Project Summary/Abstract The body?s largest collection of immune cells underlies the single layer epithelium lining the GI tract and monitors the luminal contents to maintain tolerance to dietary antigens in the steady-state and to induce immunity to pathogens during infection. How the immune system?s exposure to luminal antigens is controlled in the steady-state to promote tolerance and during infection to avoid inappropriate responses and promote immunity remains a significant gap in our understanding. We propose that luminal antigen delivery to the immune system is tightly controlled, has regional variation through the GI tract, and serves to supply antigens to the immune system and guide the phenotype of immune responses. Goblet cells (GCs) can take up luminal substances deliver them to antigen presenting cells in the underlying lamina propria (LP) in a manner capable of inducing adaptive immune responses, in a process termed Goblet cell-associated Antigen Passages (GAPs). GAP formation is induced by acetylcholine acting on GCs, is linked with secretion, and is inhibited by GC intrinsic sensing of the gut microbiota. We propose that the pathways controlling GAP formation and the properties of GAPs in various regions of the GI tract allow for a regional antigen delivery supporting tolerance to dietary antigens in the steady-state and limiting immune responses to innocuous luminal antigens and shifting the phenotype of the immune response during enteric infection. These observations give rise to the overarching hypothesis that GC-mediated antigen delivery occurs via bulk endocytosis (BE) in GCs, is a major and closely regulated mechanism promoting tolerance in the steady-state, and inhibition of this pathway shifts immune responses to promote pathogen clearance and immunity during infection. To explore this hypothesis we propose the following specific aims: Aim 1 will define the ultrastructural basis and cellular biology of GAP formation using state-of-the-art imaging approaches, and genetic and pharmacologic manipulation of endocytosis and GAPs. Aim 2 will define the role of GC/GAPs in the induction and maintenance of tolerance to luminal antigens in the steady-state using genetic manipulation of GAP formation. Aim 3 will define if altering GCs/GAPs results in inappropriate inflammatory responses to dietary antigens and/or worsened outcomes during infection using genetic manipulation of GAPs in Salmonella typhimurium infection.