PROJECT SUMMARY Coordination of Innate and Adaptive Immunity in Intestinal Barrier Defense. Intestinal barrier defense requires the actions of both innate and adaptive immune cells on the mucosal epithelium. Tissue-resident and mobile innate and adaptive immune cells each contribute to barrier defense in the intestines, but how these cell populations are coordinated under conditions of pathogen threat are not fully understood. Interleukin-22 (IL-22) is a cytokine of the IL-10 family produced by type 3 immune cells, such as group 3 innate lymphoid cells (ILC3s) and cells of the Th17 pathway that acts on epithelial cells of barrier tissues to prevent invasion of extracellular pathogens. How IL-22 acts to coordinate intestinal barrier function remains undefined. Like many immune cytokines that participate in host defense, IL-22 is upregulated in chronic immune-mediated diseases, and it appears to play a protective role in inflammatory bowel disease (IBD), presumably by restraining epithelial damage caused by dysregulated T cell responses to constituents of the intestinal microbiome. However, pro- proliferative actions of IL-22 have also been implicated in malignant transformation of colonic epithelial cells that leads to colorectal cancer (CRC). We and others have shown that during infectious colitis modeled by the enteropathogen, C. rodentium, there are two phases of IL-22 production that can be distinguished: an early phase dominated by IL-22+ innate immune cells, which is followed by a late phase dominated by IL-22+ T cells. While both innate and adaptive immune cells produce IL-22 during infection, the respective contributions to barrier protection are unknown, as are details of the mechanisms by which IL-22 acts. In preliminary studies that have employed novel IL-22 reporter/conditional knockout (cKO) mice with which to track and/or delete specific subsets of IL-22-producing immune cells, we have found that the locations and functions of IL-22–producing cells are distinct during C. rodentium infection. Innate immune cells, dominated by ILC3s, are primarily located in and restricted to isolated lymphoid follicles, and their release of IL-22 activated by IL-23 acts long-range to activate surface colonic epithelial cells at initial sites of bacterial colonization. Remarkably, however, ILC3s fail to protect the intestinal crypts, which are invaded by bacteria in mice with IL-22 deficiency targeted to T cells. Thus, IL-22–producing T cells are indispensable for protection of the intestinal crypts via their activation of crypt- lining epithelium. Moreover, we have discovered new heterogeneity within colonic absorptive enterocytes and find that IL-22-producing T cells differentially activate these populations for increased shedding and production of neutrophil-recruiting chemokines. In this proposal we will define mechanisms by which innate and adaptive immune cells are specialized for distinct IL-22–dependent actions on different subsets of colonic e...