TCR Analyses of Two Vast and Shared Melanoma Antigen-Specific T Cell Repertoires: Common and Specific Features

Among Immunotherapeutic approaches for cancer treatment, the adoptive transfer of antigen specific T cells is still a relevant approach, that could have higher efficacy when further combined with immune check-point blockade. A high number of adoptive transfer trials have been performed in metastatic melanoma, due to its high immunogenic potential, either with polyclonal TIL or antigen-specific polyclonal populations. In this setting, the extensive characterization of T cell functions and receptor diversity of infused polyclonal T cells is required, notably for monitoring purposes. We developed a clinical grade procedure for the selection and amplification of polyclonal CD8 T cells, specific for two shared and widely expressed melanoma antigens: Melan-A and MELOE-1. This procedure is currently used in a clinical trial for HLA-A2 metastatic melanoma patients. In this study, we characterized the T-cell diversity (T-cell repertoire) of such T cell populations using a new RNAseq strategy. We first assessed the added-value of TCR receptor sequencing, in terms of sensitivity and specificity, by direct comparison with cytometry analysis of the T cell populations labeled with anti-Vß-specific antibodies. Results from these analyzes also confirmed specific features already reported for Melan-A and MELOE-1 specific T cell repertoires in terms of V-alpha recurrence usage, on a very high number of T cell clonotypes. Furthermore, these analyses also revealed undescribed features, such as the recurrence of a specific motif in the CDR3α region for MELOE-1 specific T cell repertoire. Finally, the analysis of a large number of T cell clonotypes originating from various patients revealed the existence of public CDR3α and ß clonotypes for Melan-A and MELOE-1 specific T cells. In conclusion, this method of high throughput TCR sequencing is a reliable and powerful approach to deeply characterize polyclonal T cell repertoires, and to reveal specific features of a given TCR repertoire, that would be useful for immune follow-up of cancer patients treated by immunotherapeutic approaches

Double Positive CD4CD8 αβ T Cells: A New Tumor-Reactive Population in Human Melanomas

BACKGROUND: Double positive (DP) CD4CD8 Talphabeta cells have been reported in normal individuals as well as in different pathological conditions including inflammatory diseases, viral infections and cancer, but their function remains to be elucidated. We recently reported the increased frequency of DP Talphabeta cells in human breast pleural effusions. This manuscript addresses the question of the existence and above all the role of this non-conventional DP sub-population among tumor associated lymphocytes in melanomas. METHODOLOGY/PRINCIPAL FINDINGS: We analyzed the intratumoral cell infiltrate in solid metastasis (n = 6) and tumor invaded lymph nodes (n = 26) samples from melanomas patients by multiparametric cytometry. Here we documented for the first time significant increased frequency of DP T cells in about 60% of melanoma tumors compared to blood samples. Interestingly, a high proportion of these cells produced TNF-alpha in response to autologous melanoma cell lines. Besides, they are characterized by a unique cytokine profile corresponding to higher secretion of IL-13, IL-4 and IL-5 than simple positive T cells. In deep analysis, we derived a representative tumor-reactive DP T cell clone from a melanoma patient's invaded lymph node. This clone was restricted by HLA-A*2402 and recognized both autologous and allogeneic tumor cells of various origins as well as normal cells, suggesting that the target antigen was a ubiquitous self antigen. However, this DP T cell clone failed to kill HLA-A*2402 EBV-transformed B cells, probably due to the constitutive expression of immunoproteasome by these cells. CONCLUSIONS/SIGNIFICANCE: In conclusion, we can postulate that, according to their broad tumor reactivity and to their original cytokine profile, the tumor associated DP T cells could participate in immune responses to tumors in vivo. Therefore, the presence of these cells and their role will be crucial to address in cancer patients, especially in the context of immunotherapies

Non-Coding RNAs in Cutaneous Melanoma Development, Progression and Dissemination

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ROLE OF miRNA FROM MELANOMA CELL-DERIVED EXOSOMES IN TUMOR IMMUNE ESCAPE

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A long peptide from MELOE-1 contains multiple HLA class II T cell epitopes in addition to the HLA-A*0201 epitope: an attractive candidate for melanoma vaccination

