Pinpointing the tumor-specific T-cells via TCR clusters

Adoptive T cell transfer (ACT) is a promising approach to cancer immunotherapy, but its efficiency fundamentally depends on the extent of tumor-specific T-cell enrichment within the graft. This can be estimated via activation with identifiable neoantigens, tumor-associated antigens (TAAs), or living or lyzed tumor cells, but these approaches remain laborious, time-consuming, and functionally limited, hampering clinical development of ACT. Here, we demonstrate that homology cluster analysis of T cell receptor (TCR) repertoires efficiently identifies tumor-reactive TCRs allowing to: 1) detect their presence within the pool of tumor-infiltrating lymphocytes (TILs); 2) optimize TIL culturing conditions, with IL-2low/IL-21/anti-PD-1 combination showing increased efficiency; 3) investigate surface marker-based enrichment for tumor-targeting T cells in freshlyisolated TILs (enrichment confirmed for CD4+ and CD8+ PD-1+/CD39+ subsets), or re-stimulated TILs (informs on enrichment in 4-1BB-sorted cells). We believe that our approach to the rapid assessment of tumor-specific TCR enrichment should accelerate T cell therapy development. Dataset consists of 336 files. CD39+PD-1+ (double-positive or DP) and not-DP CD4+ and CD8+ TILs were freshly isolated and sorted from metastasized lymph nodes of 8 melanoma patients (2 biological replicas per sample). For one patient, CD8+ DP and not-DP TILs were expanded ex vivo and stimulated with tumor-associated antigens mix. CD137-high cells were sorted for NGS (1 biological replica per sample). Additionally, bulk TILs TCR repertoires were obtained from uncultured TILs of one melanoma patient (8 biological replicas), and TILs of 3 melanoma patients cultured in different expansion conditions (4,6, and 12 biological replicas per condition respectively).