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B cell activating factor (BAFF) has been shown to play a key role in regulating B cell function, but little is known about whether BAFF affects the function of fibroblast-like synoviocyte (FLS), an effector cell of rheumatoid arthritis (RA). CP-25, a new ester derivative of paeoniflorin, could alleviate the arthritis symptoms of collagen-induced arthritis (CIA) mice by inhibiting BAFF-mediated abnormal activation of B cells. In this study, we aimed to understand the mechanism by which BAFF activates FLS and the effect of CP-25 on FLS function. Therefore, the proliferation and migration abilities of FLS and key proteins on the non-canonical NF-κB pathway were examined. The results showed that compared with the FLS of normal rats/OA patients, the expression of BAFF-R, TRAF2, NIK, p-IKKα, P100, and P52 was higher in the FLS of AA rats/RA patients, while the expression of TRAF3 was lower. And, BAFF promotes FLS activation by activating the non-canonical NF-κB signaling pathway. Meanwhile, BAFFR-siRNA inhibited the proliferation of FLS and the activation of non-canonical NF-κB signaling in FLS induced by BAFF. Additionally, CP-25 could inhibit abnormal proliferation and migration of FLS by regulating non-canonical NF-κB signaling. We concluded that BAFF may act as an important role in facilitating the function of FLS through the BAFFR-mediated non-canonical NF-κB pathway, which would be useful for revealing the pathological mechanism of RA. And CP-25 may become a potential new drug for the treatment of RA, providing a scientific basis for the development of new drugs to treat RA., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Senescent cells release a variety of cytokines, proteases, and growth factors collectively known as the senescence-associated secretory phenotype (SASP). Sustained SASP contributes to a pattern of chronic inflammation associated with aging and implicated in many age-related diseases. Here, we investigated the expression and function of the immunomodulatory cytokine BAFF (B-cell activating factor; encoded by the TNFSF13B gene), a SASP protein, in multiple senescence models. We first characterized BAFF production across different senescence paradigms, including senescent human diploid fibroblasts (WI-38, IMR-90) and monocytic leukemia cells (THP-1), and tissues of mice induced to undergo senescence. We then identified IRF1 (interferon regulatory factor 1) as a transcription factor required for promoting TNFSF13B mRNA transcription in senescence. We discovered that suppressing BAFF production decreased the senescent phenotype of both fibroblasts and monocyte-like cells, reducing IL6 secretion and SA-β-Gal staining. Importantly, however, the influence of BAFF on the senescence program was cell type-specific: in monocytes, BAFF promoted the early activation of NF-κB and general SASP secretion, while in fibroblasts, BAFF contributed to the production and function of TP53 (p53). We propose that BAFF is elevated across senescence models and is a potential target for senotherapy., Competing Interests: MR, CA, MI, RM, CS, SD, DT, AH, JM, XY, YP, KM, JF, LF, PJ, SD, KA, MG No competing interests declared

Background: Rituximab is widely used for treatment of pemphigus patients. B-cell activating factor (BAFF) and a proliferation-inducing ligand (APRIL) play key roles in B cell survival, maturation, and differentiation. Here, the effect of rituximab on BAFF and APRIL in patients with pemphigus vulgaris (PV) was studied., Methods: Fifty PV cases and 56 healthy individuals were recruited. Patients received rituximab for a period of 6 months. The levels of BAFF and APRIL were measured in the serum samples. The frequency of CD19+ B cells was measured by flow cytometry., Results: The level of BAFF was significantly higher in the patients at the baseline level than controls (P = 0.0005). The level of BAFF was significantly higher at the 3rd month follow-up compared to the baseline (P = 0.033). There was a significant increase in the BAFF level at the 6th month follow-up compared to baseline (P = 0.0134). There was no significant difference in the CD19+ B cells/total lymphocytes ratio in the PV patients between the 3rd and 6th month follow-ups., Conclusions: Elevated BAFF in the sera could be associated with PV immunopathogenesis. Inhibition of BAFF after rituximab therapy might interfere with repopulation of B cells and confer a therapeutic approach in PV., (© 2022 the International Society of Dermatology.)

