Detection and functional resolution of soluble multimeric immune complexes by a comprehensive FcγR reporter cell panel

Fc-gamma receptor (FcγR) activation by soluble IgG immune complexes (sICs) represents a major mechanism of inflammation in certain autoimmune diseases such as systemic lupus erythematosus (SLE). A robust and scalable test system allowing for the detection and quantification of sIC bioactivity is missing. Previously described FcγR interaction assays are limited to certain FcγRs, lack scalability and flexibility, are not indicative of receptor activation or lack sensitivity towards sIC size. We developed a comprehensive reporter cell panel detecting individual activation of FcγRs from humans and the mouse. The reporter cell lines were integrated into an assay format that provides flexible read-outs enabling the quantification of sIC reactivity via ELISA or a fast detection using flow cytometry. This identified FcγRIIA(H) and FcγRIIIA as the most sIC-sensitive FcγRs in our test system. Applying the assay we demonstrate that sICs versus immobilized ICs are fundamentally different FcγR-ligands with regard to FcγR preference and signal strength. Reaching a detection limit in the very low nanomolar range, the assay proved also to be sensitive to sIC stoichiometry and size enabling for the first time a complete reproduction of the Heidelberger-Kendall precipitation curve in terms of immune receptor activation. Analyzing sera from SLE patients and mouse models of lupus and arthritis proved that sIC-dependent FcγR activation has predictive capabilities regarding severity of SLE disease. The new methodology provides a sensitive, scalable and comprehensive tool to evaluate the size, amount and bioactivity of sICs in all settings. One Sentence Summary In this study we established a comprehensive FcγR reporter cell assay enabling the detection and quantification of soluble immune complexes generated in experimental and clinical settings.