Functional memory T cells are derived from exhausted clones and expanded by checkpoint blockade [scRNATCR_rechallenge]

Immune checkpoint blockade can facilitate tumor clearance by T cells, resulting in long term patient survival. However, the capacity of exhausted CD8+ T cells (Tex), present during chronic antigen exposure, to form memory after antigen clearance remains unclear. Here, we performed longitudinal single cell RNA/T cell receptor sequencing and ATAC-sequencing on antigen-specific T cells after the peripheral clearance of chronic lymphocytic choriomeningitis virus infection. These data revealed the formation of a robust population of memory CD8+ T cells that transcriptionally, epigenetically, and functionally resemble central memory T cells (Tcm) that form after clearance of acute infection. To lineage trace the origin and memory recall response of Tex-derived memory clones, we utilized T cell receptor sequencing over the course of primary infection and rechallenge. We show that chronic Tcm are a clonally distinct lineage of Tex derived from progenitor exhausted cells, persist long-term in the absence of antigen, and undergo rapid clonal expansion during rechallenge. Finally, we demonstrate that aPD-L1 immune checkpoint blockade selectively expands clones which form Tcm after clearance. Together, these data support the concept that chronically stimulated T cells form bona fide functional memory T cells through an analogous differentiation pathway to acutely stimulated T cells, which may have significant implications for enhancing immune memory to cancer through checkpoint blockade and vaccination. Overall design: To track T cell clones during rechallenge, CD8+ T cells were isolated from the spleens of mice that cleared LCMV-Cl13 or LCMV-Arm infection. Half of the cells were used for bulk TCR sequencing prior to rechallenge. The remaining cells were transferred into new host mice, challenged with LCMV-Arm. At 8 days post-infection transferred T cells were sorted, and scRNA/TCR-seq was performed. Pre-rechallenge TCR-seq can be found at doi.org/10.5281/zenodo.14648171