Background/Purpose: Rheumatoid factors (RFs) are associated with disease activity and joint damage in rheumatoid arthritis (RA) and thought to play a pathogenic role in disease. RFs have been measured for decades by nephelometry and/or solid-phase immunoassay, but these techniques are unable to resolve RF immunoglobulin heavy-chain variable (IGHV) composition at the level of the serum proteome. Here, we have performed the first quantitative mass spectrometry (MS) analysis of serum (secreted) IgH-RF repertoires in RA patients and reveal dynamic patterns of RF production after treatment.

Methods: Serum IgM-RFs from 16 patients with RF-positive newly diagnosed RA were purified by immunoprecipitation (1). IgH-RF V-region peptides were sequenced by LC-MS/MS followed by combined de novo amino acid sequencing and database matching using Peaks 8.0 software to determine IGHV composition. Full-scan multiple reaction monitoring (MRM) was used to quantitate expression of individual IGHV subfamilies in paired serum samples at baseline and after 6 months of tDMARD (n=9) or TNFi (n=7).

Results: High resolution MS sequencing of serum IgH-RFs demonstrated shared germline-encoded oligoclonal patterns with convergent usage of IGHV1-3, 1-69, 3-15, 3-23, 3-64, 3-7, 3-74, 4-34, and 5-51 subfamilies, although each patient had an individual IgH-RF peptide expression landscape. Six-month serum RF levels were decreased in 15/16 patients by routine nephelometry and correlated with falling IgH-RF expression levels by MRM/MS (P < 0.05 by Spearman rank). Molecular profiling of IgH-RFs by MS proteomics revealed that RF responses in each patient comprised two clonal populations: treatment-responsive (classified as a reduction of > 30% in expression levels), and treatment-resistant (classified as steady, an increase of > 30%, or newly emergent) (Figure). Commensurate with global reductions in RF levels after treatment, responsive IgH-RF clones comprised over 2/3^rds of the RF clonal repertoire. On the other hand, patients co-expressed sizable numbers of resistant IgH-RF clones indicating molecular heterogeneity of RF responses. Newly emergent (de novo) clonal populations in treated patients were encoded by IGHV3 subfamilies not expressed at baseline.

Conclusion: RF production in RA is a dynamic, oligoclonal process comprising shared sets of IGHV subfamilies across unrelated patients. While most IgH-RF clonal populations are reduced by conventional therapies, the finding of resistant IgM-RF clones may reflect their production in protected niches in joints or tissues; or differences in selection and clonal expansion by immune complexes. MS-based quantitative proteomics of serum RFs offers a powerful approach for molecular characterisation of these prototypic diagnostic and pathogenic biomarkers in RA.

1.     Wang et al., Arthritis Rheumatol. (2018) doi: 10.1002/art.40539