PROJECT SUMMARY Colorectal cancer (CRC) is profoundly affected by the intestinal immune system and the intestinal microbiome, which can exert both pro- and anti-cancer effects that operate alongside cell-intrinsic mutational events. Immune cells produce reactive molecules and cytokines which contribute to epithelial mutagenesis and proliferation, and immune cells infiltrate developing tumors and influence response to chemotherapy and anti-tumor immunotherapy. Therapeutically modulating the immune system holds potential for transforming CRC outcomes, but such interventions must be precisely targeted to specific signaling pathways, immune cell types and, ideally, delivered locally. One promising source for such precision immunomodulatory targets are metabolites produced by the intestinal microbiome, which have recently been shown to exert powerful effects on specific immune cell types in the intestine. Exploiting the therapeutic potential of microbial metabolites requires: 1) basic knowledge of the diet-microbe-metabolite-immune signaling network, 2) safe and effective means for localized metabolite delivery, and 3) validated targets for defined therapeutic contexts. In this proposal, we will address these three key gaps, with a unifying focus on a key population of anti-inflammatory T cells in the intestine: RORgt+ regulatory T cells (RORgt+ Tregs). In Aim 1, we will dissect the mechanism of a previously unknown microbome-metabolite- immune pathway by which the microbiome and dietary tryptophan regulate intestinal RORgt+ Treg populations. In Aim 2, we will engineer a probiotic strain of E coli to serve as a general platform for localized delivery of therapeutic metabolites; our initial goal will be to boost RORgt+ Tregs by overproducing the short-chain fatty acid butyrate. In Aim3, we will evaluate the efficacy of elevating RORgt+ Tregs (via dietary butyrate) on key outcomes for sporadic vs colitis-associated CRC in vivo. This project will deepen our understanding of metabolic immunoregulation, develop novel probiotic strains for therapeutic metabolite delivery, and evaluate the role of an important anti-inflammatory cell type in CRC.