Analysis of Complement Activation and Regulation by Mass Cytometry

通过质谱流式细胞术分析补体激活和调节

• 批准号: 9235239
• 负责人: DENNIS EMIL HOURCADE
• 金额: $ 20.51万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-03-15 至 2019-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9235239
• 关键词: Address; Adjuvant; Age related macular degeneration; Alpha Cell; Anaphylatoxins; Antibodies; Antibody Formation; Antigen-Antibody Complex; Autoimmunity; B-Cell Activation; Binding Proteins; CD4 Positive T Lymphocytes; Cell Death; Cell Differentiation process; Cell Line; Cell Survival; Cells; Clinical; Collection; Complement; Complement 3 Convertase; Complement Activation; Complement Membrane Attack Complex; Computer software; Cytolysis; Cytometry; Dangerousness; Development; Diagnostic; Disease; Disease Progression; Effector Cell; Emerging Technologies; Epithelial Cells; Failure; Feedback; Flow Cytometry; Generations; Genomics; Goals; Heavy Metals; Hemolytic-Uremic Syndrome; Human; Immune system; Individual; Infectious Agent; Inflammatory; Inflammatory Response; Injury; Instruction; Investigation; Isotope Labeling; Isotopes; Lupus; Mass Spectrum Analysis; Measures; Mediating; Membrane; Methods; Nature; Onset of illness; Opsonin; Pathway interactions; Phenotype; Play; Proteins; Reagent; Recruitment Activity; Regulation; Reporter; Resistance; Role; Signal Transduction; Site; Structure of retinal pigment epithelium; Surface; T-Lymphocyte; Therapeutic Agents; Therapeutic Intervention; Time; Tissues; Work; antibody conjugate; base; cell type; design; human disease; immune clearance; inflammatory milieu; membrane assembly; new technology; novel; novel diagnostics; novel therapeutic intervention; pathogen; public health relevance; response; tool

 DESCRIPTION (provided by applicant): Analysis of complement activation and regulation by mass cytometry SUMMARY Complement (C) is a collection of 50-60 soluble and membrane-bound proteins that constitutes the first line of defense of the intravascular space. Complement marks infectious agents for immune clearance or lysis, promotes the local inflammatory response, and facilitates B cell activation and antibody production (i.e. nature's adjuvant) as wel as T cell effector responses. Complement is also a principal cause of tissue damage: Complement-related disease and injury states can be traced to inappropriate complement activation (humoral autoimmunity), inadequate complement regulation (PNH, atypical hemolytic uremic syndrome, age-related macular degeneration), or a failure to effectively clear immune-complexes and/or cell debris (lupus). Therapeutic agents have begun to emerge in the clinical setting to inhibit systemic complement activity. The three complement activation pathways converge with the assembly of C3 convertases on a target surface. C3 convertase generates opsonins that promote target clearance, release anaphylatoxins that activate and recruit inflammatory cells, initiate the assembly of the membrane attack complex (MAC, C5b-9), and, importantly, engage a positive feedback mechanism that amplifies all of these activities. The MAC forms a pore that compromises membrane integrity and commonly leads to pathogen lysis. Host cells are protected from complement activity by surface regulators that inhibit convertase formation, compromise convertase stability and obstruct MAC assembly and function. Nucleated cells respond to complement attack by engaging intracellular pathways that further determine cell survival, cell differentiation or cell death. Mass cytometry (MC) is an emerging technology that provides the unique ability to measure >40 proteins on a single cell basis. We propose to use MC to profile the complement activators, regulators, and intracellular responses as a cell undergoes complement attack. We have selected two vastly different cell types of translational significance for hypothesis driven analyses, retinal pigment epithelial (RPE) cells and T cells, and propose the following Specific Aims: 1. To construct and validate a panel of complement-specific heavy isotope-labeled reporter antibodies; 2. To profile by mass cytometry the responses of a human RPE cell line to sublytic C attack, and compare to those findings to the responses of other epithelial cell types; 3. To profile by mass cytometry the responses of CD4+ T cells undergoing C activation. We anticipate the proposed work will generate a set of novel, unique and critical tools to delineate in unprecedented detail the activation and regulation of complement at the membrane and intracellular levels and to define the impact that intracellular responses play on comlement-dependent disease. Further, the reagents/methods we propose to generate/establish will facilitate the development of a new generation of C-based diagnostics in subsequent investigations and likely drive a paradigm shift in C-based therapeutic intervention.

 描述（由申请人提供）：通过质谱流式细胞术分析补体激活和调节概要补体（C）是50-60种可溶性膜结合蛋白的集合，构成血管内空间的第一道防线。补体标记感染因子以进行免疫清除或裂解，促进局部炎症反应，并促进 B 细胞活化和抗体产生（即天然佐剂）以及 T 细胞效应反应。补体也是组织损伤的主要原因：补体相关疾病和损伤状态可归因于补体激活不当（体液自身免疫）、补体调节不足（PNH、非典型溶血性尿毒症综合征、年龄相关性黄斑变性）或未能有效清除免疫复合物和/或细胞碎片（狼疮）。临床上已经开始出现抑制全身补体活性的治疗药物。 三种补体激活途径与 C3 转化酶在靶标表面的组装汇聚在一起。 C3 转化酶产生调理素，促进靶标清除，释放过敏毒素，激活和募集炎症细胞，启动膜攻击复合物（MAC、C5b-9）的组装，并且重要的是，参与放大所有这些活动的正反馈机制。 MAC 形成的孔会损害膜的完整性，通常会导致病原体裂解。表面调节剂可抑制转化酶的形成、损害转化酶的稳定性并阻碍 MAC 的组装和功能，从而保护宿主细胞免受补体活性的影响。有核细胞通过参与细胞内途径来响应补体攻击，进一步决定细胞存活、细胞分化或细胞死亡。 质谱流式细胞术 (MC) 是一项新兴技术，具有在单细胞基础上测量 > 40 种蛋白质的独特能力。我们建议使用 MC 来分析细胞受到补体攻击时的补体激活剂、调节剂和细胞内反应。我们选择了两种对于假设驱动分析具有翻译意义的截然不同的细胞类型：视网膜色素上皮 (RPE) 细胞和 T 细胞，并提出以下具体目标： 1. 构建并验证一组补体特异性重同位素标记的报告抗体； 2. 通过质谱流式分析分析人 RPE 细胞系对亚裂解 C 攻击的反应，并将这些结果与其他上皮细胞类型的反应进行比较； 3. 通过质谱流式分析来分析经历 C 激活的 CD4+ T 细胞的反应。 我们预计拟议的工作将产生一套新颖、独特和关键的工具，以前所未有的细节描述补体在膜和细胞内水平的激活和调节，并确定细胞内反应对补体依赖性疾病的影响。此外，我们建议生成/建立的试剂/方法将有助于在后续研究中开发新一代基于 C 的诊断方法，并可能推动基于 C 的治疗干预的范式转变。