Divergent clonal differentiation trajectories of T cell exhaustion

Chronic antigen exposure during viral infection or cancer promotes an exhausted T cell (Tex) state with reduced effector function. However, whether all antigen-specific T cell clones follow the same Tex differentiation trajectory remains unclear. Here, we generate a single-cell multiomic atlas of T cell exhaustion in murine chronic viral infection that redefines Tex phenotypic diversity, including two late-stage Tex subsets with either a terminal exhaustion (Tex term ) or a killer cell lectin-like receptor-expressing cytotoxic (Tex KLR ) phenotype. We use paired single-cell RNA and T cell receptor sequencing to uncover clonal differentiation trajectories of Tex term -biased, Tex KLR -biased or divergent clones that acquire both phenotypes. We show that high T cell receptor signaling avidity correlates with Tex term , whereas low avidity correlates with effector-like Tex KLR fate. Finally, we identify similar clonal differentiation trajectories in human tumor-infiltrating lymphocytes. These findings reveal clonal heterogeneity in the T cell response to chronic antigen that influences Tex fates and persistence. Daniel, Yost, Hsiung, et al. generate a single-cell multiomic atlas of T cell exhaustion in chronic viral infection, which reveals molecular programs of exhausted T cell subsets, identifies divergent clonal exhausted T cell differentiation trajectories and nominates TCR signal strength as a driver of clonal fate.