Rheumatoid arthritis (RA) is a chronic inflammatory disease with predominantly joint involvement. If left untreated, RA leads to joint damage and eventually irreversible disability [1]. Approximately 50–70% of RA patients present with autoantibodies, rheumatoid factor (RF) and/or anti-citrullinated protein antibody (ACPA) [2]. Autoantibody-positive patients have a slightly different clinical presentation, a generally worse prognosis, and a higher risk for joint destruction compared with autoantibody-negative patients [3,4]. On a microscopic level, differences are found in histology and synovial fluid cytokine profiles [5,6]. In addition, distinct genetic and environmental risk factors have been reported for the two groups [[7], [8], [9]]. Therefore, autoantibody-positive and -negative RA have been proposed as different disease entities with distinct disease-underlying mechanisms [4]. However, the exact differences in pathogenesis between autoantibody-positive and -negative RA have yet to be discovered.

B cells are thought to contribute to RA pathogenesis in multiple ways, one of which is through the production of ACPA and RF. These can be present already up to 10 years before symptoms occur, indicating B cell involvement early in disease development [10]. B cells might therefore have a different role in the pathogenesis of autoantibody-positive and -negative RA. This is supported by the finding that treatment with Rituximab, a monoclonal antibody selectively targeting CD20-positive B cells, is more effective in autoantibody-positive patients [11]. We have previously shown increased protein expression and enhanced basal phosphorylation of the B cell receptor (BCR) signaling molecule Bruton's tyrosine kinase (BTK) in circulating B cells from ACPA-positive RA patients, compared with ACPA-negative RA patients and healthy controls (HCs) [12]. In line with these findings, others also reported enhanced spleen tyrosine kinase (SYK) activation in circulating B cells from ACPA-positive RA patients [13]. Following these findings in RA, we showed the presence of enhanced BCR signaling specifically in the naïve B cell population in other diseases with autoimmune involvement [14,15]. However, comprehensive studies on BCR signaling in individual B cell subsets in RA are largely lacking. In addition, differences in B cell phenotype and BCR signaling between autoantibody-positive and -negative RA are largely unknown.

We therefore aimed to identify differences in B cell phenotype and BCR signaling between autoantibody-positive and -negative RA patients compared with HCs. Previously published methods either did not differentiate between all individual B cell subsets or did not allow for BCR isotype-specific analysis [[16], [17], [18], [19]]. Therefore, we first optimized a phosphoflow protocol to study BCR signaling in the primary human B cell compartment, both in resting B cells and following isotype-specific stimulation, in a healthy donor (HD) cohort. This protocol was then used to comprehensively study activation of a range of BCR signaling proteins in circulating B cells from autoantibody-positive RA and autoantibody-negative RA patients.