Dissecting the intestinal niche for regulatory T cells

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1. Objective: Inflammatory bowel diseases (IBD), including Crohns Disease and Ulcerative Colitis, arise in genetically-susceptible hosts when intestinal epithelial defects and heightened responsiveness towards commensal organisms lead to excessive activation of CD4+ T cells and tissue damage. IBD is also associated with loss of regulatory pathways including defects in regulatory T cells (Tregs). Tregs, a subset of Foxp3- positive CD4+ T cells, are central to intestinal tolerance; they prevent the development of IBD disease and suppress inflammation during infection. In this application, we seek to understand the mechanisms that maintain intestinal Tregs during health and inflammation. Regulatory T cells include two developmentally distinct subsets: thymus-derived (tTreg or natural (nTreg) and peripherally-derived (pTreg) that differentiate from conventional naïve CD4+ T cells. Human and mouse intestine contain cells of both lineages and both are necessary to maintain tolerance. Foxp3-deficient IPEX patients, lacking natural Tregs, develop autoimmune enteritis as infants. Separately, loss of the genomic region that controls peripheral differentiation of pTregs also leads to intestinal inflammation. Thus, both murine and human studies demonstrate a requirement for both populations for the maintenance of intestinal tolerance. There are no data defining distinct mechanistic or anatomic requirements for the two populations. Treg localization and phenotypes were examined in mice lacking peripheral TCR-MHCII interactions and peripheral conversion of Tregs (pTregs). Thymically-generated nTregs entered the small intestine lamina propria (siLP) to fill the compartment in the absence of MHCII. Imaging of intact tissue shows that Tregs in the siLP have two distinct anatomic locations: individually localized within the siLP villus or clustered within organized structures resembling isolated lymphoid follicles (ILFs). Our data suggest that a subset of Tregs preferentially localize to ILFs; this association is altered during infection. Additionally, nTregs and pTregs occupy an overlapping niche despite having different requirements for costimulatory signals. Thus, we present the hypotheses that 1. thymic-derived and peripheral Tregs occupy distinct physiologic niches in the small intestine, and 2 ILFs provide a niche for Tregs that is malleable during inflammation. 2. Research Design: In our first Aim, we will utilize murine models and healthy human tissue to determine if ILFs are required for the maintenance of siLP Tregs, define the costimulatory signals present in the ILF, and then ask if the ILF specifically attracts and maintains thymic Tregs in contrast to pTregs. Multiple studies in mice and men find that both infectious and autoimmune intestinal inflammation is associated with expansion of ILFs and altered Treg dynamics. Yet, there are no data on how these changes affect the positioning and maintenance of Tregs and recovery from inflammation. In our second Aim, we will examine murine models of inflammation and specimens from IBD patients to define how inflammation alters ILF-Treg interactions. 3. Methodology: Mice with altered MHCII expression will be compared with wildtype mice. Small intestine from healthy Our data suggest thymic Tregs preferentially localize to ILFs.and IBD patients will also be examined. The immune system will be altered utilizing blocking antibodies, antibiotics, genetic manipulations, and infections. Methods will include molecular and cellular analyses of cell populations and responses by flow cytometry, expression profiling, and imaging techniques. 4. Findings: This proposal not been funded. 5. Clinical Relationships: The prevalence of inflammatory bowel disease has been increasing amongst veterans. The current study is directed towards defining pathways that can be manipulated therapeutically to increase the number of localized regulatory T cells to improve IBD outcomes.