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B cells are postulated to be central in seropositive rheumatoid arthritis (RA). Here, we use exploratory mass cytometry (n = 23) and next-generation sequencing (n = 19) to study B-cell repertoire shifts in RA patients. Expression of several B-cell markers were significantly different in ACPA(+) RA compared to healthy controls, including an increase in HLA-DR across subsets, CD22 in clusters of IgM(+) B cells and CD11c in IgA(+) memory. Moreover, both IgA(+) and IgG(+) double negative (IgD(-) CD27(-)) CD11c(+) B cells were increased in ACPA+ RA, and there was a trend for elevation in a CXCR5/CCR6(high) transitional B-cell cluster. In the RA BCR repertoire, there were significant differences in subclass distribution and, notably, the frequency of VH with low somatic hypermutation (SHM) was strikingly higher, especially in IgG1 (p < 0.0001). Furthermore, both ACPA(+) and ACPA(-) RA patients had significantly higher total serum IgA and IgM compared to controls, based on serology of larger cohorts (n = 3494 IgA; n = 397 IgM). The observed elevated Ig-levels, distortion in IgM(+) B cells, increase in double negative B cells, change in B-cell markers, and elevation of unmutated IgG(+) B cells suggests defects in B-cell tolerance in RA. This may represent an underlying cause of increased polyreactivity and autoimmunity in RA.

Objective: Antibodies against posttranslationally modified proteins are a hallmark of rheumatoid arthritis (RA), but the emergence and pathogenicity of these autoantibodies are still incompletely understood. The aim of this study was to analyze the antigen specificities and mutation patterns of monoclonal antibodies (mAb) derived from RA synovial plasma cells and address the question of antigen cross-reactivity. Methods: IgG-secreting cells were isolated from RA synovial fluid, and the variable regions of the immunoglobulins were sequenced (n = 182) and expressed in full-length mAb (n = 93) and also as germline-reverted versions. The patterns of reactivity with 53,019 citrullinated peptides and 49,211 carbamylated peptides and the potential of the mAb to promote osteoclastogenesis were investigated. Results: Four unrelated anti-citrullinated protein autoantibodies (ACPAs), of which one was clonally expanded, were identified and found to be highly somatically mutated in the synovial fluid of a patient with RA. The ACPAs recognized >3,000 unique peptides modified by either citrullination or carbamylation. This highly multireactive autoantibody feature was replicated for Ig sequences derived from B cells from the peripheral blood of other RA patients. The plasma cell-derived mAb were found to target distinct amino acid motifs and partially overlapping protein targets. They also conveyed different effector functions as revealed in an osteoclast activation assay. Conclusion: These findings suggest that the high level of cross-reactivity among RA autoreactive B cells is the result of different antigen encounters, possibly at different sites and at different time points. This is consistent with the notion that RA is initiated in one context, such as in the mucosal organs, and thereafter targets other sites, such as the joints.

QC 20171031

Background: Interleukin-10 is a pivotal immunoregulatory cytokine with pleiotropic effects on the immune system. IL-10 promoter polymorphisms have been associated with disease susceptibility and the ability to secrete IL-10 in vitro. We suspected that the association of the widely studied -819C/T and -592C/A polymorphisms with the IL-10 production might vary between ethnic groups. Therefore, we examined the association of -819 C/T and -592 C/A promoter polymorphisms with in vitro LPS stimulated secretion of IL-10 in normal healthy Indian volunteers. Methods: Peripheral blood was collected from 103 healthy volunteers and diluted whole blood cultures were set up with 100 ng/ml of LPS as stimulant: supernatant was collected at 24 h and IL-10 levels were assayed by ELISA. Genotyping was done for -819C/T polymorphism in 101 individuals and -592C/A polymorphism in 68 individuals by polymerase chain reaction followed by RFLP. The differences in IL-10 production between the genotypes were analysed by ANOVA. Results: There were 30, 47 and 24 individuals with the CC, CT and TT genotypes with a minor allele (T) frequency of 47% for the -819C/T polymorphism. The CC and TT genotypes at position -819 were strongly associated with CC and AA genotypes at -592 position suggestive of strong linkage disequilibrium. There was no association between the -819 genotype and the in vitro LPS stimulated IL-10 levels. Conclusions: The -819C/T and the -592 C/A polymorphisms of the IL-10 promoter region are not significantly associated with LPS stimulated IL-10 production healthy Indian subjects. Disclosure statement: All authors have declared no conflicts of interest

