Background CD22 is a trans-membrane protein exclusively expressed on B cells which regulates adhe... more Background CD22 is a trans-membrane protein exclusively expressed on B cells which regulates adhesion/migration and inhibits cellular responses to B-cell receptor (BCR) stimulation[1]. Spleen tyrosine kinase (Syk) and subsequent phospholipase C (PLC)-γ2-triggered Ca2+ fluxes are rapidly activated after BCR stimulation. CD22 binds specifically to α-2,6-sialic acid residues present on a broad number of proteins, including CD22 itself. Sialo-interactions appear to be crucial for optimal function of CD22 and can occur in cis (to ligands on the same cell surface) or trans (to ligands on neighboring cells). Objectives The humanized anti-CD22 monoclonal antibody epratuzumab, currently being tested in clinical trials, modulates adhesion molecule expression and B cell migration in vitro[2] however, the potential of CD22-ligation with cis and trans sialic acid to regulate BCR signaling has not been delineated. Methods In vitro effects of epratuzumab on BCR-induced signaling were evaluated by analyzing the phosphorylation of BCR-signaling molecules (Syk/PLC-γ2) and Ca2+ flux. The influence of epratuzumab on the phosphorylation of the BCR signaling molecules Syk and PLC-γ2 was studied in vitro by flow cytometry in PBMCs from healthy donors. To investigate the effect of sialo-interaction on the effects by epratuzumab, PBMCs were treated with neuraminidase (to remove sialic acic) and BCR induced phosphorylation (Syk and PLC-γ2) was detected with or without epratuzumab incubation. To monitor the concentration of intracellular Ca2+, B cells pre-loaded with Indo-1AM were incubated with or without F(ab’)2-epratuzumab for 60 minutes at 37°C and were subsequently stimulated with F(ab’)2 anti-IgM/IgG. Intracellular Ca2+ concentrations were monitored by flow cytometry over 10 minutes after activation. Results Epratuzumab inhibited Syk and PLC-γ2 phosphorylation after in vitro BCR stimulation by 23±9% and 33±5, respectively. Interestingly, preventing sialo-interactions of CD22 partially reduced the inhibitory effect of epratuzumab on Syk and PLC-γ2 phosphorylation to 12±6% and 22±4%, respectively. The inhibition of kinase phosphorylation was demonstrated in both CD27- and CD27+ memory B cells and appeared to be independent of FcR signaling since a F(ab’)2 fragment of epratuzumab induced comparable results to the full antibody. In addition, a F(ab’)2 fragment of epratuzumab reduced the BCR-induced calcium flux. These data are consistent with the potential of targeting CD22 to raise the threshold of BCR activation which also provides additional control of B cell function. Conclusions Intracellular BCR signals can be partially inhibited by CD22 ligation with epratuzumab, providing further understanding of targeting CD22 biology in human autoimmune diseases. This study was supported by Sonderforschungsbereich 650 and DFG491/7-1 and in part by UCB Pharma Inc. References Nitschke L. The role of CD22 and other inhibitory co-receptors in B-cell activation. Current opinion in immunology 2005;17:290–7. Daridon C, Blassfeld D, Reiter K, et al. Epratuzumab targeting of CD22 affects adhesion molecule expression and migration of B-cells in systemic lupus erythematosus. Arthritis Research & Therapy 2010;12:R204. Disclosure of Interest N. Sieger: None Declared, S. Fleischer: None Declared, H. Mei: None Declared, K. Reiter: None Declared, A. Shock Employee of: UCB Pharma inc., G. Burmester: None Declared, C. Daridon: None Declared, T. Dörner Grant/Research support from: Immunomedics
The anti-CD20 antibody rituximab depletes human B cells from peripheral blood, but it remains con... more The anti-CD20 antibody rituximab depletes human B cells from peripheral blood, but it remains controversial to what extent tissueresident B cells are affected. In representative patients with rheumatoid arthritis, we here demonstrate that recently activated presumably short-lived plasmablasts expressing HLA-DR high and Ki-67 continuously circulate in peripheral blood after B-cell depletion by rituximab at 26%-119% of their initial numbers. They circulate independent of splenectomy, express immunoglobulin A (IgA),  7 integrin, and CC motif receptor 10 (CCR10) and migrate along CCL28 gradients in vitro, suggesting their mucosal origin. These plasmablasts express somatically hypermutated V H gene rearrangements and spontaneously secrete IgA, exhibiting binding to microbial antigens. Notably, IgA ؉ plasmablasts and plasma cells were identified in the lamina propria of patients treated with rituximab during peripheral B-cell depletion. Although a relation of these "steady state"-like plasmablasts with rheumatoid arthritis activity could not be found, their persistence during B-cell depletion indicates that their precursors, that is, B cells resident in the mucosa are not deleted by this treatment. These data suggest that a population of mucosal B cells is self-sufficient in adult humans and not replenished by CD20 ؉ B cells immigrating from blood, lymphoid tissue, or bone marrow, that is, B cells depleted by rituximab. (Blood. 2010; 116(24):5181-5190) Methods A detailed description of the patients, samples, and methods are available as supplemental Methods (available on the Blood Web site; see the Supplemental Materials link at the top of the online article).
Background: RA is an autoimmune condition characterized by chronic joint inflammation. Tfh cells,... more Background: RA is an autoimmune condition characterized by chronic joint inflammation. Tfh cells, a recently described Th subset localized in lymphoid organs, help B cell differentiation and function. Circulating central memory CD4 T cells expressing CXCR5 can be subdivided into three different subpopulations based on the combined expression of surface CXCR5 with or without CCR6 and/or CXCR3: CXCR5+CXCR3+CCR6-(Tfh-Th1), CXCR5+CXCR3-CCR6+ (Tfh-Th17) and CXCR5+CXCR3-CCR6-(Tfh-Th2). Only Tfh-Th17 and Tfh-Th2 cells have been demonstrated to display functional properties of Tfh cells; in contrast Tfh-Th1 cells are unable to provide B cell help. An altered proportion of these subpopulations has been associated with autoimmune diseases. Objectives: To study the frequency of circulating CXCR5+CD4+ T cell subsets together with the frequency of circulating plasmablasts (CD19+CD20-CD27+CD38high B cells) in patients with RA. Methods: Peripheral blood was drawn from healthy controls (n=27) and RA patients (n=27). After isolation by Ficoll-Hypaque gradient, PBMCs were stained with antibodies to CD3, CD4, CXCR5, CCR6, CXCR3, CD19, CD20, CD27, and CD38, and examined by flow cytometry. The percentages of CXCR5+CXCR3+CCR6-(Tfh-Th1), CXCR5+CXCR3-CCR6+ (Tfh-Th17) and CXCR5+CXCR3-CCR6-(Tfh/Th2) cells were calculated after gating for CD3, CD4 and CXCR5+. The percentage of CD20-CD38high cells was calculated after gating for CD19+ and CD27+. Results: The frequency of circulating CXCR5+ cells gated for CD4+ T cells was not different among the studied groups. In contrast, in RA patients, the frequency of Tfh-Th1 cells was significantly decreased and the frequency of Tfh-Th17 and Tfh-Th2 cells was significantly increased as compared with controls. Subsequently, the ratio (Tfh-Th17+Tfh-Th2)/Tfh-Th1 was increased in RA patients. That is, RA patients demonstrate a higher proportion of Tfh cells bearing a phenotype associated with B cell helping capacity. At the same time, the frequency of circulating plasmablasts was increased in RA patients as compared with controls. Interestingly, there was a significantly positive correlation between the percentage of circulating plasmablasts and the ratio (Tfh-Th17+Tfh-Th2)/Tfh-Th1, together with a significantly negative correlation between the percentage of circulating plasmablasts and the percentage of circulating Tfh-Th1 cells. Conclusions: RA patients demonstrate altered proportions of circulating Tfh subpopulations, with overrepresentation of subsets bearing a phenotype associated with B cell helping capacity. At the same time, an increased proportion of circulating plasmablasts is apparent in RA patients.
