Background/Purpose: Autoantibodies against 60-kD Ro (Ro60)/SSA have been detected in the saliva and serum of patients with primary Sjögren’s syndrome (SS) by routine methods that are unable to resolve molecular characteristics of interacting antibodies. Accordingly, it is unknown whether the parallel glandular and systemic anti-Ro60 responses derive from related or independent antibody repertoires. In the present study, we have identified a Ro60-specific salivary IgA repertoire by analysing immunoglobulin variable region (IGV) subfamily composition and mutational profiles of matched salivary and serum proteomes.

Methods: Anti-Ro60 autoantibodies were purified and sequenced from Ro-specific precipitins prepared by electrophoresing native Ro60 protein against whole saliva or serum collected from 9 patients with seropositive primary SS. Microgram amounts of precipitating anti-Ro60 Igs were separated by SDS-PAGE, and in-gel chymotrypsin digests performed on heavy (H) and light (L) chain bands. VH/VL and constant-region peptides were subjected to nano-high performance liquid chromatography-mass spectrometry (nHPLC-MS/MS) followed by combined de novo amino acid sequencing and database matching using Peaks 8.0 software utilising ImMunoGeneTics (IMGT) and Uniprot databases.

Results: High-resolution MS sequencing of purified salivary anti-Ro60 H- and L-chains revealed a common (9/9 patients) oligoclonal IgA Ro60 repertoire dominated by IGHV3-23 and IGKV3-20 subfamily expression. Paired serum anti-Ro60 proteomes expressed a more diversified IgG1 repertoire with expression of additional IGHV1 and IGHV3 families in the systemic compartment. IGHV3-23-encoded H-chains were present in matched saliva and serum samples but were distinct in terms of their patterns of somatic mutations, suggesting independent pathways of affinity maturation. Three of 9 patients showed a less abundant IgG1 salivary anti-Ro60 proteome that was similar to the paired serum proteome.

Conclusion: Proteomic profiling of salivary and serum anti-Ro60 autoantibodies in primary SS reveals a unique salivary IgA repertoire with specific V-region peptide profiles, consistent with a parallel yet distinct salivary gland pathway of somatically selected anti-Ro60 autoantibodies. The novel proteomic workflow reported herein will allow analysis of clonal turnover of mucosal and systemic autoantibody repertoires in early versus established disease and provide molecular biomarkers to assess responses to therapy in the glandular and peripheral compartments.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/molecular-identification-of-a-ro-specific-salivary-iga-repertoire-with-unique-clonal-signatures-in-primary-sjogrens-syndrome/