Abstract
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.
Highlights
Among solid tumors, metastatic melanoma is a relevant model for immunotherapeutic approaches because of a high immunogenicity, partly due to a high mutation rate, favoring the development of specific T cell immune responses [1]
We analyzed the TCR diversity of 6 Melan-A and 4 MELOE-1 specific CD8+ T cell polyclonal populations, derived from the specific sorting of HLA-A2 patient Peripheral blood mononuclear cells (PBMC) stimulated with the cognate peptides [3]
For Melan-A-specific T cell repertoires, we observed high numbers of CDR3α and CDR3ß clonotypes with the highest amount of starting RNA, consistent with increased sensitivity in detecting rare clonotypes when starting with more sample
Summary
Metastatic melanoma is a relevant model for immunotherapeutic approaches because of a high immunogenicity, partly due to a high mutation rate, favoring the development of specific T cell immune responses [1]. Recently it was reported that the immunodominant HLA-A2 Melan-A26−35 epitope is not presented by human medullary thymic epithelial cells, due to a misinitiation of gene transcription [4], and leading to the evasion of central self-tolerance toward this epitope. This finding, together with the strong bias documented in Vα usage [5, 6] could explain the abundance of this specific-T cell repertoire and the presence of high avidity T cells among this repertoire
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