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The promiscuous nature of T-cell receptors (TCRs) allows T cells to recognize a large variety of pathogens, but makes it challenging to understand and control T-cell recognition. Existing technologies provide limited information about the key requirements for T-cell recognition and the ability of TCRs to cross-recognize structurally related elements. Here we present a 'one-pot' strategy for determining the interactions that govern TCR recognition of peptide-major histocompatibility complex (pMHC). We measured the relative affinities of TCRs to libraries of barcoded peptide-MHC variants and applied this knowledge to understand the recognition motif, here termed the TCR fingerprint. The TCR fingerprints of 16 different TCRs were identified and used to predict and validate cross-recognized peptides from the human proteome. The identified fingerprints differed among TCRs recognizing the same epitope, demonstrating the value of this strategy for understanding T-cell interactions and assessing potential cross-recognition before selection of TCRs for clinical development.

Background: Merkel cell carcinoma (MCC) is a rare but very aggressive skin tumor that develops after integration of a truncated form of the large T-antigen (truncLT) of the Merkel cell polyomavirus (MCV) into the host's genome. Therapeutic vaccination with dendritic cells (DCs) loaded with tumor antigens is an active form of immunotherapy, which intends to direct the immune system towards tumors which express the respective vaccination antigens. Methods: Cytokine-matured monocyte-derived DCs of healthy donors and MCC patients were electroporated with mRNA encoding the truncLT. To permit major histocompatibility complex (MHC) class II next to class I presentation, we used an RNA construct in which the antigen was fused to a DCLamp sequence in addition to the unmodified antigen. To further improve their immunogenicity, the DCs were additionally activated by co-transfection with the constitutively active nuclear factor (NF)-κB activator caIKK. These DCs were used to stimulate autologous CD8+ T-cells or a mixture of CD4+ and CD8+ T-cells. Then the percentage of T-cells, specific for the truncLT, was quantified by interferon (IFN)γ ELISpot assays.Results: Both the truncLT and its DCLamp-fusion were detected within the DCs by flow cytometry, albeit the latter required blocking of the proteasome. The transfection with caIKK upregulated maturation markers and induced cytokine production. After 2-3 rounds of stimulation, the T-cells from 11 out of 13 healthy donors recognized the antigen. DCs without caIKK appeared in comparison less potent in inducing such responses. When using cells derived from MCC patients, we could induce responses for 3 out of 5 patients; however, here the caIKK-transfected DCs did not display their superiority.Conclusion: These results show that optimized DCs are able to induce MCV-antigen-specific T-cell responses. Therapeutic vaccination with such transfected DCs could direct the immun

Infusion of highly heterogeneous populations of autologous tumor-infiltrating lymphocytes (TILs) can result in tumor regression of exceptional duration. Initial tumor regression has been associated with persistence of tumor-specific TILs one month after infusion, but mechanisms leading to long-lived memory responses are currently unknown. Here we studied the dynamics of bulk tumor-reactive CD8+ T cell populations in patients with metastatic melanoma following treatment with TILs. Experimental Design: We analyzed the function and phenotype of tumor-reactive CD8+ T cells contained in serial blood samples of sixteen patients treated with TILs. Results: Polyfunctional tumor-reactive CD8+ T cells accumulated over time in the peripheral lymphocyte pool. Combinatorial analysis of multiple surface markers (CD57, CD27, CD45RO, PD-1 and LAG-3) showed a unique differentiation pattern of polyfunctional tumor-reactive CD8+ T cells, with highly specific PD-1 upregulation early after infusion. The differentiation and functional status appeared largely stable for up to 1 year post-infusion. Despite some degree of clonal diversification occurring in vivo within the bulk tumor-reactive CD8+ T cells, further analyses showed that CD8+ T cells specific for defined tumor-antigens had similar differentiation status. Conclusions: We demonstrated that tumor-reactive CD8+ T cell subsets which persist after TIL therapy are mostly polyfunctional, display a stable partially differentiated phenotype and express high levels of PD-1. These partially differentiated PD-1+ polyfunctional TILs have a high capacity for persistence and may be susceptible to PD-L1/PD-L2-mediated inhibition.

