High-throughput identification and transcriptional analysis of autoreactive T cells in individuals with membranous nephropathy.

膜性肾病患者自身反应性 T 细胞的高通量鉴定和转录分析。

• 批准号: 10725558
• 负责人: Paolo Cravedi
• 金额: $ 26.9万
• 依托单位: ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-16 至 2025-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10725558
• 关键词: Adult; Affect; Aftercare; Alloantigen; Antibodies; Antigen Targeting; Antigens; Autoantibodies; Autoimmune; Autoimmune Diseases; Autologous; B-Lymphocytes; Biological; Biological Markers; Blood Volume; Blood specimen; CD4 Positive T Lymphocytes; Cell Compartmentation; Cells; Characteristics; Circulation; Clinical; Clinical Research; Cloning; Complement; Couples; DNA Sequence; Data; Deposition; Development; Diagnosis; Disease; Disease remission; End stage renal failure; Epitope spreading; Epitopes; Exposure to; Fc Receptor; Functional disorder; Gene Expression Profile; Gene Library; Generations; Genetic Transcription; Human; Human Herpesvirus 4; Immune Tolerance; Immune response; Immunologic Monitoring; In Vitro; Individual; Inflammatory; Injury; Libraries; Link; Maps; Mediating; Membranous Glomerulonephritis; Molecular; Nephrotic Syndrome; Outcome; Pathogenicity; Pathway interactions; Patients; Peptide Receptor; Peptides; Peripheral Blood Mononuclear Cell; Phospholipase; Phospholipase A2; Production; Receptor Cell; Recombinants; Renal glomerular disease; Research; Research Design; Role; Sampling; Severity of illness; Signal Transduction; Specificity; T cell regulation; T cell response; T-Cell Immunologic Specificity; T-Cell Receptor; T-Cell Receptor Genes; T-Lymphocyte; T-cell receptor repertoire; Techniques; Technology; Testing; Therapeutic; Therapeutic Uses; Tumor Antigens; United States National Institutes of Health; Up-Regulation; active method; alpha-beta T-Cell Receptor; autoreactive T cell; cost effective; design; experience; glomerular basement membrane; high reward; high risk; innovation; innovative technologies; kidney biopsy; novel strategies; pathogen; patient screening; patient subsets; podocyte; prevent; prophylactic; receptor; rituximab; screening; single-cell RNA sequencing; therapeutic target; tool

SUMMARY Membranous nephropathy (MN) is a glomerular disease due to the deposition of anti-podocyte antibodies in the subepithelial space of the glomerular basement membrane. This leadis to complement-mediated podocyte injury and, in ~30% of patients, to the development of end stage kidney disease within 10 years from diagnosis. Most of the research in MN has focused on the autoantibody specificities and has led to the identification of phospholipase 2A receptor (PLA2R) as the main target antigen in 70%-80% of the patients. Conversely, little is known on autoreactive T cells, despite the evidence from other antibody-mediated autoimmune diseases clearly showing a critical pathogenic role of autoreactive T cells, efficacy of T cell targeting treatments, and our preliminary data documenting that autoreactive T cells are present in the circulation of MN patients. Aim 1 of the present project will develop a new strategy to capture the T cell receptor (TCR) repertoire of PLA2R-reactive T cells in MN patients. We will apply high-throughput paired T cell receptor alpha and beta chain DNA sequence capture for renewable TCR gene library generation and functional screening from patient T cells. We will physically link TCR gene sequences for cloning into cellular display libraries, enabling repeated in vitro functional analyses of TCRs in ways that are impossible with alternative available techniques. Aim 2 will apply these technologies for the study of the single-cell transcriptional profile of CD4+ T cells. These data will reveal the characteristics of autoreactive TCRs in a previously collected samples from MN patients with remission versus active disease, setting the stage for expanded clinical studies. Overall, this project will collect and analyze immune response data from MN patients with unprecedented molecular scope and scale. Our approach is innovative because it couples carefully designed clinical sample sets with new high-throughput approaches in TCR analysis to comprehensively interrogate the molecular mechanisms of T cell responses in MN patients. The proposed research is significant because it will provide important tools for mechanistic studies aimed at identifying new biomarkers and therapeutic targets for MN patients.

概括 膜性肾病（MN）是一种由于抗足细胞抗体沉积引起的肾小球疾病 位于肾小球基底膜的上皮下空间。这导致补体介导的足细胞 损伤，约 30% 的患者在诊断后 10 年内发展为终末期肾病。 MN 的大部分研究都集中在自身抗体特异性上，并导致了以下疾病的鉴定： 70%-80%的患者以磷脂酶2A受体（PLA2R）为主要靶抗原。反之则少之又少 尽管其他抗体介导的自身免疫性疾病的证据清楚，但在自身反应性 T 细胞上已知 显示了自身反应性 T 细胞的关键致病作用、T 细胞靶向治疗的功效以及我们的研究 初步数据表明，MN 患者的循环中存在自身反应性 T 细胞。 本项目的目标 1 将开发一种新策略来捕获 T 细胞受体 (TCR) 库 MN 患者中的 PLA2R 反应性 T 细胞。我们将应用高通量配对T细胞受体α和β链 DNA 序列捕获，用于从患者 T 细胞生成可再生 TCR 基因库和功能筛选。 我们将物理连接 TCR 基因序列以克隆到细胞展示库中，从而实现体外重复 以替代现有技术无法实现的方式对 TCR 进行功能分析。目标 2 将适用 这些技术用于研究 CD4+ T 细胞的单细胞转录谱。这些数据将揭示 先前从缓解的 MN 患者中收集的样本中自身反应性 TCR 的特征 与活动性疾病的比较，为扩大临床研究奠定了基础。 总体而言，该项目将收集并分析来自 MN 患者的免疫反应数据，这些数据是前所未有的 分子范围和尺度。我们的方法是创新的，因为它结合了精心设计的临床样本 在 TCR 分析中采用新的高通量方法来全面探究分子 MN 患者 T 细胞反应机制。拟议的研究意义重大，因为它将提供 旨在识别 MN 新生物标志物和治疗靶点的机制研究的重要工具 患者。