Single-cell clonal lineage tracing identifies the transcriptional program controlling the cell fate decisions by...

Neoantigen-specific T cells specifically recognize tumor cells and are critical for cancer immunotherapies. However, the transcriptional program controlling the cell fate decisions by neoantigen-specific T cells is incompletely understood. Here, using joint single-cell transcriptome and TCR profiling, we mapped the clonal expansion and differentiation of neoantigen-specific CD8 ⁺ T cells in the tumor and draining lymph node in mouse prostate cancer. Compared to other antitumor CD8 ⁺ T cells and bystanders, neoantigen-specific CD8 ⁺ tumor-infiltrating lymphocytes (TILs) upregulated gene signatures of T cell activation and exhaustion. In the tumor draining lymph node, we identified TCF1 ⁺ TOX ^- T _SCM , TCF1 ⁺ TOX ⁺ T _PEX , and TCF1 ^- TOX ⁺ effector-like T _EX subsets among neoantigen-specific CD8 ⁺ T cells. Clonal tracing analysis of neoantigen-specific CD8 ⁺ T cells revealed greater clonal expansion in divergent clones and less expansion in clones biased towards T _EX, T _PEX , or T _SCM . The T _PEX subset had greater clonal diversity and likely represented the root of neoantigen-specific CD8 ⁺ T cell differentiation, whereas highly clonally expanded effector-like T _EX cells were positioned at the branch point where neoantigen-specific clones exited the lymph node and differentiated into T _EX TILs. Notably, T _SCM differentiation of neoantigen-specific CD8 ⁺ clones in the lymph node negatively correlated with exhaustion and clonal expansion of the same clones in the tumor. In addition, the gene signature of neoantigen-specific clones biased toward tumor infiltration relative to lymph node residence predicted a poorer response to immune checkpoint inhibitor. Together, we identified the transcriptional program that controls the cell fate choices by neoantigen-specific CD8 ⁺ T cells and correlates with clinical outcomes in cancer patients.