Atlas-Guided Discovery of Transcription Factors for T Cell Programming.

CD8+ T cells differentiate into diverse states that shape immune outcomes in cancer and chronic infection. To systematically define the transcription factors (TFs) driving these states, we built a comprehensive atlas integrating transcriptional and epigenetic data across nine CD8+ T cell states and inferred TF activity profiles. Our analysis catalogued TF activity fingerprints, uncovering regulatory mechanisms governing selective cell state differentiation. Leveraging this platform, we focused on two transcriptionally similar but functionally opposing states critical in tumor and viral contexts: terminally exhausted T cells (TEXterm), which are dysfunctional, and tissue-resident memory T cells (TRM), which are protective. Global TF community analysis revealed distinct biological pathways and TF-driven networks underlying protective versus dysfunctional states. Through in vivo CRISPR screening integrated with single-cell RNA sequencing (in vivo Perturb-seq), we delineated that TFs selectively govern TEXterm. We identified HIC1 and GFI1 as shared regulators of TEXterm and TRM differentiation and KLF6 as a unique regulator of TRM. Importantly, we discovered novel TEXterm single-state TFs, including ZSCAN20 and JDP2 with no prior known function in T cells. Targeted deletion of these TFs enhanced tumor control and synergized with immune checkpoint blockade. Consistently, their depletion in human T cells reduces the expression of inhibitory receptors and improves effector function. By decoupling exhaustion-selective from protective TRM programs, our platform enables more precise engineering of T cell states, advancing rational design of effective immunotherapies.