Graft versus host disease (GVHD) is the major complication associated with allogeneic hematopoietic stem cell transplantation (HSCT). During the acute phase of this disease, a restricted set of organs is affected of which the gastrointestinal (GI) tract is the most clinically significant. Proinflammatory cytokines play a critical role in the pathophysiology of intestinal GVHD, in part, by activating donor innate and T cell populations which subsequently induce tissue damage. We previously demonstrated that CD4+ T cell production of granulocyte- macrophage colony stimulating factor (GM-CSF) plays a critical inflammatory role in the pathophysiology of GI GVHD. More recently, we have identified two CD4+ GM-CSF+ T cell populations within the GVHD colon microenvironment that possess distinct transcriptional profiles, employ non-overlapping gene regulatory units, emerge with divergent temporal kinetics, have differential responsiveness to IL-7, and are distinguishable by the presence or absence of IFN-γ co-expression, suggesting that they may have different pathogenic roles in mediating GVHD in the GI tract. In addition, our preliminary studies have uncovered a previously unappreciated interleukin 34 (IL-34)-mediated regulatory pathway that inhibits the emergence of CD4+ GM-CSF+ T cells in the GI tract, indicating the existence of a potential therapeutically targetable cytokine to reduce GM-CSF- mediated inflammation in this tissue site. The overall goal of this proposal is therefore to delineate mechanistic pathways by which CD4+ GM-CSF+ T cells promote pathological damage in the GI tract and define how these populations are regulated by IL-34. Our overall hypothesis is that there exist discrete CD4+ GM- CSF+ T cell populations that have distinct functional roles in mediating pathological damage in the GI tract and that are regulated by the host production of IL-34. Studies in Specific Aim 1 will define the pathogenicity of CD4+ GM-CSF+ T cell populations during GVHD. To address this question, we will employ a recently created GM-CSF fate reporter mouse model that faithfully identifies viable GM-CSF- expressing CD4+ T cells in GVHD target organs. Experiments in Specific Aim 2 will identify key transcription factors and define the role of IL-7 in the development of CD4+ GM-CSF+ T cells. We will employ genetically modified murine models that will ascertain the extent to which specific transcription factors are required for the development and pathogenicity of CD4+ GM-CSF+ T cells and determine the dependency of these cells on IL-7 receptor signaling. Studies in Specific Aim 3 will characterize how IL-34 regulates GM-CSF-responsive innate immune cell populations in the GI tract during GVHD and affects the emergence of CD4+ GM-CSF+ T cells. Experiments will identify mechanistic pathways by which IL-34 regulates GVHD and determine whether these pathways can be therapeutically targeted to reduce disease severity. The overall objective of this proposal is to...