Local antibody responses in human cardiac allograft vasculopathy

项目总结 同种异体心脏移植物血管病(CAV)是心脏术后发病率和死亡率的主要原因之一。 移植。虽然CAV的病理生理学仍不明确，但有一系列证据表明 移植物组织的局部免疫反应在这一并发症中的关键作用。在人类中，CAV一直是 与B细胞和产生抗体的浆细胞相关，在冠状动脉内或周围有浸润性改变。这些 对浸润性细胞的研究一直很少。特别是，局部的抗原特异性和效应器功能 产生的抗体目前尚不清楚。了解这些抗体是如何促进肿瘤机制的 CAV无疑将促进治疗这种形式的排斥反应的治疗剂的发展。这里, 我们建议使用最先进的IGHV谱系分析，单细胞RNA-SEQ结合CITE-SEQ 序列号和成对单细胞免疫球蛋白L测序获得浆细胞的全面特征 CAV过程中心脏移植物的渗入。单项抗体的功能特性和致病性 还将使用体外细胞分析和体内实验对原位生产的产品进行评估 移植物内浆细胞产生重组单抗后的移植模型。 目的1.研究人CAV移植物内浆细胞浸润的特征。目标1的研究将结合IGHV 用于确定克隆组成和转录组图谱的谱系和单细胞RNA-序列分析 CAV期间直接在移植物部位发现浆细胞。这些实验还将确定主要的 克隆原位扩增。利用表达克隆平台，我们将产生重组单抗 移植物中存在的大量浆细胞的抗体(MAb)渗入并鉴定其反应性。 目的2鉴定FCR介导的原位产生抗体对CAV的作用机制。 我们在这里将重点放在原位分泌的抗体形成免疫复合体(IC)并激活Fc的能力 移植物中表达受体(FCR)的细胞。目标2中的实验将使用scRNA-seq相结合的方法。 用CITE-SEQ系统地定位移植物中所有表达FCR的免疫细胞和非免疫细胞 因此能够对IC作出响应。然后我们将调查是否通过刺激这些细胞 特异性FCR导致与CAV相关的促炎和促纤维化途径的参与。 最后，我们将寻找在CAV的情况下体内发生类似过程的证据。 目的3.确定体内抗体促进血管病变的FCR依赖机制。在……里面 目的3，我们将使用小鼠同种异体主动脉移植模型来评估分泌抗体的能力 移植物浸润性PC在体内促进移植血管病变。此外，我们还将使用一系列 结构性或条件性基因敲除菌株，以确定哪些FCR与该效应有关并识别 表达这些单个受体的细胞。我们将特别调查新生儿FC的参与情况 受体FcRN由移植物内皮细胞和平滑肌细胞表达。

PROJECT SUMMARY Cardiac allograft vasculopathy (CAV) is one of the leading causes of morbidity and mortality following heart transplantation. While the pathophysiology of CAV is still poorly defined, converging lines of evidence point to a critical role of local immune responses in the graft tissue in this complication. In humans, CAV is consistently associated with B cells and antibody-producing plasma cell infiltrates in or around coronary arteries. These infiltrating cells have been poorly studied. In particular, the antigen specificity and effector functions of locally produced antibodies are currently unknown. Understanding how these antibodies contribute to mechanisms of CAV would undoubtedly facilitate the development of therapeutic agents to treat this form of rejection. Here, we are proposing to use state-of-the-art IGHV repertoire analysis, single-cell-RNA-seq combined with CITE- seq and paired single-cell-IgH+L sequencing to obtain a comprehensive characterization of plasma cells infiltrating cardiac allografts during CAV. The functional properties and pathogenicity of individual antibodies produced in situ will also be evaluated using both in vitro cell-based assays and in vivo experimental transplantation models after generation of recombinant monoclonal antibodies from intragraft plasma cells. Aim 1. To characterize intragraft plasma cell infiltrates in human CAV. Studies in aim 1 will combine IGHV repertoire and single-cellRNA-seq analyses to determine the clonal composition and transcriptome profile of plasma cells found directly at the graft site during CAV. These experiments will also identify predominant clones expanded in situ. Using an expression-cloning platform, we will generate recombinant monoclonal antibodies (mab) from a large number of plasma cells present in the graft infiltrates and identify their reactivity. Aim 2 To identify FcR-mediated mechanisms whereby antibodies produced in situ contribute to CAV. We will focus here on the ability of antibodies secreted in situ to form immune complexes (IC) and activate Fc receptor (FcR)-expressing cells in the graft. Experiments in aim 2 will use a scRNA-seq approach combined with CITE-seq to systematically map all immune and non-immune cells expressing FcR in the graft and therefore capable of responding to IC. We will then investigate whether stimulation of these cells through specific FcR leads to the engagement of pro-inflammatory and pro-fibrotic pathways associated with CAV. Lastly we will look for evidence that a comparable process occurs in vivo in the context of CAV. Aim 3. To determine FcR-dependent mechanisms whereby antibodies promote vasculopathy in vivo. In aim 3, we will use a mouse aortic allotransplantation model to assess the capacity of antibodies secreted by graft-infiltrating PC to contribute to transplant vasculopathy in vivo. Moreover, we will use a series of constitutive or conditional knockout strains to determine which FcR are implicated in the effect and identify cells expressing these individual receptors. We will particularly investigate the involvement of the neonatal Fc receptor FcRn expressed by graft endothelial cells and smooth muscle cells.