Background/Purpose: Rheumatoid Arthritis (RA) autoimmunity starts years before clinically apparent inflammatory arthritis (IA) develops, and elevated disease-specific autoantibodies, including anti-citrullinated protein antibodies (ACPA) and rheumatoid factor (RF), can be detected in this period. Early responses to known and/or novel autoantigens likely drive the eventual production of pathogenic autoantibodies. We hypothesized that through characterization of circulating plasmablasts one can identify the specific antigens to which early tolerance has been broken in at-risk individuals who exhibit autoantibodies associated with future RA onset but who are currently without IA.

Methods: We have investigated the antibody-secreting plasmablasts of a characterized cohort of seropositive (Ab+) individuals (ACPA and/or RF) in comparison to seronegative (Ab-) individuals, patients recently diagnosed seropositive RA (<1 yr), and healthy controls. Plasmablasts were identified by flow cytometric analysis and defined as CD19+CD3-CD33-CD14-CD20-CD27+CD38^hi. As IgG-producing plasmablasts have low surface BCR expression, ‘IgG+’ cells were identified by the absence of IgA and IgM staining, while IgA+ plasmablasts were identified by IgM-IgA+ surface staining. The antibody repertoires of at-risk subjects were analyzed using a DNA barcode-based method of paired heavy- and light-chain gene sequencing. Cells were single-cell sorted into 96-well plates prior to adding cell-specific DNA barcodes by template switching during reverse transcription, followed by adding plate-specific DNA barcodes by PCR. Samples underwent Roche 454 sequencing or 2 x 300 MiSeq analysis. Data were demultiplexed using a custom software pipeline to separate reads from each single cell according to its unique compound (plate + well) barcode ID. The isotype of sorted cells was confirmed by gene-specific PCR and by identification of isotype-specific sequences in the final data.

Results: Total plasmablast levels were not elevated in Ab+ individuals (1%) compared to controls (0.4-1.6%). However, fitting with prior studies supporting a potential role for mucosal immune responses in RA initiation in the preclinical period, we observed markedly increased frequencies of IgA+ vs. IgG+ plasmablasts in Ab+ individuals (39% IgA+, 37% IgG+ plasmablasts) as compared to all other groups (1-9% IgA+, 71-87% IgG+ plasmablasts). Furthermore, sequencing of paired antibody heavy and light chains from Ab+ subjects revealed the presence of cross-isotype clonal families as well as similar sequence characteristics between the IgA and IgG plasmablast repertoires of seropositive subjects. 

Conclusion: The IgA plasmablast dominance in these Ab+ individuals suggests that a subset of antibodies may arise from both systemic as well as mucosal immune responses which may drive clinical disease development in individuals who are at-risk for developing RA. We intend to identify and characterize antigens to which these dual isotype antibodies react with the immediate goal of identifying known and novel autoantigens, and a potential future goal of using tolerizing therapies to these autoantigens that could be effective in prevention or early treatment strategies.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/increased-iga-plasmablasts-are-associated-with-rheumatoid-arthritis-related-autoantibodies-in-the-absence-of-inflammatory-arthritis/