A distinct gene module uncouples dysfunction from activation in tumor-infiltrating T cells (part 2)

Reversing the dysfunctional T cell state that arises in cancer and chronic viral infections is the focus of therapeutic interventions; however, current therapies are effective in only some patients and some tumor types. To gain a deeper molecular understanding of the dysfunctional T cell state, we analyzed population and single-cell RNA profiles of CD8+ tumor-infiltrating lymphocytes (TILs) and used genetic perturbations to identify a distinct gene module for T cell dysfunction that can be uncoupled from T cell activation. This distinct dysfunction module is downstream of intracellular metallothioneins that regulate zinc metabolism and can be identified at single-cell resolution. We further identify Gata-3, a zinc-finger transcription factor in the dysfunctional module, as a regulator of dysfunction, and use CRISPR/Cas9 genome editing to show that it drives a dysfunctional phenotype in CD8+ TILs. Our results open novel avenues for targeting dysfunctional T cell states, while leaving activation programs intact. CD8 TILs sorted on PD1 and Tim3 to subpopulations were analyzed from two batches: batch 1: 2 WT and 2 MTKO mice , batch 2: 2 WT and 3 MTKO mice.

逆转癌症与慢性病毒感染中出现的功能失调性T细胞状态，是当前治疗干预的核心方向；然而现有疗法仅在部分患者与部分肿瘤类型中取得疗效。为深入解析功能失调性T细胞状态的分子机制，我们对CD8+肿瘤浸润淋巴细胞（tumor-infiltrating lymphocytes, TILs）的群体转录组与单细胞转录组图谱开展了分析，并借助遗传扰动实验，鉴定出一个可与T细胞激活程序解耦联的独特T细胞功能失调基因模块。该独特的功能失调基因模块定位于调控锌代谢的细胞内金属硫蛋白的下游，且可通过单细胞分辨率进行识别。我们进一步鉴定出功能失调模块中的锌指转录因子Gata-3为功能失调的调控因子，并通过CRISPR/Cas9基因组编辑实验证实，该因子可诱导CD8+ TILs产生功能失调表型。本研究结果为靶向功能失调性T细胞状态同时保留T细胞激活程序提供了全新的干预途径。本研究对两批次经程序性死亡受体1（programmed cell death protein 1, PD1）与T细胞免疫球蛋白粘蛋白3（T-cell immunoglobulin and mucin-domain containing-3, Tim3）分选得到的CD8+ TILs亚群进行了分析：批次1包含2只野生型（wild type, WT）与2只金属硫蛋白敲除（metallothionein knockout, MTKO）小鼠样本，批次2包含2只野生型与3只金属硫蛋白敲除小鼠样本。