ABSTRACT The production of donor specific HLA class I and class II antibodies is associated with development of chronic antibody-mediated rejection (cAMR) and transplant vasculopathy (TV). Two key features of cAMR are endothelial cell (EC) dysfunction and infiltration of immune cells, particularly macrophages. The proposed research will explore the novel paradigm that antibody crosslinking of HLA class I with ITGB4 and TLR4 and HLA class II with ITGB1 orchestrate divergent intracellular signaling programs in allograft endothelium inducing functions that promote monocyte recruitment to the graft and macrophage polarization. We propose infiltrating monocytes encounter local EC-derived factors and IgG-Fc?R signals that prime them for differentiation. In the graft, the proangiogenic/profibrotic macrophages drive neointimal thickening. Antagonizing EC activation and/or monocyte recruitment may represent viable therapeutic targets to arrest cAMR and protect grafts from TV. We will employ a primary human EC culture model, a mouse cardiac allograft model of TV and human cardiac explants with TV to dissect the HLA I and II signaling pathways and mechanisms of monocyte recruitment and polarization. Aim 1: Define the structural requirements for complex formation and functional crosstalk between HLA Class I and its co-receptors ITGB4 and TLR4 in mediating endothelial cell activation, monocyte recruitment and macrophage polarization in cardiac TV. We will: (1a) determine how HLA I Ab- activated EC poise infiltrating monocytes to polarize to macrophages with distinct phenotypes and functions in vitro; (1b) characterize monocyte recruitment, TV lesion formation and phenotypes of infiltrating macrophages during MHC I antibody-mediated allograft injury and development of TV; (1c) define the structural requirements and functional effects of molecular crosstalk between ITGB4, TLR4 and HLA I on monocyte recruitment and macrophage polarization by Ab-activated EC in vitro; and (1d) characterize the requirement for MHC I co- receptors TLR4 and ITGB4 in monocyte recruitment, macrophage polarization and TV lesion formation in murine cardiac transplants. Aim 2. Define the structural requirements for complex formation and functional crosstalk between HLA class II and its co-receptor ITGB1 in mediating endothelial cell activation, monocyte recruitment and macrophage polarization in cardiac TV. We will: (2a) determine how HLA II Ab-activated EC poise infiltrating monocytes to polarize to macrophages with divergent phenotypes and functions in vitro; (2b) characterize monocyte recruitment, vascular lesion formation and phenotypes of infiltrating macrophages in a murine cardiac transplant model of MHC II cAMR and TV; (2c) define the structural requirements and functional effects of molecular crosstalk between ITGB1 and HLA II on monocyte recruitment and macrophage polarization by Ab-activated EC in vitro; (2d) characterize the requirement for MHC II coreceptor ITGB1 in monocyte recruitment, macrophage polarization and TV lesion formation in murine cardiac transplants; and (2e) define the phenotype profile of intragraft macrophages in human cardiac transplants with TV.