Abstract
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 (TEXterm), which are dysfunctional, and tissue-resident memory T cells (TRM), 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 TEXterm differentiation while preserving beneficial TRM characteristics. Through in vivo CRISPR screening combined with single-cell RNA sequencing (Perturb-seq), we validated the specific TFs driving the TEXterm state and confirmed the accuracy of TF specificity predictions. Importantly, we discovered novel TEXterm-specific TFs such as ZSCAN20, JDP2, and ZFP324. The deletion of these TEXterm-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 TEXterm and TRM states. Furthermore, our global TF community analysis and Perturb-seq experiments revealed how TFs differentially regulate key processes in TRM and TEXterm cells, uncovering new biological pathways like protein catabolism that are specifically linked to TEXterm 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.
Competing Interest Statement
The authors have declared no competing interest.
Footnotes
Perturb-seq data were included in Figure 3 and Figure 4. New TF community analysis and in vivo validation were included in Figure 4. Two more authors were updated and author affiliations were undated.
Data availability
ATAC-sequencing data from this paper will be deposited in the GEO database. Taiji v2.0 output of this study (TF activity atlas, TF-TF interaction maps, and TF activity on genome browser view) will be available at our CD8+ T cell TF atlas portal (https://github.com/apotato123/CD8-TF-Atlas-Portal). All other raw data are available from the corresponding author upon request.