Figure 4.

IgG4 activates complement via the classical pathway. (A) C3b deposition by anti-biotin IgG1, IgG4 and IgG1 PG-LALA (eliminated Fc-mediated effector functions) in presence or absence of either C1q, mannose-binding lectin (MBL), or complement factor B (FB) inhibitors, biotinylated human serum albumin (HSA; 240 μM biotin) and 2.5% human serum. Antibodies with different glycan profiles were tested: high mannose (HM), high galactose (HG), normal galactose (NH) and low galactose (LG). The molar ratios of target to inhibitor are 1:4.5 for C1q, 1:8.5 for MBL, and 1:3.5 for FB. (B) The percentage of complement-mediated lysis of biotinylated human red blood cells (2.5 mM biotin) by IgG1, IgG4 and IgG1 PG-LALA in presence or absence of C1q, MBL, or factor D (FD) inhibitors and 10% human serum. The molar ratios for target to inhibitor were 1:3 for C1q, 1:3 for MBL, and 1:2 for FD. The percentage of lysis was calculated based on the 100% lysis control (red blood cells with saponin). (C) The relative C3b deposition and (D) percentage of complement-mediated lysis of biotinylated human red blood cells induced by IgG4 glycovariants are shown as bar graphs with an antibody concentration of either (C) 8 µg/mL, or (D) 15 µg/mL. (E) To test binding of purified C1q (600 μg/mL) to the mAbs, IgG1, IgG4 and IgG1 PG-LALA were tested in the presence of biotinylated human serum albumin (HSA; 240 μM biotin). Kruskal-Wallis test with Dunn’s multiple comparisons test was performed for statistical comparison of the IgG4 glycovariants per inhibition condition: *p < 0.05; **p < 0.01. All antibodies were tested in a two-fold serial dilution starting from 8 µg/mL for C3b deposition and 15 µg/mL for CDC in at least three independent experiments.