Context: Teprotumumab, an IGF-I receptor (IGF-IR) inhibitor, is effective in thyroid-associated ophthalmopathy (TAO). The drug can modulate induction by TSH of IL-6 and IL-8 in CD34+ fibrocytes and their putative derivatives, CD34+ orbital fibroblasts (CD34+ OF). Fibrocytes express multiple thyroid autoantigens and cytokines implicated in TAO, which are downregulated by Slit2. Inflammation and disordered hyaluronan (HA) accumulation occur in TAO. Whether teprotumumab alters these processes directly in fibrocytes/CD34+ OF remains uncertain., Objective: Determine teprotumumab effects on expression/synthesis of several TAO-relevant molecules in fibrocytes and GD-OF., Design/setting/participants: Patients with TAO and healthy donors were recruited from an academic endocrine and oculoplastic practice., Main Outcome Measures: Real-time PCR, specific immunoassays., Results: Teprotumumab attenuates basal and TSH-inducible autoimmune regulator protein, thyroglobulin, sodium iodide symporter, thyroperoxidase, IL-10, and B-cell activating factor levels in fibrocytes. It downregulates IL-23p19 expression/induction while enhancing IL-12p35, intracellular and secreted IL-1 receptor antagonists, and Slit2. These effects are mirrored by linsitinib. HA production is marginally enhanced by teprotumumab, the consequence of enhanced HAS2 expression., Conclusion: Teprotumumab affects specific gene expression in fibrocytes and GD-OF in a target-specific, nonmonolithic manner, whereas IGF-IR control of these cells appears complex. The current results suggest that the drug may act on cytokine expression and HA production systemically and locally, within the TAO orbit. These findings extend our insights into the mechanisms through which IGF-IR inhibition might elicit clinical responses in TAO, including a potential role of Slit2 in attenuating inflammation and tissue remodeling., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Tumor-associated neutrophils (TANs) are the major cellular component of the tumor microenvironment and have been shown to release of different bioactive molecules such as B-cell activating factor (BAFF). The data on the interactions between OSCC cells and neutrophils are limited and do not explain the actual role of the BAFF in the development of the OSCC. In the present study we examined the direct effect of neutrophils-derived BAFF on the OSCC cell line CAL-27 proliferation and apoptosis. PMNs of OSCC patients and healthy control were isolated from whole blood and separated by magnetic selection with monoclonal anti-human CD16 antibodies. CD-16 - positive neutrophils were incubated in the presence of TGF-β and/or LPS as well as flavonoids (luteolin and quercetin). CAL-27 cells were co-incubated with supernatants of neutrophils. BAFF expression in neutrophils, BAFF-R expression on CAL-27 cells and apoptosis of CAL-27 cells were assessed by flow cytometry. To determine the CAL-27 cells proliferation, the MTT test was used. Expression of select mitochondrial proteins in CAL-27 cells were measured by Western blot. Neutrophils from OSCC patients showed significantly higher expression of BAFF than those from the healthy controls. The results obtained revealed upregulation of the proliferation and downregulation of the apoptosis of the CAL-27 cells in the presence of the supernatants of TGF-β-treated neutrophils. Flavonoids reduced BAFF expression in neutrophils of patients with OSCC and control group. Lower intensity of apoptosis in CAL-27 cells was associated with the increased expression of anti-apoptotic Bcl-2, Mcl-1 and activated form of PI3K kinase (pPI3K) and simultaneously reduced expression of pro-apoptotic Bax protein in the presence of rhBAFF, as well as of supernatants of neutrophils derived from OSCC patients. In conclusion, the data presented confirm the previously suggested role of neutrophil-derived BAFF in OSCC development. The favorable effects of examined flavonoids on tumor-promoting BAFF expression in neutrophils suggest that they might be promising candidates as chemo-preventive agents in the therapy of patients with OSCC., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier GmbH. All rights reserved.)

Therapeutically targeting B cells has received great attention in the treatment of B-cell malignancies and autoimmune diseases. The B-cell activating factor (BAFF) is critical to the survival of normal and neoplastic B cells, and excess production of BAFF contributes to autoimmune diseases. Resveratrol, a natural polyphenolic compound, has a positive effect on the treatment of autoimmune diseases. However, how resveratrol affects BAFF-stimulated B-cell proliferation and survival is poorly understood. Here, we show that resveratrol increased autophagosome formation and ATG5/LC3-II levels and decreased p62 level, promoting autophagic flux/autophagy and thereby suppressing the basal or human soluble BAFF (hsBAFF)-stimulated proliferation and survival of normal and B-lymphoid (Raji) cells. This is supported by the findings that inhibition of autophagy with 3-methyladenine (3-MA, an inhibitor of Vps34) or ATG5 shRNA attenuates resveratrol-induced autophagy and -reduced proliferation/viability in B-cells. Inhibition of mTOR with rapamycin or knockdown of mTOR potentiated resveratrol-induced autophagy and inhibition of hsBAFF-stimulated B-cell proliferation/viability, while overexpression of wild-type mTOR conferred resistance to the actions of resveratrol. Similarly, inhibition of Akt with Akt inhibitor X or ectopic expression of dominant negative Akt reinforced resveratrol-induced autophagy and inhibition of hsBAFF-stimulated B-cell proliferation/viability, whereas expression of constitutively active Akt conferred resistance to the actions of resveratrol. Taken together, these results indicate that resveratrol induces autophagy impeding BAFF-stimulated proliferation and survival via blocking the Akt/mTOR signaling pathway in normal and neoplastic B cells. Our findings highlight that resveratrol has a great potential for prevention and treatment of excessive BAFF-elicited aggressive B-cell disorders and autoimmune diseases., (Copyright © 2022 Elsevier Inc. All rights reserved.)

