Unraveling complement activation profiles on endothelial cells in healthy donors using an <i>ex vivo</i> model

Functional approaches to properly examine individual's susceptibility to complement dysregulation are limited. We assessed ex vivo complement activation induced by sera from 38 healthy donors on resting microvascular endothelial cells with or without complement dysregulation (OX-24 monoclonal antibody). Following incubation, immunofluorescence was used to quantify membrane-bound C3b/iC3b and C5b-9 with a computer-assisted method (H-score). C3a and C5a anaphylatoxins were quantified in supernatants by ELISA. Genetic sequencing of 32 donors was also performed to identify variants in alternative pathway genes. Elevated complement deposition was defined by H-scoreC3c > 50 and/or H-scoreC5b-9 > 30. Combined analysis of C3b/iC3b and C5b-9 deposition in 35 donors showed that one donor (2.8%) had an isolated increase in C3b/iC3b deposits, three (8.6%) had an isolated increase in C5b-9 deposits, and one (2.8%) had both increased C3b/iC3b and C5b-9 deposition. Genetic analysis revealed three heterozygous rare/low frequency missense variants in CFH (p.N1050Y, p.R1210C) and CFI (p.A76G) in 3/5 donors with increased complement deposition. This model revealed distinct patterns of complement activation among healthy individuals and identified a genetic basis for dysregulation in three cases. This assay offers a promising tool to study complement activity and its mechanisms in research.