αvβ3-dependent cross-presentation of matrix metalloproteinase–2 by melanoma cells gives rise to a new tumor antigen

A large array of antigens that are recognized by tumor-specific T cells has been identified and shown to be generated through various processes. We describe a new mechanism underlying T cell recognition of melanoma cells, which involves the generation of a major histocompatibility complex class I–restricted epitope after tumor-mediated uptake and processing of an extracellular protein—a process referred to as cross-presentation—which is believed to be restricted to immune cells. We show that melanoma cells cross-present, in an αvβ3-dependent manner, an antigen derived from secreted matrix metalloproteinase–2 (MMP-2) to human leukocyte antigen A*0201-restricted T cells. Because MMP-2 activity is critical for melanoma progression, the MMP-2 peptide should be cross-presented by most progressing melanomas and represents a unique antigen for vaccine therapy of these tumors

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

Faecalibacterium prausnitzii Skews Human DC to Prime IL10-Producing T Cells Through TLR2/6/JNK Signaling and IL-10, IL-27, CD39, and IDO-1 Induction

The human colonic mucosa contains regulatory type 1-like (Tr1-like, i.e., IL-10-secreting and Foxp3-negative) T cells specific for the gut Clostridium Faecalibacterium prausnitzii (F. prausnitzii), which are both decreased in Crohn's disease patients. These data, together with the demonstration, in mice, that colonic regulatory T cells (Treg) induced by Clostridium bacteria are key players in colon homeostasis, support a similar role for F. prausnitzii-specific Treg in the human colon. Here we assessed the mechanisms whereby F. prausnitzii induces human colonic Treg. We demonstrated that F. prausnitzii, but not related Clostridia, skewed human dendritic cells to prime IL-10-secreting T cells. Accordingly, F. prausnitzii induced dendritic cells to express a unique array of potent Tr1/Treg polarizing molecules: IL-10, IL-27, CD39, IDO-1, and PDL-1 and, following TLR4 stimulation, inhibited their up-regulation of costimulation molecules as well as their production of pro-inflammatory cytokines IL-12 (p35 and p40) and TNFα. We further showed that these potent tolerogenic effects relied on F. prausnitzii-induced TLR2/6 triggering, JNK signaling and CD39 ectonucleotidase activity, which was induced by IDO-1 and IL-27. These data, together with the presence of F. prausnitzii-specific Tr1-like Treg in the human colon, point out to dendritic cells polarization by F. prausnitzii as the first described cellular mechanism whereby the microbiota composition may affect human colon homeostasis. Identification of F. prausnitzii-induced mediators involved in Tr1-like Treg induction by dendritic cells opens therapeutic avenues for the treatment of inflammatory bowel diseases

CD4CD8αα Lymphocytes, A Novel Human Regulatory T Cell Subset Induced by Colonic Bacteria and Deficient in Patients with Inflammatory Bowel Disease

It has become evident that bacteria in our gut affect health and disease, but less is known about how they do this. Recent studies in mice showed that gut Clostridium bacteria and their metabolites can activate regulatory T cells (Treg) that in turn mediate tolerance to signals that would ordinarily cause inflammation. In this study we identify a subset of human T lymphocytes, designated CD4CD8αα T cells that are present in the surface lining of the colon and in the blood. We demonstrate Treg activity and show these cells to be activated by microbiota; we identify F. prausnitzii, a core Clostridium strain of the human gut microbiota, as a major inducer of these Treg cells. Interestingly, there are fewer F. prausnitzii in individuals suffering from inflammatory bowel disease (IBD), and accordingly the CD4CD8αα T cells are decreased in the blood and gut of patients with IBD. We argue that CD4CD8αα colonic Treg probably help control or prevent IBD. These data open the road to new diagnostic and therapeutic strategies for the management of IBD and provide new tools to address the impact of the intestinal microbiota on the human immune system

Folding of Matrix Metalloproteinase-2 Prevents Endogenous Generation of MHC Class-I Restricted Epitope

