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

Caractérisation de lymphocytes infiltrant les tumeurs coliques et implication du récepteur CD94/NKG2A dans l'immunité anti-tumorale

Dans la perspective de développer des immunothérapies, j'ai étudié les lymphocytes T infiltrants les tumeurs coliques, en particulier 2 clones T, g9.2 et .b, spécifiques de la lignée tumorale autologue. J'ai montré: 1/ que le clone g9 2 tue spontanément une majorité de lignées tumorales coliques, de façon TCR, LFA-3 et NKG2D dépendante et que tous les lymphocytes g9 2 partagent cette propriété, suggérant l'intérêt de ces cellules en immunothérapie et 2/ que le clone .b co-exprime CD8 b CD4 et sécrète un panel de cytokines potentiellement régulateur (TNF , IL-2, IL-4, TGF ). Parallèlement, j'ai étudié le rôle de HLA-E, ligand du récepteur CD94/NKG2A, dans la réponse anti-mélanome. J'ai montré 1/ que HLA-E est exprimé par les mélanocytes et les mélanomes primaires mais est perdu ou fortement diminué dans les mélanomes métastatiques et 2/ la production, par les mélanocytes et les mélanomes métastatiques, d'une forme soluble de HLA-E, augmentée par l'IFN , mais dont le rôle est inconnu.With the perspective to develop immunotherapies of colorectal cancer, I studied the lymphocyte infiltrate (TIL) of these tumors, especially the phenotype and function of 2 clones: a 9 2 and an , specific for the autologous tumor cell line. I showed that : 1/ the 9 2 clone killed spontaneously the majority of colon carcinoma cell lines in a TCR- LFA-3- and NKG2D-dependant manner; 2/ all the 9 2 T cells shared this property, suggesting their interest for immunotherapy; 3/ the clone co-expressed CD8 and CD4 and secreted an array of cytokine of potential regulatory function (TGF- , IL-4, IL-2, TNF- ). I also studied the role of HLA-E, the CD94/NKG2A receptor ligand, in the anti melanoma response. I showed that 1/ HLA-E is expressed in vivo by melanocytes and most primary melanoma tumor cells but fe metastatic ones; 2/ melanocytes and melanoma cell lines spontaneously shed a soluble form of HLA-E undescribed so far, of unknown function and upregulated by IFN-gNANTES-BU Sciences (441092104) / SudocSudocFranceF

Étude des réponses lymphocytaires T à l'antigène de mélanome Melan-A/MART-1

La protéine Melan-A exprimée par la majorité des mélanomes contient deux épitopes chevauchants de forte antigénicité restreints HLA-A2. Ces épitopes sont donc souvent ciblés dans les immunothérapies de ce cancer. L efficacité clinique de ces traitements reste décevante. Au moins deux causes peuvent l expliquer: l incapacité de ces approches à induire une présentation croisée des épitopes CD8 par des cellules dendritiques et/ou la non stimulation de réponse CD4 helper. Mon travail de thèse aborde ces questions par la recherche de nouveaux épitopes de Melan-A et par l étude de la capacité d un glycociblage à induire la présentation croisée de cet antigène. J ai montré 1) l existence, dans la région 51-73 de Melan-A, d un nouvel epitope CD4 et d un épitope CD8, 2) l internalisation de glycoconjugués liés au peptide Melan-A16-40 27L par les récepteurs au mannose et DC-SIGN exprimés à la surface des cellules dendritiques, 3) que cette internalisation induit la présentation croisée par les cellules dendritiques de l épitope 26-35 et l expansion de lymphocytes T CD8+ spécifiques de forte avidité. Enfin, j ai observé la capacité intrinsèque du peptide 16-40 à induire la présentation croisée de l épitope 26-35 et l expansion de T CD8 et CD4 spécifiques de Melan-A. La stimulation de PBMC par ce long peptide devrait permettre de caractériser les réponses CD4 spécifiques et de déterminer l intérêt potentiel en vaccination de ce peptide seul ou conjugué à un système de ciblage aux cellules dendritiquesThe Melan-A protein, expressed by a majority of melanoma tumor cells, contains two highly antigenic HLA-A2 restricted epitopes. So, these epitopes are frequently targeted in melanoma immunotherapy. The clinical efficiency of these treatments is still disappointing. At least two major reasons can explain that: the incapacity of these strategies to induce CD8 epitope cross-presentation by dendritic cells and/or the non-stimulation of CD4+ helper T cell responses. My PhD work addressed these questions by looking for new Melan-A epitopes and by studying the ability of a glycotargeting to induce antigen cross-presentation. I demonstrated 1) the presence, in Melan-A 51-73 region, of a novel CD4+ epitope and of a CD8+ epitope, 2) the internalization of glycoclusters conjugated to Melan-A16-40 27L via Mannose Receptor and DC-SIGN expressed on dendritic cells, 3) that this internalization induce the epitope 26-35 cross-presentation by dendritic cells and the expansion of high avidity specific CD8+ T lymphocytes. Finally, I observed the intrinsic capacity of the long peptide Melan-A16-40 to induce epitope 26-35 cross-presentation and the expansion of specific CD4+ and CD8+ T lymphocytes. PBMC stimulation by this long peptide should allow to characterize Melan-A specific CD4+ T cell responses and to determine the potential interest to use this peptide in vaccination, either alone or combined with a system to specifically target dendritic cellsNANTES-BU Sciences (441092104) / SudocSudocFranceF

