Local antibody responses in human cardiac allograft vasculopathy

项目摘要 心脏移植物血管病（cardiac allograft vasculopathy，CAV）是心脏移植术后并发症的主要原因之一 移植虽然CAV的病理生理学仍不清楚，但证据表明， 移植组织中的局部免疫反应在该并发症中的关键作用。在人类中，CAV一直是 与冠状动脉内或周围的B细胞和产生抗体的浆细胞浸润有关。这些 对浸润细胞的研究很少。特别地，局部免疫抑制剂的抗原特异性和效应子功能是不稳定的。 产生的抗体目前是未知的。了解这些抗体如何有助于 CAV无疑将促进治疗药物的发展，以治疗这种形式的排斥。在这里， 我们建议使用最先进的IGHV库分析，单细胞RNA-seq结合CITE， seq和配对单细胞IgH+L测序，以获得浆细胞的全面表征 在CAV期间浸润心脏移植物。单个抗体的功能特性和致病性 还将使用基于细胞的体外测定和体内实验测定来评估原位产生的细胞。 在从移植物内浆细胞产生重组单克隆抗体后的移植模型。 目标1。描述人CAV移植物内浆细胞浸润的特征。目标1中的研究将结合联合收割机IGHV 库和单细胞RNA测序分析，以确定克隆组成和转录组谱 CAV期间直接在移植部位发现浆细胞。这些实验还将确定主要的 克隆原位扩增。使用表达克隆平台，我们将产生重组单克隆抗体， 来自存在于移植物浸润物中的大量浆细胞的抗体（mAb），并鉴定它们的反应性。 目的2探讨原位产生的抗体对CAV的作用机制。 我们将在这里集中在原位分泌的抗体形成免疫复合物（IC）和激活Fc的能力 受体（FcR）表达细胞在移植。目标2中的实验将使用scRNA-seq方法结合 用CITE-seq系统地定位移植物中表达FcR的所有免疫和非免疫细胞， 因此能够响应IC。然后，我们将研究是否刺激这些细胞通过 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，我们将寻找证据，一个类似的过程中发生在体内的CAV的背景下。 目标3。确定抗体促进体内血管病变的FcR依赖性机制。在 目的3，我们将使用小鼠主动脉同种异体移植模型来评估抗体分泌的能力。 移植物浸润PC有助于体内移植血管病变。此外，我们将使用一系列 组成型或条件性敲除菌株，以确定哪些FcR与该效应有关，并鉴定 表达这些受体的细胞。我们将特别研究新生儿Fc的参与， 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.