Abnormalities of memory B cells seem to be closely involved in the pathogenesis of primary Sjögre... more Abnormalities of memory B cells seem to be closely involved in the pathogenesis of primary Sjögrens Syndrome (pSS) and its malignant complication, B cell lymphoma. Recent studies on B cells in pSS add to our understanding of the distinct memory B cell subsets in pSS. Reduction of peripheral memory CD27 + B cells, most strikingly of the CD27 + IgM + subset, may indicate a lack of appropriate censoring mechanisms and incomplete differentiation processes within the ectopic lymphoid tissues in pSS. This ectopically formed lymphoid tissue might protect autoreactive memory B cells from deletion by physiological checkpoints and, thereby, may contribute to the perpetuation of the disease as well as to an enhanced lymphoma risk. Thus, B cells may be potential targets of direct or indirect treatment in pSS.
Introduction: Systemic lupus erythematosus (SLE) is an autoimmune disease associated with a break... more Introduction: Systemic lupus erythematosus (SLE) is an autoimmune disease associated with a break in self-tolerance reflected by a production of antinuclear autoantibodies. Since autoantibody production can be activated via nucleic acid Toll-like receptor 9 (TLR9), the respective pathway has been implicated in the development of SLE and pathogenic B cell responses. However, the response of B cells from SLE patients to TLR9 stimulation remains incompletely characterized. Methods: In the current study, the response of B cells from SLE patients and healthy donors upon TLR9 stimulation was analyzed in terms of proliferation and cytokine production and correlated with the lupus disease activity and anti-dsDNA titers. Results: B cells from SLE patients showed a reduced response to TLR9 agonist compared to B cells from healthy donors in terms of proliferation and activation. B cells from SLE patients with higher disease activity produced less interleukin (IL)-6, IL-10, vascular endothelial growth factor, and IL-1ra than B cells from healthy donors. Further analyses revealed an inverse correlation of cytokines produced by TLR9-stimulated B cells with lupus disease activity and anti-dsDNA titer, respectively. Conclusion: The capacity of B cells from lupus patients to produce cytokines upon TLR9 engagement becomes less efficient with increasing disease activity, suggesting that they either enter an exhausted state or become tolerant to TLR stimulation for cytokine production when disease worsens.
Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by a breakdown of self-... more Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by a breakdown of self-tolerance, autoantibody production, B cell hyperreactivity and an impaired B cell homeostasis. Recent data suggested that an impaired B cell response could also lead to autoimmunity. In this study, the BCR downstream signaling spleen tyrosine kinase Syk of healthy donors (HD), SLE, rheumatoid arthritis (RA) and primary Sjögren’s syndrome (pSS) patients was analyzed in detail and revealed new insights into peripheral B cell abnormalities based on intracellular characteristics. Interestingly, SLE B cells showed a significantly lower but long-lasting Syk (Y352) phosphorylation after BCR engagement compared to HD (p<0.05). Furthermore, a significant enlarged frequency of Syk(bright) B cell population has been identified within the CD27(-) B cell subset in the blood of SLE patients (p<0.001), but no correlation with the disease activity was observed. Notably, the frequency of Syk(bright) B cells was not increased in RA or pSS patients. Further characterization showed that Syk(bright) cells exhibit an increased basal level of phosphorylated Syk, expressed higher levels of CD19 and CD95 and mainly do not express CD38 as compared to Syk(dim) cells. Thus, SLE patients show a diminished but long-lasting BCR response and exhibit an enlarged unique Syk(bright) B cell subset that could be responsible for the hyperactivity of SLE B cells.
During mucosal immune responses, IgA-secreting plasmablasts are generated from mucosal B cells. W... more During mucosal immune responses, IgA-secreting plasmablasts are generated from mucosal B cells. We previously demonstrated that at steady-state, most human peripheral blood antibody-secreting cells produce IgA and express mucosal homing receptors, and suggested their generation in mucosal immune responses. In the current study, we analyzed the blood of patients who received B cell depletion therapy with rituximab (anti-CD20) for treatment of rheumatoid arthritis before and at 2-9months after rituximab infusion by flow cytometry. While CD20+ B cells were reduced to <0.02% of their intial numbers before therapy, CD19+/CD27hi/CD20- antibody-secreting cells remained detectable in the blood at 26-119% of their initial numbers. These circulating antibody-secreting cells expressed IgA that bound to bacterial antigens, beta7 integrin and CCR10 before and during B cell depletion, suggesting their mucosal origin. High expression of HLA-DR and Ki-67 and in vitro migration towards CCL28 and CXCL12 qualified these cells as recently activated plasmablasts, reflecting the survival of functional B cells during rituximab treatment. Consistently, IgA+ plasmablasts and plasma cells were identified in the lamina propria during B cell depletion. Our data implicate that a population of mucosal B cells is resistant to rituximab and self-sufficient in adult humans and not replenished by CD20+ B cells immigrating from blood, lymphoid tissue, or bone marrow, that is, B cells depleted by rituximab.
Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by a breakdown of self-... more Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by a breakdown of self-tolerance, autoantibody production, B cell hyperreactivity and an impaired B cell homeostasis. Recent data suggested that an impaired B cell response could also lead to autoimmunity. In this study, the BCR downstream signaling spleen tyrosine kinase Syk of healthy donors (HD), SLE, rheumatoid arthritis (RA) and primary Sjögren’s syndrome (pSS) patients was analyzed in detail and revealed new insights into peripheral B cell abnormalities based on intracellular characteristics. Interestingly, SLE B cells showed a significantly lower but long-lasting Syk (Y352) phosphorylation after BCR engagement compared to HD (p<0.05). Furthermore, a significant enlarged frequency of Syk(bright) B cell population has been identified within the CD27(-) B cell subset in the blood of SLE patients (p<0.001), but no correlation with the disease activity was observed. Notably, the frequency of Syk(brigh...
Background: RA is an autoimmune condition characterized by chronic joint inflammation. Tfh cells,... more Background: RA is an autoimmune condition characterized by chronic joint inflammation. Tfh cells, a recently described Th subset localized in lymphoid organs, help B cell differentiation and function. Circulating central memory CD4 T cells expressing CXCR5 can be subdivided into three different subpopulations based on the combined expression of surface CXCR5 with or without CCR6 and/or CXCR3: CXCR5+CXCR3+CCR6-(Tfh-Th1), CXCR5+CXCR3-CCR6+ (Tfh-Th17) and CXCR5+CXCR3-CCR6-(Tfh-Th2). Only Tfh-Th17 and Tfh-Th2 cells have been demonstrated to display functional properties of Tfh cells; in contrast Tfh-Th1 cells are unable to provide B cell help. An altered proportion of these subpopulations has been associated with autoimmune diseases. Objectives: To study the frequency of circulating CXCR5+CD4+ T cell subsets together with the frequency of circulating plasmablasts (CD19+CD20-CD27+CD38high B cells) in patients with RA. Methods: Peripheral blood was drawn from healthy controls (n=27) and RA patients (n=27). After isolation by Ficoll-Hypaque gradient, PBMCs were stained with antibodies to CD3, CD4, CXCR5, CCR6, CXCR3, CD19, CD20, CD27, and CD38, and examined by flow cytometry. The percentages of CXCR5+CXCR3+CCR6-(Tfh-Th1), CXCR5+CXCR3-CCR6+ (Tfh-Th17) and CXCR5+CXCR3-CCR6-(Tfh/Th2) cells were calculated after gating for CD3, CD4 and CXCR5+. The percentage of CD20-CD38high cells was calculated after gating for CD19+ and CD27+. Results: The frequency of circulating CXCR5+ cells gated for CD4+ T cells was not different among the studied groups. In contrast, in RA patients, the frequency of Tfh-Th1 cells was significantly decreased and the frequency of Tfh-Th17 and Tfh-Th2 cells was significantly increased as compared with controls. Subsequently, the ratio (Tfh-Th17+Tfh-Th2)/Tfh-Th1 was increased in RA patients. That is, RA patients demonstrate a higher proportion of Tfh cells bearing a phenotype associated with B cell helping capacity. At the same time, the frequency of circulating plasmablasts was increased in RA patients as compared with controls. Interestingly, there was a significantly positive correlation between the percentage of circulating plasmablasts and the ratio (Tfh-Th17+Tfh-Th2)/Tfh-Th1, together with a significantly negative correlation between the percentage of circulating plasmablasts and the percentage of circulating Tfh-Th1 cells. Conclusions: RA patients demonstrate altered proportions of circulating Tfh subpopulations, with overrepresentation of subsets bearing a phenotype associated with B cell helping capacity. At the same time, an increased proportion of circulating plasmablasts is apparent in RA patients.