CD40 ligand (CD40L) and B-cell activating factor (BAFF) play important roles in the function of B cells. However, the difference of their regulatory effects remains obscure. In this study, we used anti-CD40 to imitate CD40L and investigated the different regulatory effects of CD40L and BAFF on the function of B cells. In the functional analyses, both anti-CD40 and BAFF significantly enhanced the survival and differentiation of B cells, and slightly increased the activation and proliferation. However, in the transcriptome analysis, anti-CD40 and BAFF exerted very different regulation on the gene expression profile of B cells. Anti-CD40 upregulated the expression of genes related to the adaptive immune function of B cells, but BAFF enhanced the genes associated with the innate immune function. Furthermore, the effect analysis of the combination of anti-CD40 or BAFF with anti-IgM also demonstrated that anti-CD40 could cooperate with anti-IgM to promote the proliferation of B cells, but BAFF could not do it. The mechanism study revealed that the different effects of anti-CD40 and BAFF on B cells were resulting from the different modulation on NF-кB, ERK1/2, and PI3K-AKT signaling pathways. Collectively, the results suggest that CD40L mainly promotes adaptive immune function of B cells, but BAFF primarily enhances innate immune function., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Background: Hyperactivation of B cells by activators has been demonstrated to play a central role in the pathogenesis of Sjögren's syndrome (SS). In this study, we found that artesunate (ART) can attenuate BAFF-induced B cell hyperactivation and SS-like symptoms in NOD/Ltj mice., Purpose: To determine the efficacy of ART in attenuating SS-like symptoms in vivo and explore the underlying mechanism in vitro., Study Design: ART was intragastrically injected into SS-like NOD/Ltj mice. The cytokine hsBAFF was used to activate Raji and Daudi B cells to mimic B cell hyperactivation in vitro., Methods: The efficacy of ART in inhibiting SS progression was studied in NOD/Ltj mice. Salivary flow rate, the number of lymphocytic infiltration foci, the level of autoantibodies and the extent of B cell infiltration were measured in the indicated groups. CCK-8 assays, flow cytometry-based EdU staining and Annexin V/PI staining were also used to detect the effect of ART on the survival and proliferation mechanism in BAFF-induced Raji and Daudi cells. Further studies determined that TRAF6 degradation is a potential mechanism by which ART determines B cell fate., Results: Treatment with ART inhibited lymphocytic foci formation, B cell infiltration and autoantibody secretion in SS-like NOD/Ltj mice. In vitro assay results indicated that ART effectively inhibited BAFF-induced viability, survival and proliferation of neoplastic B cells. Mechanistically, ART targeted BAFF-activated NFκB by regulating the proteasome-mediated degradation of TRAF6 in Raji and Daudi cells., Conclusion: ART ameliorated murine SS-like symptoms and regulated TRAF6-NFκB signaling, thus determining survival and proliferation of B cells., (Copyright © 2020 Elsevier GmbH. All rights reserved.)

