Project Summary/Abstract Acute gastroenteritis, attributable to enteric intracellular pathogens, is a significant cause of morbidity and mortality, particularly in children and the elderly. By itself, norovirus is accountable for nearly 20% of worldwide cases and upwards of 200,000 deaths annually. Vaccines may mediate protection against intracellular pathogens by establishing memory CD8 T cell populations that are permanently situated within the intestinal tissue parenchyma. This subset of memory T cells, known as tissue-resident memory T cells (TRM), could mediate rapid clearance of infected cells before the pathogen disseminates and manifests clinically. Indeed, emerging evidence supports a role for CD8 TRM in local pathogen protection in animal models of intestinal infection. However, it is unknown whether TRM are a transient population. For rational vaccine design to successfully exploit CD8 TRM in a clinical context, the longevity of CD8 TRM must be addressed. Extensive preliminary analysis now indicates that the intestinal TRM population undergoes exponential decay, in contrast to bloodborne memory T cells which remain stable. Specific Aim 1 will test this question through a novel PCR-based quantification of intestinal TRM and potentially substantiate preliminary findings. Additionally, we will evaluate the anatomic origin of long-lived intestinal memory T cells. Specific Aim 2 will address the extent to which preexisting CD8 TRM cells are displaced by new CD8 TRM cells. Collectively, these studies will address the long-standing questions of longevity, durability, and functionality of CD8 TRM cells. The protective capacity of long-lived intestinal TRM will be tested against a physiologically relevant pathogen. Results will have a direct impact on vaccine strategies to protect against intracellular enteric pathogens. Tissue-resident memory T cells have largely been understudied with preference given to bloodborne lymphocyte populations. There is now a growing consensus that the interface between cell- mediated immunity and the tissue parenchyma warrants renewed focus. In the long-term, the studies outlined here will have broader implications as to how CD8 TRM cells mediate protective immunity, autoimmune pathogenesis, hypersensitivity reactions, and tumor control, not only in the intestinal tract, but throughout the body.