Objective. Systemic lupus erythematosus (SLE) is characterized by B cell hyperactivity and autoan... more Objective. Systemic lupus erythematosus (SLE) is characterized by B cell hyperactivity and autoantibody production. As spleen tyrosine kinase (Syk) is pivotal in B cell activation, these experiments aimed to examine the extent to which Syk was abnormally expressed in SLE B cells and the nature of the B cell subset that differently expressed Syk. Methods. B cells from healthy donors and SLE patients were analyzed by flow cytometry to assess basal expression of Syk and phosphorylated Syk. B cell subsets expressing higher levels of Syk were found, and their detailed phenotype, in vitro differentiation into plasmablasts/plasma cells, and Syk induction by cytokines were determined. Results. Syk expression was higher in CD27؉ memory B cells than in naive B cells from SLE patients. However, a significantly increased frequency of CD27؊ B cells with bright expression of Syk (Syk؉؉) was found in SLE patients. CD27؊Syk؉؉ B cells showed enhanced basal expression of p-Syk and stronger Syk phosphorylation upon B cell receptor (BCR) engagement as compared to CD27؊Syk؉ B cells. CD27؊Syk؉؉ B cells were CD38؊ as well as CD19؉؉, CD20؉؉, and mainly CD21؊, with decreased ABCB1 transporter activity. In contrast to CD27؊Syk؉ B cells, CD27؊Syk؉؉ B cells exhibited enhanced differentiation into CD27؉؉ IgG-secreting cells and expressed somatically mutated BCR gene rearrangements. Syk؉؉ B cells were inducible in vitro by stimulation with interferon-␥, lipopolysaccharide, or tumor necrosis factor ␣. Conclusion. SLE patients exhibit an increased frequency of hitherto unknown CD27؊Syk؉؉ memorylike B cells, indicating that intracellular Syk density could distinguish CD27؊ memory B cells from truly naive B cell subsets. Furthermore, the CD27؊Syk؉؉ subset is a candidate for a source of increased plasma cells in SLE.
Abnormalities of memory B cells seem to be closely involved in the pathogenesis of primary Sjögre... more Abnormalities of memory B cells seem to be closely involved in the pathogenesis of primary Sjögrens Syndrome (pSS) and its malignant complication, B cell lymphoma. Recent studies on B cells in pSS add to our understanding of the distinct memory B cell subsets in pSS. Reduction of peripheral memory CD27 + B cells, most strikingly of the CD27 + IgM + subset, may indicate a lack of appropriate censoring mechanisms and incomplete differentiation processes within the ectopic lymphoid tissues in pSS. This ectopically formed lymphoid tissue might protect autoreactive memory B cells from deletion by physiological checkpoints and, thereby, may contribute to the perpetuation of the disease as well as to an enhanced lymphoma risk. Thus, B cells may be potential targets of direct or indirect treatment in pSS.
ObjectiveTo identify the cells that produce BAFF in the salivary glands of patients with primary ... more ObjectiveTo identify the cells that produce BAFF in the salivary glands of patients with primary Sjögren's syndrome (SS), and to analyze BAFF receptor expression by local T and B lymphocytes.MethodsWe used 3 methods to identify the source of BAFF: in situ hybridization of the transcripts for BAFF combined with staining of membrane markers, regular and real‐time reverse transcription–polymerase chain reaction (RT‐PCR) of cultured epithelial cells, and RT‐PCR of sorted single‐cell T and B lymphocytes eluted from salivary glands. Cells expressing TACI, BCMA, and B lymphocyte stimulator receptor 3 (BR‐3) were disclosed by combining each specific staining of the receptors with each specific staining of the cells. The function of BAFF generated by epithelial cells on B lymphocytes was determined in short‐term cocultures.ResultsTranscripts for BAFF were seen in epithelial cells and infiltrating T lymphocytes and, for the first time, were detected in local B cells. It is interesting tha...
Background CD22, a transmembrane protein exclusively expressed on B cells, mediates migration by ... more Background CD22, a transmembrane protein exclusively expressed on B cells, mediates migration by cell–cell interaction and negatively regulates B-cell receptor (BCR) signaling. CD22 acts as an inhibitory co-receptor of the BCR via de-phosphorylation of signaling molecules such as spleen tyrosine kinase (Syk) and subsequent phospholipase C (PLC)-γ2-triggered Ca2+ fluxes. The humanized anti-CD22 monoclonal antibody epratuzumab modulates adhesion molecule expression and B-cell migration in vitro. However, the potential of CD22 to regulate BCR signaling has not been fully delineated. Objectives As toll-like receptor (TLR)-dependent activation of B cells represents an important pathway of B-cell hyperactivity in autoimmunity, our studies addressed whether epratuzumab might be able to modulate BCR signaling pathways in TLR9 pre-activated B cells or whether TLR9 activation abrogates the inhibitory effect of epratuzumab on the BCR signaling. Methods Our study focused on the influence of epratuzumab on the B cells’ response to BCR alone or in combination with TLR9. The recruitment of CD22 to the BCR (CD79α) after epratuzumab incubation on B cells from healthy volunteers was analyzed by confocal microscopy. The in vitro effects of epratuzumab on BCR-induced signaling were evaluated by analyzing the phosphorylation status of BCR-signaling molecules, Syk and PLC-γ2 by flow cytometry with or without TLR9 pre-activation. The concentration of intracellular Ca2+ was monitored by flow cytometry after BCR stimulation. Finally, to evaluate the origin of the Ca2+ flux, extracellular Ca2+ was chelated with 1 mM ethylene glycol tetraacetic acid (EGTA). Results B cells incubated with epratuzumab showed a specific co-localization of CD22 to the BCR-associated molecule CD79α on B cells. In addition, pre-treatment with epratuzumab led to a reduction in the MFI of phosphorylated Syk and PLC-γ2 induced by BCR stimulation compared with IgG1 isotype control. Similar results were observed when the cells were pre-incubated with F(ab’)2 fragment of epratuzumab, which excludes an inhibitory effect dependent on FcR signaling. Interestingly, the reduction of BCR induced kinase phosphorylation was demonstrated in both CD27- and CD27+ memory B cells. In addition, pre-activation of B cells by TLR9 did not circumvent the influence of epratuzumab on BCR signaling, as shown by a reduction in the MFI of phosphorylated Syk and PLC-γ2 in comparison to non-activated cells. Finally, a F(ab’)2 fragment of epratuzumab reduced BCR-induced calcium mobilization. Conclusions Intracellular BCR signals can be modulated by CD22 ligation using epratuzumab, and pre-activation with TLR9 did not circumvent this effect. These data expand the potential mechanisms of action of epratuzumab. Disclosure of Interest N. Sieger Grant/research support from: UCB Pharma, S. Fleischer Grant/research support from: UCB Pharma, K. Reiter Grant/research support from: UCB Pharma, H. Mei Grant/research support from: UCB Pharma, A. Shock Employee of: UCB Pharma, G. Burmester Grant/research support from: UCB Pharma, C. Daridon Grant/research support from: UCB Pharma, T. Dörner Grant/research support from: UCB Pharma, Consultant for: UCB Pharma
Background:Glucocorticoid (GC) therapy has strong anti-inflammatory effects and helps slow radiog... more Background:Glucocorticoid (GC) therapy has strong anti-inflammatory effects and helps slow radiographic progression in RA1; however, GCs can be associated with adverse events (AEs) such as infection, especially with long-term use and higher doses.Objectives:To evaluate the impact of baseline GCs on the efficacy and safety of upadacitinib (UPA) with or without concomitant conventional synthetic DMARDs (csDMARDs).Methods:In this ad hoc analysis of three Phase 3 studies, patients with inadequate response to MTX (MTX-IR) receiving UPA 15 mg once daily (QD) or placebo (PBO) + csDMARDs in SELECT-NEXT, and MTX-IR/MTX-naïve patients receiving UPA 15 mg QD monotherapy or MTX monotherapy in SELECT-MONOTHERAPY/SELECT-EARLY, respectively, were included. Efficacy outcomes, including measures of remission and low disease activity (LDA) determined by DAS in 28 joints using CRP (DAS28[CRP]; <2.6/≤3.2) and Clinical Disease Activity Index (CDAI; ≤2.8/≤10), were assessed and stratified by baseline ...
