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

Tumor infiltrating lymphocytes (TIL) with a CD8+ T cell tissue-resident memory (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 ß (TGF-ß) 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. Overall design: Oligo-hashtag barcoded CD45+CD3+CD8+ TIL were single cell sorted from n=3 wild-type female 10wk old C57BL/6J mice bearing orthotopically implanted KPC-4662 pancreatic cancer at day 15 post tumor implant. Cells were sequenced using 10X Genomics 5' GEX+VDJ using a NextSeq2000 P3 flow cell and were processed using standard CellRanger multi and Seurat pipelines.