Multi-Omics Atlas-Assisted Discovery of Transcription Factors for Selective T Cell State Programming.

Transcription factors (TFs) regulate the differentiation of T cells into diverse states with distinct functionalities. To precisely program desired T cell states in viral infections and cancers, we generated a comprehensive transcriptional and epigenetic atlas of nine CD8 T cell differentiation states for TF activity prediction. Our analysis catalogued TF activity fingerprints of each state, uncovering new regulatory mechanisms that govern selective cell state differentiation. Leveraging this platform, we focused on two critical T cell states in tumor and virus control: terminally exhausted T cells (TEX ), which are dysfunctional, and tissue-resident memory T cells (T ), which are protective. Despite their functional differences, these states share significant transcriptional and anatomical similarities, making it both challenging and essential to engineer T cells that avoid TEX differentiation while preserving beneficial T characteristics. Through CRISPR screening combined with single-cell RNA sequencing (Perturb-seq), we validated the specific TFs driving the TEX state and confirmed the accuracy of TF specificity predictions. Importantly, we discovered novel TEX -specific TFs such as ZSCAN20, JDP2, and ZFP324. The deletion of these TEX -specific TFs in T cells enhanced tumor control and synergized with immune checkpoint blockade. Additionally, this study identified multi-state TFs like HIC1 and GFI1, which are vital for both TEX and T states. Furthermore, our global TF community analysis and Perturb-seq experiments revealed how TFs differentially regulate key processes in T and TEX cells, uncovering new biological pathways like protein catabolism that are specifically linked to TEX differentiation. In summary, our platform systematically identifies TF programs across diverse T cell states, facilitating the engineering of specific T cell states to improve tumor control and providing insights into the cellular mechanisms underlying their functional disparities.