The binding of antigen to the B cell receptor (BCR) results in a cascade of signalling events tha... more The binding of antigen to the B cell receptor (BCR) results in a cascade of signalling events that ultimately drive B cell activation. Uncontrolled B cell activation is regulated by negative feedback loops that involve inhibitory co-receptors such as CD22 and CD32B that exert their functions following phosphorylation of immunoreceptor tyrosine-based inhibition motifs (ITIMs). The CD22-targeted antibody epratuzumab has previously been shown to inhibit BCR-driven signalling events, but its effects on ITIM phosphorylation of CD22 and CD32B have not been properly evaluated. The present study therefore employed both immunoprecipitation and flow cytometry approaches to elucidate the effects of epratuzumab on direct phosphorylation of key tyrosine (Tyr) residues on both these proteins, using both transformed B cell lines and primary human B cells. Epratuzumab induced the phosphorylation of Tyr(822) on CD22 and enhanced its co-localisation with SHP-1. Additionally, in spite of high basal ph...
Objective. Systemic lupus erythematosus (SLE) is associated with hyperactivity of B cells and abn... more Objective. Systemic lupus erythematosus (SLE) is associated with hyperactivity of B cells and abnormalities of B cell receptor (BCR) signaling. To address the linkage between dysregulated BCR signaling and increased B cell function, we assessed immediate phosphorylation events in lupus B cells. Methods. B cells from SLE patients and healthy donors were analyzed by flow cytometry to assess phosphorylated CD22, Syk, and Akt as well as the basal expression of the BCR coreceptors CD22 and CD19. Confocal microscopy studies determined the recruitment of CD22 and the tyrosine phosphatase SH2 domain-containing phosphatase 1 to the activated BCR complex. Additionally, phosphatase activity in SLE versus healthy donor B cells was measured. Results. B cells from SLE patients showed diminished Syk phosphorylation and reduced intracellular calcium release after BCR activation as compared to B cells from healthy donors. This was related to an enhanced activity of tyrosine, but not serine/threonine, phosphatases and was corrected by inhibition of tyrosine phosphatase activity. In contrast to reduced Syk phosphorylation after BCR activation, phosphorylation of Akt was significantly increased in SLE B cells. The disturbed balance between Syk and Akt phosphorylation was significantly correlated with B cell survival following BCR engagement. Furthermore, CD272, but not CD271, B cells from SLE patients displayed increased expression and phosphorylation of the inhibitory BCR coreceptor CD22. Conclusion. These results indicate that an imbalance between serine and tyrosine phosphatases in SLE contributes to an intrinsically disturbed balance of BCR-initiated signaling pathways, resulting in enhanced survival of lupus B cells and differentiation into plasma cells. The development, maturation, and selection of B cells are tightly regulated by the affinity and activation threshold of the B cell receptor (BCR). Besides its important role during B cell differentiation and survival, the BCR plays a pivotal role in avoiding autoreactivity by initiating a variety of negative-selection events (1,2). After BCR crosslinking, conformation changes induced by cytoskeleton reorganization lead to a Lyn-dependent phosphorylation of the intracellular Iga/b immunoreceptor tyrosine-based activation motif (ITAM) of the BCR and its coreceptors (e.g., CD19 and CD22), followed by the recruitment and activation of the spleen tyrosine kinase (Syk). Subsequently, signaling molecules, such as Bruton's tyrosine kinase (BTK), phosphatidylinositol 3-kinase (PI3K) and phospholipase Cg2 (PLCg2) accumulate in a signalosome to initiate internalization, antigen processing, calcium release, and a specific gene expression profile (3). Even though Syk can directly activate PI3K (4), a Syk-independent PI3K/ Akt activation has been described (5), subdividing the BCR-associated signaling pathway into two axes: Syk activation linked to PLCg2 phosphorylation and calcium influx, and CD19 phosphorylation followed by activation Supported by the DFG (SFB 650 project TP12, SFB 650 project TP16, SFB 633 A14, SPP ImmunoBone [Do491/8-2], and project Do491/7-3).
Introduction: Cytokines produced by B cells are believed to play important roles in autoimmune di... more Introduction: Cytokines produced by B cells are believed to play important roles in autoimmune diseases. CD22 targeting by epratuzumab has been demonstrated to inhibit phosphorylation of B cell receptor (BCR) downstream signaling in B cells. It has been shown that other sialoadhesin molecules related to CD22 have immunoregulatory functions; therefore, in the present study, we addressed the role of epratuzumab on the production of key cytokines by B cells of patients with systemic lupus erythematosus (SLE) and of healthy donors (HD). Methods: Peripheral blood B cells were purified and activated by BCR with or without Toll-like receptor 9 (TLR9) stimulation in the presence or absence of epratuzumab. Cytokine production by B cells (interleukin [IL]-6, tumor necrosis factor [TNF]-α and IL-10) in the supernatant and the induction of IL-10 + B cells from patients with SLE and HD were analyzed. Results: The secretion of the proinflammatory cytokines TNF-α and IL-6 by anti-BCR and BCR-and/or TLR9-activated B cells from HD and patients with SLE was inhibited by epratuzumab. In contrast, the production of IL-10 by B cells was not affected by epratuzumab under either stimulation condition. Consistently, the induction of IL-10-producing B cells in culture was not affected by epratuzumab. Conclusions: Epratuzumab, by targeting CD22, was able to inhibit the production of the proinflammatory cytokines IL-6 and TNF-α by B cells, in contrast to IL-10, in vitro. These data suggest that targeting CD22 alters the balance between proinflammatory cytokines (TNF-α, IL-6) and the regulatory cytokine IL-10 as another B cell effector mechanism.
Conclusion.-Les patients avec PR ont fréquemment une fonction diastolique ventriculaire gauche al... more Conclusion.-Les patients avec PR ont fréquemment une fonction diastolique ventriculaire gauche altérée et ces variations sont beaucoup mieux analysées au DT qu'en échocardiographie conventionnelle. Le DT semble être un examen précis et fiable permettant d'évaluer l'atteinte myocardique dans la PR et la prédictivité des anomalies détectées est en cours d'évaluation [1].
