Functionally heterogeneous intratumoral CD4+CD8+ double positive T cells can give rise to single positive T cells [scRNA-seq + scTCR-seq]

Conventional single positive (SP) CD4+ and CD8+ T cells recognize tumor antigens and help mediate clinical responses with cancer immunotherapy. Double positive CD4+CD8+ (DP) T cells have also been described in human cancers, but their role in the tumor microenvironment (TME) remains unclear. By generating a multi-omic single cell atlas of DP and SP T cells, we find that DP T cells possess phenotypic heterogeneity similar to SP T cells that includes multiple clonally expanded populations of cytotoxic DP T cells in human renal cell carcinoma (RCC). These intratumoral DP T cells can mediate by both MHC class I- and class II-dependent killing of autologous tumor cells. In addition, transcriptional profiling of DP TCR-bearing T cells revealed a gene signature enriched for clinical responders to PD-1 blockade in advanced RCC. We confirm prior observations of SP T cells transitioning into DP T cells and more notably, demonstrate that intratumoral T cells are capable of bidirectional differentiation in which DP T cells serve as precursors to SP T cells in vivo. In the latter scenario, intratumoral DP T cells are shown to express Rag2, suggesting that the tumor may act as an extrathymic site of T cell development. These findings reveal the multiple roles that DP T cells can possess in anti-tumor immunity. Overall design: To characterize T-cell heterogeneity in renal cell carcinoma (RCC), we performed single-cell RNA sequencing (scRNA-seq) and single-cell TCR sequencing (scTCR-seq) using the 10x Genomics platform. Samples were collected from each patient's tumor tissue, adjacent normal tissue, and peripheral blood mononuclear cells (PBMCs), along with PBMCs from healthy donors. To assign cells to their respective individuals following sequencing, we applied demuxlet, which leverages donor-specific genetic variants derived from bulk RNA-seq to enable genotype-based sample demultiplexing.