B cell activating factor (BAFF) is recognized as a member of the TNF superfamily proteins that mediate the immune responses. In this study, BAFF from rockfish (Sebastes schlegelii) (SsBAFF) was characterized based on its functional aspects. The open reading frame of SsBAFF is 804 bp in length and encodes a 267 long amino acid residue protein with predicted molecular weight of 29.48 kDa. The deduced protein sequence comprises with transmembrane domain, furin cleavage site and TNF domain carrying Flap binding site that unique to TNF family. Recombinant SsBAFF (rSsBAFF) significantly enhanced rockfish lymphocytes proliferation and viability in a concentration dependent-manner according to the results from water soluble tetrazolium salt (WST-1) assay and flow cytometric assay. rSsBAFF also modulated the expression of genes involved in anti-inflammatory (IL-10 and NFκB-2) and anti-apoptotic (Bcl-2 and Bax) signal pathways. SsBAFF mRNA expression was detected ubiquitously in all analyzed rockfish tissues, with the highest levels in the spleen and head kidney. Further, the expression of SsBAFF in spleen were significantly induced following LPS, poly (I:C) and Streptococcus iniae challenges. These findings strongly suggest that SsBAFF might play an important role in rockfish immune system through regulating the inflammatory response and proliferation of immune cells., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Epstein-Barr virus (EBV) reactivation can lead to serious complications in kidney transplant patients, including post-transplant lymphoproliferative disorder (PTLD). Here, we have assessed the impact of EBV on B cell homeostasis at cellular and humoral level. In a multicenter study monitoring 540 kidney transplant patients during the first post-transplant year, EBV reactivation was detected in 109 patients. Thirteen soluble factors and B cell counts were analyzed in an EBV + sub-cohort (N = 54) before, at peak and after EBV clearance and compared to a control group (N = 50). The B cell activating factor (BAFF) was significantly elevated among EBV + patients. No additional soluble factors were associated with EBV. Importantly, in vitro experiments confirmed the proliferative effect of BAFF on EBV-infected B cells, simultaneously promoting EBV production. In contrast, elevated levels of BAFF in EBV + patients did not lead to B cell expansion in vivo. Moreover, diminished positive inter-correlations of soluble factors and alterations of the bi-directional interplay between B cell and soluble factors were observed in EBV + patients at peak and after clearance. Our data suggest that such alterations may counteract the proliferative effect of BAFF, preventing B cell expansion. The role of these alterations in lymphoma development should be analyzed in future studies.

Background: It has been reported that B cell activating factor belonging to the tumor necrosis factor family (BAFF) expression is increased in chronic obstructive pulmonary disease (COPD). However its role in this chronic inflammatory disease is not fully understood. Previous studies have suggested that BAFF also affects T cell function. We therefore investigated the effects of BAFF on T lymphocytes in COPD., Methods: BAFF was detected in the cells of sputum and the plasma. Peripheral blood mononuclear cells (PBMCs) were isolated from COPD patients and treated with BAFF or BAFF plus BR3-Fc (BAFF antagonist). The apoptosis of CD4 + cells and CD8 + cells was analyzed by flow cytometry. CD4 + cells and CD8 + cells were isolated from peripheral blood of COPD patients respectively and treated with BAFF or BAFF plus BR3-Fc. Interferon-γ (IFN-γ) and interleukin-4 (IL-4) were detected in the CD4 + cells, and perforin and granzyme B were detected in the CD8 + cells., Results: BAFF expression was increased in the cells of sputum and the plasma from COPD patients compared with control subjects. The plasma BAFF levels were inversely correlated with FEV 1 percentage of predicted in patients with COPD. BAFF did not significantly alter the apoptosis of CD4 + cells, however it significantly inhibited the apoptosis of CD8 + cells from COPD patients. BAFF increased IFN-γ expression in the CD4 + cells from COPD patients, while it did not significantly alter the expresson of IL-4 in these cells. BAFF increased the expression of perforin and granzyme B in the CD8 + cells from COPD patients., Conclusions: Our findings indicate that BAFF may be involved in the inflammatory response in COPD via affecting T lymphocytes, suggesting a possible role of BAFF in the pathogenesis of COPD.