Background CD22 is a trans-membrane protein exclusively expressed on B cells which regulates adhe... more Background CD22 is a trans-membrane protein exclusively expressed on B cells which regulates adhesion/migration and inhibits cellular responses to B-cell receptor (BCR) stimulation[1]. Spleen tyrosine kinase (Syk) and subsequent phospholipase C (PLC)-γ2-triggered Ca2+ fluxes are rapidly activated after BCR stimulation. CD22 binds specifically to α-2,6-sialic acid residues present on a broad number of proteins, including CD22 itself. Sialo-interactions appear to be crucial for optimal function of CD22 and can occur in cis (to ligands on the same cell surface) or trans (to ligands on neighboring cells). Objectives The humanized anti-CD22 monoclonal antibody epratuzumab, currently being tested in clinical trials, modulates adhesion molecule expression and B cell migration in vitro[2] however, the potential of CD22-ligation with cis and trans sialic acid to regulate BCR signaling has not been delineated. Methods In vitro effects of epratuzumab on BCR-induced signaling were evaluated by analyzing the phosphorylation of BCR-signaling molecules (Syk/PLC-γ2) and Ca2+ flux. The influence of epratuzumab on the phosphorylation of the BCR signaling molecules Syk and PLC-γ2 was studied in vitro by flow cytometry in PBMCs from healthy donors. To investigate the effect of sialo-interaction on the effects by epratuzumab, PBMCs were treated with neuraminidase (to remove sialic acic) and BCR induced phosphorylation (Syk and PLC-γ2) was detected with or without epratuzumab incubation. To monitor the concentration of intracellular Ca2+, B cells pre-loaded with Indo-1AM were incubated with or without F(ab’)2-epratuzumab for 60 minutes at 37°C and were subsequently stimulated with F(ab’)2 anti-IgM/IgG. Intracellular Ca2+ concentrations were monitored by flow cytometry over 10 minutes after activation. Results Epratuzumab inhibited Syk and PLC-γ2 phosphorylation after in vitro BCR stimulation by 23±9% and 33±5, respectively. Interestingly, preventing sialo-interactions of CD22 partially reduced the inhibitory effect of epratuzumab on Syk and PLC-γ2 phosphorylation to 12±6% and 22±4%, respectively. The inhibition of kinase phosphorylation was demonstrated in both CD27- and CD27+ memory B cells and appeared to be independent of FcR signaling since a F(ab’)2 fragment of epratuzumab induced comparable results to the full antibody. In addition, a F(ab’)2 fragment of epratuzumab reduced the BCR-induced calcium flux. These data are consistent with the potential of targeting CD22 to raise the threshold of BCR activation which also provides additional control of B cell function. Conclusions Intracellular BCR signals can be partially inhibited by CD22 ligation with epratuzumab, providing further understanding of targeting CD22 biology in human autoimmune diseases. This study was supported by Sonderforschungsbereich 650 and DFG491/7-1 and in part by UCB Pharma Inc. References Nitschke L. The role of CD22 and other inhibitory co-receptors in B-cell activation. Current opinion in immunology 2005;17:290–7. Daridon C, Blassfeld D, Reiter K, et al. Epratuzumab targeting of CD22 affects adhesion molecule expression and migration of B-cells in systemic lupus erythematosus. Arthritis Research & Therapy 2010;12:R204. Disclosure of Interest N. Sieger: None Declared, S. Fleischer: None Declared, H. Mei: None Declared, K. Reiter: None Declared, A. Shock Employee of: UCB Pharma inc., G. Burmester: None Declared, C. Daridon: None Declared, T. Dörner Grant/Research support from: Immunomedics
The anti-CD20 antibody rituximab depletes human B cells from peripheral blood, but it remains con... more The anti-CD20 antibody rituximab depletes human B cells from peripheral blood, but it remains controversial to what extent tissueresident B cells are affected. In representative patients with rheumatoid arthritis, we here demonstrate that recently activated presumably short-lived plasmablasts expressing HLA-DR high and Ki-67 continuously circulate in peripheral blood after B-cell depletion by rituximab at 26%-119% of their initial numbers. They circulate independent of splenectomy, express immunoglobulin A (IgA),  7 integrin, and CC motif receptor 10 (CCR10) and migrate along CCL28 gradients in vitro, suggesting their mucosal origin. These plasmablasts express somatically hypermutated V H gene rearrangements and spontaneously secrete IgA, exhibiting binding to microbial antigens. Notably, IgA ؉ plasmablasts and plasma cells were identified in the lamina propria of patients treated with rituximab during peripheral B-cell depletion. Although a relation of these "steady state"-like plasmablasts with rheumatoid arthritis activity could not be found, their persistence during B-cell depletion indicates that their precursors, that is, B cells resident in the mucosa are not deleted by this treatment. These data suggest that a population of mucosal B cells is self-sufficient in adult humans and not replenished by CD20 ؉ B cells immigrating from blood, lymphoid tissue, or bone marrow, that is, B cells depleted by rituximab. (Blood. 2010; 116(24):5181-5190) Methods A detailed description of the patients, samples, and methods are available as supplemental Methods (available on the Blood Web site; see the Supplemental Materials link at the top of the online article).
Background: RA is an autoimmune condition characterized by chronic joint inflammation. Tfh cells,... more Background: RA is an autoimmune condition characterized by chronic joint inflammation. Tfh cells, a recently described Th subset localized in lymphoid organs, help B cell differentiation and function. Circulating central memory CD4 T cells expressing CXCR5 can be subdivided into three different subpopulations based on the combined expression of surface CXCR5 with or without CCR6 and/or CXCR3: CXCR5+CXCR3+CCR6-(Tfh-Th1), CXCR5+CXCR3-CCR6+ (Tfh-Th17) and CXCR5+CXCR3-CCR6-(Tfh-Th2). Only Tfh-Th17 and Tfh-Th2 cells have been demonstrated to display functional properties of Tfh cells; in contrast Tfh-Th1 cells are unable to provide B cell help. An altered proportion of these subpopulations has been associated with autoimmune diseases. Objectives: To study the frequency of circulating CXCR5+CD4+ T cell subsets together with the frequency of circulating plasmablasts (CD19+CD20-CD27+CD38high B cells) in patients with RA. Methods: Peripheral blood was drawn from healthy controls (n=27) and RA patients (n=27). After isolation by Ficoll-Hypaque gradient, PBMCs were stained with antibodies to CD3, CD4, CXCR5, CCR6, CXCR3, CD19, CD20, CD27, and CD38, and examined by flow cytometry. The percentages of CXCR5+CXCR3+CCR6-(Tfh-Th1), CXCR5+CXCR3-CCR6+ (Tfh-Th17) and CXCR5+CXCR3-CCR6-(Tfh/Th2) cells were calculated after gating for CD3, CD4 and CXCR5+. The percentage of CD20-CD38high cells was calculated after gating for CD19+ and CD27+. Results: The frequency of circulating CXCR5+ cells gated for CD4+ T cells was not different among the studied groups. In contrast, in RA patients, the frequency of Tfh-Th1 cells was significantly decreased and the frequency of Tfh-Th17 and Tfh-Th2 cells was significantly increased as compared with controls. Subsequently, the ratio (Tfh-Th17+Tfh-Th2)/Tfh-Th1 was increased in RA patients. That is, RA patients demonstrate a higher proportion of Tfh cells bearing a phenotype associated with B cell helping capacity. At the same time, the frequency of circulating plasmablasts was increased in RA patients as compared with controls. Interestingly, there was a significantly positive correlation between the percentage of circulating plasmablasts and the ratio (Tfh-Th17+Tfh-Th2)/Tfh-Th1, together with a significantly negative correlation between the percentage of circulating plasmablasts and the percentage of circulating Tfh-Th1 cells. Conclusions: RA patients demonstrate altered proportions of circulating Tfh subpopulations, with overrepresentation of subsets bearing a phenotype associated with B cell helping capacity. At the same time, an increased proportion of circulating plasmablasts is apparent in RA patients.
