Background Antibodies against citrullinated proteins antigens (ACPA) are the most relevant prognostic and diagnostic biomarker for rheumatoid arthritis (RA). ACPA positive patients are characterized by progressive disease, a high rate of joint destruction, and a low chance to achieve remission. Despite these strong and well-defined associations with clinical phenotype, however, molecular mechanisms underlying the citrulline-specific immune response are incompletely understood. Of note, this immune response is highly polyclonal with considerable cross-reactivity, develops and matures prior to disease onset, and is of remarkably low avidity. These features indicate that the underlying B cell response is different from “conventional” antibody responses, but also different from other autoantibody responses such as the high affinity double-stranded DNA antibody response in SLE.
Objectives To study molecular characteristics of ACPA in order to obtain insight into the properties of the underlying B cell response.
Methods Serum of ACPA positive RA patients was fractionated by size exclusion chromatography and analysed for the presence of ACPA-IgG by ELISA. In addition, ACPA-IgG and non-citrulline-specific IgG were affinity purified from serum and synovial fluid and analysed by gel electrophoresis. Electrophoresis bands were excised, enzymatically digested and subsequently analysed by HPLC and mass-spectrometry. Recombinant monoclonal ACPA were used to study binding affinity for multiple antigens by surface plasmon resonance.
Results In almost all donors studied (23 out of 24), ACPA-IgG molecules were found to have a higher molecular weight than non auto-reactive IgG. Structural analysis pinpointed this observation to a high frequency of N-glycans in the ACPA variable regions, which was found on up to 90% of all ACPA. These Fab-glycans were linked to N-glycosylation sites generated by somatic hypermutation, were absent from other disease-specific autoantibodies, were characterized by a high degree of sialylation, and clearly differentiated ACPA from “conventional” IgG molecules. Moreover, ACPA Fab glycans modulated reactivity to citrullinated antigens.
Conclusions These data describe a unique and completely novel feature of the citrulline-specific immune response in RA, with important implications for ACPA pathogenicity and disease prognostication. The high frequency of variable domain Fab glycans points to specific developmental abnormalities of the underlying B cell response.
Disclosure of Interest None declared
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