A lineage-specific methylation pattern controls the transcription of the polycistronic mRNA coding MELOE melanoma antigens

International audienceWe recently characterized two melanoma antigens MELOE-1 and MELOE-2 derived from a polycistronic RNA overexpressed in the melanocytic lineage. This transcription profile was because of hypomethylation of the meloe proximal promoter in melanomas and melanocytes. Here, we investigated whether this demethylation was restricted to the meloe promoter or was linked to a general lack of methylation at the meloe locus in the melanocytic lineage. We established the methylation pattern of the locus spanning more than 40 kbp, focusing on CpG islands, using DNA bisulfite conversion and pyrosequencing. The study was carried out on cultured cell lines (melanoma, melanocyte, colon cancer, and mesothelioma cell lines), healthy tissues (skin and colon), and melanoma tumors. Demethylation, specifically observed in the melanocytic lineage, involves a large promoter area and not the entire meloe locus. This enables updating a tight regulation of meloe transcription in this lineage, suggesting tissue-specific epigenetic mechanisms. Associated with the previously described translational mechanisms, leading to the specific expression of MELOE-1 and MELOE-2 in melanomas, this makes MELOE-derived antigens a relevant candidate for immunotherapy of melanoma. Melanoma Res 00:000–00

Expression and Release of HLA-E by Melanoma Cells and Melanocytes: Potential Impact on the Response of Cytotoxic Effector Cells

International audienceHLA-E are nonclassical MHC molecules with poorly characterized tissue distribution and functions. Because of their capacity to bind the inhibitory receptor, CD94/NKG2A, expressed by NK cells and CTL, HLA-E molecules might play an important role in immunomodulation. In particular, expression of HLA-E might favor tumor cell escape from CTL and NK immunosurveillance. To address the potential role of HLA-E in melanoma immunobiology, we assessed the expression of these molecules ex vivo in human melanoma biopsies and in melanoma and melanocyte cell lines. Melanoma cell lines expressed no or low surface, but significant intracellular levels of HLA-E. We also report for the first time that some of them produced a soluble form of this molecule. IFN-␥ significantly increased the surface expression of HLA-E and the shedding of soluble HLA-E by these cells, in a metalloproteinase-dependent fashion. In contrast, melanocyte cell lines constitutively expressed HLA-E molecules that were detectable both at the cell surface and in the soluble form, at levels that were poorly affected by IFN-␥ treatment. On tumor sections, a majority of tumor cells of primary, but a low proportion of metastatic melanomas (30-70 and 10-20%, respectively), expressed HLA-E. Finally, HLA-E expression at the cell surface of melanoma cells decreased their susceptibility to CTL lysis. These data demonstrate that HLA-E expression and shedding are normal features of melanocytes, which are conserved in melanoma cells of primary tumors, but become dependent on IFN-␥ induction after metastasis. The biological significance of these findings warrants further investigation

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

p53 dysregulation in B-cell malignancies: More than a single gene in the pathway to hell

International audienceTP53 deletion or mutation is frequent in B-cell malignancies and is associated with a low response rate. We describe here the p53 landscape in B-cellmalignancies, from B-Acute Lymphoblastic Leukemia to Plasma Cell Leukemia, by analyzing incidence of gain or loss of function of actors both upstream and within the p53 pathway, namely MYC, RAS, ARF, MDM2, ATM and TP53. Abnormalities are not equally distributed and their incidence is highly variable among malignancies. Deletion and mutation, usually associated, of ATM or TP53 are frequent in Diffuse Large B-Cell Lymphoma and Mantle Cell Lymphoma. MYC gain, absent in post-GC malignancies, is frequent in B-Prolymphocytic-Leukemia,MultipleMyeloma and Plasma Cell Leukemias. RAS mutations are rare except in MM and PCL. Multiple Factorial Analysis notes that MYC deregulation is closely related to TP53 status. Moreover, MYC gain, TP53 deletion and RAS mutations are inversely correlated with survival. Based on this landscape, we further propose targeted therapeutic approaches for the different B-cell malignancies