Divergent clonal differentiation trajectories of T cell exhaustion (scRNA-seq)

T cells activated by chronic antigen exposure in the setting of viral infections or cancer can adopt an exhausted T cell (Tex) state, characterized by reduced effector function and proliferative capacity, and the upregulation of inhibitory receptors. However, whether all antigen-specific T cell clones follow the same molecular and cellular Tex differentiation trajectory remains unclear. Here, we generate a single-cell multi-omic atlas of T cell exhaustion that redefines the phenotypic diversity and molecular regulation of Tex phenotypes. Longitudinal analysis during chronic viral infection identifies an early effector phenotype that is epigenetically primed for Tex differentiation and two late-stage Tex cell states with either a terminal exhaustion or a killer cell lectin-like receptor (KLR)-expressing cytotoxic gene signature. We define clonal trajectories of antigen-specific T cells using paired single-cell RNA and T cell receptor sequencing and reveal distinct differentiation trajectories resulting in terminal Tex-biased, KLR Tex-biased, or divergent clones that differentiate into both phenotypes. Comparison of Tex phenotypes among shared T cell clones that traffic to multiple organs reveals that clonal differentiation trajectories are maintained across tissues. Finally, we show that differences in clonal differentiation trajectory are driven by TCR signaling avidity, whereby high-avidity T cell clones preferentially adopt a terminal Tex fate, while low-avidity clones adopt an effector-like KLR Tex fate. These findings reveal clonal heterogeneity in the T cell response to chronic antigen and genomic programs that underlie Tex fates and persistence. T cells from 5, 8, or 21 dpi with LCMV-c13 or LCMV-Armstrong were sorted to obtain either gp-33 positive or negative CD8+ T cells. Sorted cells were subjected to single cell RNA and TCR sequencing on the droplet based 10X Genomics platform.