BACKGROUND: We previously demonstrated that the matrix metalloproteinase-2 (MMP-2) contained an antigenic peptide recognized by a CD8 T cell clone in the HLA-A*0201 context. The presentation of this peptide on class I molecules by human melanoma cells required a cross-presentation mechanism. Surprisingly, the classical endogenous processing pathway did not process this MMP-2 epitope. METHODOLOGY/PRINCIPAL FINDINGS: By PCR directed mutagenesis we showed that disruption of a single disulfide bond induced MMP-2 epitope presentation. By Pulse-Chase experiment, we demonstrated that disulfide bonds stabilized MMP-2 and impeded its degradation. Finally, using drugs, we documented that mutated MMP-2 epitope presentation used the proteasome and retrotranslocation complex. CONCLUSIONS/SIGNIFICANCE: These data appear crucial to us since they established the existence of a new inhibitory mechanism for the generation of a T cell epitope. In spite of MMP-2 classified as a self-antigen, the fact that cross-presentation is the only way to present this MMP-2 epitope underlines the importance to target this type of antigen in immunotherapy protocols

Rôle du récepteur CD94/NKG2-A dans la réponse T anti-tumorale et recherche d'antigènes reconnus par les lymphocytes T CD8+ infiltrant les tumeurs du sein

La première partie porte sur le rôle du récepteur inhibiteur CD94/NKG2-A dans la réponse T anti-tumorale. Les résultats montrent que 1) l'IL-12 induit l'expression de ce récepteur à la surface des lymphocytes T CD8+, 2) la plupart des lignées de mélanome exprime le ligand de ce récepteur : la molécule HLA-E, à différents niveaux, 3) l'expression de HLA-E est augmentée par l'IFN-g. De plus, nous avons observé l'existence d'une balance entre le signal d'activation dépendant du TCR et le signal d'inhibition médié par l'interaction CD94/NKG2-A/HLA-E. Ces résultats suggèrent que le récepteur CD94/NKG2-A et la molécule HLA-E, dont l'expression est régulée par différents facteurs, participeraient au phénomène d'échappement tumoral. Dans une deuxième partie, nous avons recherché la présence de lymphocytes T CD8+ spécifiques d'antigènes de tumeurs de sein parmi les TIL (Tumor Infiltrating Lymphocytes). Sur les 34 populations de TIL testés contre 48 antigènes de cancer du sein, quatre répondent aux antigènes P53, GAGE-6 et LAGE-1a dans quatre contextes HLA-A, B, ou Cw différents. Nous recherchons à définir les épitopes reconnus, dont la caractérisation permettra de réaliser de nouveaux protocoles d'immunothérapies visant ces antigènes.In the first part, we have studies the role of the CD94/NKG2-A receptor in anti-tumoral T cell response The results show that 1) IL-12 induce the expression of this receptor in CD8 T cell, 2) the ligand of this NKR : HLA-E is expressed, although at different levels, by human melanoma cell lines, 3) IFN-gamma-treatment up-regulates this expression. We further show that inhibition of the response of melanoma antigen specific T cell clones was correlated with the density of HLA-E on melanoma cells and the density of CD94/NKG2-A on the CTL, but inversely correlated with the strength of the TCR-dependant. In the second part, 34 tumor infiltrating lymphocytes (TIL) cultures established from breast tumor or invaded lymph nodes were screened for recognition of 48 breast associated antigens. Four TIL lines reacted to three antigens (P53, GAGE-6 and LAGE-1a) in association with four differents HLA molecules. The caraterisation of this new epitopes could lead to realise new immunotherapy protocols for breast cancer patients targeting this antigens.NANTES-BU Sciences (441092104) / SudocSudocFranceF

La présentation croisée de la métalloprotéase matricielle-2 par les cellules de mélanome génère un épitope T spécifique de ce cancer

