The body's largest collection of immune cells underlies the single layer epithelium lining the gastrointestinal (GI) tract and monitors the luminal contents, which includes trillions of microbes, their products, and substances from the diet. The basal tone of the healthy gut immune system is tolerogenic, despite being exposed to trillions of microbes and their products. While this strong tolerogenic capacity is beneficial to the host to avoid inflammatory responses to innocuous dietary and commensal antigens in the healthy state, the inability to dampen this tolerogenic capacity could be detrimental in the setting of enteric infection and inappropriately dampening this tolerogenic capacity could underlie the pathogenesis of intestinal inflammatory diseases. We propose that the gut has a capacity to turn off tolerogenic responses and generate inflammatory responses. While great progress has been made in elucidating the role of specific immune cell subsets, cytokines, and other factors promoting tolerance or immunity, how the gut immune system switches from tolerogenic responses in the steady-state to protective immunity when needed remains a significant gap in our understanding. Completion of the studies outlined in this proposal will fill this void in our understanding by identifying how inhibiting a major pathway delivering luminal substances activates cellular and humoral immune responses at the mucosa. In prior cycles of this award we have identified how goblet cell associated antigen passages (GAPs) are formed, the stimulus inducing GAPs in the steady-state, acetylcholine (ACh), the stimuli and receptors regulating GAP formation, including the luminal microbiota, cytokines, and epidermal growth factor receptor (EGFR) ligands, and the properties of GAPs in various regions of the GI tract. Further we have identified roles for GAPs, when physiologically present, in supporting tolerance to luminal substances including dietary and commensal microbial antigens. Moreover, we have now assembled genetic and pharmacologic models for the manipulation of GAPs and are poised to dissect the role of GAP inhibition in promoting protective/inflammatory immunity in the absence of enteric infection or overt changes in the gut microbiota. Based upon our prior studies and preliminary observations we hypothesize that when GAPs form in the steady state, they act to imprint the immune system to promote tolerance and when small intestine (SI) GAPs are inhibited they participate in a cascade of events promoting protective immunity. To explore this hypothesis we propose to (Aim 1) define the LP-APCs phenotypes, the origins of Th17 and TFH cells that expand, and the durability of the response that occurs when SI GAPs are inhibited, (Aim 2) define the drivers and specificities of the B cell responses arising when SI GAPs are inhibited and (Aim 3) determine if SI GAP inhibition improves outcomes and is required for appropriate responses during enteric infection.