Abnormalities of memory B cells seem to be closely involved in the pathogenesis of primary Sjögre... more Abnormalities of memory B cells seem to be closely involved in the pathogenesis of primary Sjögrens Syndrome (pSS) and its malignant complication, B cell lymphoma. Recent studies on B cells in pSS add to our understanding of the distinct memory B cell subsets in pSS. Reduction of peripheral memory CD27 + B cells, most strikingly of the CD27 + IgM + subset, may indicate a lack of appropriate censoring mechanisms and incomplete differentiation processes within the ectopic lymphoid tissues in pSS. This ectopically formed lymphoid tissue might protect autoreactive memory B cells from deletion by physiological checkpoints and, thereby, may contribute to the perpetuation of the disease as well as to an enhanced lymphoma risk. Thus, B cells may be potential targets of direct or indirect treatment in pSS.
Introduction: Systemic lupus erythematosus (SLE) is an autoimmune disease associated with a break... more Introduction: Systemic lupus erythematosus (SLE) is an autoimmune disease associated with a break in self-tolerance reflected by a production of antinuclear autoantibodies. Since autoantibody production can be activated via nucleic acid Toll-like receptor 9 (TLR9), the respective pathway has been implicated in the development of SLE and pathogenic B cell responses. However, the response of B cells from SLE patients to TLR9 stimulation remains incompletely characterized. Methods: In the current study, the response of B cells from SLE patients and healthy donors upon TLR9 stimulation was analyzed in terms of proliferation and cytokine production and correlated with the lupus disease activity and anti-dsDNA titers. Results: B cells from SLE patients showed a reduced response to TLR9 agonist compared to B cells from healthy donors in terms of proliferation and activation. B cells from SLE patients with higher disease activity produced less interleukin (IL)-6, IL-10, vascular endothelial growth factor, and IL-1ra than B cells from healthy donors. Further analyses revealed an inverse correlation of cytokines produced by TLR9-stimulated B cells with lupus disease activity and anti-dsDNA titer, respectively. Conclusion: The capacity of B cells from lupus patients to produce cytokines upon TLR9 engagement becomes less efficient with increasing disease activity, suggesting that they either enter an exhausted state or become tolerant to TLR stimulation for cytokine production when disease worsens.
Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by a breakdown of self-... more Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by a breakdown of self-tolerance, autoantibody production, B cell hyperreactivity and an impaired B cell homeostasis. Recent data suggested that an impaired B cell response could also lead to autoimmunity. In this study, the BCR downstream signaling spleen tyrosine kinase Syk of healthy donors (HD), SLE, rheumatoid arthritis (RA) and primary Sjögren’s syndrome (pSS) patients was analyzed in detail and revealed new insights into peripheral B cell abnormalities based on intracellular characteristics. Interestingly, SLE B cells showed a significantly lower but long-lasting Syk (Y352) phosphorylation after BCR engagement compared to HD (p&lt;0.05). Furthermore, a significant enlarged frequency of Syk(bright) B cell population has been identified within the CD27(-) B cell subset in the blood of SLE patients (p&lt;0.001), but no correlation with the disease activity was observed. Notably, the frequency of Syk(bright) B cells was not increased in RA or pSS patients. Further characterization showed that Syk(bright) cells exhibit an increased basal level of phosphorylated Syk, expressed higher levels of CD19 and CD95 and mainly do not express CD38 as compared to Syk(dim) cells. Thus, SLE patients show a diminished but long-lasting BCR response and exhibit an enlarged unique Syk(bright) B cell subset that could be responsible for the hyperactivity of SLE B cells.
During mucosal immune responses, IgA-secreting plasmablasts are generated from mucosal B cells. W... more During mucosal immune responses, IgA-secreting plasmablasts are generated from mucosal B cells. We previously demonstrated that at steady-state, most human peripheral blood antibody-secreting cells produce IgA and express mucosal homing receptors, and suggested their generation in mucosal immune responses. In the current study, we analyzed the blood of patients who received B cell depletion therapy with rituximab (anti-CD20) for treatment of rheumatoid arthritis before and at 2-9months after rituximab infusion by flow cytometry. While CD20+ B cells were reduced to &lt;0.02% of their intial numbers before therapy, CD19+/CD27hi/CD20- antibody-secreting cells remained detectable in the blood at 26-119% of their initial numbers. These circulating antibody-secreting cells expressed IgA that bound to bacterial antigens, beta7 integrin and CCR10 before and during B cell depletion, suggesting their mucosal origin. High expression of HLA-DR and Ki-67 and in vitro migration towards CCL28 and CXCL12 qualified these cells as recently activated plasmablasts, reflecting the survival of functional B cells during rituximab treatment. Consistently, IgA+ plasmablasts and plasma cells were identified in the lamina propria during B cell depletion. Our data implicate that a population of mucosal B cells is resistant to rituximab and self-sufficient in adult humans and not replenished by CD20+ B cells immigrating from blood, lymphoid tissue, or bone marrow, that is, B cells depleted by rituximab.
Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by a breakdown of self-... more Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by a breakdown of self-tolerance, autoantibody production, B cell hyperreactivity and an impaired B cell homeostasis. Recent data suggested that an impaired B cell response could also lead to autoimmunity. In this study, the BCR downstream signaling spleen tyrosine kinase Syk of healthy donors (HD), SLE, rheumatoid arthritis (RA) and primary Sjögren’s syndrome (pSS) patients was analyzed in detail and revealed new insights into peripheral B cell abnormalities based on intracellular characteristics. Interestingly, SLE B cells showed a significantly lower but long-lasting Syk (Y352) phosphorylation after BCR engagement compared to HD (p<0.05). Furthermore, a significant enlarged frequency of Syk(bright) B cell population has been identified within the CD27(-) B cell subset in the blood of SLE patients (p<0.001), but no correlation with the disease activity was observed. Notably, the frequency of Syk(brigh...
Background: RA is an autoimmune condition characterized by chronic joint inflammation. Tfh cells,... more Background: RA is an autoimmune condition characterized by chronic joint inflammation. Tfh cells, a recently described Th subset localized in lymphoid organs, help B cell differentiation and function. Circulating central memory CD4 T cells expressing CXCR5 can be subdivided into three different subpopulations based on the combined expression of surface CXCR5 with or without CCR6 and/or CXCR3: CXCR5+CXCR3+CCR6-(Tfh-Th1), CXCR5+CXCR3-CCR6+ (Tfh-Th17) and CXCR5+CXCR3-CCR6-(Tfh-Th2). Only Tfh-Th17 and Tfh-Th2 cells have been demonstrated to display functional properties of Tfh cells; in contrast Tfh-Th1 cells are unable to provide B cell help. An altered proportion of these subpopulations has been associated with autoimmune diseases. Objectives: To study the frequency of circulating CXCR5+CD4+ T cell subsets together with the frequency of circulating plasmablasts (CD19+CD20-CD27+CD38high B cells) in patients with RA. Methods: Peripheral blood was drawn from healthy controls (n=27) and RA patients (n=27). After isolation by Ficoll-Hypaque gradient, PBMCs were stained with antibodies to CD3, CD4, CXCR5, CCR6, CXCR3, CD19, CD20, CD27, and CD38, and examined by flow cytometry. The percentages of CXCR5+CXCR3+CCR6-(Tfh-Th1), CXCR5+CXCR3-CCR6+ (Tfh-Th17) and CXCR5+CXCR3-CCR6-(Tfh/Th2) cells were calculated after gating for CD3, CD4 and CXCR5+. The percentage of CD20-CD38high cells was calculated after gating for CD19+ and CD27+. Results: The frequency of circulating CXCR5+ cells gated for CD4+ T cells was not different among the studied groups. In contrast, in RA patients, the frequency of Tfh-Th1 cells was significantly decreased and the frequency of Tfh-Th17 and Tfh-Th2 cells was significantly increased as compared with controls. Subsequently, the ratio (Tfh-Th17+Tfh-Th2)/Tfh-Th1 was increased in RA patients. That is, RA patients demonstrate a higher proportion of Tfh cells bearing a phenotype associated with B cell helping capacity. At the same time, the frequency of circulating plasmablasts was increased in RA patients as compared with controls. Interestingly, there was a significantly positive correlation between the percentage of circulating plasmablasts and the ratio (Tfh-Th17+Tfh-Th2)/Tfh-Th1, together with a significantly negative correlation between the percentage of circulating plasmablasts and the percentage of circulating Tfh-Th1 cells. Conclusions: RA patients demonstrate altered proportions of circulating Tfh subpopulations, with overrepresentation of subsets bearing a phenotype associated with B cell helping capacity. At the same time, an increased proportion of circulating plasmablasts is apparent in RA patients.
