Deletion of a state-specific PD-1 enhancer modulates exhausted T cell fate and function [RNA-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 WT P14+ CD8+ T cells were isolated from donor mice, mixed at a 50:50 ratio, and transferred to congenically-distinct recipient mice (n = 8). Recipients were infected with LCMV Clone 13. Transferred CD8+ T cells were isolated from spleens at day 9. Genotypes (Enhancer-deleted, WT) and cell types (progenitor exhausted SLAMF6+TIM3-, terminally exhausted SLAMF6-TIM3+CX3CR1-) were then isolated by sorting, pooled in pairs to create four samples, and processed for bulk RNA-seq. We then performed differential gene expression analysis using DESeq2 in R to compare the transcriptomic profiles of EnhDel and wild type CD8+ T cells at the D9 time point, controlling for exhausted subset.