Project Summary: Allogeneic hematopoietic cell transplantation (Allo-HCT) can cure hematological malignancies, but this treatment causes graft-versus-host disease (GVHD), an immune response of donor cells against the recipient. Anti-inflammatory corticosteroid medications can often control GVHD, but in some cases, GVHD is resistant to this treatment, especially when it affects the intestinal tract. The goal of this project is to understand the mechanisms that cause steroid-resistant (SR) gut GVHD and to develop effective approaches to prevent or treat SR-Gut-GVHD. Results with a newly established murine model have demonstrated that SR-Gut- GVHD is induced by prolonged administration of corticosteroids. Corticosteroid treatment induces expansion of IL-22-producing Th/Tc22-like cells, IL-22-dependent abnormalities in gut microorganisms, and reduced numbers of anti-inflammatory CX3CR1hi mononuclear phagocytes (MNP) that regulate T cell expansion and control bacterial translocation. Information from our preliminary studies and other investigators suggests that corticosteroid treatment inhibits the polyamine-hypusine pathway in T and MNP cells and regulates the fidelity of T cell lineage differentiation and the expansion of pro- and anti-inflammatory MNP cells. Our studies also suggest that steroid treatment increases Ceacam-1 expression by intestinal epithelial cells, thereby increasing bacterial transcytosis and unfavorably altering the balance between proinflammatory CX3CR1lo MNP and anti- inflammatory CX3CR1hi MNP cells. These changes trigger a feedforward pathogenic loop consisting of expanding IL-22-producing Th/Tc22-like cells, IL-22-dependent dysbiosis, and increased numbers of proinflammatory CX3CR1lo MNP with decreased numbers of regulatory CX3CR1hi MNP, leading to full-blown SR-Gut-GVHD. To test this hypothesis, this application proposes 3 aims. Aim 1 will determine whether corticosteroid inhibition of the polyamine-hypusine pathway in T cells leads to expansion of Th/Tc22-like cells with lineage infidelity. Aim 2 will determine whether corticosteroid inhibition of polyamine-hypusine pathway in CX3CR1+ MNP cells augment expansion of proinflammatory CX3CR1lo MNP with reduced numbers of anti- inflammatory CX3CR1hi MNP in response to challenge by dysbiosis. Aim 3 will determine whether observations in murine models reflect the pathogenesis of SR-Gut-GVHD in humans and whether reversal of abnormalities in the polyamine-hypusine pathway and whether blocking bacterial interaction with Ceacam-1 on intestinal epithelial cells by anti-Ceacam-1 mAb prevents and reverses SR-Gut-GVHD. Relevance: By elucidating in-depth mechanistic understanding of the pathogenesis leading to SR-Gut-GVHD in a well characterized murine model and by assessing the relevance of the experimental results in colon tissue from patients with SR-Gut-GVHD, results of this project could identify potentially effective approaches for translational testing in humans.