Background and Objective: Atacicept is an inhibitor of the B lymphocyte stimulator (BLyS) and a proliferation-inducing ligand (APRIL), and is being studied in relation to immunological disease. Currently, limited data on atacicept are available in non-Caucasian subjects. Pharmacokinetic data from earlier studies of atacicept were derived using an enzyme-linked immunosorbent assay (ELISA), which was subsequently found to have inadequacies. Hence, a new bioanalytical ELISA for total atacicept was developed and validated. We conducted this randomized, double-blind, placebo-controlled phase I study to compare the safety, tolerability, pharmacokinetics, and pharmacodynamics of atacicept in healthy Japanese and Caucasian subjects while generating pharmacokinetic data using the new ELISA., Methods: Japanese subjects aged ≥ 18 to ≤ 55 years (n  = 24) were randomized (1:1:1:1) to a single subcutaneous dose of atacicept 25, 75, or 150 mg or placebo. Caucasian subjects were then enrolled to match the Japanese subjects' gender, body weight (± 20%), and height (± 15%)., Results: Atacicept was well tolerated and there were no clinically significant differences in treatment-emergent adverse events (TEAEs), vital signs, or laboratory parameters between the Japanese and Caucasian subjects. Most (90%) TEAEs were mild; no severe or serious TEAEs or deaths occurred. Weight-adjusted atacicept exposure was comparable between ethnicities and across doses: the Japanese/Caucasian ratio of the area under the serum concentration-time curve from time zero to the last sampling point (AUC 0-t ) was 107.21% (90% CI 93.42-123.02%) and the Japanese/Caucasian ratio of maximum serum concentration (C max ) was 95.74% (90% CI 74.26-123.43%; ANCOVA). Median time to reach C max (t max ) was 20-60 h across all subjects. Dose-exposure relationships were comparable for the two ethnicities, with dose-normalized AUC 0-t decreasing with increasing dose, indicating nonlinear pharmacokinetics for the doses examined. There were no statistically significant differences between ethnicities in the pharmacokinetics-dose relationship. Some transient dose-related decreases in mean serum immunoglobulin (Ig)A and IgM, but not IgG, were observed after atacicept administration. There were small transient increases in peripheral B cell numbers in the first 4 days after dosing that were larger with atacicept than with placebo, with no apparent dose relationship. No anti-atacicept antibodies were detected., Conclusion: The safety, pharmacokinetic, and pharmacodynamic profiles of atacicept in healthy Japanese subjects were comparable to those in healthy Caucasian subjects. EudraCT-ID: 2013-002703-34.

Background: Circulating endothelial progenitor cells (EPCs) are biologic markers of endothelial function. In patients with systemic lupus erythematosus (SLE), the numerical reduction and functional impairment of EPCs contribute to the endothelial dysfunction. Through ex vivo and in vitro studies, we aimed at evaluating the effects of B lymphocyte stimulator (BLyS) on EPC colonies and endothelial cells and also investigating BLyS receptor expression on these cells., Methods: EPCs were isolated from peripheral blood mononuclear cells (PBMC). In order to evaluate their ability to form colonies, EPCs were cultured on fibronectin-coated dishes and incubated with BlyS alone or BlyS and belimumab. Apoptosis of EPCs and endothelial cell line EA.hy926 was evaluated after 6, 12, and 24 h of incubation with BLyS and after 6 h with BLyS and belimumab. The expression of B cell activating factor-receptor (BAFF-R), B cell maturation antigen (BCMA), and transmembrane activator and calcium modulator and cyclophilin ligand (CAML) interactor (TACI) on EPCs and EA.hy926 was analyzed by cytofluorimetry., Results: The number of EPC colonies was lower in patients than in controls. Moreover, the colonies from SLE patients were poorly organized compared to controls; the addition of belimumab restored the colony structure. Incubation with BLyS induced apoptosis of EPCs and EA.hy926 that was inhibited by the co-incubation with belimumab. BAFF-R and BCMA were expressed on both EPCs and EA.hy926, while TACI was expressed only on EPCs., Conclusions: EPCs and endothelial cells preferentially express BAFF-R which could be involved in the pro-apoptotic effect of BlyS. Belimumab administration seems to restore the quantitative and qualitative changes of EPC colonies both ex vivo and in vitro.