Objective. Systemic lupus erythematosus (SLE) is characterized by B cell hyperactivity and autoan... more Objective. Systemic lupus erythematosus (SLE) is characterized by B cell hyperactivity and autoantibody production. As spleen tyrosine kinase (Syk) is pivotal in B cell activation, these experiments aimed to examine the extent to which Syk was abnormally expressed in SLE B cells and the nature of the B cell subset that differently expressed Syk. Methods. B cells from healthy donors and SLE patients were analyzed by flow cytometry to assess basal expression of Syk and phosphorylated Syk. B cell subsets expressing higher levels of Syk were found, and their detailed phenotype, in vitro differentiation into plasmablasts/plasma cells, and Syk induction by cytokines were determined. Results. Syk expression was higher in CD27؉ memory B cells than in naive B cells from SLE patients. However, a significantly increased frequency of CD27؊ B cells with bright expression of Syk (Syk؉؉) was found in SLE patients. CD27؊Syk؉؉ B cells showed enhanced basal expression of p-Syk and stronger Syk phosphorylation upon B cell receptor (BCR) engagement as compared to CD27؊Syk؉ B cells. CD27؊Syk؉؉ B cells were CD38؊ as well as CD19؉؉, CD20؉؉, and mainly CD21؊, with decreased ABCB1 transporter activity. In contrast to CD27؊Syk؉ B cells, CD27؊Syk؉؉ B cells exhibited enhanced differentiation into CD27؉؉ IgG-secreting cells and expressed somatically mutated BCR gene rearrangements. Syk؉؉ B cells were inducible in vitro by stimulation with interferon-␥, lipopolysaccharide, or tumor necrosis factor ␣. Conclusion. SLE patients exhibit an increased frequency of hitherto unknown CD27؊Syk؉؉ memorylike B cells, indicating that intracellular Syk density could distinguish CD27؊ memory B cells from truly naive B cell subsets. Furthermore, the CD27؊Syk؉؉ subset is a candidate for a source of increased plasma cells in SLE.
Abnormalities of memory B cells seem to be closely involved in the pathogenesis of primary Sjögre... more Abnormalities of memory B cells seem to be closely involved in the pathogenesis of primary Sjögrens Syndrome (pSS) and its malignant complication, B cell lymphoma. Recent studies on B cells in pSS add to our understanding of the distinct memory B cell subsets in pSS. Reduction of peripheral memory CD27 + B cells, most strikingly of the CD27 + IgM + subset, may indicate a lack of appropriate censoring mechanisms and incomplete differentiation processes within the ectopic lymphoid tissues in pSS. This ectopically formed lymphoid tissue might protect autoreactive memory B cells from deletion by physiological checkpoints and, thereby, may contribute to the perpetuation of the disease as well as to an enhanced lymphoma risk. Thus, B cells may be potential targets of direct or indirect treatment in pSS.
ObjectiveTo identify the cells that produce BAFF in the salivary glands of patients with primary ... more ObjectiveTo identify the cells that produce BAFF in the salivary glands of patients with primary Sjögren's syndrome (SS), and to analyze BAFF receptor expression by local T and B lymphocytes.MethodsWe used 3 methods to identify the source of BAFF: in situ hybridization of the transcripts for BAFF combined with staining of membrane markers, regular and real‐time reverse transcription–polymerase chain reaction (RT‐PCR) of cultured epithelial cells, and RT‐PCR of sorted single‐cell T and B lymphocytes eluted from salivary glands. Cells expressing TACI, BCMA, and B lymphocyte stimulator receptor 3 (BR‐3) were disclosed by combining each specific staining of the receptors with each specific staining of the cells. The function of BAFF generated by epithelial cells on B lymphocytes was determined in short‐term cocultures.ResultsTranscripts for BAFF were seen in epithelial cells and infiltrating T lymphocytes and, for the first time, were detected in local B cells. It is interesting tha...
Background CD22, a transmembrane protein exclusively expressed on B cells, mediates migration by ... more Background CD22, a transmembrane protein exclusively expressed on B cells, mediates migration by cell–cell interaction and negatively regulates B-cell receptor (BCR) signaling. CD22 acts as an inhibitory co-receptor of the BCR via de-phosphorylation of signaling molecules such as spleen tyrosine kinase (Syk) and subsequent phospholipase C (PLC)-γ2-triggered Ca2+ fluxes. The humanized anti-CD22 monoclonal antibody epratuzumab modulates adhesion molecule expression and B-cell migration in vitro. However, the potential of CD22 to regulate BCR signaling has not been fully delineated. Objectives As toll-like receptor (TLR)-dependent activation of B cells represents an important pathway of B-cell hyperactivity in autoimmunity, our studies addressed whether epratuzumab might be able to modulate BCR signaling pathways in TLR9 pre-activated B cells or whether TLR9 activation abrogates the inhibitory effect of epratuzumab on the BCR signaling. Methods Our study focused on the influence of epratuzumab on the B cells’ response to BCR alone or in combination with TLR9. The recruitment of CD22 to the BCR (CD79α) after epratuzumab incubation on B cells from healthy volunteers was analyzed by confocal microscopy. The in vitro effects of epratuzumab on BCR-induced signaling were evaluated by analyzing the phosphorylation status of BCR-signaling molecules, Syk and PLC-γ2 by flow cytometry with or without TLR9 pre-activation. The concentration of intracellular Ca2+ was monitored by flow cytometry after BCR stimulation. Finally, to evaluate the origin of the Ca2+ flux, extracellular Ca2+ was chelated with 1 mM ethylene glycol tetraacetic acid (EGTA). Results B cells incubated with epratuzumab showed a specific co-localization of CD22 to the BCR-associated molecule CD79α on B cells. In addition, pre-treatment with epratuzumab led to a reduction in the MFI of phosphorylated Syk and PLC-γ2 induced by BCR stimulation compared with IgG1 isotype control. Similar results were observed when the cells were pre-incubated with F(ab’)2 fragment of epratuzumab, which excludes an inhibitory effect dependent on FcR signaling. Interestingly, the reduction of BCR induced kinase phosphorylation was demonstrated in both CD27- and CD27+ memory B cells. In addition, pre-activation of B cells by TLR9 did not circumvent the influence of epratuzumab on BCR signaling, as shown by a reduction in the MFI of phosphorylated Syk and PLC-γ2 in comparison to non-activated cells. Finally, a F(ab’)2 fragment of epratuzumab reduced BCR-induced calcium mobilization. Conclusions Intracellular BCR signals can be modulated by CD22 ligation using epratuzumab, and pre-activation with TLR9 did not circumvent this effect. These data expand the potential mechanisms of action of epratuzumab. Disclosure of Interest N. Sieger Grant/research support from: UCB Pharma, S. Fleischer Grant/research support from: UCB Pharma, K. Reiter Grant/research support from: UCB Pharma, H. Mei Grant/research support from: UCB Pharma, A. Shock Employee of: UCB Pharma, G. Burmester Grant/research support from: UCB Pharma, C. Daridon Grant/research support from: UCB Pharma, T. Dörner Grant/research support from: UCB Pharma, Consultant for: UCB Pharma
Background:Glucocorticoid (GC) therapy has strong anti-inflammatory effects and helps slow radiog... more Background:Glucocorticoid (GC) therapy has strong anti-inflammatory effects and helps slow radiographic progression in RA1; however, GCs can be associated with adverse events (AEs) such as infection, especially with long-term use and higher doses.Objectives:To evaluate the impact of baseline GCs on the efficacy and safety of upadacitinib (UPA) with or without concomitant conventional synthetic DMARDs (csDMARDs).Methods:In this ad hoc analysis of three Phase 3 studies, patients with inadequate response to MTX (MTX-IR) receiving UPA 15 mg once daily (QD) or placebo (PBO) + csDMARDs in SELECT-NEXT, and MTX-IR/MTX-naïve patients receiving UPA 15 mg QD monotherapy or MTX monotherapy in SELECT-MONOTHERAPY/SELECT-EARLY, respectively, were included. Efficacy outcomes, including measures of remission and low disease activity (LDA) determined by DAS in 28 joints using CRP (DAS28[CRP]; <2.6/≤3.2) and Clinical Disease Activity Index (CDAI; ≤2.8/≤10), were assessed and stratified by baseline ...
