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.