There are global efforts in developing therapeutic strategies for central nervous system (CNS) injuries using multimodal approaches. Nogo receptor type 1 (NgR1) has been known as a primary molecule limiting neuronal regeneration in the adult CNS. We identified lateral olfactory tract usher substance (LOTUS) as an endogenous NgR1 antagonist. Membrane-bound LOTUS interacts with NgR1 and inhibits its function by blocking its ligand binding. Five molecules including Nogo, myelin-associated glycoprotein (MAG), oligodendrocyte myelin glycoprotein (OMgp), B lymphocyte stimulator (BLyS) and chondroitin sulfate proteoglycans (CSPGs) have been identified as NgR1 ligands. These ligands bind to NgR1 and activate NgR1 signaling, leading to axon growth inhibition such as growth cone collapse and neurite outgrowth inhibition. We have recently reported that the soluble form of LOTUS (s-LOTUS) also suppressed NgR1-mediated signaling induced by myelin axonal inhibitors (MAIs) including Nogo, MAG and OMgp by binding with both NgR1 and its co-receptor p75 neurotrophin receptor (p75 NTR ). Though s-LOTUS has been reported to suppress MAIs, whether s-LOTUS also suppresses NgR1 signaling induced by BLyS and CSPGs remains to be elucidated. Here, we show that s-LOTUS inhibits NgR1-mediated signaling induced by BLyS and CSPGs. Although treatment with s-LOTUS did not suppress BLyS-NgR1 interaction, s-LOTUS inhibited growth cone collapse and neurite outgrowth inhibition induced by BLyS and CSPGs in chick dorsal root ganglion (DRG) neurons. Furthermore, s-LOTUS compensated for the suppressive function of endogenous LOTUS in NgR1-mediated signaling in olfactory bulb neurons of lotus-knockout mice. These findings suggest that s-LOTUS is a potent therapeutic agent for neuronal regeneration in the CNS injuries., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Chondroitin sulfate proteoglycans (CSPGs) are axon growth inhibitors in the glial scar, and restrict axon regeneration following damage to the adult mammalian central nervous system. CSPGs have recently been identified as functional ligands for Nogo receptor-1 (NgR1), which is the common receptor for Nogo proteins, myelin-associated glycoprotein (MAG), oligodendrocyte myelin glycoprotein (OMgp) and B lymphocyte stimulator (BLyS). We have previously reported that through its binding to NgR1, lateral olfactory tract usher substance (LOTUS) suppresses Nogo, MAG, OMgp, and BLyS-induced axon growth inhibition. However, it remains unknown whether LOTUS also exerts this suppressive action on CSPG-induced axon growth inhibition. LOTUS overexpression rescued CSPG-induced growth cone collapse and neurite outgrowth inhibition in cultured dorsal root ganglion neurons, which only weakly express endogenous LOTUS. In cultured olfactory bulb neurons, which endogenously express LOTUS, the growth cone was insensitive to CSPG-induced collapse, but was sensitive to collapse induced by CSPGs in lotus-deficient mice. Our data demonstrate that LOTUS suppresses CSPG-induced axon growth inhibition, suggesting that LOTUS may represent a promising therapeutic agent for promoting axon regeneration., (Copyright © 2017 IBRO. Published by Elsevier Ltd. All rights reserved.)

Disturbances of plasma cell homeostasis and auto-antibody production are hallmarks of systemic lupus erythematosus. The aim of this study was to explore the presence of circulating anti-ENA and anti-dsDNA antibody-secreting cells, to determine their dependence on plasma cell-niche cytokines and to analyze their clinical value. The study was performed in SLE patients with serum anti-ENA and/or anti-dsDNA antibodies (n = 57). Enriched B-cell fractions and sorted antibody-secreting cells (CD19 low CD38 high ) were obtained from blood. dsDNA- and ENA-specific antibody-secreting cells were identified as cells capable of active auto-antibody production in culture. The addition of a combination of IL-6, IL-21, BAFF, APRIL, and CXCL12 to the cultures significantly augmented auto-antibody production and antibody-secreting cell proliferation, whereas it diminished apoptosis. The effect on auto-antibody production was dependent on STAT-3 activation as it was abrogated in the presence of the JAK/STAT-3 pathway inhibitors ruxolitinib and stattic. Among patients with serum anti-dsDNA antibodies, the detection of circulating anti-dsDNA-antibody-secreting cells was associated with higher disease activity markers. In conclusion, auto-antibody production in response to plasma cell-niche cytokines that are usually at high levels in SLE patients is dependent on JAK/STAT-3 activation. Thus, patients with circulating anti-dsDNA antibody-secreting cells and active disease could potentially benefit from therapies targeting the JAK/STAT3 pathway., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

CD72 is a co-receptor of B cells and regulates B cell activation. Although aberrant expression of CD72 has been reported in primary immune thrombocytopenia (ITP), it is uncertain whether this aberrant expression is restricted to specific B cell subsets. Furthermore, the mechanisms that regulate CD72 expression are unknown. In this study, we found higher frequency of CD19 + B cells, CD19 + CD27 + memory B cells and lower frequency of CD19 + CD27 - naive B cells in active ITP patients compared with controls and patients in remission. CD72 expression on CD19 + CD27 + cells was upregulated in active ITP patients and correlated with platelet count and anti-platelet autoantibodies. In vitro, CD40L could specifically induce CD72 upregulation on CD19 + CD27 + B cells. In combination with CD40L, interleukin (IL) 10 and BAFF (also termed TNFSF13B) further enhanced CD72 expression on CD19 + CD27 + B cells, whereas IL21 reduced CD72 upregulation. CD72mRNA expression after CD40L stimulation was increased in ITP patients and controls. Significant increase of CD40L on CD4 + T cells was correlated with CD72 expression on CD19 + CD27 + B cells in ITP patients. In conclusion, upregulation of CD72 expression on CD27 + memory B cells might take part in the pathogenesis of ITP. Elevated CD40L on CD4 + cells combined with cytokines might contribute to the upregulation of CD72 expression on CD27 + memory B cells., (© 2017 John Wiley & Sons Ltd.)

