The binding of mouse IgG3 to Fcγ receptors (FcγR) and the existence of a mouse IgG3-specific receptor have been discussed for 40 years. Recently, integrin beta-1 (ITGB1) was proposed to be a part of an IgG3 receptor involved in the phagocytosis of IgG3-coated pathogens. We investigated the interaction of mouse IgG3 with macrophage-like J774A.1 and P388D1 cells. The existence of an IgG3-specific receptor was verified using flow cytometry and a rosetting assay, in which erythrocytes clustered around the macrophage-like cells coated with an erythrocyte-specific IgG3. Our findings confirmed that receptors binding antigen-free IgG3 are present on J774A.1 and P388D1 cells. We demonstrated for the first time that the removal of N-glycans from IgG3 completely abolished its binding to the cells.
Fig: Deglycosylation of mIgG3 prevents its binding to J774A.1 and P388D1 cells. (a) Erythrocyte rosetting around J774A.1 or P388D1 cells coated with the M18 mIgG3 antibody specific to antigen B of the human ABO blood group system. The cells were coated with native or deglycosylated M18 (degM18). The small cells are group B human erythrocytes, whereas the large bright cells are J774A.1 or P388D1 cells. The arrows point to exemplary rosettes. Representative microscope images of three independent experiments are shown. The pie charts display the percentage of rosetting and non-rosetting cells observed in three different fields in the same representative experiment. (b,c) Binding of M18-488 to J774A.1 or P388D1 cells analyzed using flow cytometry. The cells were stained with fluorescently labeled native or degM18. The dot plots show representative results of three independent experiments. (c) Average values ± SD of the three independent experiments described in (b).
Moreover, we discovered that the cells treated with Accutase did not bind IgG3, indicating that IgG3-specific receptors are substrates of this enzyme. The results of antibody-mediated blocking of putative IgG3 receptors suggested that apart from previously proposed ITGB1, FcγRII, FcγRIII, also additional, still unknown, receptor is involved in IgG3 binding. These findings indicate that there is a complex network of glycan-dependent interactions between mouse IgG3 and the surface of effector immune cells.
Karabasz, A., Bzowska, M., Bereta, J. et al. Mouse IgG3 binding to macrophage-like cells is prevented by deglycosylation of the antibody or by Accutase treatment of the cells. Sci Rep 11, 10295 (2021). https://doi.org/10.1038/s41598-021-89705-3