Supplementary Figure 5: Role of CD32b, C1q, CRP and glycosylation in non-aggregated IgG3 binding to B cells of HIV-negative individuals.

(a) Immunoblot analysis of C1q, IgG3 and CRP content in PEG precipitate (PEG), SEC-1, SEC-2 or SEC-2 depleted of C1q (C1q-d SEC-2) using serum of an HIV-viremic individual with moderate-intensity IgG3 on IgM⁺ B cells. The gels are shown in the order the membrane was sequentially stained and stripped. Lanes marked IgG3 and C1q indicate respective protein controls. (b) Flow cytometry of CD20-gated B cells of an HIV-negative individual incubated without PEG-IgG3 (no treatment), with PEG-IgG3 from serum of an HIV-viremic individual with moderate-intensity IgG3 on IgM⁺ B cells treated with control rabbit IgG or rabbit anti-C1q or rabbit anti-CRP antibody. Right, cumulative pattern from sera of chronically infected HIV-viremic individuals with moderate- to high-intensity IgG3 on IgM⁺ B cells. (c) Flow cytometry of CD20-gated B cells of an HIV-negative individual stained for IgG3 and IgM. The B cells were untreated or treated with antibody to CD32b or control goat IgG prior to addition of PEG-IgG3 from an HIV-viremic individual with high-intensity IgG3 on IgM⁺ B cells. Graph: cumulative effect of pre-treating B cells with anti-CD32b prior to addition of PEG-IgG3 from HIV-viremic individuals with moderate- to high-intensity IgG3 on IgM⁺ B cells (n = 6). (d) Effect of treating PEG-IgG3 prepared from individuals with moderate- to high-intensity IgG3 on IgM⁺ B cells with PNGase F or EndoS2 on binding to B cells of HIV-negative individuals. Each color coded symbol (b,d) represents an individual (b, n = 7; d, n = 6); black horizontal bars represent medians; * P < 0.05; n.s., not significant (two-tailed Wilcoxon matched-pairs signed rank test, after (b,d) obtaining significance by Friedman ANOVA test on full set). Data are representative of eight independent experiments (a).