PROJECT SUMMARY Graft-versus-host-disease (GVHD) is the most serious complication of allogeneic bone marrow transplantation (allo-BMT). GVHD pathogenesis is mediated by alloreactive T cells present in the graft that recognize alloantigens and damage host tissues. Most GVHD therapies rely on global immunosuppression and are associated with increased risks of infection and decreased graft-versus-tumor effects. New strategies for GVHD prevention that protect host tissues while simultaneously preserving the tumor-killing activity of alloimmune T cells are needed. Our laboratory identified Notch signaling and Delta-like1/4 (Dll1/Dll4) Notch ligands as critical regulators of the pathogenic allo-T cell response, and attractive new therapeutic targets. In both mouse and non-human primate models, Notch inhibition in donor T cells leads to long-term protection from GVHD morbidity and mortality. Short-term in vivo Notch blockade using monoclonal antibodies against specific Notch receptors or ligands blunts the pathogenicity of alloreactive effector T cells without eliciting broad immunosuppression, reducing GVT activity, or triggering toxic side effects of chronic Notch inhibition. While a single injection of anti-Dll1/Dll4 Notch ligand antibodies at the time of transplant was sufficient for long- term GVHD control, Dll1/Dll4 blockade administered >48 hours after transplant provided no protection against GVHD. Thus, an early pulse of Notch signaling during the initial stages of T cell activation is critical for establishing a pathogenic T cell state. However, we do not currently understand how short-term Notch inhibition exerts long-lasting GVHD protection. Alloreactive T cells rely on two central functions to mediate disease: trafficking to peripheral target organs and producing proinflammatory cytokines. Our preliminary data show that Notch inhibition preserves initial T cell IL-2 production and expansion in secondary lymphoid organs while impairing secretion of multiple inflammatory cytokines and homing to the gastrointestinal tract. Therefore, I hypothesize that Notch signals delivered to allo-T cells during early stages of T cell priming and activation are critical for trafficking to the gut and initiating a sustained inflammatory cytokine response. I speculate that these two key mechanisms explain why short-term Notch inhibition prevents many of the severe, pathological consequences of GVHD. To explore this hypothesis, I will use MHC-mismatched models of GVHD to investigate how Notch inhibition influences allo-T cell gut tropism. Moreover, I will use a combination of RNA sequencing, Assay for Transposase-Accessible Chromatin sequencing (ATAC-seq), and Cleave Under Targets and Release using Nuclease (CUT&RUN) chromatin profiling to provide a genome-wide view of the transcriptional and epigenetic effects of Notch signaling during early stages of allo-T cell priming/activation. Altogether, this proposal will elucidate the cellular and molecular mechanis...