Background/Purpose: Anti-citrullinated protein antibodies, the hallmarking autoantibodies in Rheumatoid Arthritis (RA), are characterized by N-linked glycans in the variable domain (V-domain). The occurrence of these glycans results from the selective introduction of N-glycosylation sites during somatic hypermutation and their presence is predictive for the transition from pre-disease autoimmunity towards RA. We have now determined the biological implication of V-domain glycans on antigen-binding and B-cell activation.

Methods: ACPA crystal structures, including the starting monosaccharides of the V-domain glycans, were generated. These crystal structures were used as a basic framework and the complete glycan structure modelled onto the V-domain. Fab crystal structures with modelled V-domain glycans were subsequently used for molecular dynamics simulations. Antigen binding assays using monoclonal ACPA IgG including V-domain glycans and their non-glycosylated counterparts were performed. Further, we generated human Ramos B cells lines including V-domain glycosylated and non-glycosylated mIgG B cell receptors and identified their activation upon stimulation.

Results: Autoantibody crystal structures show that V-domain glycans are positioned in the vicinity of the binding-pocket and dynamic modelling shows their potential to interact with antigen-binding, which is confirmed by binding assays. Noteworthy, human Ramos B cells carrying V-domain glycosylated B cell receptors undergo increased signalling after stimulation compared to their non-glycosylated counterparts.

Conclusion: Our data indicate that autoreactive B cells in RA are selected for the presence of V-domain glycans that convey a signalling advantage, potentially explaining the outgrowth of autoreactive B cells and the increase of autoantibody levels towards disease-onset. These findings are relevant for the understanding of how autoreactive B cells escape immune checkpoints in humans and indicate that the introduction of V-domain glycans enables B cells to breach tolerance in the prominent autoimmune disease RA.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/n-linked-glycosylation-of-the-immunoglobulin-variable-domain-affects-antigen-binding-and-autoreactive-b-cell-activation/