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. Overall design: 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.

T细胞耗竭（T cell exhaustion）是由慢性抗原暴露与炎症诱导的CD8+ T细胞功能失调状态，可出现于癌症及慢性病毒感染两类情境中。共抑制受体PD-1在介导T细胞耗竭过程中发挥关键作用，但通过基因敲除（gene knock-out）完全敲除PD-1，会导致更为严重的功能缺陷以及T细胞存活能力下降。本研究提出假说：PD-1的中等表达水平或许可使耗竭CD8+ T细胞的功能达到更优平衡，因此我们敲除了一个与T细胞耗竭相关的PD-1基因增强子，实验结果确实使PD-1的表达水平有所降低。我们通过单细胞RNA测序（single-cell RNA-Seq）对增强子敲除型（EnhDel）、野生型（WT）以及PD-1敲除型（PD-1 KO）T细胞进行比较分析，结果发现仅PD-1敲除型细胞而非增强子敲除型细胞，会显著偏向终末耗竭细胞亚群。增强子敲除型细胞还会特异性富集效应相关基因及基因特征集。然而在匹配耗竭细胞亚群的实验条件下，通过ATAC测序（ATAC-Seq）分析显示，三种基因型细胞的染色质可及性景观并无显著差异。上述数据表明，对PD-1表达水平的精准调控，或许可特异性维持耗竭CD8+ T细胞的“效应性”转录谱型。实验整体设计：将增强子敲除型与PD-1敲除型P14+细胞，或增强子敲除型与野生型P14+细胞分别以75:25与50:50的比例共同移植到受体动物体内（每组样本量n=15~30）。受体动物经淋巴细胞脉络丛脑膜炎病毒克隆13株（LCMV Clone 13）感染。于感染后第30天，将脾脏混合为两份生物学重复样本，通过磁珠富集法分离得到移植的CD8+ T细胞。随后通过流式细胞分选分别分离各基因型的祖细胞耗竭与终末耗竭亚群，对样本进行ATAC测序建库与分析。