Merkel Cell Carcinoma is an aggressive human skin cancer induced by Merkel Cell Polyomavirus (MCPyV). MCPyV is commonly found in human, but the oncogenic transformation takes place during immunosuppression. Two mutation events allow the clonal integration of the viral genome into the host genome and translation of the two viral genes large T (LTA) and small T antigen (STA). Standard treatment with chemotherapy shows poor clinical outcome instead immunotherapy offers new potential treatment strategies. The use of PD-1 checkpoint inhibi-tors has shown promising results (>50% response rates, RECIST). However, not all patients are able to mount an immune response. Instead adoptive transfer of MCPyV-reactive T cells is an attractive strategy for this cohort. We have previously identified T cell epitopes from the MCPyV-derived proteins LTA, STA and VP1. Here we aim to expand the knowledge about T cell epitopes by including a broader range of HLA restrictions. We analyzed 31 patients’ peripheral blood mononuclear cells through enrichment of low frequency clones, followed by revealing of T cell reactivity using combinatorial color-encoded peptide-MHC multimers. 28 T cell responses against 18 MCPyV-derived peptides were detected. Addi-tional testing has confirmed functional T cell reactivity against one of these epitopes. We analyzed 3 patients’ tumor infiltrating lymphocytes by direct ex-vivo detection of T cell reactivity using combinatorial color-encoded peptide-MHC multimers. 5 T cell responses against 5 peptides were detected. The functional T cell response towards detected epitopes is under investigation in order to characterize them as potential T cell targets in a new therapy.

Introduction: Tumour infiltrating lymphocyte (TIL) based adoptive cell therapy (ACT) is a promising treatment for patients with advanced melanoma. Retrospective studies suggested an association between previous treatment with anti-CTLA-4 antibodies and long term survival after subsequent ACT. Thus, we hypothesized that treatment with anti-CTLA-4 antibodies can induce favourable changes to be detected in TILs.Results: Expanded T cells from Ipilimumab treated patients had a higher proportion of cells expressing CD27, intracellular CTLA-4, TIM-3 and LAG-3. In addition, broader and more frequent T cell responses against common tumour antigens were detected in patients treated with Ipilimumab as compared to anti-CTLA-4 naive patients.Materials and methods: Expanded TILs were obtained from patients with advanced melanoma who had received Ipilimumab in the previous six months, or had not received any type of anti-CTLA-4 antibody. T cell specificity and expression of phenotypic and exhaustion markers were scrutinized as well as functional properties.Conclusions: Ipilimumab may induce tumor-infiltration of T cells of a more naive phenotype expressing markers related to activation or exhaustion. Additionally, Ipilimumab may increase the frequency of T cells recognizing common tumour associated antigens.

Type 1 diabetes (T1D) is characterized by a CD8+ lymphocyte-mediated selective destruction of theinsulin-producing β-cells causing clinical diabetes. Several autoantigens including glutamic acid decarboxylase 65kDa (GAD65), insulin, protein tyrosine phosphatase (IA-2) and zinc transporter 8 (ZnT8) have been identified based on reactivity in sera from T1D individuals. Here we investigate if post-translational deamination of arginine in the form of citrullination plays a role in T cell recognition of T1D autoantigens. Citrullination may lead to generation of neo-epitopes, which has been described as T cell targets in other autoimmune diseases. We used netMHC prediction algorithm to identify 764 epitopes from Insulin, GAD65, IA-2 and ZnT8 restricted to HLA-A2, A24, B8 and B15. Among these 91 peptide sequences were susceptible for citrullination. We evaluate the MHC-affinity of both the citrullinated and non-citrullinated library, to identify potential neo-epitopes and to understand the impact of citrullination on MHC affinity. In parallel we will analyse peripheral blood lymphocytes from 50 T1D patients for immune reactivity against the full library. The large library screen will be conducted applying a novel technology where the selection of MHC-multimer binding T cells is followed by amplification and sequencing of MHC multimer-associated DNA barcodes revealing their recognition. This technique enables simultaneous detection of >1000 specificities. Identifying post translational modifications capable of eliciting autoreactive T cell responses in T1D patients is highly relevant for understanding the underlying mechanisms leading to T1D.