Immunological prevention of infectious disease, especially viral, is based on antigen-specific long-lived memory B cells. To test for cellular proliferation and differentiation factors in swine, an outbred model for humans, CD21+ B cells were activated in vitro with CD40L and stimulated with purported stimulatory cytokines to characterize functional responses. IL-21 induced a 3-fold expansion in total cell numbers with roughly 15% of all B cells differentiating to IgM or IgG antibody secreting cells (ASCs.) However, even with robust proliferation, cellular viability rapidly deteriorated. Therefore, a proliferation inducing ligand (APRIL) and B cell activating factor (BAFF) were evaluated as survival and maintenance factors. BAFF was effective at enhancing the viability of mature B cells as well as ASCs, while APRIL was only effective for ASCs. Both cytokines increased approximately two-fold the amount of IgM and IgG which was secreted by IL-21 differentiated ASCs. Mature B cells from porcine reproductive and respiratory virus (PRRSV) immune and naïve age-matched pigs were activated and treated with IL-21 and then tested for memory cell differentiation using a PRRSV non-structural protein 7 ELISPOT and ELISA. PRRSV immune pigs were positive on both ELISPOT and ELISA while naïve animals were negative on both assays. These results highlight the IL-21-driven expansion and differentiation of memory B cells in vitro without stimulation of the surface immunoglobulin receptor complex, as well as the establishment of a defined memory B cell culture system for characterization of vaccine responses in outbred animals., Competing Interests: The authors have declared that no competing interests exist.

Ethnopharmacological Relevance: Total glucosides of paeony (TGP) is the first anti-inflammatory immune regulatory drug approved for the treatment of rheumatoid arthritis in China. A novel compound, paeoniflorin-6'-O-benzene sulfonate (code CP-25), comes from the structural modification of paeoniflorin (Pae), which is the effective active ingredient of TGP. The aim of the present study is to investigate the effect of CP-25 on adjuvant arthritis (AA) fibroblast-like synoviocytes (FLS) co-cultured with BAFF-activated CD4(+) T cells and the expression of BAFF-R in CD4(+) T cells., Methods: The mRNA expression of BAFF and its receptors was assessed by qPCR. The expression of BAFF receptors in CD4(+) T cells was analyzed by flow cytometry. The effect of CP-25 on AA rats was evaluated by their joint histopathology. The cell culture growth of thymocytes and FLS was detected by cell counting kit (CCK-8). The concentrations of IL-1β, TNF-α, and IL-6 were measured by Enzyme-linked immunosorbent assay (ELISA)., Results: The mRNA expression levels of BAFF and BAFF-R were enhanced in the mesenteric lymph nodes of AA rats, TACI expression was reduced, and BCMA had no change. The expression of BAFF-R in CD4(+) T cells was also enhanced. CP-25 alleviated the joint histopathology and decreased the expression of BAFF-R in CD4(+) T cells from AA rats in vivo. In vitro, CP-25 inhibited the abnormal cell culture growth of BAFF-stimulated thymocytes and FLS. In the co-culture system, IL-1β, IL-6 and TNF-α production was enhanced by FLS co-cultured with BAFF-activated CD4(+) T cells. Moreover, BAFF-stimulated CD4(+) T cells promoted the cell culture growth of FLS. The addition of CP-25 decreased the expression of BAFF-R in CD4(+) T cells and inhibited the cell culture growth and cytokine secretion ability of FLS co-cultured with BAFF-activated CD4(+) T cells., Conclusion: The present study indicates that CP-25 may repress the cell culture growth and cytokine secretion ability of FLS, and its inhibitory effects might be associated with its ability to inhibit the expression of BAFF-R in CD4(+) T cells in a co-culture. These observations might provide a scientific basis for the development of new drugs for the treatment of autoimmune diseases by CP-25., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Unlabelled: ESSENTIALS: Dysfunctional B-cell-activating factor (BAFF) system is related to many autoimmune diseases. The regulatory functions of BAFF/BAFF receptors were investigated in an in vitro coculture system. Different regulatory roles of BAFF were investigated via different receptors in immune thrombocytopenia. The upregulated BAFF receptors on autoreactive lymphocytes lead to their hypersensitivity to BAFF., Background: The pathogenesis of immune thrombocytopenia (ITP) remains enigmatic. B-cell-activating factor (BAFF) and its receptors (BAFF receptor [BAFF-R], transmembrane activator and calcium modulator and cyclophilin ligand interactor [TACI], and B-cell maturation antigen) play central roles in the integrated homeostatic regulation of lymphocytes., Objectives: To investigate the pathologic roles of BAFF receptors in regulating the bioactivities of lymphocytes in ITP., Methods: An in vitro culture system was established by stimulating CD14(-) peripheral lymphocytes with platelet-preloaded dendritic cells in the presence of recombinant human BAFF (rhBAFF; 20 ng mL(-1)). The functions of BAFF receptors were specifically blocked with blocking antibodies., Results: BAFF-R, besides prolonging the survival of B cells in both patients and healthy controls, prominently promoted the survival of CD8(+) T cells and the proliferation of B cells in patients with ITP. TACI, as a positive regulator, not only promoted the proliferation of CD4(+) and CD8(+) T cells, but also significantly enhanced the secretion of interleukin-4 in patients with ITP, but not in controls. Besides revealing the pathologic roles of BAFF receptors, these results also indicate that lymphocytes of patients with ITP have enhanced antiapoptotic or proliferative capacity as compared with those from healthy controls when exposed under similar stimulation of rhBAFF. Further study demonstrated that activated autoreactive B cells and CD4(+) T cells from patients with ITP showed significantly higher expression of BAFF-R or TACI than those from healthy controls., Conclusions: Both BAFF-R and TACI are pathogenic participants in ITP. Their dysregulated expression in patients with ITP may lead to hyperreactivity of activated autoreactive lymphocytes in response to rhBAFF, and thus is highly significant in the pathogenesis of ITP., (© 2016 International Society on Thrombosis and Haemostasis.)

