PROJECT SUMMARY. Maintenance of an intact intestinal barrier is critical to prevent microbial activation of mucosal immunity. In inflammatory bowel disease, this barrier is compromised, thus exposing the mucosal immune system to the contents of the intestinal lumen. A substantial increase in the shedding or extrusion of epithelial cells into the lumen has been shown to be a predictor of relapse in Crohn’s disease (CD) patients. gd intraepithelial lymphocytes (IEL) migrate extensively within the epithelial compartment to serve as a first line of defense against invasive microorganisms and facilitate apoptotic cell shedding. Although gd IELs are protective in mouse models of colitis, the involvement of these sentinel lymphocytes in the pathogenesis of chronic ileitis is less clear. Published reports provide conflicting evidence regarding the contribution of gd IELs in the pathogenesis of chronic ileitis; however, we now show that inducible depletion of gd T cells prior to disease initiation increases lethality. Further, detailed immunoprofiling of the IEL compartment in mice that develop spontaneous CD-like ileitis indicates that the loss of gd IELs coincides with the histological onset of ileal inflammation, suggesting that loss of gd IELs may be an initiating event. In support of this, we observe a reduction in the frequency of CD39+ gd Tregs, while less activated, peripheral Vg1+ T cells infiltrate the IEL compartment prior to disease development. Therefore, we propose to interrogate the contribution of gd IELs in the pathogenesis of chronic ileitis and elucidate the cellular and molecular mechanisms involved in the dysregulation of the gd IEL compartment during the development of CD-like ileitis. To address these questions, we will take advantage of unique gd T-cell-specific mouse models and intravital microscopy to define how gd IEL motility and effector function are regulated in the events leading up to the onset of chronic ileitis. By combining temporal and cell-specific gene targeting, cutting-edge live imaging techniques, and novel models to analyze gd IEL function ex vivo, we expect to clearly elucidate the contribution of gd IELs to the development of ileal disease and further define the functional dysregulation within gd IEL subpopulations in the context of chronic inflammation. Developing a better understanding of the cellular and molecular mechanisms involved in gd IEL immunosurveillance and immunoregulation may elucidate additional targets for future therapies designed to reinforce the epithelial barrier and prevent disease relapse.