The role of HLA and its coreceptors in endothelial cell activation and leukocyte recruitment in antibody-mediated transplant rejection

摘要 供体特异性HLA I类和II类抗体的产生与慢性淋巴细胞白血病的发展相关。 抗体介导的排斥反应（cAMR）和移植血管病变（TV）。cAMR的两个关键特征是 内皮细胞（EC）功能障碍和免疫细胞，特别是巨噬细胞的浸润。拟议 研究将探索新的范例，即HLA I类与ITGB 4和TLR 4的抗体交联， HLA II类与ITGB 1在同种异体移植物内皮诱导中协调不同的细胞内信号传导程序 促进单核细胞向移植物募集和巨噬细胞极化的功能。我们建议潜入 单核细胞遇到局部EC衍生因子和IgG-FcγR信号，使其分化。在 在移植物中，促血管生成/促纤维化巨噬细胞驱动新生内膜增厚。拮抗EC活化 和/或单核细胞募集可能代表了阻止cAMR和保护移植物免于 电视我们将采用原代人EC培养模型、TV的小鼠心脏同种异体移植模型和人EC培养模型。 心脏移植与电视解剖HLA I和II信号通路和机制的单核细胞 招募和两极分化。目标1：确定复合物形成和功能的结构要求 HLA I类与其共受体ITGB 4和TLR 4在介导内皮细胞活化中的相互作用， 心脏TV中单核细胞募集和巨噬细胞极化。我们将：（1）确定HLA I Ab- 活化的EC将浸润的单核细胞平衡到具有不同表型和功能的巨噬细胞， 体外;（1b）表征单核细胞募集、TV病变形成和浸润巨噬细胞的表型 在MHC I抗体介导的同种异体移植物损伤和TV的发展过程中;（1c）定义结构要求 以及ITGB 4、TLR 4和HLA I之间的分子串扰对单核细胞募集的功能影响， 体外通过Ab激活的EC的巨噬细胞极化;和（1d）表征对MHC I共刺激的需要。 受体TLR 4和ITGB 4在单核细胞募集、巨噬细胞极化和TV损伤形成中的作用 小鼠心脏移植。目标2.确定复杂结构的形成和功能的结构要求 HLA-II及其辅助受体ITGB 1在介导内皮细胞活化、单核细胞 募集和巨噬细胞极化。我们将：（2a）确定HLA Ⅱ Ab激活EC 在体外将浸润的单核细胞与具有不同表型和功能的巨噬细胞平衡;（2b） 表征单核细胞募集、血管病变形成和浸润性巨噬细胞的表型， MHC II cAMR和TV的鼠心脏移植模型;（2c）定义结构要求， ITGB 1和HLA II分子间的相互作用对单核细胞募集和巨噬细胞功能的影响 （2d）表征在体外通过Ab激活的EC的极化; 小鼠心脏移植物中单核细胞募集、巨噬细胞极化和TV损伤形成;以及 (2e)定义了TV的人心脏移植物中移植物内巨噬细胞的表型特征。

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-FcγR 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.