Highlights d Ly108 and CD69 define four Tex subsets linked in a hierarchical developmental pathway d Two TCF1+ subsets, effector-like and terminally exhausted subsets, are identified d Key transcriptional, epigenetic, and biological changes define subset transitions d TCF1, T-bet, and Tox coordinate Tex subset development and dynamics

TCF-1 is a key transcription factor in progenitor exhausted CD8 T cells (Tex). Moreover, this Tex cell subset mediates responses to PD-1 checkpoint pathway blockade. However, the role of the transcription factor TCF-1 in early fate decisions and initial generation of Tex cells is unclear. Single-cell RNA sequencing (scRNA-seq) and lineage tracing identified a TCF-1 + Ly108 + PD-1 + CD8 T cell population that seeds development of mature Tex cells early during chronic infection. TCF-1 mediated the bifurcation between divergent fates, repressing development of terminal KLRG1 Hi effectors while fostering KLRG1 Lo Tex precursor cells, and PD-1 stabilized this TCF-1 + Tex precursor cell pool. TCF-1 mediated a T-bet-to-Eomes transcription factor transition in Tex precursors by promoting Eomes expression and drove c-Myb expression that controlled Bcl-2 and survival. These data define a role for TCF-1 in early-fate-bifurcationdriving Tex precursor cells and also identify PD-1 as a protector of this early TCF-1 subset.

Exhausted CD8 T cells (T EX ) are a distinct state of T cell differentiation associated with failure to clear chronic viruses and cancer. Immunotherapies like PD-1 blockade can re-invigorate T EX cells, but re-invigoration is not durable. A major unanswered question is whether T EX cells differentiate into functional durable memory T cells (T MEM ) upon antigen clearance. Here, using a mouse model, we found that upon eliminating chronic antigenic stimulation, T EX cells partially (re)acquire phenotypic and transcriptional features of T MEM cells. These “recovering” T EX cells originated from the T-cell factor (TCF-1 + ) T EX progenitor subset. Nevertheless, the recall capacity of these recovering-T EX cells remained compromised compared to T MEM cells. Chromatin-accessibility profiling revealed failure to recover core memory epigenetic circuits and maintenance of a largely exhausted open chromatin landscape. Thus, despite some phenotypic and transcriptional recovery upon antigen clearance, exhaustion leaves durable epigenetic scars constraining future immune responses. These results support epigenetic remodeling interventions for T EX cell targeted immunotherapies.