Notch Signaling in Alloimmunity

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ABSTRACT Allogeneic T cell responses against foreign host antigens mediate graft-versus-host disease, the most serious complication of allogeneic hematopoietic cell transplantation (allo-HCT). During the course of this proposal we defined a critical role for Notch signaling in the regulation of pathogenic alloreactive T cells that mediate graft-versus-host disease (GVHD) in multiple mouse models of allo-HCT. Notch inhibition in donor T cells led to long-term protection from GVHD morbidity and mortality. Using monoclonal antibodies, we identified a critical role for Notch1/2 receptors in T cells and Delta-like1/4 (Dll1/4) Notch ligands in the host, with dominant effects of Notch1 and Dll4. Dll1/4 blockade with a short course of antibodies emerged as the most promising strategy to target Notch signaling while avoiding systemic side effects of pan-Notch inhibition. We recently uncovered several remarkable features of Notch regulation in T cell alloimmunity that warrant further investigation. First, we identified specialized radioresistant stromal cells lineage-traced with a Ccl19-Cre transgene as the critical source of Notch ligands in secondary lymphoid organs at the onset of GVHD. These findings uncover a central role for fibroblastic stromal cell subsets in GVHD. Second, short-term inhibition of Delta-like Notch ligands within days after allo-HCT was essential to confer long- term protection from GVHD in multiple mouse models. Within this early time window, Notch induced unique transcriptional effects during the activation of alloantigen-specific T cells that impacted selected aspects of their differentiation. Third, we studied Notch ligand inhibition in a non-human primate allo-HCT model that mimics human transplantation. A single dose of anti-DLL4 antibodies had marked single agent activity to prevent GVHD, showing highly conserved effects of Notch signaling from mice to non-human primates. In both models, we observed an increased ratio of regulatory to conventional T cells in the gut and striking protection from intestinal GVHD, the most dangerous component of acute GVHD. We hypothesize that alloantigen-specific T cells engage in early interactions with specialized subsets of fibroblastic stromal cells expressing Delta-like Notch ligands, inducing a Notch-driven pathogenic and gut-homing program in T cells that promotes GVHD. To explore this hypothesis, we will identify individual subsets of fibroblastic stromal cells that present Dll1 and/or Dll4 Notch ligands to donor-derived T cells early after allo-HCT, map the anatomical sites that support Notch activation in alloreactive T cells, and define the impact of immune- mediate injury on the subsequent integrity of stromal networks in secondary lymphoid organs. In addition, we will identify mechanisms that blunt the accumulation of Notch-deficient T cells in the gut, thus preventing intestinal GVHD, and investigate the early transcriptional effects of Notch signaling in alloreactive T cells. These studies will bring novel insights into the regulation of alloimmunity and might lead to the development of new approaches to limit damaging consequences of T cell reactivity after allogeneic transplantation.