Smad4 and Eomes converge on a shared transcriptional program associated with inflammatory CD8 T-cell differentiation.

The ability of CD8 T cells to mediate protective immunity while limiting immunopathology depends on critical transcription factors such as Smad4 and Eomes that regulate differentiation of activated CTLs. Disruption of transcriptional programs mediated by Eomes and Smad4 in activated CTLs has been implicated in viral infections, chronic inflammation and cancer. Smad4 and Eomes have been shown to be critical for differentiation of memory cells, particularly TRM cells. To understand the gene regulation programs mediated by Smad4 and Eomes, we have done an integrated RNAseq and ChIPseq analysis using CD8 T cells that are isolated from Smad4KO (S4KO), EomesKO, and Smad3/Smad4-DKO (S34-DKO) mice. We identified overlapping and distinct gene expression programs mediated by Smad4 and Eomes that are associated with shaping CD8 T cell identity and functional bias. They act as critical regulators of genes involved in T cell differentiation, metabolic programming and epigenetic control in CD8 T cells. Smad4 and Eomes regulate the genes involved in tissue residency and OXPHOS-linked mitochondrial bioenergetics. Finally, we showed that canonical TGF-beta signaling is active in the absence of Smad4 or Eomes and abrogated only when both Smad3 and Smad4 are deleted. Surprisingly, CD8 T cells lacking both Smad3 and Smad4 are more enriched for TRM signature genes compared to Smad4 or Eomes-deficient CD8 T cells.