T cell receptor gene-therapy has entered the clinic and shown potential for successful cancer treatment. However, the clinical evaluation has also highlighted the need for selection of truly cancerspecific targets. Merkel cell carcinoma (MCC) is a highly aggressive skin cancer associated with Merkel cell polyomavirus (MCPyV). Due to the clear viral correlation CD8+ T cells specific for viral epitopes could potentially form cancer-specific targets in MCC patients. We have identified MCPyV specific T cells using a high-throughput platform for T-cell enrichment and combinatorial encoding offluorescence-labeled major histocompatibility complex (MHC) class I multimers. We identified 35 T cell epitopes among 398 MCPyV derived peptides analyzed. Strikingly, T-cell responses against the two oncogenic MCPyV proteins Large T antigen and small T antigen were exclusively present in blood of MCC patients when compared to healthy donors. We demonstrate both the processing and presentation of oncoprotein-derived epitopes, as well as lytic activity of specific T cells towards MHC matched MCC cells. Demonstrating the presence of oncoprotein-specific T cells among tumorinfiltrating lymphocytes ex vivo further substantiated the relevance of the identified epitopes. The viralepitopes represents specific targets and should be ideal for TCR-gene therapy approaches. We have isolated and sequenced MCPyV oncogenic protein specific T cell receptors and are currently testing in vitro transduction systems with the purpose of introducing the TCRs into human PBMC, injecting them into immune deficient NOG mice carrying HLA matched MCPyV positive tumor to investigate the tumor rejection capacity of these gene-modified T cells.

As tumors grow, they acquire mutations, some of which create neoantigens that influence the response of patients to immune checkpoint inhibitors. We explored the impact of neoantigen intratumor heterogeneity (ITH) on antitumor immunity. Through integrated analysis of ITH and neoantigen burden, we demonstrate a relationship between clonal neoantigen burden and overall survival in primary lung adenocarcinomas. CD8(+)tumor-infiltrating lymphocytes reactive to clonal neoantigens were identified in early-stage non-small cell lung cancer and expressed high levels of PD-1. Sensitivity to PD-1 and CTLA-4 blockade in patients with advanced NSCLC and melanoma was enhanced in tumors enriched for clonal neoantigens. T cells recognizing clonal neoantigens were detectable in patients with durable clinical benefit. Cytotoxic chemotherapy-induced subclonal neoantigens, contributing to an increased mutational load, were enriched in certain poor responders. These data suggest that neoantigen heterogeneity may influence immune surveillance and support therapeutic developments targeting clonal neoantigens.

Immune therapy has provided a significant breakthrough in the treatment of metastatic melanoma. Despite the remarkable clinical efficacy and established involvement of effector CD8 T cells, the knowledge of the exact peptide-MHC complexes recognized by T cells on the tumor cell surface is limited. Many melanoma-associated T-cell epitopes have been described, but this knowledge remains largely restricted to HLA-A2, and we lack understanding of the T-cell recognition in the context of other HLA molecules. We selected six melanoma-associated antigens (MAGE-A3, NY-ESO-1, gp100, Mart1, tyrosinase and TRP-2) that are frequently recognized in patients with the aim of identifying novel T-cell epitopes restricted to HLA-A1, -A3, -A11 and -B7. Using in silico prediction and in vitro confirmation, we identified 127 MHC ligands and analyzed the T-cell responses against these ligands via the MHC multimer-based enrichment of peripheral blood from 39 melanoma patients and 10 healthy donors. To dissect the T-cell reactivity against this large peptide library, we used combinatorial-encoded MHC multimers and observed the T-cell responses against 17 different peptide-MHC complexes in the patient group and four in the healthy donor group. We confirmed the processing and presentation of HLA-A3-restricted T-cell epitopes from tyrosinase (TQYESGSMDK) and gp100 (LIYRRRLMK) and an HLA-A11-restricted T-cell epitope from gp100 (AVGATKVPR) via the cytolytic T-cell recognition of melanoma cell lines and/or K562 cells expressing the appropriate antigen and HLA molecule. We further found T-cell reactivity against two of the identified sequences among tumor-infiltrating lymphocytes from melanoma patients, suggesting a potential clinical relevance of these sequences.