Project Summary Infectious diseases pose a significant public health burden, accounting for nearly one-fifth of deaths globally per annum. Most infections are initiated from a restricted mucosal tissue, such as the intestine. To fight intestinal infections, gut-resident immune cells are superior to circulating ones. There is a surge of recent interest in tissue-resident memory T (TRM) cells that have been shown to be a critical adaptive immune component of mucosal immunity. These cells have been flagged as an ideal cell population to be generated in T cell-based vaccines. However, the factors involved in the differentiation and maintenance of gut TRM cells remain poorly defined. Distinct from other T cell populations in the periphery, gut TRM cells harbor a unique transcription network. We propose that simultaneously suppressing the transcription factor Eomes and inducing the transcription factor Runx3 are required for the proper differentiation and maintenance of gut TRM cells following acute infections. Based on our previous findings, we hypothesize that TGF-β mediates TRM cell differentiation and maintenance in the gut through controlling Eomes- and Runx3-dependent transcription programs. Eomes and Runx3 are relatively independent nodules downstream of TGF-β signaling in gut TRM cells. Using different genetic models (conditional and inducible knockout mice that specifically delete TGF-β receptor on T cells, and TGF-β receptor/Eomes double conditional knockout mice in T cells), three major questions will be addressed: 1) When do gut TRM precursor or gut TRM cells receive TGF-β signaling? Whether continuous TGF-β signaling is required for the maintenance of gut TRM cells? What is the transcription program maintained by TGF-β signaling in gut TRM cells? 2) What is the function of TGF-β-dependent down-regulation of transcription factor Eomes in the differentiation and maintenance of gut TRM cells? What is the TGF-β-dependent transcription program that is prevented by Eomes in gut TRM cells? 3) What are the functions of TGF-β dependent induction of transcription factor Runx3? Whether Eomes and Runx3 together control the majority of TGF-β-dependent transcription program in gut TRM cells? The results from these studies will elucidate the transcription program underlying TGF-β-controlled differentiation and maintenance of gut TRM cells. These studies will substantially further our understanding of gut TRM cell biology and lay the basis for future translational works.