Cross-reactivity of IgM anti-modified protein antibodies in rheumatoid arthritis despite limited mutational load.

BACKGROUND: Anti-modified protein antibodies (AMPA) targeting citrullinated, acetylated and/or carbamylated self-antigens are hallmarks of rheumatoid arthritis (RA). Although AMPA-IgG cross-reactivity to multiple post-translational modifications (PTMs) is evident, it is unknown whether the first responding B cells, expressing IgM, display similar characteristics or if cross-reactivity is crucially dependent on somatic hypermutation (SHM). We now studied the reactivity of (germline) AMPA-IgM to further understand the breach of B cell tolerance and to identify if cross-reactivity depends on extensive SHM. Moreover, we investigated whether AMPA-IgM can efficiently recruit immune effector mechanisms. METHODS: Polyclonal AMPA-IgM were isolated from RA patients and assessed for cross-reactivity towards PTM antigens. AMPA-IgM B cell receptor sequences were obtained by single cell isolation using antigen-specific tetramers. Subsequently, pentameric monoclonal AMPA-IgM, their germline counterparts and monomeric IgG variants were generated. The antibodies were analysed on a panel of PTM antigens and tested for complement activation. RESULTS: Pentameric monoclonal and polyclonal AMPA-IgM displayed cross-reactivity to multiple antigens and different PTMs. PTM antigen recognition was still present, although reduced, after reverting the IgM into germline. Valency of AMPA-IgM was crucial for antigen recognition as PTM-reactivity significantly decreased when AMPA-IgM were expressed as IgG. Furthermore, AMPA-IgM was 15- to 30-fold more potent in complement-activation compared to AMPA-IgG. CONCLUSIONS: We provide first evidence that AMPA-IgM are cross-reactive towards different PTMs, indicating that PTM (cross-)reactivity is not confined to IgG and does not necessarily depend on extensive somatic hypermutation. Moreover, our data indicate that a diverse set of PTM antigens could be involved in the initial tolerance breach in RA and suggest that AMPA-IgM can induce complement-activation and thereby inflammation.

Light chain skewing in autoantibodies and B-cell receptors of the citrullinated antigen-binding B-cell response in rheumatoid arthritis.

Rheumatoid arthritis (RA) is a chronic autoimmune disease affecting 1% of the world population. RA is associated with the presence of autoantibodies, of which anti-citrullinated protein antibodies (ACPA) are most prominent. ACPA are produced by citrullinated antigen-binding B cells that have presumably survived tolerance checkpoints. So far, it is unclear how and when such autoreactive B cells emerge. Light chain (LC) rearrangement and mutation rates can be informative with regard to selection steps during B-cell development. Therefore, we studied LC characteristics of ACPA-expressing B cells and secreted ACPA with the aim to better understand the development of this disease-specific, autoreactive B-cell response. Paired ACPA-IgG and ACPA-depleted IgG were isolated from serum (n = 87) and synovial fluid (SF, n = 21) of patients with established RA. We determined the LC composition for each fraction by ELISA using kappa(Igκ)- and lambda(Igλ) LC-specific antibodies. Cellular LC expression was determined using flow cytometry. In addition, we used a B-cell receptor (BCR)-specific PCR to obtain LC variable region sequences of citrullinated antigen- and tetanus toxoid (TT)-binding B cells. In serum, we observed an increased frequency of lambda LC in ACPA-IgG (1.64:1) compared to control IgG (2.03:1) and to the κ/λ ratio reported for healthy individuals (2:1). A similar trend towards higher frequencies of lambda LCs was observed for ACPA-IgG in SF (1.84:1). Additionally, the percentage of Igλ-expressing B cells was higher for citrullinated antigen-binding B cells (51%) compared to TT-specific (43%) and total CD19+CD20+ B cells (36%). Moreover, an increased Igλ percentage was observed in BCR-sequences derived from ACPA-expressing (49%) compared to TT-specific B cells (34%). Taken together, we report an enhanced frequency of lambda LCs in the secreted ACPA-IgG repertoire and, on the cellular level, in BCR sequences of ACPA-expressing B cells compared to control. This skewing in the autoreactive B-cell repertoire could reflect a process of active selection.