PROJECT 2 SUMMARY/ABSTRACT Acute graft versus host disease (GVHD) is the major cause of non-relapse mortality (NRM) following allogeneic bone marrow transplantation (BMT). The GI tract is the GVHD target organ that is most difficult to treat. Studies from this P01 grant have identified and validated Regenerative 3 alpha (REG3ΓA) as a plasma biomarker specific for the GVHD of the GI tract. REG3ΓA (and its mouse homologue Reg3γγ) are antimicrobial peptides produced by intestinal epithelial cells that protect the barrier function and integrity of the intestinal mucosa. Our long term objective is to develop novel strategies to reduce the morbidity and mortality of GVHD. Our central hypothesis is that REG3ΓA functions as a master regulator of GI GVHD. We have formulated our hypothesis based on our own published data that show that the destruction of Paneth cells by GVHD correlates with NRM. Unpublished data show that GVHD suppresses production of Reg3γγ in mice, and that mice deficient in Reg3γγ suffer more severe GVHD than normal mice. Further preliminary data show that administration of IL-22 which induces Reg3γγ, restores endogenous Reg3γγ and reduces GI GVHD. But in the absence of Reg3γγ, IL-22 loses its ability to protect mice from GVHD. Thus REG3ΓA/Reg3γγ appears to be a master (negative) regulator of GVHD. Once the role of Reg3γγ in the pathophysiology of GI GVHD is better understood, these mechanistic insights will lay the foundation for innovative approaches to prevent and treat GVHD through modulation of the epimmunome. We will test our central hypothesis and achieve the objective of this project by pursing the following two specific aims: 1. To define the mechanisms by which Reg3γ reduces GVHD. We will examine effects of Reg3γ on both intestinal epithelium and hematopoietic cells that drive T cell responses. In collaboration with Project 1 we will examine the role of butyrate in modulating Reg3γ in its prevention of GVHD. We will also evaluate the effects of Reg3γ on GVL effects. 2. To determine if Enterococcus drives GVHD. Using the gnotobiotic expertise in revised Core 2, have demonstrated that germ free (GF) mice do not develop severe GVHD after allogeneic BMT. Given the predominance of enterococcus in both severe GVHD in both humans and experimental models is both necessary and sufficient to cause GVHD using customized human microbial laboratories to customize GF mice. We will use advanced metagonomic sequencing techniques to correlate shifts in the microbiota with evolving GVHD status. 3. To determine the effects of the antibiotic treatment and therapeutic probiotics on GVHD severity. Early systemic antibiotic treatment is associated with more severe GVHD in humans and mice. Experiments in Core 2 have now shown that systemic antibiotics dramatically decrease Reg3γ in mice. This SA we will test the hypothesis that the increased severity of GVHD after antibiotic treatment is mediated by a loss of Reg3γ. The second subaim will test probio...