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.
Complement, a part of the immune system, presents a first line defense against dangerous infectious agents. Complement is also a principal cause of tissue damage in human diseases and therapeutic agents designed to inhibit harmful complement activity have begun to emerge in the clinical setting. The goal of this project is to use a new technology to measure for the first time simultaneously the many factors that take part in a complement attack and the variety of cellular responses that may result. This information will be used to devise new diagnostic and therapeutic strategies to address complement-related disease.
