PROJECT SUMMARY/ABSTRACT FOXP3+ CD4+ cells are expanded within the inflamed mucosa of Crohn’s disease (CD) patients; yet the persistence of intestinal inflammation suggests these CD associated CD4+FOXP3+ cells lack typical regulatory T cell (TREG) suppressive function. The functional and therapeutic implications of senescence in FOXP3+ TREG cells is unknown. The long-term goal of this application is to dissect the epigenetic mechanisms driving the differentiation and function of CD4+FOXP3+ cells in the setting of intestinal inflammation. Consequently, the overall objective of this proposal is to identify the mechanisms leading to senescent FOXP3+ cells in the setting of GI inflammation and test therapeutic opportunities to restore regulatory function. The central hypothesis is that CD associated FOXP3+ cells represent a senescent state and contribute to intestinal inflammation. Furthermore, this new cellular phenotype (senescent FOXP3+ cell, snFOXP3) represents a potential therapeutic target. This hypothesis was formulated through single cell transcriptomic analysis of CD4+FOXP3+ cells from the lamina propria of CD patients, produced by this applicant’s laboratory. The rationale is that defining the mechanisms critical to the development and function of snFOXP3 cells in the setting of inflammation will uncover targets for TREG-directed therapies, including the advanced engineering of human TREGS for adoptive cell therapy trials. Guided by strong preliminary data, this hypothesis will be tested by pursuing three specific aims: 1) Why do FOXP3+ cells become senescent in the absence of BMI1?; 2) Why are FOXP3+ cells senescent in Crohn’s disease?; and 3) Why are snFOXP3 cells proinflammatory? Under the first aim, the applicant will utilize epigenetic assays, which have been established as feasible in the applicant’s hands, to establish BMI1 repression on an ETS1-regulated senescence program in FOXP3+ cells. Antibodies and assay reagents that are already on hand will be used. Under the second aim, the applicant will utilize a biochemistry approach with treatment of cultured TREGS to determine a TNFα-induced disruption of BMI1-mediated repression of senescence. Finally, under the third aim, the applicant will use a novel in vitro co-culture system to establish the proinflammatory capacity of snFOXP3 cells and establish therapeutic targets. The approach is innovative because it departs from the status quo of functional characterization of CD4+FOXP3+ cells utilizing single cell approaches, and a mechanistic approach to define the epigenetic events that lead to a pathogenic snFOXP3 cell. Upon conclusion, the applicant will understand the epigenetic mechanisms leading to the development of snFOXP3 cells in the setting of intestinal inflammation. This contribution is significant as the snFOXP3 is expanded in the Crohn’s lesion, is proinflammatory, and represents an important epigenetic drug target. This project is the first to articulate both signature and ...