized only HLA-A2+ target cells loaded with 1E-8-1E-6M peptide both in IFNg release assays and cytotoxicity assays. Especially peptide-loaded target cells with an APC phenotype like EBVs were recognized efficiently, whereas normal peripheral blood cells were only recognized when loaded with 1E-6M peptide. In contrast, the HLA-A2 negative WT1-specific T cells efficiently recognized normal peripheral blood cells upon loading of <1E-9M WT1 peptide, indicating that these T cells express TCRs with a higher affinity for the WT1 peptide compared to the HLA-A2+ WT1-specific T cells. Strikingly, EBV-LCL as well as TAP-deficient T2 cells were recognized by the HLA-A2 negative T cells even in the absence of exogenously loaded WT1 peptide. This might be due to the efficient recognition by the high avidity T cells of low levels of WT1 presented in HLA-A2 processed from the endogenous WT1 expression. However, due to the absence of HLA-A2 mediated thymic selection in HLA-A2 negative donors, cross-recognition of other peptides in HLA-A2 may also occur. In conclusion, high avidity WT1 specific CD8+ T cells could be exclusively isolated from HLA-A2 negative donor cells. These high avidity T cells are capable of recognizing target cells expressing very low levels of WT1. These T cell responses will gain more insight into the correlation of WT1 expression in different normal and malignant cell types and the recognition by high avidity WT1 T cells, and the clinical applicability of T cells directed against self antigens.

Human papilloma virus specific T cells infiltrating cervical cancer and draining lymph nodes show remarkably frequent use of HLA‐DQ and –DP as a restriction element

Human papillomavirus (HPV)-induced malignancies are frequently infiltrated by lymphocytes. To comprehend the contribution of HPV-specific T cells in this anti-tumor response we developed a method that allowed the analysis of the presence and specificity of cervix-infiltrating and draining lymph node resident T cells in a group of 74 patients with cervical malignancies, 54 of which were induced by HPV16 or HPV18. We detected the presence of HPV16 or HPV18-specific T cells in at least 23 of the 54 HPV-16 or -18 positive patients, and not in the 20 controls. Detailed studies resulted in the identification of 17 novel CD4+ and CD8+ T cell epitopes and their HLA-restriction elements, and also revealed that the HPV-specific immune response was aimed at both E6 and E7 and showed no preferential recognition of immunodominant regions. Unexpectedly, the vast majority of the CD4+ T cell epitopes were presented in the context of the less abundantly expressed HLA-DQ and HLA-DP molecules. Since the identified T cell epitopes constitute physiological targets in the immune response to HPV16 and HPV18 positive tumors they will be valuable for detailed studies on the interactions between the tumor and the immune system. This is crucial for the optimization of cancer immunotherapy in patients with pre-existing tumor-immunity.

Activation-induced expression of CD137 permits detection, isolation, and expansion of the full repertoire of CD8+ T cells responding to antigen without requiring knowledge of epitope specificities

CD137 is a member of the TNFR-family with costimulatory function. Here we show that it also has many favorable characteristics as a surrogate marker for antigen-specific activation of human CD8(+) T cells. Although undetectable on unstimulated CD8(+) T cells, it is uniformly up-regulated 24 hours after stimulation on virtually all responding cells regardless of differentiation stage or profile of cytokine secretion, which circumvents limitations of current surrogate markers for defining the repertoire of responding cells based on only individual functions. Antibody-labeled responding CD137(+) cells can be easily and efficiently isolated by flow sorting or magnetic beads to substantially enrich antigen-specific T cells. To test this approach for epitope discovery, we examined in vitro priming of naive T cells from healthy donors to Wilms tumor antigen 1 (WT1), a protein overexpressed in various malignancies. Two overlapping pentadecamers were identified as immunogenic, and further analysis defined WT1((286-293)) as the minimal amino acid sequence and HLA-Cw07 as the HLA restriction element. In conclusion, this approach appears to be an efficient and sensitive in vitro technique to rapidly identify and isolate antigen-specific CD8(+) T cells present at low frequencies and displaying heterogeneous functional profiles, and does not require prior knowledge of the specific epitopes recognized or the HLA-restricting elements.

Mature CD83(+) dendritic cells infected with recombinant gp100 vaccinia virus stimulate potent antimelanoma T cells.

Mature dendritic cells (DCs) are potent antigen-presenting cells that activate naive T lymphocytes and initiate cellular immune responses. The ability of CD83(+) mature DCs infected with vaccinia virus encoding the gp100 melanoma transgene (rV-gp100) to stimulate an antimelanoma CD8(+) T-cell response was investigated. Monocyte-derived immature or CD83(+) mature DCs were infected with rV-gp100. The activation state of the DCs and the expression of gp100 protein were evaluated by flow cytometry. The reactivity of antimelanoma CD8(+) T cells was confirmed by measuring specific interferon gamma secretion by using enzyme-linked immunosorbent assay in a mixed-tumor lymphocyte culture. Both immature and CD83(+) mature DCs expressed gp100 protein when the DCs were infected with rV-gp100. Calcium-signaling agents were required to induce maturation of both infected and noninfected immature DCs. Only rV-gp100-infected CD83(+) DCs induced CD8(+) T cells, after a single stimulation that recognized both peptide-pulsed target cells to multiple gp100 epitopes and a melanoma cell line that endogenously expressed gp100 antigen. CD83(+) DCs transduced with rV-gp100 are capable of generating a strong CD8(+) T-cell response against melanoma tumor cells. Expression of melanoma antigens by mature DCs offers the potential advantage of presenting multiple endogenously processed T-cell epitopes and using multiple HLA restriction elements for antimelanoma vaccine therapy.