The activation of leukocytes during inflammation results in the engagement of many related intracellular signalling pathways. The role of two key proteins families, phosphodiesterase (PDE) and Src kinase, has been assessed in these signalling pathways using specific inhibitors. Inhibition of phosphodiesterase type 4 (PDE4) in lipopolysaccharide (EPS) stimulated human peripheral blood mononuclear cells (PBMC) resulted in the inhibition of TNF-a by an IL-10-independent mechanism. This is in contrast to other agents that elevate cAMP which in turn inhibit TNF-a synthesis by an IL-10-dependent mechanism. When PBMC were stimulated with EPS plus IFN-y, inhibition of PDE4 resulted in an altered effect on cytokine production, elevating IE-10 while still inhibiting TNF-a. Inhibition of PDE4 in activated T cells only weakly inhibited cellular proliferation. In contrast, PDE4 inhibitors were potent but non-selective inhibitors of T-cell cytokine production. In contrast to studies in murine cells, inhibition of T-cell activation correlates with inhibition of binding to a high-affinity Rolipram binding site on the PDE4 protein. Kinases from the Src family (Eck, Fyn and Eyn) have been implicated in signalling via the T-cell receptor (TCR) on lymphocytes and the high affinity IgE receptor (FceRI) on mast cells. Potent inhibitors of Src kinases blocked T-cell proliferation and cytokine generation in response to specific ligation of the TCR. However, activation of T-cells by multiple costimulatory pathways was much more resistant to Src kinase inhibition. Inhibition of Src kinase in human cord blood-derived mast cells inhibited IgE-dependent degranulation of these cells. This indicates that Src kinase inhibitors may be useful in down-regulating allergic responses. Conversely, inhibitors of PDE4 and PDE type 7 (PDE7) did not prevent mast cell degranulation. The observation that PDE4 inhibitors can regulate mo

The activation of leukocytes during inflammation results in the engagement of many related intracellular signalling pathways. The role of two key proteins families, phosphodiesterase (PDE) and Src kinase, has been assessed in these signalling pathways using specific inhibitors. Inhibition of phosphodiesterase type 4 (PDE4) in lipopolysaccharide (EPS) stimulated human peripheral blood mononuclear cells (PBMC) resulted in the inhibition of TNF-a by an IL-10-independent mechanism. This is in contrast to other agents that elevate cAMP which in turn inhibit TNF-a synthesis by an IL-10-dependent mechanism. When PBMC were stimulated with EPS plus IFN-y, inhibition of PDE4 resulted in an altered effect on cytokine production, elevating IE-10 while still inhibiting TNF-a. Inhibition of PDE4 in activated T cells only weakly inhibited cellular proliferation. In contrast, PDE4 inhibitors were potent but non-selective inhibitors of T-cell cytokine production. In contrast to studies in murine cells, inhibition of T-cell activation correlates with inhibition of binding to a high-affinity Rolipram binding site on the PDE4 protein. Kinases from the Src family (Eck, Fyn and Eyn) have been implicated in signalling via the T-cell receptor (TCR) on lymphocytes and the high affinity IgE receptor (FceRI) on mast cells. Potent inhibitors of Src kinases blocked T-cell proliferation and cytokine generation in response to specific ligation of the TCR. However, activation of T-cells by multiple costimulatory pathways was much more resistant to Src kinase inhibition. Inhibition of Src kinase in human cord blood-derived mast cells inhibited IgE-dependent degranulation of these cells. This indicates that Src kinase inhibitors may be useful in down-regulating allergic responses. Conversely, inhibitors of PDE4 and PDE type 7 (PDE7) did not prevent mast cell degranulation. The observation that PDE4 inhibitors can regulate mo

Background: Interleukin-10 is a pivotal immunoregulatory cytokine with pleiotropic effects on the immune system. IL-10 promoter polymorphisms have been associated with disease susceptibility and the ability to secrete IL-10 in vitro. We suspected that the association of the widely studied -819C/T and -592C/A polymorphisms with the IL-10 production might vary between ethnic groups. Therefore, we examined the association of -819 C/T and -592 C/A promoter polymorphisms with in vitro LPS stimulated secretion of IL-10 in normal healthy Indian volunteers. Methods: Peripheral blood was collected from 103 healthy volunteers and diluted whole blood cultures were set up with 100 ng/ml of LPS as stimulant: supernatant was collected at 24 h and IL-10 levels were assayed by ELISA. Genotyping was done for -819C/T polymorphism in 101 individuals and -592C/A polymorphism in 68 individuals by polymerase chain reaction followed by RFLP. The differences in IL-10 production between the genotypes were analysed by ANOVA. Results: There were 30, 47 and 24 individuals with the CC, CT and TT genotypes with a minor allele (T) frequency of 47% for the -819C/T polymorphism. The CC and TT genotypes at position -819 were strongly associated with CC and AA genotypes at -592 position suggestive of strong linkage disequilibrium. There was no association between the -819 genotype and the in vitro LPS stimulated IL-10 levels. Conclusions: The -819C/T and the -592 C/A polymorphisms of the IL-10 promoter region are not significantly associated with LPS stimulated IL-10 production healthy Indian subjects. Disclosure statement: All authors have declared no conflicts of interest