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）：通过质谱细胞术分析补体激活和调节。概述补体（C）是50-60种可溶性和膜结合蛋白的集合，其构成血管内空间的第一道防线。补体标记用于免疫清除或裂解的感染因子，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细胞效应子反应。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以追溯到不适当的补体激活（体液自身免疫），不充分的补体调节（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）。治疗剂已开始出现在临床环境中，以抑制全身补体活性。 三种补体激活途径与靶表面上的C3转化酶的组装会聚。C3转化酶产生调理素，其促进靶清除，释放过敏毒素，其激活和募集炎性细胞，启动膜攻击复合物（MAC，C5 b-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形成损害膜完整性并通常导致病原体裂解的孔。通过抑制转化酶形成、损害转化酶稳定性并阻碍MAC组装和功能的表面调节剂保护宿主细胞免受补体活性的影响。有核细胞通过参与进一步决定细胞存活、细胞分化或细胞死亡的细胞内途径来响应补体攻击。 质谱流式细胞术（MC）是一种新兴技术，提供了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的轮廓的补体激活剂，调节剂，和细胞内的反应作为一个细胞经历补体攻击。我们选择了两种具有翻译意义的细胞类型，视网膜色素上皮（RPE）细胞和T细胞，用于假设驱动分析，并提出以下具体目的：1.构建并验证补体特异性重同位素标记的报告抗体; 2.通过质谱细胞术分析人RPE细胞系对亚溶解性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.通过质谱细胞术分析经历C活化的CD 4 + T细胞的反应。 我们预计，拟议的工作将产生一套新的，独特的和重要的工具，以前所未有的细节描绘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，并可能推动基于C的治疗干预的范式转变。