Systematic perturbation screening and multimodal single-cell profiling reveal that RUNX functions as a master collaborator in CD8+ T cells, with RUNX2:KLF2 cooperative binding at composite DNA motifs specifically controlling exhaustion programs through direct protein-protein interactions that can be synthetically enhanced by molecular tethering to amplify therapeutic reprogramming of T cell states.

Transcription factors (TFs) collaborate to regulate gene expression programs that define cell fate. In CD8+ T cells, this coordinated regulation underlies exhaustion, a dysfunctional state that constrains immunity in chronic infection and cancer. Here, we screen for cell state-specific TFs by performing pooled overexpression screens of 3,548 TF and TF isoforms in primary T cells across multiple CD8+ T cell states. We identify 82 regulators that collaborate with exhaustion-specific programs and profile their effects using perturb-SHARE-seq, connecting perturbations to changes in chromatin accessibility and gene expression across 702,314 single cells. We identify 38 reproducible regulatory programs and construct a map of 12,616 TF-program connections that shape CD8+ T cell states, nominating KLF2 as predictive of positive response to CAR-T therapy. Using seq2PRINT, a deep learning framework that predicts functional TF interactions, we identify RUNX as a “master collaborator”, a TF that broadly collaborates with other factors, and uncover a RUNX2:KLF2 interaction that specifies exhaustion-associated programs. Mutation of the RUNX2:KLF2 protein interface attenuates KLF2-mediated repression of exhaustion, while synthetic tethering of RUNX2 to KLF2 leads to an amplification of the phenotype. More broadly, we identify the collaborative action of RUNX as a driver in CD8+ T cell states, and show that tethering TFs enables the rational engineering of cell state identity for cell and gene therapies.