Anticitrullinated protein antibodies (ACPA) are highly specifi c for rheumatoid arthritis (RA) and have been implicated in disease pathogenesis. We have recently shown that IgG1 ACPA of RA patients harbour different glycan moieties on their Fc-tail, as compared to total sera IgG1. Given the crucial roles of Fc-linked N-glycans for the structure and biological activity of IgG, Fc-glycosylation of antibodies and especially ACPA is receiving considerable interest. However, little is known about the signals and factors that could infl uence the composition of these carbohydrate structures on secreted IgG produced by B lymphocytes. Here, we investigated modifi cation of Fc-glycosylation by treatment of B cells with different immune modulatory stimulants. CD19 B cells were isolated from peripheral blood mononuclear cells obtained from blood of healthy donors, and cultured with specifi c supplements for 7-9 days. Subsequently, IgG was purifi ed by protein A beads from the cell culture supernatants and digested with trypsin. Finally, the Fc-glycans were analysed by mass spectrometry. We show that both factors belonging to the innate immune system including the toll-like receptor 9 ligand CpG, as well as factors of the adaptive immune system, like the T cell derived cytokine interleukin 21 (IL-21), can modulate IgG Fc-glycosylation. In addition, also 'environmental' factors, such as retinoic acid, a natural metabolite of vitamin A, modulated the glycosylation of IgG Fc. These factors affect Fc-glycan profi les in different ways; CpG and IL-21 increase Fc-linked galactosylation and reduce bisecting N-acetylglucosamine levels, while retinoic acid signifi cantly decreases galactosylation and sialylation levels. Moreover, these effects appeared to be specifi c for immunoglobulins as no signifi cant changes in the overall glycoforms of cellular proteins were observed. Interestingly, several other cytokines and molecules known to affect B cell biology and antibody production did not have an impact on IgG Fc-coupled glycan profi les. Together, these results indicate that different stimuli received by B cells during their activation and differentiation can modulate the Fc-linked glycosylation of secreted IgG, without affecting the general cellular glycosylation machinery. These data implicate that modifi cation of Fc-glycans of ACPA secreted by B cells of RA patients could be a promising therapeutic tool to reduce the pathogenicity of ACPA in ACPA-positive RA patients.
Anti-citrullinated protein antibodies (ACPA) are specifi c for rheumatoid arthritis (RA), their presence is predictive for development to RA and have been implicated in diseasepathogenesis in animal studies. Despite the important implications for diagnoses and etiology, the natural development and biology of the ACPA response is poorly studied. Here the authors determined the avidity and avidity maturation of the ACPA response and compared this to the avidity of antibodies against recall-antigens.Our data show that the avidity of ACPA against several citrullinated antigens is considerably lower as compared to the avidity of antibodies against recall antigens such as tetanus toxoid or diphtheria toxoid. Intriguingly, despite high titers and extensive isotype switching, no avidity maturation in the ACPA response was observed during longitudinal follow-up.Our data indicate that the natural evolution of ACPA differs from the development of antibodies against prototype T cell dependent recall antigens. Moreover, these data point to an independency of avidity maturation and isotype-switching where full-isotype switching is taking place in the face of restricted and/or hampered avidity maturation.As avidity maturation of ACPA differs from avidity maturation of antibodies to recall antigens, our data are of relevance to the design of anti-B cell therapeutics.
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