Local antibody responses in human cardiac allograft vasculopathy

人心脏同种异体移植血管病中的局部抗体反应

• 批准号: 10457560
• 负责人: Emmanuel Zorn
• 金额: $ 54.26万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-08 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10457560
• 关键词: Allogenic; Animal Model; Antibodies; Antibody Response; Antigen-Antibody Complex; Antigens; Aorta; Apoptotic; Autoimmune Diseases; B-Lymphocytes; Binding; Biological Assay; Biopsy; Blood; Blood Vessels; Blood specimen; Cardiac; Cells; Cellular Indexing of Transcriptomes and Epitopes by Sequencing; Clone Cells; Complication; Coronary artery; Elements; Endothelial Cells; Fc Receptor; Functional disorder; Generations; Heart Transplantation; Heavy-Chain Immunoglobulins; Human; Humoral Immunities; Hyperplasia; Immune; Immune response; In Situ; In Vitro; Individual; Inflammation; Inflammatory; Injections; Location; MS4A1 gene; Maps; Mediating; Modeling; Monoclonal Antibodies; Morbidity - disease rate; Mus; Pathogenicity; Pathway interactions; Patients; Plasma Cells; Play; Process; Property; Recombinants; Research; Role; Series; Site; Smooth Muscle Myocytes; Somatic Mutation; Specificity; Specimen; Time; Tissue Grafts; Tissues; Transplantation; Tumor-infiltrating immune cells; Vascular Diseases; allotransplant; base; conditional knockout; deep sequencing; experimental study; expression cloning; heart allograft; in vivo; molecular phenotype; mortality; neonatal Fc receptor; programs; receptor; response; single-cell RNA sequencing; therapeutic development; transcriptome; transplant model; Allogenic; Animal Model; Antibodies; Antibody Response; Antigen-Antibody Complex; Antigens; Aorta; Apoptotic; Autoimmune Diseases; B-Lymphocytes; Binding; Biological Assay; Biopsy; Blood; Blood Vessels; Blood specimen; Cardiac; Cells; Cellular Indexing of Transcriptomes and Epitopes by Sequencing; Clone Cells; Complication; Coronary artery; Elements; Endothelial Cells; Fc Receptor; Functional disorder; Generations; Heart Transplantation; Heavy-Chain Immunoglobulins; Human; Humoral Immunities; Hyperplasia; Immune; Immune response; In Situ; In Vitro; Individual; Inflammation; Inflammatory; Injections; Location; MS4A1 gene; Maps; Mediating; Modeling; Monoclonal Antibodies; Morbidity - disease rate; Mus; Pathogenicity; Pathway interactions; Patients; Plasma Cells; Play; Process; Property; Recombinants; Research; Role; Series; Site; Smooth Muscle Myocytes; Somatic Mutation; Specificity; Specimen; Time; Tissue Grafts; Tissues; Transplantation; Tumor-infiltrating immune cells; Vascular Diseases; allotransplant; base; conditional knockout; deep sequencing; experimental study; expression cloning; heart allograft; in vivo; molecular phenotype; mortality; neonatal Fc receptor; programs; receptor; response; single-cell RNA sequencing; therapeutic development; transcriptome; transplant model

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.

项目摘要 心脏同种异体血管病（CAV）是心脏发病和死亡的主要原因之一 移植。虽然CAV的病理生理学仍然很差，但融合的证据线表明 局部免疫反应在移植组织中的关键作用在这种并发症中。在人类中，骑士一直是 与B细胞和产生抗体的浆细胞浸润有关，冠状动脉动脉或周围。这些 浸润细胞的研究很少。特别是，局部的抗原特异性和效应子功能 生产的抗体目前未知。了解这些抗体如何促进 CAV无疑会促进治疗剂的发展以治疗这种排斥。这里， 我们建议使用最先进的IGHV曲目分析，单细胞-RNA-Seq与Cite-seq结合 SEQ和配对的单细胞+L测序以获得血浆细胞的全面表征 在CAV期间浸润心脏同种异体移植。单个抗体的功能特性和致病性 也将使用基于体外细胞的测定和体内实验进行原位生产 移植模型，一年后产生的重组单克隆抗体，该抗体是从内部血浆细胞中的。 目的1。表征人类CAV中的内部浆细胞浸润。 AIM 1的研究将结合IGHV 曲目和单细胞序列分析，以确定克隆组成和转录组轮廓 在CAV期间，血浆细胞直接发现在移植物位置。这些实验还将确定主要的 克隆原位扩展。使用表达式克隆平台，我们将生成重组单克隆 来自移植物中的大量浆细胞的抗体（MAB）并鉴定其反应性。 AIM 2以鉴定FCR介导的机制，从而使原位产生的抗体有助于CAV。 我们将重点关注原位分泌形成免疫复合物（IC）并激活FC的抗体的能力 受体（FCR）表达移植物中的细胞。 AIM 2中的实验将使用SCRNA-SEQ方法组合 用Cite-seq系统地绘制所有在移植物中表达FCR的免疫和非免疫细胞 因此能够响应IC。然后，我们将研究这些细胞是否通过 特定的FCR导致与CAV相关的促炎和促纤维途径的参与。 最后，我们将寻找证据表明，在CAV的背景下，体内发生了可比的过程。 目的3。确定抗FCR依赖性机制，抗体在体内促进血管病。在 AIM 3，我们将使用小鼠主动脉同异种移植模型来评估分泌的抗体的能力 移植浸入PC有助于体内移植血管病。此外，我们将使用一系列 构成或条件敲除菌株，以确定哪个FCR与效果有关 表达这些单独受体的细胞。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。