Project Summary Inflammatory bowel disease (IBD) affects an estimated 1.4 million Americans. A major component of IBD pathogenesis is the breakdown of immune tolerance toward commensal microbes normally present in the gastrointestinal tract. CD4+ regulatory T(reg) cells are essential for maintaining normal mucosal tolerance to commensal antigens and it has been suggested that disrupting the balance of T cells from an immunosuppressive Treg cell phenotype toward an inflammatory T helper (Th)17 cell phenotype can lead to colitis. Interestingly, despite the fact that the lineage defining transcription factors for Treg and Th17 cells induce opposing phenotypes and inhibit each other's function in vitro, Foxp3+RORt+ cells that co-express these factors can be readily identified in vivo, particularly in mucosal tissues. Thus, the mechanisms that mediate tolerogenic and effector T cell development during mucosal homeostasis remain unclear. In this proposal, we will investigate the developmental relationships between Treg, Th17, and Foxp3+RORt+ T cell subsets. A critical barrier in the understanding of T cell plasticity stems from the use of polyclonal populations of cells, which cannot readily distinguish between true phenotypic conversion of individual cells and the selective outgrowth of minor populations. In order to circumvent this, we will analyze T cell development at the level of individual clones through the use of T cell receptor (TCR) sequence analysis in Aim 1. Using this approach, we will be able to determine if all cells in a given population are capable of adopting an alternative phenotype or if only a small number of TCRs are capable of driving interconversion. We will also determine the ability of individual TCR specificities to drive T cell differentiation. In Aim 2, we will focus on the developmental hierarchy of Treg, Th17, and Foxp3+RORt+ cells, as well as their specific antigen presentation requirements. These experiments will demonstrate which CD4+ populations, if any, are capable of generating other effector populations and identify the antigen presenting cells that are involved in this phenotypic transition. Together, the findings generated by these aims will expand the understanding of effector T cell development and plasticity in the gut, elucidate the impact of this plasticity in the early stages of IBD pathogenesis, and facilitate the development of Treg cell based therapeutics.