T-bet and Runx3 orchestrate effector CD8 T cell differentiation and lineage fidelity through cooperative and distinct chromatin regulatory mechanisms

Effector CD8⁺T cells must rapidly acquire cytotoxic functions while maintaining lineage fidelity and suppressing alternative fates. T-bet and Runx3 are key transcription factors that drive this process, yet their integrated regulatory logic has remained unclear due to the lack of comprehensive binding and chromatin accessibility data. Here, we systematically dissect the cooperative and distinct roles of T-bet and Runx3 in shaping the transcriptional and epigenetic landscape of antiviral CD8⁺ T cells. Using CUT&RUN, ATAC-seq, RNA-seq and scRNA-seq across single and double knockout models, we reveal that T-bet and Runx3 coordinate effector differentiation through both chromatin remodeling–dependent and –independent mechanisms. T-bet preferentially binds promoters and restrains Th17-like programs, while Runx3 targets distal elements to suppress Tfh-associated genes and sustain cytotoxic identity. Our findings uncover a cis-regulatory logic wherein these factors act combinatorially to balance activation of effector programs with repression of alternative lineages, ensuring functional precision during antiviral responses. This work establishes a comprehensive framework for understanding lineage enforcement by transcription factors in effector T cells.