Project Summary Early life exposure to microbes is necessary for normal immune development, and clinical studies link alterations in early life bacterial composition to subsequent development of inflammatory disorders such as inflammatory bowel disease (IBD) and atopy such as asthma and dermatitis. In intestinal disease, microbiota- specific T cells can drive tissue pathology. Therefore, it is crucial to understand the homeostatic mechanisms that establish immune tolerance to commensal microbes during infancy in order to address inflammation in adulthood. As antigen presenting cells (APCs) initiate the T-cell response, we want to elucidate how they respond to microbes during early life. This work will have broad implications for development of therapies that address the root of inflammatory disease including reshaping of pathogenic T cell responses. The proposed project aims to describe how microbial colonization during early life drives the phenotype and function of mononuclear phagocytes (MNP), which are a lineage of APCs known to coordinate host-microbe responses. This lineage consists of monocytes that circulate in the blood, and the dendritic cells (DC) and macrophages that they differentiate into upon entering tissue. While the factors that dictate this fate decision remain undefined, these diverging cell fates lead to functionally different outcomes. DCs migrate to lymph nodes to initiate T-cell responses while macrophages remain resident in the tissue to clear microbes and support barrier function. Data from our lab shows that in early life but not adulthood, intestinal DCs expand and bring microbial antigens to the thymus where they induce proliferation of microbe-specific T-cells. Interestingly in the early life mesenteric lymph node, we see similar DC expansion and microbial trafficking but do not see the corresponding T-cell response. This data led to our hypothesis that during early life, mononuclear phagocyte differentiation is skewed towards a DC fate. We further hypothesize that early life lymphoid organs provide signals to instruct microbial antigen processing and presentation resulting in life span and organ restriction of expansion of microbiota recognizing T cells. To address our hypothesis, in Aim 1 we will utilize single-cell RNA sequencing and adoptive monocyte transfer to understand cell intrinsic and tissue regulation of monocyte fate over the lifespan. In Aim 2, by using functional readouts, we will interrogate changes in DC function over the lifespan. Together, our work will reveal how MNPs mediate T-cell responses to microbes during development, further advancing our ability to address inflammation during adulthood.