Enhancer-driven gene regulatory networks reveal transcription factors governing T cell adaptation and differentiation in the tumor microenvironment

Tumor-infiltrating lymphocytes (TILs) with a tissue-resident memory CD8⁺ T cell (Trm) phenotype are associated with improved patient outcomes in solid malignancies. To define programs governing the formation of Trm-like TIL, we performed paired single-cell RNA sequencing and single-cell ATAC sequencing of T cell receptor (TCR)-matched CD8⁺ T cells in models of infection and cancer. Enhancer-driven regulons assembled from multiomic profiling data revealed epigenetic and transcriptional programs regulating the formation of Trm-like TIL in relation to canonical exhausted and memory T cell states. The transcriptional regulator KLF2 repressed the formation of CD69⁺CD103⁺ Trm-like TIL and limited anti-tumor activity. Conversely, sustained expression of the transcription factor BATF enhanced formation of CD69⁺CD103⁺ TIL, contingent upon downregulation of KLF2. Transforming growth factor β (TGF-β) signaling and CD103 expression were necessary for Trm-like TIL formation, but BATF overexpression was sufficient to drive formation of CD69⁺CD103⁺ TIL in TGFBR2-silenced cells. These findings reveal mechanisms of Trm-like TIL differentiation and provide a framework for considering tissue residency in the context of CD8⁺ T cell heterogeneity in the tumor microenvironment.