Organ transplantation is a life-saving therapy for patients with end stage organ failure. However, long-term graft survival is limited by the complications of acute and chronic antibody-mediated rejection. The production of donor specific HLA class I and class II antibodies is associated with development of acute and chronic antibody-mediated rejection and transplant associated vasculopathy. Our central hypothesis is that DSA against class I and class II HLA molecules elicit the key features of transplant vasculopathy by acting on the endothelium of the allograft via two mechanisms: a) directly activating survival and proliferative signal transduction in the endothelium; and b) promoting recruitment and polarization of macrophages to elicit chronic inflammation and fibrosis. We propose that these two mechanisms act synergistically during chronic antibody- mediated rejection to provoke proliferation of the graft vasculature, culminating in transplant vasculopathy We will conduct the proposed experiments using an in vitro endothelial cell culture model to dissect in detail the class I and class II signaling pathways and mechanisms of monocyte recruitment. We will then apply what we learned in two clinically relevant models of antibody-mediated rejection, including a mouse vascularized heterotopic cardiac allograft model and human endomyocardial transplant biopsies from recipients producing donor specific antibodies to translate our mechanistic findings into treatment strategies to suppress antibody- mediated rejection and macrophage recruitment.
Aim 1 will determine the HLA class II signal transduction pathways leading to endothelial cell proliferation and monocyte recruitment. We will characterize role of mTOR and ERK in HLA class II-mediated cell proliferation. We will establish the class II intracellular signaling cascades that promote cytoskeletal remodeling and monocyte recruitment. We will investigate the proximal molecular events regulating HLA class II dependent signaling in endothelium.
Aim 2 will characterize the Fc receptor-mediated functions of HLA I and II antibody on monocyte recruitment in vitro and in vivo. We will characterize the effect of the HLA antibody isotype and recipient monocyte Fc?RIIa polymorphisms on HLA-mediated endothelial activation and recruitment of monocytes. We will determine whether different donor specific antibody subclasses differentially promote macrophage trafficking to the graft, and whether modulation of anti-donor MHC antibody glycosylation by EndoS and IdeS treatments can suppress AMR in vivo.
Aim 3 will characterize HLA class I and class II antibody-mediated macrophage polarization. We will determine whether Fc-independent endothelial activation and/or Fc?R-dependent monocyte activation drive monocyte polarization into M1, M2 and/or DC subsets in response to HLA class I and class II signaling in vitro. We will assess whether markers of polarized M1, M2 macrophage and dendritic cell macrophage subsets are differentially distributed in murine cardiac allografts undergoing AMR. Understanding the mechanisms of antibody- mediated rejection will reveal novel therapies for its treatment and prevention.
Characterization of the HLA class I and class II signaling pathways in the proposed in-vitro and in-vivo models will permit us to establish the role of HLA antibodies in acute and chronic allograft rejection. These studies will identify key signaling molecules and pathways involved in endothelial cell activation and leukocyte recruitment and will provide insight into mechanisms underlying antibody-mediated rejection which will permit the identification of new treatment strategies.
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