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.