Fc-glycosylation of IgG1 is modulated by B-cell stimuli

Abstract

We have recently shown that IgG1 directed against antigens thought to be involved in the pathogenesis of rheumatoid arthritis harbor different glycan moieties on their Fc-tail, as compared with total sera IgG1. Given the crucial roles of Fc-linked N-glycans for the structure and biological activity of IgG, Fc-glycosylation of antibodies is receiving considerable interest. However, so far little is known about the signals and factors that could influence the composition of these carbohydrate structures on secreted IgG produced by B lymphocytes. Here we show that both "environmental" factors, such as all-trans retinoic acid (a natural metabolite of vitamin A), as well as factors stimulating the innate immune system (i.e. CpG oligodeoxynucleotide, a ligand for toll-like receptor 9) or coming from the adaptive immune system (i.e. interleukin-21, a T-cell derived cytokine) can modulate IgG1 Fc-glycosylation. These factors affect Fc-glycan profiles in different ways. CpG oligodeoxynucleotide and interleukin-21 increase Fc-linked galactosylation and reduce bisecting N-acetylglucosamine levels, whereas all-trans retinoic acid significantly decreases galactosylation and sialylation levels. Moreover, these effects appeared to be stable and specific for secreted IgG1 as no parallel changes of the corresponding glycans in the cellular glycan pool were observed. Interestingly, several other cytokines and molecules known to affect B-cell biology and antibody production did not have an impact on IgG1 Fc-coupled glycan profiles. Together, these data indicate that different stimuli received by B cells during their activation and differentiation can modulate the Fc-linked glycosylation of secreted IgG1 without affecting the general cellular glycosylation machinery. Our study, therefore, furthers our understanding of the regulation of IgG1 glycosylation at the cellular level.

Fig. 1.

Purification and differentiation of human B cells in vitro. Human CD19⁺ B cells were purified with anti-CD19 beads and subsequently cultured with anti-IgM and CD40L-transfected cells in the absence or presence of additional IL-2 and IL-10 (IL-2/10). Following 7 days, cells were collected and processed for the analysis of CD27 and CD38 expression. A, a schematic overview of the signals required for the T-cell dependent antibody production from human naive B cells. B, representative dot plots showing the CD19 versus CD20 (top left panel) and CD27 versus CD38 (top right and bottom panels) expressions on purified B cells before (D0, top panel) and following 7 days culture (D7, bottom panel). Numbers indicate the percentage of CD19⁺CD20⁺ cells (top left panel) or CD27^highCD38^high antibody-secreting cells (ASCs) within circles. C, levels of IgG in supernatants collected on day 7 were determined by ELISA. Results are expressed as mean ± S.E. of data obtained from three different donors. Th, CD4⁺ helper T cells.

Fig. 2.

A representative mass spectrometric picture for the Fc glycosylation of IgG1 purified from in vitro B-cell cultures. Purified human CD19⁺ B cells were cultured as described in the legend to Fig. 1 in the absence (M) (A) or presence of additional IL-21 (B) or ATRA (C) for 7–9 days. IgG1 were subsequently purified from the culture supernatants and their Fc glycosylation profiles were analyzed as described in the Materials and Methods. Mass spectrometric signals are shown for the triple protonated glycopeptides with neutral N-glycan chains (left panels) and acidic N-glycan chains (right panels). *, nonglycopeptide signal.

Fig. 3.

Fc-linked glycosylation of IgG1 produced by B cells among different culture conditions. Purified human CD19⁺ B cells were cultured as described in the legend to Fig. 1 in the absence (M) or presence of additional CpG (2.5 μg/ml), ATRA (10 nm) and cytokines (50 ng/ml) as indicated. Following 7–9 days, galactosylation patterns of IgG1 in the culture supernatant were analyzed. Results were expressed as mean ± S.E. of the relative degree of galactosylation from 2–4 different experiments. The abundance of Fc-attached galactose residues in IgG1 purified from cultures with M (black bar) in each donor was set as 100%.

Fig. 4.

Fc-coupled galactosylation and sialylation levels of IgG1 purified from B-cell cultures with IL-21 or ATRA. Purified human CD19⁺ B cells were cultured as described in the legend to Fig. 1 in the absence (M)/presence of additional IL-21 or ATRA as indicated. Following 7–9 days, glycosylation patterns of IgG1 in the culture supernatant were analyzed. A and C, comparison of the galactosylation (A) or sialylation (C) levels of IgG1 purified from indicated culture conditions. B and D, comparison of the incidence of IgG1 lacking galactose residues but with core fucose (G0F, B) or the percent of sialylated galactose residues on IgG1 purified from indicated cultures (D). Each dot represents an independent experiment with B cells isolated from different healthy individuals. The missing values indicate that corresponding conditions were not tested in these donors. *, p < 0.05; **, p < 0.01.

Fig. 5.

IL-21 reduces the percentage of IgG1 with an additional bisecting GlcNAc. Purified human CD19⁺ B cells were cultured as described in the legend to Fig. 1 in the absence (M)/presence of IL-21 or ATRA as indicated. Following 7–9 days, the incidence of IgG1 in culture supernatants with bisecting GlcNAc was quantified. Each dot represents an independent experiment with B cells isolated from different healthy individuals. The missing values indicate that corresponding conditions were not tested in these donors. **, p < 0.01; n.s, not significant.

Fig. 6.

Comparison of the glycoforms between secreted IgG1 and cellular proteins. Purified human CD19⁺ B cells were cultured as described in the legend to Fig. 1 in the absence (M)/presence of additional IL-21 or ATRA. Following 7–9 days, glycan profiles of IgG1 purified from culture supernatants (A) or proteins extracted from cell pellets (B) were analyzed. Only those glycan signals that were found to be shared between secreted IgG1 and the cellular proteins were considered in this comparison. Results are expressed as mean ± S.D. of the relative intensity of each glycoform from three different individuals.

Fig. 7.

Long-lasting effect of IL-21 and ATRA on IgG1 glycan profiles. Purified human CD19⁺ B cells were cultured as described in the legend to Fig. 1 in the absence or presence of additional IL-21 or ATRA (indicated by IL-21 and ATRA before “/”). Following 5 days, cells were collected, washed extensively and then cultured in medium with/without IL-21 or ATRA for an additional 5 days (indicated by M, IL-21, and ATRA after “/”). Supernatants were collected and glycan profiles of IgG1 were analyzed. Galactosylation (A), sialylation (B) and bisecting GlcNAc (C) levels are given following normalization (samples cultured continuously in medium for 10 days were set as 100%).