PROJECT SUMMARY. Under homeostatic conditions, there is a balance between pro- and anti-inflammatory responses in the intestinal mucosa to provide a rapid response to pathogen while preventing aberrant tissue damage. In inflammatory bowel disease (IBD), disruption of this balance can lead to harmful inflammation resulting in disease initiation or relapse. gd intraepithelial lymphocytes (IEL) provide the first line of defense against luminal microorganisms and exhibit immunoregulatory capacity through the expression of CD39, which contributes to the hydrolysis of extracellular ATP to adenosine. We and others have reported that the total number of gd IELs, and CD39 expression on these cells, is reduced both in IBD patients and in a mouse model of Crohn’s disease-like ileitis; however, the regulation of CD39 and its influence on gd IEL effector function remains unclear. We now show that the frequency of immunoregulatory CD39+ gd IELs is decreased a month prior to the onset of Crohn’s disease-like ileitis and the microbiota contributes to gd IEL CD39 expression. Further, we recently described a novel transmissible gd IEL hyperproliferative (gdHYP) phenotype, in which the presence of a unique microbiota increased gd IEL number and surveillance behavior. Our preliminary data show that WT mice exhibiting the gdHYP phenotype also exhibit increased CD39 expression that is accompanied by a shift toward mitochondrial metabolism and reduced cytokine production. Therefore, we propose to interrogate the mechanisms by which CD39 expression is regulated in gd IELs, elucidate the extent to which CD39 is coupled to gd IEL bioenergetic capacity, and investigate the intersection between antibiotic treatment and gd IEL functionality in pouchitis. To address these questions, we will use a combination of ex vivo IEL culture, IEL/enteroid co-culture, single cell metabolomics assays, and unique gd T cell-specific mouse strains to dissect the molecular mechanisms involved in the upregulation of CD39 and immunometabolism in response to the microbiota. Moreover, we will leverage access to retrospective and prospective longitudinal biobank samples to both translate findings from animal studies and investigate the potential link between antibiotic therapy and gd IEL CD39 expression in the context of pouch inflammation. These studies will further our understanding of the cellular and molecular mechanisms involved in amplifying gd IEL regulatory function to maintain mucosal homeostasis with the ultimate goal of preventing disease relapse in IBD patients.