International audienceCD4(+) T cells contribute importantly to the antitumor T cell response, and thus, long peptides comprising CD4 and CD8 epitopes may be efficient cancer vaccines. We have previously identified an overexpressed antigen in melanoma, MELOE-1, presenting a CD8(+) T cell epitope, MELOE-1(36-44), in the HLA-A*0201 context. A T cell repertoire against this epitope is present in HLA-A*0201+ healthy subjects and melanoma patients and the adjuvant injection of TIL containing MELOE-1 specific CD8(+) T cells to melanoma patients was shown to be beneficial. In this study, we looked for CD4(+) T cell epitopes in the vicinity of the HLA-A*0201 epitope. Stimulation of PBMC from healthy subjects with MELOE-1(26-46) revealed CD4 responses in multiple HLA contexts and by cloning responsive CD4(+) T cells, we identified one HLA-DRβ1*1101-restricted and one HLA-DQβ1*0603-restricted epitope. We showed that the two epitopes could be efficiently presented to CD4(+) T cells by MELOE-1-loaded dendritic cells but not by MELOE-1+ melanoma cell-lines. Finally, we showed that the long peptide MELOE-1(22-46), containing the two optimal class II epitopes and the HLA-A*0201 epitope, was efficiently processed by DC to stimulate CD4(+) and CD8(+) T cell responses in vitro, making it a potential candidate for melanoma vaccination

Overexpression of meloe gene in melanomas is controlled both by specific transcription factors and hypomethylation.

The melanoma antigens MELOE-1 and MELOE-2 are encoded by a messenger, called meloe, overexpressed in melanomas compared with other tumour cell types and healthy tissues. They are both able to elicit melanoma-specific T cell responses in melanoma patients, and MELOE-1-specific CD8 T cells have been involved in melanoma immunosurveillance. With the aim to develop immunotherapies targeting this antigen, we investigated the transcriptional mechanisms leading to the preferential expression of meloe messenger in the melanocytic lineage. We defined the minimal promoter region of meloe gene and identified binding motifs for a set of transcription factors. Using mutagenesis, co-transfection experiments and chromatin immunoprecipitation, we showed that transcription factors involved in meloe promoter activity in melanomas were the melanocytic specific SOX9 and SOX10 proteins together with the activated P-CREB protein. Furthermore, we showed that meloe promoter was hypomethylated in melanomas and melanocytes, and hypermethylated in colon cancer cell lines and mesotheliomas, thus explaining the absence of P-CREB binding in these cell lines. This was a second key to explain the overerexpression of meloe messenger in the melanocytic lineage. To our knowledge, such a dual transcriptional control conferring tissue-specificity has never been described for the expression of tumour antigens

PD-1 expression conditions T cell avidity within an antigen-specific repertoire

International audienceDespite its negative regulatory role on tumor specific T cells, PD-1 is also a marker ofactivated tumor-infiltrating T cells. In cancer, PD-1 blockade partially reverses T celldysfunction allowing the amplification of tumor reactive T cells. Here we investigated therole of PD-1 signaling on effector/memory human T cells specific for shared melanomaantigens, derived from blood. We documented for the first time the existence of melanomaspecific T cell clones unable to express PD-1. This stable feature was due to the persistentmethylation of the PDCD1 promoter. PD-1pos counterparts, suggesting that high affinity specific T cell clones unable to express PD-1 are not or rarely present in peripheral blood, as they are probably eliminated by negativeselection, due to their high reactivity. We also documented the existence of such PD-1neg Tcell clones in melanoma TIL, which also exhibited a lower functional avidity than PD-1pos TILclones. This clearly shows that PD-1 expression identifies antigen-specific T cell clonotypes ofhigh functional avidity. Finally, we demonstrated that PD-1 blockade during the in vitroselection process of Melan-A specific T cells favored the amplification of higher avidity T cellclonotypes. This preferential amplification of high avidity memory T cells upon PD-1blockade resonates with the expansion of reactive T cells, including neo-antigen-specific Tcells observed in anti-PD-1 treated patients. This feature should also be a useful biomarkerof clinical efficiency, while providing new insights for adoptive transfer treatments

