Broadening the repertoire of melanoma-associated T-cell epitopes

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. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s00262-015-1664-x) contains supplementary material, which is available to authorized users

Ex vivo modulation of intact tumor fragments with anti-PD-1 and anti-CTLA-4 influences the expansion and specificity of tumor-infiltrating lymphocytes

Checkpoint inhibition (CPI) therapy and adoptive cell therapy with autologous tumor-infiltrating lymphocytes (TIL-based ACT) are the two most effective immunotherapies for the treatment of metastatic melanoma. While CPI has been the dominating therapy in the past decade, TIL-based ACT is beneficial for individuals even after progression on previous immunotherapies. Given that notable differences in response have been made when used as a subsequent treatment, we investigated how the qualities of TILs changed when the ex vivo microenvironment of intact tumor fragments were modulated with checkpoint inhibitors targeting programmed death receptor 1 (PD-1) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4). Initially, we show that unmodified TILs from CPI-resistant individuals can be produced, are overwhelmingly terminally differentiated, and are capable of responding to tumor. We then investigate these properties in ex vivo checkpoint modulated TILs finding that that they retain these qualities. Lastly, we confirmed the specificity of the TILs to the highest responding tumor antigens, and identified this reactivity resides largely in CD39+CD69+ terminally differentiated populations. Overall, we found that anti-PD-1 will alter the proliferative capacity while anti-CTLA4 will influence breadth of antigen specificity

The metabolic enzyme arginase-2 is a potential target for novel immune modulatory vaccines

One way that tumors evade immune destruction is through tumor and stromal cell expression of arginine-degrading enzyme arginase-2 (ARG2). Here we describe the existence of pro-inflammatory effector T-cells that recognize ARG2 and can directly target tumor and tumor-infiltrating cells. Using a library of 34 peptides covering the entire ARG2 sequence, we examined reactivity toward these peptides in peripheral blood mononuclear cells from cancer patients and healthy individuals. Interferon-γ ELISPOT revealed frequent immune responses against several of the peptides, indicating that ARG2–specific self-reactive T-cells are natural components of the human T-cell repertoire. Based on this, the most immunogenic ARG2 protein region was further characterized. By identifying conditions in the microenvironment that induce ARG2 expression in myeloid cells, we showed that ARG2-specific CD4T-cells isolated and expanded from a peripheral pool from a prostate cancer patient could recognize target cells in an ARG2-dependent manner. In the ‘cold’ in vivo tumor model Lewis lung carcinoma, we found that activation of ARG2-specific T-cells by vaccination significantly inhibited tumor growth. Immune-modulatory vaccines targeting ARG2 thus are a candidate strategy for cancer immunotherapy