La plupart des tumeurs expriment des antigènes susceptibles d'être reconnus par des lymphocytes T CD8 cytotoxiques. Néanmoins, les essais d'immunothérapies réalisés chez l'homme, ciblant différents antigènes reconnus par des lymphocytes T, ont montré une efficacité limitée. L'une des voies possibles pour améliorer ces traitements consiste à identifier de nouveaux antigènes tumoraux. Dans ce but, nous avons étudié la spécificité de lymphocytes T ayant infiltré les mélanomes de 22 patients. Ce criblage a permis d'identifier un nouvel antigène de tumeur reconnu par un clone T CD8 dans le contexte HLA-A*0201 : la métalloprotéase matricielle-2 (MMP-2). Nous avons également montré que l'épitope MMP-2560-568 n'était pas présenté par la voie endogène classique, mais par présentation croisée. L'apprêtement original de MMP-2, utilisé exclusivement par les cellules de mélanome, et ses fonctions pro-tumorales en font un candidat idéal à cibler en immunothérapie anti-mélanome.Most tumor cells express antigens that can be recognized by cytotoxic T CD8 lymphocytes. Nevertheless, immunotherapeutic trials, targeting various human tumor cell antigens recognized by T lymphocytes, have shown limited efficacy. To improve these treatments, it is necessary to identify new tumor antigens. With this aim, we studied the specificity of melanoma infiltrating lymphocytes from 22 patients. This screening allowed us to identify a new tumor antigen recognized by a CD8 T cell clone in the HLA-A*0201 context : the matrix metalloproteinase-2 (MMP-2). We also showed that the MMP-2560-568 epitope is not processed by the classical endogenous pathway, but by cross-presentation. The original processing of the MMP-2 epitope, only used by melanoma cells, and the pro-tumoral functions of MMP-2 make of this new antigen an ideal target for immunotherapy, and opens the way to new innovative therapeutic strategies to treat HLA-A*0201 melanoma patients.NANTES-BU Sciences (441092104) / SudocSudocFranceF

Etude de l'immunogénicité de longs peptides d'antigènes de mélanomes humains in vitro, et in vivo chez des souris HLA-A2 transgéniques ( implication en vaccinothérapie )

Les échecs des vaccinations du mélanome utilisant des peptides épitopiques peuvent s expliquer par leur incapacité à générer des réponses CD8 durables et CD4 efficaces, ou par la faible liaiso n au HLA de certains épitopes CD8, tels que les épitopes immunodominants des antigènes Melan-A et NY-ESO-1. Deu x solutions, ont été proposées: 1/ l utilisation de longs peptides (LP) incluant des épitopes CD4 et CD8, afin de restreindre la présentation des antigènes aux cellules présentatrices et permettre la coopération CD4/ CD8, 2/ l utilisation d épitopes optimisés pour l ancrage au HLA . Le potentiel immunogénique de LP d antigènes de tumeurs hu maines, n ayant pas été étudié, nous avons choisi de poser ce tte question pour des LP Melan-A et NY-ESO-1 contenant l épitope immunodominant, optimisé ou non pour la liaison au HLA -A 2, et pour deu x LP contenant l épitope NA17A1-10. Les résultats majeurs montrent que ces LP induisent tous une présentation croisée durable, restreinte aux cellules dendritiques myéloïdes ou à leurs précurseurs. Cependant, pour Melan-A, une telle présentation de l épitope naturel est ineffica ce à induire une réponse CD8 a lors que, de façon majeu re, la présentation croisée de l épitope opt imisé à part ir du LP modifié induit des réponses CD8 anti-tumorales fortes et durables parmi les PBL de donneurs sains et de patients. De plus, chez certains donneurs, cette réponse est amplifiée par les lymphocytes CD4 et le LP modifié induit une réponse CD4 spécifique du Melan-A naturel. Cette étude établit l intérêt vaccina l unique du LP Melan -A modifié pour le traitement des mélanomes HLA-A2 et l impact de l affinité de liaison d un épitope au HLA sur l immunogénicité des LP.NANTES-BU Sciences (441092104) / SudocSudocFranceF

Adoptive transfer with high-affinity TCR to treat human solid tumors: how to improve the feasibility?

International audienceThe adoptive transfer of tumor antigen-specific T cells recently achieved clinical efficacy for a fraction of melanoma patients refractory to other therapies. Unfortunately , the application of this strategy to the remaining melanoma and most other cancer patients is hampered by the difficulty to generate high-affinity tumor-reactive T cells. Two strategies are currently developed to extend the feasibility of this therapeutic approach: clinical grade tool production for MHC-peptide multimer-driven sorting of antigen-specific T cells from the endogenous peripheral T cell repertoire and de novo engineering of the missing repertoire by genetic transfer of cloned specific T cell receptor (TCR) into T cells. The expected multiplication of adoptive transfer treatments, by these strategies, and their careful evaluation should enable the cure of a number of otherwise compromised cancer patients and to gain insight into the characteristics of transferred T cells best fitted to eradicate tumor cells, in terms of antigen specificities, phenotype, and functions. In particular, identification of tumor-rejection antigens by this approach would improve the design and efficacy of all immuno-therapeutic approaches