Single-cell CRISPR screens in vivo map T cell fate regulomes in cancer

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^3-7. 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.