Project Summary Over the millennia humans have coevolved with a large number of microorganisms, collectively known as the microbiota, which provide many key signals that aid in the development and proper functioning of the immune system. It is vital to human health that the immune system remains tolerant to the microbiota maintaining a symbiotic relationship. To limit pathological immune responses against the microbiota, immune tolerance to the microbiota must be established early in life. A breakdown in tolerance to commensal microbes can result in chronic inflammatory disorders such as inflammatory bowel disease, asthma and dermatitis. Homeostasis with the microbiota is achieved by generation of antigen specific regulatory T cells that develop in both the thymus and periphery. Exposure to the microbiota, especially during the neonatal period is thought to be critical for this Treg induction. It has been found in humans as well as in animal models of disease, that compositional changes in the microbiota, also known as dysbiosis, correlate with increased susceptibility to subsequent induction of inflammatory disease. Notably, changes early in life are thought to have the most significant impact on disease susceptibility. In animal models, colonization with select intestinal microbes sensitizes to disease. It remains unclear how tolerance against the microbiota is achieved, and further, if the ability to induce tolerance to intestinal microbes is limited to a specific early life developmental window. Using a mouse model whose T cell receptor recognizes intestinal microbes, we find intestinal colonization with this microbe leads to selection of T cells specific for that organism. We hypothesize that intestinal antigen presenting cells (APCs) that encounter these organisms traffic to the thymus where they induce T cell selection. In Aim 1 of the proposed work, we will use in vivo models to define how introduction of specific microbes leads to alterations of T cell populations. We will also define whether there are differential impacts on T cell development depending on the developmental window of when microbial colonization occurs. We will further define if timing of colonization with select microbes promotes or limits development of inflammatory disease. In Aim 2 we will define how the microbe or microbial product travels from the intestine to the thymus. We will determine the intestinal cell population required for trafficking of this microbe as well as define transcriptional profiles that allow for T cell selection. This will identify pathways that we will be able to manipulate to limit or rescue from the development of autoimmunity.