Allogeneic hematopoietic cell transplantation (allo-HCT) is a therapy with curative intent for a variety of malignant and non-malignant diseases. One of the major complications after allo-HCT is graft-versus-host disease (GVHD), especially of the gastrointestinal tract. Our group was one of the first to explore the role of the intestinal microbiome in allo-HCT in patients and preclinical models by combining next-generation sequencing with immunological, microbial and metabolomic approaches. We found relationships between the intestinal microbiome and GVHD, relapse, infections, engraftment, and immune reconstitution. The clinical and preclinical studies we performed in the previous funding period resulted in 19 manuscripts (including NEJM, Nature, Science, and Nature Med). We observed in large multi-center studies that higher diversity of intestinal microbiota was associated with better overall survival, reconstitution of CD4, mucosal-associated invariant and V2 T cells. Expansion of particular Enterococci was not only associated with GVHD in patients but exacerbated disease in mice. We showed that loss of Clostridia was associated with the onset of acute GVHD, whereas lower serum levels of butyrate were associated with an increased risk for chronic GVHD. In addition to their role in digestive physiology, bile acids (BAs) exert wide-ranging biological effects, including antimicrobial activity, intestinal epithelial homeostasis, and modulation of immunity. Primary BAs (PBA) are synthesized in the liver and transformed by the intestinal microbiota into a diverse pool of secondary bile acids (SBAs) with unique biochemical properties. BAs can bind to Farnesoid X Receptor (FXR), a nuclear receptor that is a critical regulator of BA homeostasis, inflammation, and GI barrier function. Our preliminary preclinical studies demonstrate that allo-HCT recipients with GVHD have: a) a decrease in unconjugated BAs and SBAs in cecal contents and plasma, b) a reduced abundance of bile salt hydrolase genes (critical for SBA transformation) in the intestinal microbiome, and c) decreased BA synthesis in the liver. In addition, we found that a) most BAs can counteract FXR activation by the PBA chenodeoxycholic acid and b) donor T cells deficient of FXR induce less GVHD. Our preliminary clinical studies corroborate the experimental results in that fecal α-diversity is associated with increased abundance of SBAs, which are in turn correlated to less acute GVHD and better overall survival. Based on studies demonstrating that BAs can inhibit Th17 polarization and enhance Treg generation and our preliminary data in mouse models and allo-HCT patients, we hypothesize that BA metabolism is an important modulator of alloreactive donor T cells and GVHD in allo-HCT recipients. All our research is performed through perpetual dialogue between investigation in mice and humans. Therefore, we propose to study the role of bile acids in GVHD after allo-HCT in preclinical models (Aim 1)...