Analysis of Complement Activation and Regulation by Mass Cytometry

 描述(申请人提供)：补体激活和调节的质控分析摘要补体(C)是由50-60种可溶性和膜结合的蛋白质组成的集合，构成了血管内空间的第一道防线。补体标记感染因子以清除或溶解免疫，促进局部炎症反应，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：补体相关的疾病和损伤状态可追溯到补体激活不当(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体和/或细胞碎片(狼疮)。治疗药物已经开始出现在临床环境中，以抑制全身补体活性。这三条补体激活途径与C3转换酶在靶表面的组装汇聚在一起。C3转换酶产生调理素，促进靶标清除，释放激活和招募炎症细胞的过敏性毒素，启动膜攻击复合体(MAC，C5b-9)的组装，重要的是，参与放大所有这些活动的正反馈机制。MAC形成一个孔，影响膜的完整性，通常导致病原体溶解。宿主细胞受到表面调节剂的保护，使其不受补体活性的影响，表面调节剂抑制转换酶的形成，损害转换酶的稳定性，并阻碍MAC的组装和功能。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。质量细胞术(MC)是一项新兴的技术，它提供了在单细胞基础上测量&GT；40蛋白质的独特能力。我们建议使用MC来描述当细胞经历补体攻击时补体激活物、调节器和细胞内的反应。我们选择了两种非常不同的翻译意义细胞类型，视网膜色素上皮(RPE)细胞和T细胞，并提出了以下具体目标：1.构建和验证一组补体特异性重同位素标记的报告抗体；2.通过质量细胞仪分析人RPE细胞系对亚溶血性C攻击的反应，并将这些发现与其他类型的上皮细胞的反应进行比较；3.通过质量细胞仪分析经历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的、独特的和关键的工具，以前所未有的详细描述补体在膜和细胞内水平的激活和调节，并确定细胞内反应在补体依赖性疾病中所起的影响。此外，我们建议产生/建立的试剂/方法将在随后的研究中促进新一代基于C的诊断的发展，并可能推动基于C的治疗干预的范式转变。

 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.