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

Abstract

Fcγ-receptor (FcγR) activation by antibody derived soluble immune complexes (sICs) is a major contributor to inflammation in autoimmune diseases such as systemic lupus erythematosus (SLE). A robust and scalable test system allowing for the detection and quantification of sICs with regard to receptor activation is missing. We developed a comprehensive cell-based reporter system capable of measuring the sIC-mediated activation of individual human and mouse FcγRs. We show that compared to human FcγRs IIB and III, human FcγRs I and IIA lack sensitivity to sICs. Further, the assay proved to be sensitive to sIC size enabling us to demonstrate for the first time a complete translation of the Heidelberger-Kendall precipitation curve to FcγR responsiveness. The assay also proved useful to quantify sICs-mediated FcγR activation using sera from SLE patients and mouse models of lupus and arthritis. Thus, in clinical practice, it might be employed to measure FcγR activation as a biomarker for disease activity in immune-complex mediated disease.

Circulating multimeric immune complexes drive immunopathology in COVID-19

“…We thus set out to characterize our patient cohort regarding the presence of sICs in serum samples taken at various time points during disease and after hospitalization. To this end, we deployed a novel cell-based reporter assay developed to quantify CD16 (158V) activation by IgG-containing sICs 17 . As recently shown, the assay does not react to monomeric IgG or small dimeric complexes in solution, but specifically identifies multimeric sICs and has been successfully used to identify sICs in patients with systemic lupus erythematosus (SLE) 16 .…”

“…Based on the herein presented results identifying CD16 activation to be a major correlate of disease severity, we further explored this using an adapted FcγR activation assay optimized to measure soluble immune complexes (sICs) 17 . Indeed, we provide first evidence of such circulating sICs in the serum of COVID-19 patients and experimentally confirm previous hypotheses suggesting immune complexes as potential drivers of disease progression in COVID-19 [33][34][35] .…”

“…For all activation assays, mouse IL-2 secretion was quantified by anti-IL-2 ELISA, as described earlier. FcγRIIIA (CD16A) activation by sICs was measured by a recently developed cell-based assay 17 . Briefly, 2x10 5 BW5147-CD16 reporter cells were incubated with SARS-CoV-2 sera in a total volume of 200 µl for 16 h at 37°C, 5% CO2.…”

“…ICs. Solubility of sICs is achieved by pre-blocking an ELISA plate with PBS supplemented with 10% FCS as previously described 17 . CD16 activation by A) S-, B) N-and C) RBD-specific IgG in 9 representatively selected serum samples and one SARS-CoV-2 negative serum (dark blue bars).…”

“…A prototypical activating FcγR in this regard is FcγRIII (CD16) expressed by NK cells and monocyte-derived macrophages (CD16A) 13 or neutrophils (CD16B, 98% sequence identical ectodomains). Specifically, CD16 is able to recognize circulating soluble immune complexes (sICs) as they are formed in certain autoimmune diseases such as lupus [14][15][16][17] and viral infections 18 . Overstimulation of activating FcγRs in these cases is associated with disease severity [18][19][20] and thus an FcγR-driven overshooting inflammatory response 12 might be an explanation for the pronounced immunopathology observed during severe courses of COVID-19 21 .…”