CD8⁺ cytotoxic T cells (CTLs) orchestrate antitumour immunity and exhibit inherent heterogeneity^1,2, with precursor exhausted T (T_pex) cells but not terminally exhausted T (T_ex) cells capable of responding to existing immunotherapies^3,4,5,6,7. The gene regulatory network that underlies CTL differentiation and whether T_ex cell responses can be functionally reinvigorated are incompletely understood. Here we systematically mapped causal gene regulatory networks using single-cell CRISPR screens in vivo and discovered checkpoints for CTL differentiation. First, the exit from quiescence of T_pex cells initiated successive differentiation into intermediate T_ex cells. This process is differentially regulated by IKAROS and ETS1, the deficiencies of which dampened and increased mTORC1-associated metabolic activities, respectively. IKAROS-deficient cells accumulated as a metabolically quiescent T_pex cell population with limited differentiation potential following immune checkpoint blockade (ICB). Conversely, targeting ETS1 improved antitumour immunity and ICB efficacy by boosting differentiation of T_pex to intermediate T_ex cells and metabolic rewiring. Mechanistically, TCF-1 and BATF are the targets for IKAROS and ETS1, respectively. Second, the RBPJ–IRF1 axis promoted differentiation of intermediate T_ex to terminal T_ex cells. Accordingly, targeting RBPJ enhanced functional and epigenetic reprogramming of T_ex cells towards the proliferative state and improved therapeutic effects and ICB efficacy. Collectively, our study reveals that promoting the exit from quiescence of T_pex cells and enriching the proliferative T_ex cell state act as key modalities for antitumour effects and provides a systemic framework to integrate cell fate regulomes and reprogrammable functional determinants for cancer immunity.

CD8 ⁺细胞毒性 T 细胞 (CTL) 协调抗肿瘤免疫并表现出固有的异质性^1,2 ，前体耗尽 T (T _pex ) 细胞但不是终末耗尽 T (T _ex ) 细胞能够对现有免疫疗法作出反应^{3,4,5, 6,7} . CTL 分化背后的基因调控网络以及 T _ex细胞反应是否可以在功能上重振尚不完全清楚。在这里，我们使用体内单细胞 CRISPR 筛选系统地绘制了因果基因调控网络，并发现了 CTL 分化的检查点。首先，T _pex细胞从静止状态退出开始连续分化为中间 T _ex细胞。这一过程受到 IKAROS 和 ETS1 的差异调节，它们的缺陷分别抑制和增加 mTORC1 相关的代谢活动。 IKAROS 缺陷细胞作为代谢静止的 T _pex细胞群积累，在免疫检查点阻断 (ICB) 后分化潜力有限。相反，靶向 ETS1 通过促进 T _pex向中间 T _ex细胞的分化和代谢重连，改善了抗肿瘤免疫和 ICB 功效。从机制上讲，TCF-1 和 BATF 分别是 IKAROS 和 ETS1 的目标。其次，RBPJ-IRF1 轴促进中间 T _ex细胞向末端 T _ex细胞的分化。因此，靶向 RBPJ 增强了 T _ex细胞向增殖状态的功能和表观遗传重编程，并改善了治疗效果和 ICB 功效。 总的来说，我们的研究表明，促进 T _pex细胞退出静止状态并丰富增殖性 T _ex细胞状态是抗肿瘤作用的关键方式，并为整合细胞命运调节组和癌症免疫的可重编程功能决定因素提供了系统框架。