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FRI0004 Modulating FC-linked glycosylation patterns of human IGG1
  1. J. Wang1,
  2. C.I. Balog2,
  3. K. Stavenhagen2,
  4. C.A. Koeleman2,
  5. M.H. Selman2,
  6. A.M. Deelder2,
  7. T.W. Huizinga3,
  8. R.E. Toes3,
  9. M. Wuhrer2,
  10. H.U. Scherer3
  1. 1Department of Human Genetics
  2. 2Biomolecular Mass Spectrometry Unit, Department of Parasitology
  3. 3Department of Rheumatology, Leiden University Medical Center, Leiden, Netherlands

Abstract

Background Glycosylation of the Fc tail of human IgG strongly influences Fc-mediated effector functions. Recently, we have shown that anti-citrullinated protein antibodies (ACPA) isolated from serum and synovial fluid of patients with rheumatoid arthritis (RA) exhibit a specific, pro-inflammatory Fc-linked glycosylation profile. This glycan pattern is distinct from the glycan residues attached to the Fc tail of non-specific total IgG. As these data indicate a potential role of Fc-linked glycans for the biological effects that ACPA exert within the pathogenesis of RA, it is of interest to understand the mechanisms that regulate the Fc-glycosylation machinery of B cells.

Objectives To study the effect of various mediators (cytokines, vitamins, microbial products) with relevance to adaptive and innate immune responses on Fc-linked glycan residues of human IgG.

Methods Peripheral blood mononuclear cells were obtained from healthy human donors. CD19 expressing B cells were isolated by magnetic bead-based positive selection and cultured in the presence of anti-IgM, IL-2 and IL-10 on a layer of irradiated, CD40L transfected fibroblasts to induce IgG production. In addition, mediators described to have immunomodulatory activity on B cells (IL-4/6/17/21, IFN-γ, TNF-α, LT-α, TGF-β, CpG, all-trans retinoic acid (ATRA)) were added to the cultures. After 7-9 days of culture, IgG molecules (IgG1, 2 and 4) were isolated from culture supernatants by protein A affinity chromatography and subjected to tryptic digest. IgG1-linked Fc-glycopeptides were analyzed by mass-spectrometry.

Results Whereas IL-21 and CpG significantly and consistently increased the degree of galactosylation and sialylation of the Fc-linked glycan, ATRA strongly decreased the frequency of these glycoforms. No significant effects were observed for the other mediators tested. Of interest, IL-21 additionally decreased the frequency of bisecting N-acteylglucosamine (GlcNAc), whereas ATRA left this sugar moiety unchanged. Once induced, the Fc-linked glycan profiles of secreted IgG1 remained stable, even after the added mediators were withdrawn after 5 days of culture.

Conclusions Our data indicate that several factors, present in the microenvironment of B cells during activation and differentiation, are capable of differentially regulating the Fc-glycosylation of IgG1. Once B cells differentiate into antibody secreting cells, the IgG1 Fc-linked glycan profile remained stable. These data further our understanding of the mechanisms underlying IgG1 Fc-glycosylation in the human and may open up novel approaches for therapeutic intervention.

Disclosure of Interest None Declared

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