Background/Purpose:

Rheumatoid arthritis (RA) associated anti-citrullinated protein autoantibodies (ACPA) develop before RA diagnosis, at which point a wide range of citrullinated proteins have been shown to be targeted based on different peptide assays. Intriguingly, ACPAs have been found to be multi-reactive even on monoclonal level. We set out to study the breadth of that multireactivity and try to determine what the antibodies recognize, as recent findings suggest that certain, but not all, ACPA may have pathogenic properties.

Methods:

We screened 6 different monoclonal ACPAs, originating from 3 different patients, 4 were derived from synovial fluid plasma cells and 2 were derived from peptide-tetramer captured B cells from peripheral blood. All ACPA mAbs have confirmed anti-CCP2 reactivity and have demonstrated multi-citrulline-peptide reactivity by ELISA. A custom-made peptide array (NimbleGen, Roche) was designed with 16aa peptides derived from 1610 extracellular matrix- or RA associated- proteins. Peptides containing either an arginine or lysine were synthesized in pairs with a modified version containing either a citrulline or a homocitrulline in the respective position. Altogether >53,000 citrullinated peptides, >49,000 carbamylated peptides and >70,000 native peptides were included. Monoclonal antibodies were run on the array at a concentration of 1ug/ml, while serum and synovial fluid were diluted 1/100.

Results:

The monoclonal ACPAs consistently displayed low reactivity (<0.06%) to unmodified peptides, while all 6 reacted to 1,000s (3.4-9.4%) of the citrullinated peptides. Intriguingly, 3 out of the 6 ACPAs also reacted with the carbamylated peptides to a similar broad extent (2.2-4.5% of the peptides). Based on the sequences from the positive peptides we could create LOGOs of the preferred amino acids. Intriguingly, two dominating aa-patterns were identified and only minor contributions from the flanking sequences were observed.

Serum and synovial fluid (SF) from the same patient as the synovial plasma cell-derived ACPAs were also assessed at both the time point of the cell draw and a 10-year follow-up. The SF samples displayed a robust citrulline-reactivity with only 10% divergence between the two samples taken 10 years apart. In contrast, the homocitrulline reactivity was significantly altered between the two time points as approximately 50% of the signals were lost at follow-up in both SF and peripheral blood.

Conclusion:

An ACPA serum response is a hallmark of seropositive RA and develops early, often before clinical symptoms. Our study of monoclonal ACPAs generated from established RA implicates that a truly broad citrulline-reactivity is found also on individual immunoglobulin level and may be the result of multiple antigen encounters which have selected for very focused citrulline-recognition with only modest contribution from flanking amino acids. This citrulline autoimmunity appears robust with minimal changes over time, while that was less true for the homocitrulline response.

« Back to 2018 ACR/ARHP Annual Meeting

ACR Meeting Abstracts - https://acrabstracts.org/abstract/monoclonal-anti-citrullinated-protein-antibodies-generated-from-ra-derived-b-cells-recognize-amino-acid-motifs-rather-than-specific-proteins/