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. 2018 Jun 6;9:1183. doi: 10.3389/fimmu.2018.01183

Figure 3.

Figure 3

Self- and polyreactive IgG2a and IgG2b autoantibodies (autoAbs) in 56R+/−Fcgr2b−/− mice develop T cell independently and are sialylated. (A,B) AutoAb-positive 5-month-old Fcgr2b−/− mice or 2.5-month-old 56R+/−Fcgr2b−/− mice received i.p. injections of anti-mouse CD4 (GK1.5) every 4 days for 25 days to deplete CD4+ T cells (Figure S2 in Supplementary Material). (A) Serum anti-DNA IgG2ab, IgG2b and IgG2aa levels before and after CD4 depletion. (B) The frequencies of CD138+ PCs in the spleen and bone marrow (BM) of untreated (n = 6) versus CD4-depleted (n = 4) Fcgr2b−/− mice, and untreated (spleen, n = 13; BM, n = 3) versus CD4 depleted (n = 4) 56R+/−Fcgr2b−/− mice were analyzed via FACS. One representative experiment is shown. (C) The biantennary core of the glycan structure linked to Asn 297 in the Fc region of IgG Abs consists of four N-acetylglucosamines (GlcNAc; blue) and three mannoses (Man), which can be further modified with fucose, bisecting GlcNAc and terminal galactose (G) and sialic acid (S) residues. (D) The sialic acid content in serum IgG Abs of 5–6-month-old autoAb (AB)-negative and proteinuria (PU)-negative [AB−PU− (n = 8), AB+PU− (n = 6) and AB+PU+ (n = 4)] Fcgr2b−/− mice and 56R+/−Fcgr2b−/− mice (n = 11) compared to wt mice (n = 10) were analyzed with the GlykoScreen™ Sialic Acid Quantification Kit (Prozyme) (Efl: fluorescence emission at 590 nm). (E,F) St6gal1 protein expression in splenic total and IgG+ PCs of 6–7-month-old AB−PU− (n = 4), AB+PU− (n = 5) and AB+PU+ (n = 9) Fcgr2b−/− mice and 56R+/−Fcgr2b−/− mice (n = 6), compared to wt mice (n = 5). (E) Representative intracellular staining of St6gal1 protein expression levels in splenic PCs of wt, AB+PU+Fcgr2b−/− and 56R+/−Fcgr2b−/− mice measured by flow cytometry. (F) Relative median fluorescence levels of St6gal1 protein expression. Median St6gal1 protein expression levels in total or IgG+ PCs of three independent experiments were measured by flow cytometry and normalized (fold change) to the expression in wt controls (=1) in the respective experiments. The normalized data of three independent experiments were summarized (mean) in the graphs.