Complement pathways function to identify and remove pathogens and infected cells. There are three complement pathways: the classical, lectin and alternative pathway (AP). While all pathways are activated following pathogen stimuli, the AP is constitutively active and tightly controlled by activators (e.g., Factor B, Factor D) and negative regulators (e.g., Factor H). Complement activity can be measured by well-established methods that are often used in a diagnostic setting to determine the CH50 (50% complement hemolytic activity) or AP50, specifically to measure AP activity. The protocol here has adapted the traditional AP50 method designed to measure AP activity in human sera, to measure the positive or negative AP regulatory activity within a given test sample. The assay relies on the ability of AP components in human serum to lyse rabbit erythrocytes under in vitro conditions specific for the AP with subsequent release of hemoglobin that is quantitated by measurement of optical density. Our method has added test substances, such as cell culture media with defined changes in individual complement components and determined the ability to either promote or inhibit AP activity in vitro. Thus, this protocol reflects the overall functional ability of a sample to effect AP activity and can be used in the research laboratory to determine AP regulatory activity in a complex biological sample, or to test the ability of drugs or novel biomolecules to regulate AP activity.

Regulators of complement activation (RCA) inhibit complement-induced immune responses on healthy host tissues. We present crystal structures of human RCA (MCP, DAF, and CR1) and a smallpox virus homolog (SPICE) bound to complement component C3b. Our structural data reveal that up to four consecutive homologous CCP domains (i-iv), responsible for inhibition, bind in the same orientation and extended arrangement at a shared binding platform on C3b. Large sequence variations in CCP domains explain the diverse C3b-binding patterns, with limited or no contribution of some individual domains, while all regulators show extensive contacts with C3b for the domains at the third site. A variation of ~100° rotation around the longitudinal axis is observed for domains binding at the fourth site on C3b, without affecting the overall binding mode. The data suggest a common evolutionary origin for both inhibitory mechanisms, called decay acceleration and cofactor activity, with variable C3b binding through domains at sites ii, iii, and iv, and provide a framework for understanding RCA disease-related mutations and immune evasion.

Complement is undeniably quintessential for innate immunity by detecting and eliminating infectious microorganisms. Recent work, however, highlights an equally profound impact of complement on the induction and regulation of a wide range of immune cells. In particular, the complement regulator CD46 emerges as a key sensor of immune activation and a vital modulator of adaptive immunity. In this review, we summarize the current knowledge of CD46-mediated signalling events and their functional consequences on immune-competent cells with a specific focus on those in CD4(+) T cells. We will also discuss the promises and challenges that potential therapeutic modulation of CD46 may hold and pose.