Frequent occurrence of high affinity T cells against MELOE-1 makes this antigen an attractive target for melanoma immunotherapy

International audienceWe recently showed that the infusion of tumor infiltrating lymphocytes specific for the MELOE-1 antigen was associated with a prolonged relapse-free survival for HLA-A2 1 melanoma patients who received tumor infiltrating lymphocytes therapy. Here, we characterized the MELOE-1/A2-specific T-cell repertoire in healthy donors and melanoma patients to further support an immunotherapy targeting this epitope. Using tetramer enrichment followed by multicolor staining, we found that MELOE-1-specific T cells were present in the blood of healthy donors and patients at similar frequencies (around 1 in 1 Â 10 5 CD8 1 cells). These cells mainly displayed a naïve phenotype in 4/6 healthy donors and 3/6 patients, whereas high proportions of memory cells were observed in the remaining individuals of both groups. There was a recurrent usage of the Va12.1 chain for 17/18 MELOE-1-specific T-cell clones derived from healthy donors or patients, associated with diverse Vb chains and V(D)J junctional sequences. All clones derived from melanoma patients (9/9) were reactive against the MELOE-1 36-44 peptide and against HLA-A2 1 melanoma cell lines. This study documents the existence of a large TCR repertoire specific for the MELOE-1/A2 epitope and its capacity to give rise to antitumor CTL that supports the development of immunotherapies targeting this epitope

A new tyrosinase epitope recognized in the HLA-B*4002 context by CTL from melanoma patients

International audienceMelanoma reactive CTL were obtained by stimulating PBL from a melanoma patient in remission since 1994 following adjuvant TIL immunotherapy, with the autologous melanoma cell line. They were cloned by limiting dilution. One CTL clone recognized melanoma cell lines expressing tyrosinase and the B*4002 molecule, either spontaneously or upon transfection. We demonstrated that this clone recognizes the tyrosinase-derived nonapeptide 316-324 (ADVEFCLSL) and the overlapping decapeptide 315-324 (SADVEFCLSL). We derived two distinct additional speciWc CTL clones from this same patient that were also reactive against B*4002 melanoma cell lines, suggesting a relative diversity of this speciWc repertoire in this patient. Stimulating PBMC derived from four additional B*4002 melanoma patients with the tyrosinase 316-324 nonapeptide induced the growth of speciWc cells for two of the patients, demonstrating the immunogenicity of this new epitope. Our data show that this nonapeptide is a new tool that could be used to generate melanoma-speciWc T cells for adoptive immunotherapy or serve as a peptide vaccine for HLA-B*4002 melanoma patients

MELOE-1 is a new antigen overexpressed in melanomas and involved in adoptive T cell transfer efficiency

International audienceA cytotoxic T lymphocyte (CTL) clone was derived from a tumor-infiltrating lymphocyte (TIL) population infused to a melanoma patient who remained relapse free for 10 yr after this adoptive transfer. This clone recognized all melanoma cell lines tested and, to a lower extent, melanocytes, in the context of human histocompatibility leukocyte antigen A2 (HLA-A2), but it did not recognize other tumor cell types. The gene coding for the antigen recognized by this clone was identified by the screening of a melanoma complementary DNA expression library. This antigen is overexpressed in melanomas, compared with other cancer cell lines and healthy tissues, and was thus called melanoma-overexpressed antigen (meloe). Remarkably, the structure of meloe was unusual, with multiple short open reading frames (ORFs). The peptide recognized by the CTL clone was encoded by one of these ORFs, called MELOE-1. Using a specific HLA-A2/peptide tetramer, we showed a correlation between the infusion of TILs containing MELOE-1-specific T cells and relapse prevention in HLA-A2 patients. Indeed, 5 out of 9 patients who did not relapse were infused with TILs that contained MELOE-1-specific T cells, whereas 0 out of the 21 patients who relapsed was infused with such TIL-containing lymphocytes. Overall, our results suggest that this new antigen is involved in immunosurveillance and, thus, represents an attractive target for immunotherapy protocols of melanoma