Project Summary Developing infants are colonized with trillions of bacteria within the intestine. Failure to establish tolerance within a narrow early life window leads to increased risk of immune mediated diseases in later life, including chronic inflammation and cancer. Central to the generation of intestinal tolerance is the peripheral conversion of naïve T cells into regulatory T (pTreg) cells that suppress immune responses to commensal microbes. pTreg cells arise in the intestine at the time of weaning; however, the cell types that instruct pTreg cell fate are not known, limiting our ability to modulate pTreg cells for therapeutic benefit. We recently discovered a fascinating population of antigen presenting cells (APC), enriched within the intestinal lymph nodes during early life. These cells, dubbed Thetis cells (TCs), express the autoimmune regulator Aire, known for its critical role in immune tolerance. Here we set forth the tantalizing possibility that TCs represent a dedicated lineage of tolerogenic APCs. We aim to uncover their role in instructing pTreg cell fate in neonates, and susceptibility to inflammatory disease in later life. Our proposed genetic models allow lineage-specific manipulation of TCs, including deletion of Aire. Through these studies we aim to develop a deep mechanistic understanding of TC function. In our efforts to define the biology of TCs, we seek to understand the ontogeny and development of these cells. Using state-of-the-art lineage tracing approaches and genetic models that allow us to perturb the intestinal micro-environment in a tissue- and developmental-stage-specific manner, we will dissect the cross-talk between stromal and immune cells that drives tissue-specific early life immune development. The overarching goal of this proposal is to establish a roadmap for intestinal immune tolerance, delineating the critical antigen presenting cells that direct tolerance to commensal antigens, and the environmental cues that drive their differentiation. These studies will i) provide an unprecedented view of early life immune development, ii) establish a new framework for peripheral immune tolerance, and iii) reveal potential therapeutic targets for inflammatory and immune mediated diseases.