The most important feature of B cells is the production of Abs upon activation; additionally, B cells produce pro- and anti-inflammatory cytokines in response to certain stimuli. IL-10–producing B cells represent a major subset of regulatory B cells (Bregs) that suppress autoimmune and inflammatory responses. B cells play a crucial role in the development and maintenance of the chronic inflammatory autoimmune disease rheumatoid arthritis (RA); however, controversial data are available on IL-10– producing Bregs in RA. Our aim was to identify the optimal conditions that induce IL-10⁺ Bregs and, furthermore, to shed light on the signaling pathways that are responsible for their expansion. The results show that dual stimulation by CpG and CD40L for 48 h is optimal for IL-10 induction, and this can be synergistically boosted by IL-21. We identified the CD19⁺CD27⁺ memory B cell population as the major source of IL-10⁺ Bregs. We detected significantly fewer CD19⁺CD27⁺IL-10⁺ cells in RA patients compared with healthy controls, and these were functionally defective in suppressing IFN-γ production by CD4⁺ T cells in coculture. IL-21 drastically increased the number of IL-10⁺ Bregs within the CD19⁺CD27⁺ and CD19⁺CD27⁻ populations; furthermore, it induced the appearance of IL-10⁺Blimp-1⁺ plasmablasts. Monitoring the phosphorylation of key signaling molecules revealed that activation of ERK, p38, and CREB is indispensable for the induction of IL-10 production, whereas phosphorylation of STAT3 further enhances IL-10 expression in human Bregs. We conclude that CREB and STAT3 are the key transcription factors responsible for the expansion and differentiation of human IL-10–producing Bregs.

Anti-citrullinated protein antibodies (ACPAs) are involved in the pathogenesis of rheumatoid arthritis. N-glycosylation pattern of ACPA-IgG and healthy IgG Fc differs. The aim of this study is to determine the relative sialylation and galactosylation level of ACPAs and control IgG to assess their capability of inducing TNFα production, and furthermore, to analyze the correlations between the composition of Fc glycans and inflammatory markers in RA. We isolated IgG from sera of healthy volunteers and RA patients, and purified ACPAs on a citrulline-peptide column. Immunocomplexes (IC) were formed by adding an F(ab)₂ fragment of anti-human IgG. U937 cells were used to monitor the binding of IC to FcγR and to trigger TNFα release determined by ELISA. To analyze glycan profiles, control IgG and ACPA-IgG were digested with trypsin and the glycosylation patterns of glycopeptides were analyzed by determining site-specific N-glycosylation using nano-UHPLC-MS/MS. We found that both sialylation and galactosylation levels of ACPA-IgG negatively correlate with inflammation-related parameters such as CRP, ESR, and RF. Functional assays show that dimerized ACPA-IgG significantly enhances TNFα release in an FcγRI-dependent manner, whereas healthy IgG does not. TNFα production inversely correlates with the relative intensities of the G0 glycoform, which lacks galactose and terminal sialic acid moieties.

In mice, marginal zone (MZ) B cells are found principally in the MZ of the spleen and characterized as CD23‐negative cells, primarily express polyreactive BCRs, high levels of complement receptor‐2 and TLRs. Collagen‐induced arthritis (CIA) is a commonly used animal model of human rheumatoid arthritis, considered as a Th1‐mediated disease. Although the importance of MZ B cells in the initiation of CIA is well established, their role in remission is unexplored. Besides, playing a central role in Th1 cell development, T‐box transcription factor (T‐bet) has important functions in B cells. T‐bet is regulated by IFN‐γ and through the BCR and TLR9, the signals that have an impact on regulatory IL‐10 production. In this work, we aimed to analyze the contribution of T‐bet to the function of IL‐10‐positive MZ B cells. We demonstrate that during the remission phase of CIA, MZ B cells express an elevated level of T‐bet and confirm the existence of IL‐10/T‐bet coexpressing cells. Moreover, we show that T‐bet‐expressing MZ B cells migrate toward CXCR3 ligand and secrete IL‐10 by inflammatory stimuli. Our data suggest that T‐bet might contribute to the remission of CIA by facilitating the regulatory potential of IL‐10‐positive MZ B cells.

Background: In rheumatoid arthritis (RA), anti-citrullinated protein/peptide antibodies (ACPAs) are responsible for disease onset and progression, however, our knowledge is limited on ligand binding affinities of autoantibodies with different citrulline-peptide specificity. Methods: Citrulline-peptide-specific ACPA IgGs were affinity purified and tested by ELISA. Binding affinities of ACPA IgGs and serum antibodies were compared by surface plasmon resonance (SPR) analysis. Bifunctional nanoparticles harboring a multi-epitope citrulline-peptide and a complement-activating peptide were used to induce selective depletion of ACPA-producing B cells. Results: K_D values of affinity-purified ACPA IgGs varied between 10⁻⁶ and 10⁻⁸ M and inversely correlated with disease activity. Based on their cross-reaction with citrulline-peptides, we designed a novel multi-epitope peptide, containing Cit-Gly and Ala-Cit motifs in two–two copies, separated with a short, neutral spacer. This peptide detected antibodies in RA sera with 66% sensitivity and 98% specificity in ELISA and was recognized by 90% of RA sera, while none of the healthy samples in SPR. When coupled to nanoparticles, the multi-epitope peptide specifically targeted and depleted ACPA-producing B cells ex vivo. Conclusions: The unique multi-epitope peptide designed based on ACPA cross-reactivity might be suitable to develop better diagnostics and novel therapies for RA.