Deletion of a state-specific PD-1 enhancer modulates exhausted T cell fate and function [ATAC-seq]

T cell exhaustion is a state of CD8+ T cell dysfunction elicited by chronic exposure to antigen and inflammation, arises in both cancer and chronic viral infection. The co-inhibitory receptor PD-1 plays a key role in mediating exhaustion, but complete ablation of PD-1 by gene knock-out leads to deeper functional deficits and poor T cell survival. We hypothesized that an intermediate level of PD-1 expression may confer an improved balance of exhausted CD8+ T cell functionality, so we deleted an exhaustion-associated enhancer of PD-1 which indeed resulted in a reduced expression level. We compared EnhDel, WT and PD-1 KO T cells using single-cell RNA-Seq and found that PD-1 KO but not EnhDel cells are strongly biased towards the terminally exhausted subset. EnhDel cells also uniquely enrich for effector-associated genes and gene signatures. However, all three genotypes (EnhDel, WT and PD-1 KO) exhibit a similar chromatin accessibility landscape by ATAC-Seq, controlling for exhausted subset. These data suggest that tuning of PD-1 expression may uniquely permit the maintenance of an “effector” transcriptional profile in exhausted CD8+ T cells. Enhancer-deleted and PD-1 KO P14+ cells, or enhancer-deleted and wildtype P14+ cells, were co-transferred into recipient animals (n = 15-30) at a 75:25 or 50:50 ratio, respectively. Recipients were infected with LCMV Clone 13. At day 30, spleens were pooled into two biological replicates and transferred CD8+ T cells were isolated with magnetic enrichment. Progenitor and terminally-exhausted subsets for each genotype were isolated with flow-assisted sorting, and samples were processed for ATAC-Seq.