ObjectiveAutoantibodies against antigens carrying distinct post-translational modifications (PTMs), such as citrulline, homocitrulline or acetyllysine, are hallmarks of rheumatoid arthritis (RA). The relation between these anti-modified protein antibody (AMPA)-classes is poorly understood as is the ability of different PTM-antigens to activate B-cell receptors (BCRs) directed against citrullinated proteins (CP). Insights into the nature of PTMs able to activate such B cells are pivotal to understand the ‘evolution’ of the autoimmune response conceivable underlying the disease. Here, we investigated the cross-reactivity of monoclonal AMPA and the ability of different types of PTM-antigens to activate CP-reactive BCRs.MethodsBCR sequences from B cells isolated using citrullinated or acetylated antigens were used to produce monoclonal antibodies (mAb) followed by a detailed analysis of their cross-reactivity towards PTM-antigens. Ramos B-cell transfectants expressing CP-reactive IgG BCRs were generated and their activation on stimulation with PTM-antigens investigated.ResultsMost mAbs were highly cross-reactive towards multiple PTMs, while no reactivity was observed to the unmodified controls. B cells carrying CP-reactive BCRs showed activation on stimulation with various types of PTM-antigens.ConclusionsOur study illustrates that AMPA exhibit a high cross-reactivity towards at least two PTMs indicating that their recognition pattern is not confined to one type of modification. Furthermore, our data show that CP-reactive B cells are not only activated by citrullinated, but also by carbamylated and/or acetylated antigens. These data are vital for the understanding of the breach of B-cell tolerance against PTM-antigens and the possible contribution of these antigens to RA-pathogenesis.

ObjectiveTo isolate and characterize muscle-specific kinase (MuSK) monoclonal antibodies from patients with MuSK myasthenia gravis (MG) on a genetic and functional level.MethodsWe generated recombinant MuSK antibodies from patient-derived clonal MuSK-specific B cells and produced monovalent Fab fragments from them. Both the antibodies and Fab fragments were tested for their effects on neural agrin-induced MuSK phosphorylation and acetylcholine receptor (AChR) clustering in myotube cultures.ResultsThe isolated MuSK monoclonal antibody sequences included IgG1, IgG3, and IgG4 that had undergone high levels of affinity maturation, consistent with antigenic selection. We confirmed their specificity for the MuSK Ig-like 1 domain and binding to neuromuscular junctions. Monovalent MuSK Fab, mimicking functionally monovalent MuSK MG patient Fab-arm exchanged serum IgG4, abolished agrin-induced MuSK phosphorylation and AChR clustering. Surprisingly, bivalent monospecific MuSK antibodies instead activated MuSK phosphorylation and partially induced AChR clustering, independent of agrin.ConclusionsPatient-derived MuSK antibodies can act either as MuSK agonist or MuSK antagonist, depending on the number of MuSK binding sites. Functional monovalency, induced by Fab-arm exchange in patient serum, makes MuSK IgG4 antibodies pathogenic.

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Autoreactive B cells mediate autoimmune pathology, but exactly how remains unknown. A hallmark of rheumatoid arthritis (RA), a common autoimmune disease, is the presence of disease-specific anticitrullinated protein antibodies (ACPAs). Here, we showed that ACPA-positive B cells in patients with RA strongly expressed T cell–stimulating ligands, produced abundant proinflammatory cytokines, and were proliferative while escaping inhibitory signals. This activated state was found at different degrees in different stages of disease: highest in patients with recent-onset RA, moderate in patients with established RA, and far less pronounced in ACPA-positive individuals “at risk” for developing disease. The activated autoreactive B cell response persisted in patients who achieved clinical remission with conventional treatment. ACPA-positive B cells in blood and synovial fluid secreted increased amounts of the chemoattractant interleukin-8, which attracted neutrophils, the most abundant immune cell in arthritic joints. Tetanus toxoid–specific B cells from the same patients exhibited properties of memory B cells without the activation and proliferation phenotype, but these cells transiently acquired a similar proliferative phenotype upon booster vaccination. Together, these data indicated that continuous antigenic triggering of autoreactive B cells occurs in human autoimmune disease and support the emerging concept of immunological activity that persists under treatment even in clinical remission, which may revise our current concept of treatment targets for future therapeutic interventions. In addition, our data pointed to a pathogenic role of ACPA-positive B cells in the inflammatory disease process underlying RA and favor approaches that aim at their antigen-specific inactivation or depletion.

Objective. Among autoimmune diseases, Sjögren's syndrome (SS) is most strongly associated with the development of malignant B cell lymphoma, in particular mucosa-associated lymphoid tissue (MALT)-type lymphoma. Previously, we have shown that in ϳ40% of cases of salivary gland MALT lymphoma, high-affinity stereotypic rheumatoid factor (RF) B cell receptors, specific for IgG-Fc, are expressed. This study was undertaken to investigate whether in the inflamed salivary glands of patients with SS, a similar RF-biased Ig repertoire is present.Methods. Extensive analyses of the B cell Ig V H region repertoire were performed on microdissected tissue samples from the labial salivary glands of 4 patients with SS.Results. All SS labial salivary glands harbored expanded B cell clones, of which 1 or 2 were highly expanded and detected in >50% of the microdissected samples. However, among the identified 464 distinct Ig clonotypes, only 3 stereotypic RF-expressing clones were detected. In 2 patients with SS, an RF-expressing clone was detected at low frequency in 1 of the microdissected samples, whereas 1 patient with SS harbored a highly expanded RF-expressing clone that was detected in all microdissected samples and also detected in the peripheral blood. Two years after analysis of this sample, the latter patient developed a diffuse large B cell lymphoma originating from the same RF clone.Conclusion. Inflamed labial salivary glands in patients with SS generally harbor 1 or 2 highly expanded B cell clones. The repertoire strongly biased toward stereotypic RFs in salivary gland MALT lymphomas is not a reflection of a similar repertoire in the inflamed salivary glands of patients with SS; rather, in the latter, the repertoire is based on a strong selection advantage of incidental stereotypic RF-expressing B cells.