Background/Purpose : Long-lived humoral autoimmunity to Ro60 is considered a hallmark of primary Sjögren’s syndrome (SS), but the mechanism by which this immunity is sustained over decades remains unclear. Persistence of anti-Ro60 autoantibodies could be generated either by long-lived plasma cells residing in the bone marrow or by continual production of short-lived plasma cells. In the present study, these models were tested in humans for the first time by analysing serial molecular signatures of a recently reported public Ro60-specific clonotypic autoantibody (1).

Methods: Serial serum samples were collected over weeks to years from 8 patients with primary SS who expressed stable V_H3-23/ V_K3-20 Ro60 clonotypic autoantibody levels. Clonotypic IgGs were isolated by epitope-specific affinity chromatography and subjected to high resolution Orbitrap mass spectrometry. Variable regions of heavy and light chains were analysed by combined database and de novoamino acid sequencing. Alterations in autoantibody affinity were assessed by equilibrium binding analysis (Biacore).

Results: At each time point, patients expressed a single Ro60-specific monoclonal IgG1 kappa species, specified by a V_H3-23/J_H5 and V_K3-20/J_K2 pairing signature. However, near full-length V region protein sequencing showed a subtle turnover of clonotypes that was not detected by solid-phase immunoassay. The clonal turnover was characterised by 4-6 month cycles of clonal succession, with dominant clonotypes undergoing continual replacement by new somatically mutated clonal variants. Surprisingly, earlier clones never reappear in the periphery, and each new clone has a unique molecular signature. Affinities (K_D value) of successive clonotypes did not change significantly in the face of ongoing perpetual turnover. Autoantibody levels showed a cyclical rise and fall pattern every 3-4 months in keeping with this turnover model.

Conclusion: Analysis of the secreted autoantibody proteome demonstrates a dynamic process of clonal succession that masquerades as long-term Ro60 humoral autoimmunity. Surprisingly, the selection pressure for replacement clones is not based on affinity selection, indicating that an affinity ceiling is reached early in disease.  Our findings are compatible with ongoing clonal expansion and exhaustion of short-lived autoreactive plasma cells, as opposed to a single event generation of long-lived plasma cells. The relentless generation of autoantibody clonotypes in primary SS by antigen-driven clonal selection has important therapeutic and diagnostic implications.

(1) Lindop et al. (2011), Arthritis Rheum, Vol. 63, No. 11, p.p. 3477-3486.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/long-term-humoral-autoimmunity-to-ro60-in-primary-sjogrens-syndrome-is-driven-by-clonal-succession/