Open Access
Peer-reviewed
Research Article
MELOE-1 Antigen Contains Multiple HLA Class II T Cell Epitopes Recognized by Th1 CD4+ T Cells from Melanoma Patients
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MELOE-1 is an overexpressed melanoma antigen containing a HLA-A2 restricted epitope, involved in melanoma immunosurveillance of patients adoptively transferred with tumour infiltrating lymphocytes (TIL). The use of the full-length antigen (46 aa) for anti-melanoma vaccination could be considered, subject to the presence of Th epitopes all along MELOE-1 sequence. Thus, in this study we evaluated in vitro the immunoprevalence of the different regions of MELOE-1 (i.e. their ability to induce CD4 T cell responses in vitro from PBMC). Stimulation of PBMC from healthy subjects with MELOE-1 induced the amplification of CD4 T cells specific for various regions of the protein in multiple HLA contexts, for each tested donor. We confirmed these results in a panel of melanoma patients, and documented that MELOE-1 specific CD4 T cells, were mainly Th1 cells, presumably favourable to the amplification of CD8 specific T cells. Using autologous DC, we further showed that these class II epitopes could be naturally processed from MELOE-1 whole protein and identified minimal epitopes derived from each region of MELOE-1, and presented in four distinct HLA contexts. In conclusion, vaccination with MELOE-1 whole polypeptide should induce specific Th1 CD4 responses in a majority of melanoma patients, stimulating the amplification of CD8 effector cells, reactive against melanoma cells.
Citation:Bobinet M, Vignard V, Rogel A, Khammari A, Dreno B, Lang F, et al. (2012) MELOE-1 Antigen Contains Multiple HLA Class II T Cell Epitopes Recognized by Th1 CD4+ T Cells from Melanoma Patients. PLoS ONE 7(12): e51716.
Editor:Sylvie Le Gall, Massachusetts General Hospital, United States of America
Received:September 5, 2012; Accepted:November 5, 2012; Published: December 20, 2012
Copyright: © 2012 Bobinet et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding:This work was supported by a grant from Inserm-transfer (Maturation/proof of concept projects). Mathilde Bobinet was supported by the Ligue Regionale contre le cancer (comité 85). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
In antitumor immune responses, CTL have been identified as the most powerful effector cells. As a consequence, most previous anti-cancer vaccines use class I HLA-restricted peptides derived from tumour antigens in order to stimulate CTL responses. However, the clinical impact of such peptide-based cancer vaccines remains still modest, even if a recent gp100-derived peptide vaccination was shown to increase patient survival in melanoma. In addition to a variety of immune suppressive mechanisms originating from the tumour itself, suboptimal design of vaccines used so far may explain this failure. In particular, short epitopic peptides, could induce vanishing CTL responses or tolerance towards targeted antigens. In the meanwhile, CD4 helper T cells have gained interest in anti-tumour immunity and immunotherapy. Indeed, tumour-reactive CD4+ T helper 1 cells (Th1) produce several cytokines (such as IFN-fγ, TNF-α and IL-2) essential for the induction of cell-mediated immunity against tumours. One widely accepted model demonstrates the ability of CD4+ T cells to ‘license’ dendritic cells (DCs) for efficient CD8+ T cell priming through the interaction of costimulatory receptors. The cytokines secreted by CD4+ Th1 cells also exert direct antitumor and antiangiogenic effects. Furthermore, it has been demonstrated in a mouse model that only tumour-reactive CD4+ T cells have been found to ensure efficient effector CTLs recruitment at the tumour site. From a clinical standpoint, a high density of tumour-infiltrating CD4+ Th1 cells has been recently shown as a good prognostic marker in colorectal cancer patients emphasizing the role of these cells in cancer immunosurveillance. In melanoma, tumour-reactive CD4 T cells have also been associated with a good clinical outcome, and more recently the same group showed that tumour specific CD4 T cells were present in at least 20% of metastatic melanomas, and suggested that the infusion of TIL (Tumour Infiltrating Lymphocytes) populations containing CD4 specific T cells could enhance the efficacy of adoptive cell therapy. In the same line of thought, it has been demonstrated in a melanoma patient that the adoptive cell transfer of CD4 T cells specific for NYESO-1 antigen induced durable clinical remission and led to endogenous responses against non-targeted tumour antigens, suggesting the stimulation of immune responses by transferred CD4 T cells.
In the field of peptide vaccination, it has been documented twenty years ago, in a mouse model, that the generation of a strong CD8 response against a LCMV-derived peptide depended on the presence of CD4 helper T cells. These results have been more recently confirmed in a clinical setting by the use of synthetic long peptides (SLP) in colorectal cancer, using P53 derived SLP, in vulvar intraepithelial neoplasia and cervical cancer patients using HPV16-derived SLP. In the case of vulvar neoplasia, clinical responses appeared to be correlated with the induction of strong HPV16 specific immune responses. SLPs containing immunogenic CD8 and CD4 tumour epitopes are therefore attractive tools to implement therapeutic cancer vaccine.
One of the main issues in the field of SLP vaccination in solid tumours is to identify immunogenic long peptides derived from relevant tumour associated antigens. Target antigens should be widely expressed in tumour cells, and able to induce robust CD8 and CD4 anti-tumour T cell responses. In melanoma, the Melan-A antigen fulfils these requirements and we recently reported the efficiency of a Melan-A modified SLP, to cross-prime human tumour-reactive T cells.. Another attractive target for melanoma vaccination would be the MELOE-1 antigen (46 aa), overexpressed in melanoma. Indeed, we previously reported that the infusion of TIL specific for this antigen was associated with a prolonged relapse-free survival for HLA-A2 melanoma patients who received TIL therapy. Furthermore, we documented the presence of a large and tumour reactive CD8 T cell repertoire in HLA-A2 melanoma patients and the presence of two class II epitopes in the vicinity of the class I epitope, located at the C-terminal end of the polypeptide.
In this study, our objective was to define the ability of this promising melanoma antigen to induce CD4 specific T cells in vitro, from healthy subjects and melanoma patients. This ability, called “immunoprevalence” can be defined as the frequency of responders. This property is a requirement for the design a therapeutic vaccine using full-length MELOE-1 antigen or a mixture of selected long peptides for melanoma patient’s vaccination. The proof of concept will be achieved by the characterization of class II epitopes located all along the MELOE-1 sequence and of the Th profile of CD4 T cell specific lymphocytes.
Blood samples from healthy subjects and melanoma patients were respectively obtained from the “Etablissement Français du Sang”, Nantes, France and from the department of Onco-Dermatology, Nantes Hospital, France. Patients were stage III to IV melanoma patients. Melanoma and B-EBV cell-lines were maintained in RPMI 1640 (GIBCO) containing 10% FCS (PAA). Lymphocytes were grown in RPMI 1640 8% human serum (HS) with 50 or 150 IU/ml of recombinant IL-2 (Chiron, Proleukin® Aldesleukin) and 2 nM of L-Glutamine (GIBCO). For experiments using DC, RPMI supplemented with 20 mg/mL of human albumin (LFB, Viabelex 200 mg/mL) was used to avoid peptide degradation by serum proteases.
