Abstract
Background Autoantibodies appear in the serum years before the development of clinical disease in patients ultimately diagnosed with systemic lupus erythematosus (SLE) or Sjögrens syndrome. The transition from benign to pathogenic autoimmunity is not understood. Anti-IgG rheumatoid factors provide an illuminating example of an autoantibody that persists, on average, more than four years prior to the development of systemic vasculitis and type II cryoglobuinemia. The aim of this study was to identify the pathogenic changes occurring in B cells making rheumatoid factor autoantibody during this transition.
Methods Massively parallel sequencing of peripheral blood B cell receptors (BCR) was combined with mass spectrometry peptide sequencing of serum rheumatoid factor autoantibodies to identify circulating B cells expressing IgM rheumatoid factors in 4 patients with SLE or Sjögrens syndrome. In a patient presenting with primary Sjögrens syndrome evolving to cutaneous vasculitis, blood samples before and after transition were subjected to single cell analysis of mRNA, genome methylation, and lymphocyte regulatory genes recurrently mutated in lymphoma and leukemia.
Results Circulating rheumatoid factor B cell clones and their matched serum autoantibodies were identified in 4 patients, 3 of whom, produced the stereotypic Wa-type rheumatoid factor with IGHV1–69 and IGKV3–20 variable domains. Longitudinal analysis of a patient before and after vasculitis revealed a single Wa-type rogue clone was responsible for the rheumatoid factor over a period of 6 years. Compared to the patients normal memory B cells, the rogue cells had aberrant gene expression corresponding to CD11c+CD21 low B cells observed in autoimmunity, and hypomethylation of gene regions aberrantly hypomethylated in chronic lymphocytic leukemia (CLL). A loss-of-function somatic mutation in a Kelch-like protein gene, KLHL6 identical to recurring CLL and lymphoma mutations was acquired by half of the rheumatoid factor B cells. KLHL6 mutant cells slowly accumulated antibody variable domain replacement mutations that did not alter binding affinity but caused IgM-IgG immune complexes to precipitate at temperatures below 25C.
Conclusions Transition from a benign to pathogenic rheumatoid factor was preceded by a lymphoproliferative disease mutation in the responsible B cells and resulted in accumulation of antibody somatic mutations that diminish immune complex solubility. The ability to detect clones of the rheumatoid factor that are accumulating replacement mutations years prior to the patient developing cryoglobulinemia represents an opportunity to interfere with the conversion from asymptomatic seropositivity to clinical disease.
Funding Source(s): Australian National Health and Medical Research Council and New South Wales Department of Health
