TCR Analyses of Two Vast and Shared Melanoma Antigen-Specific T Cell Repertoires: Common and Specific Features

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.

Clonality of CD4+ Blood T Cells Predicts Longer Survival With CTLA4 or PD-1 Checkpoint Inhibition in Advanced Melanoma

“…Simple differentiation between CD4 + and CD8 + T cells as done here may already improve resolution for analyzing clinical-relevant immune responses. Careful optimization by minimum pre-amplification is essential to avoid PCR errors for both immunoscope and high-throughput sequencing methods (14, 37, 38). The immunoscope analysis can directly visualize the diversity of clonal T-cell responses for each V-gene family.…”

“…It may be speculated that the more diversified clonal CD4 + T-cell responses in our TCR analyses reflect the diversification of active antigen/neoantigen epitopes, i.e., tumor antigenicity that had actually stimulated T-cell responses in context of each patient's HLA haplotypes. Each (neo-)antigenic peptide may selectively activate oligoclonal T cell populations with the same antigen specificity, which tend to share common features in rearranged Vβ-gene family and CDR3 size (24, 38). T-cell responses against each antigenic epitope may result in select peaks with a dominant length in the spectratypy of a particular Vβ-gene family (24, 39, 40).…”

PD-1 and TIGIT coexpression identifies a circulating CD8 T cell subset predictive of response to anti-PD-1 therapy

“…TCR-sequencing analysis cDNA librairies for TCR-seq analysis were prepared from 15 ng of total RNA using Human TCR Panel QIAseq Immune Repertoire RNA Library Kit (QIAGEN) according to manufacturer's instructions and was described previously. 44 Sequencing libraries were quantified with QIAseq Library Quant System (QIAGEN) and quality control was performed by capillary electrophoresis on a TapeStation System (Agilent Technologies). For sequencing, each library was diluted to 4 nM, pooled and denatured.…”

“…FASTQ files were analyzed in the QIAGEN GeneGlobe Data Analysis Center as described previously. 44 TCR sequencing data analysis TCR sequence analysis was performed using R (V.3.5.2). The input data consisted of the observed number of each TCR (alpha or beta) sequence in each sample.…”

Comprehensive single institute experience with melanoma TIL: Long term clinical results, toxicity profile, and prognostic factors of response

“…T cells genetically modified to express a chimeric antigen receptor (CAR) against the B cell marker CD19 received FDA approval in 2017 for patients with acute lymphoid leukemia and various types of non‐Hodgkin lymphoma. Another form of adoptive T cell therapy utilizes tumor‐infiltrating lymphocytes (TIL), which naturally infiltrate the tumor and have the ability to recognize shared or tumor mutation‐specific antigens, thereby causing destruction of tumor cells 1‐3 . TIL for therapy may be selected based on their ability to recognize autologous tumor cells in vitro (“Selected TIL”) or based on the identification of a fast‐growing culture, in which TIL spend minimal time in culture (“Young/Unselected” TIL).…”