The binding of antigen to the B cell receptor (BCR) results in a cascade of signalling events tha... more The binding of antigen to the B cell receptor (BCR) results in a cascade of signalling events that ultimately drive B cell activation. Uncontrolled B cell activation is regulated by negative feedback loops that involve inhibitory co-receptors such as CD22 and CD32B that exert their functions following phosphorylation of immunoreceptor tyrosine-based inhibition motifs (ITIMs). The CD22-targeted antibody epratuzumab has previously been shown to inhibit BCR-driven signalling events, but its effects on ITIM phosphorylation of CD22 and CD32B have not been properly evaluated. The present study therefore employed both immunoprecipitation and flow cytometry approaches to elucidate the effects of epratuzumab on direct phosphorylation of key tyrosine (Tyr) residues on both these proteins, using both transformed B cell lines and primary human B cells. Epratuzumab induced the phosphorylation of Tyr(822) on CD22 and enhanced its co-localisation with SHP-1. Additionally, in spite of high basal ph...
Objective. Systemic lupus erythematosus (SLE) is associated with hyperactivity of B cells and abn... more Objective. Systemic lupus erythematosus (SLE) is associated with hyperactivity of B cells and abnormalities of B cell receptor (BCR) signaling. To address the linkage between dysregulated BCR signaling and increased B cell function, we assessed immediate phosphorylation events in lupus B cells. Methods. B cells from SLE patients and healthy donors were analyzed by flow cytometry to assess phosphorylated CD22, Syk, and Akt as well as the basal expression of the BCR coreceptors CD22 and CD19. Confocal microscopy studies determined the recruitment of CD22 and the tyrosine phosphatase SH2 domain-containing phosphatase 1 to the activated BCR complex. Additionally, phosphatase activity in SLE versus healthy donor B cells was measured. Results. B cells from SLE patients showed diminished Syk phosphorylation and reduced intracellular calcium release after BCR activation as compared to B cells from healthy donors. This was related to an enhanced activity of tyrosine, but not serine/threonine, phosphatases and was corrected by inhibition of tyrosine phosphatase activity. In contrast to reduced Syk phosphorylation after BCR activation, phosphorylation of Akt was significantly increased in SLE B cells. The disturbed balance between Syk and Akt phosphorylation was significantly correlated with B cell survival following BCR engagement. Furthermore, CD272, but not CD271, B cells from SLE patients displayed increased expression and phosphorylation of the inhibitory BCR coreceptor CD22. Conclusion. These results indicate that an imbalance between serine and tyrosine phosphatases in SLE contributes to an intrinsically disturbed balance of BCR-initiated signaling pathways, resulting in enhanced survival of lupus B cells and differentiation into plasma cells. The development, maturation, and selection of B cells are tightly regulated by the affinity and activation threshold of the B cell receptor (BCR). Besides its important role during B cell differentiation and survival, the BCR plays a pivotal role in avoiding autoreactivity by initiating a variety of negative-selection events (1,2). After BCR crosslinking, conformation changes induced by cytoskeleton reorganization lead to a Lyn-dependent phosphorylation of the intracellular Iga/b immunoreceptor tyrosine-based activation motif (ITAM) of the BCR and its coreceptors (e.g., CD19 and CD22), followed by the recruitment and activation of the spleen tyrosine kinase (Syk). Subsequently, signaling molecules, such as Bruton's tyrosine kinase (BTK), phosphatidylinositol 3-kinase (PI3K) and phospholipase Cg2 (PLCg2) accumulate in a signalosome to initiate internalization, antigen processing, calcium release, and a specific gene expression profile (3). Even though Syk can directly activate PI3K (4), a Syk-independent PI3K/ Akt activation has been described (5), subdividing the BCR-associated signaling pathway into two axes: Syk activation linked to PLCg2 phosphorylation and calcium influx, and CD19 phosphorylation followed by activation Supported by the DFG (SFB 650 project TP12, SFB 650 project TP16, SFB 633 A14, SPP ImmunoBone [Do491/8-2], and project Do491/7-3).
Introduction: Cytokines produced by B cells are believed to play important roles in autoimmune di... more Introduction: Cytokines produced by B cells are believed to play important roles in autoimmune diseases. CD22 targeting by epratuzumab has been demonstrated to inhibit phosphorylation of B cell receptor (BCR) downstream signaling in B cells. It has been shown that other sialoadhesin molecules related to CD22 have immunoregulatory functions; therefore, in the present study, we addressed the role of epratuzumab on the production of key cytokines by B cells of patients with systemic lupus erythematosus (SLE) and of healthy donors (HD). Methods: Peripheral blood B cells were purified and activated by BCR with or without Toll-like receptor 9 (TLR9) stimulation in the presence or absence of epratuzumab. Cytokine production by B cells (interleukin [IL]-6, tumor necrosis factor [TNF]-α and IL-10) in the supernatant and the induction of IL-10 + B cells from patients with SLE and HD were analyzed. Results: The secretion of the proinflammatory cytokines TNF-α and IL-6 by anti-BCR and BCR-and/or TLR9-activated B cells from HD and patients with SLE was inhibited by epratuzumab. In contrast, the production of IL-10 by B cells was not affected by epratuzumab under either stimulation condition. Consistently, the induction of IL-10-producing B cells in culture was not affected by epratuzumab. Conclusions: Epratuzumab, by targeting CD22, was able to inhibit the production of the proinflammatory cytokines IL-6 and TNF-α by B cells, in contrast to IL-10, in vitro. These data suggest that targeting CD22 alters the balance between proinflammatory cytokines (TNF-α, IL-6) and the regulatory cytokine IL-10 as another B cell effector mechanism.
Conclusion.-Les patients avec PR ont fréquemment une fonction diastolique ventriculaire gauche al... more Conclusion.-Les patients avec PR ont fréquemment une fonction diastolique ventriculaire gauche altérée et ces variations sont beaucoup mieux analysées au DT qu'en échocardiographie conventionnelle. Le DT semble être un examen précis et fiable permettant d'évaluer l'atteinte myocardique dans la PR et la prédictivité des anomalies détectées est en cours d'évaluation [1].
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