PROJECT SUMMARY/ABSTRACT The intestinal immune system has evolved to tolerate most antigens while mounting an effective immune response to combat infections by pathogens. Epigenetics, defined as heritable changes in the genome without alteration of the DNA sequence, has been shown to play a critical role in regulating intestinal immunity. However, a better understanding of epigenetic modifications and how their pharmacological modulators affect these modifications is still lacking. In addition, the current approach to developing vaccines containing novel adjuvants is slow and needs improvement. Epigenetics offers an exciting opportunity towards the development of novel adjuvants by targeting small molecules as pharmacological modulators to reprogram the epigenetic landscape of innate immune memory, or trained immunity, which could confer enhanced resistance against a broad array of bacteria in humans. In Research Project 3, our overall goal is to investigate the role of epigenetic marks and their pharmacological modulators in intestinal immunity, specifically focusing on the function of epithelial cells, natural killer (NK), and innate T cells, such as Mucosal-associated invariant T (MAIT) cells, T-cell receptor gamma delta (TCR- ) cells, and natural killer T (NKT) cells, in response to two important enteric pathogens, Salmonella enterica serovar Typhi (S. Typhi) and Shigella flexneri 2a (S. flexneri). This study will build on our previous findings that MAIT subsets exhibiting specific cytokine pattern signatures were associated with protection against typhoid fever and that S. Typhi infection regulates changes in chromatin marks that depend on individual cell subsets. Additionally, compared to the placebo group, vaccination with the oral Ty21a typhoid vaccine resulted in increased levels of NK cells upon a second antigen encounter after experimental human challenge with wild-type S. Typhi, which is consistent with the presence of trained immunity driven by epigenetic control. We have assembled a unique set of human specimens and tools to test our hypotheses, including (i) cells obtained from peripheral blood of healthy individuals immunized with oral vaccines, (ii) cells isolated from the duodenum, terminal ileum, and colon, and (iii) their counterparts as in vitro culture models [explants and organoids] from adults and children.