Divergent clonal differentiation trajectories of T cell exhaustion (scRNA-seq). Divergent clonal differentiation trajectories of T cell exhaustion (scRNA-seq)

T cells activated by chronic antigen exposure in the setting of viral infections or cancer can adopt an exhausted T cell (Tex) state, characterized by reduced effector function and proliferative capacity, and the upregulation of inhibitory receptors. However, whether all antigen-specific T cell clones follow the same molecular and cellular Tex differentiation trajectory remains unclear. Here, we generate a single-cell multi-omic atlas of T cell exhaustion that redefines the phenotypic diversity and molecular regulation of Tex phenotypes. Longitudinal analysis during chronic viral infection identifies an early effector phenotype that is epigenetically primed for Tex differentiation and two late-stage Tex cell states with either a terminal exhaustion or a killer cell lectin-like receptor (KLR)-expressing cytotoxic gene signature. We define clonal trajectories of antigen-specific T cells using paired single-cell RNA and T cell receptor sequencing and reveal distinct differentiation trajectories resulting in terminal Tex-biased, KLR Tex-biased, or divergent clones that differentiate into both phenotypes. Comparison of Tex phenotypes among shared T cell clones that traffic to multiple organs reveals that clonal differentiation trajectories are maintained across tissues. Finally, we show that differences in clonal differentiation trajectory are driven by TCR signaling avidity, whereby high-avidity T cell clones preferentially adopt a terminal Tex fate, while low-avidity clones adopt an effector-like KLR Tex fate. These findings reveal clonal heterogeneity in the T cell response to chronic antigen and genomic programs that underlie Tex fates and persistence. Overall design: T cells from 5, 8, or 21 dpi with LCMV-c13 or LCMV-Armstrong were sorted to obtain either gp-33 positive or negative CD8+ T cells. Sorted cells were subjected to single cell RNA and TCR sequencing on the droplet based 10X Genomics platform.

在病毒感染或癌症情境下，经慢性抗原刺激活化的T细胞可进入耗竭性T细胞（Tex）状态，其特征为效应功能与增殖能力下降，且抑制性受体表达上调。然而，目前尚不清楚所有抗原特异性T细胞克隆是否均遵循相同的分子与细胞层面的Tex分化轨迹。
本研究构建了T细胞耗竭的单细胞多组学（single-cell multi-omic）图谱，重新定义了Tex表型的多样性及其分子调控机制。在慢性病毒感染过程中开展的纵向分析，鉴定出一种表观遗传上已预编程以走向Tex分化的早期效应表型，以及两种晚期Tex细胞状态：一类为终末耗竭状态，另一类则表达杀伤细胞凝集素样受体（KLR）相关的细胞毒性基因特征。
本研究通过配对单细胞RNA测序与T细胞受体（TCR）测序，解析了抗原特异性T细胞的克隆分化轨迹，揭示了三类不同的分化路径：分别导向终末耗竭偏向型、KLR阳性Tex偏向型，以及可分化为两种表型的双潜能克隆。
对迁移至多个器官的共享T细胞克隆的Tex表型进行比较分析后发现，克隆分化轨迹在不同组织中保持一致。
最后，本研究证实克隆分化轨迹的差异由TCR信号亲和力驱动：高亲和力T细胞克隆优先走向终末耗竭Tex命运，而低亲和力克隆则呈现效应细胞样的KLR阳性Tex命运。
这些研究结果揭示了慢性抗原刺激下T细胞应答的克隆异质性，以及支撑Tex细胞命运与存续性的基因组程序。
实验设计：分别采集淋巴细胞脉络丛脑膜炎病毒（LCMV）弱毒株（LCMV-c13）与强毒株（LCMV-Armstrong）感染后第5、8、21天的T细胞，通过分选获取gp33阳性或阴性的CD8阳性T细胞。分选后的细胞基于液滴式10× Genomics平台开展单细胞RNA测序与TCR测序。