Peripheral B lymphocyte survival requires the B cell receptor (BCR) and B cell activating factor (BAFF) binding to its receptor (BAFF-R). Deletion of the BCR, or its signal transducing chaperone Igβ, leads to rapid loss of mature B cells, indicating that signals initiated at the BCR are crucial for B cell survival. BAFF or BAFF-R deficiency also significantly reduces the numbers of mature B cells despite normal BCR expression. Together, these observations indicate that continued BCR and BAFF-R signaling are essential for the survival of mature resting B cells in the periphery. Here we demonstrate that tonic BCR signals up-regulate p100 (Nfkb2) as well as Mcl-1 protein expression at a post-transcriptional level via a PI3K-dependent pathway. p100 expression is mTOR-independent, whereas Mcl-1 expression is mTOR-dependent. BAFF treatment further elevated Mcl-1 levels by an mTOR-independent pathway, while consuming p100. Accordingly, Mcl-1 induction by BAFF is abrogated in Nfkb2-/- B cells. We propose that the cumulative effects of the BCR and BAFF-R signaling pathways increase Mcl-1 levels beyond the threshold required for B cell survival.

Unlabelled: Gammaherpesviruses (GHVs) carry homologs of cellular genes, including those encoding a viral cyclin that promotes reactivation from latent infection. The viral cyclin has reduced sensitivity to host cyclin-dependent kinase inhibitors in vitro; however, the in vivo significance of this is unclear. Here, we tested the genetic requirement for the viral cyclin in mice that lack the host inhibitors p27(Kip1) and p18(INK4c), two cyclin-dependent kinase inhibitors known to be important in regulating B cell proliferation and differentiation. While the viral cyclin was essential for reactivation in wild-type mice, strikingly, it was dispensable for reactivation in mice lacking p27(Kip1) and p18(INK4c). Further analysis revealed that genetic ablation of only p18(INK4c) alleviated the requirement for the viral cyclin for reactivation from latency. p18(INK4c) regulated reactivation in a dose-dependent manner so that the viral cyclin was dispensable in p18(INK4c) heterozygous mice. Finally, treatment of wild-type cells with the cytokine BAFF, a known attenuator of p18(INK4c) function in B lymphocytes, was also able to bypass the requirement for the viral cyclin in reactivation. These data show that the gammaherpesvirus viral cyclin functions specifically to bypass the cyclin-dependent kinase inhibitor p18(INK4c), revealing an unanticipated specificity between a GHV cyclin and a single cyclin-dependent kinase inhibitor., Importance: The gammaherpesviruses (GHVs) cause lifelong infection and can cause chronic inflammatory diseases and cancer, especially in immunosuppressed individuals. Many GHVs encode a conserved viral cyclin that is required for infection and disease. While a common property of the viral cyclins is that they resist inhibition by normal cellular mechanisms, it remains unclear how important it is that the GHVs resist this inhibition. We used a mouse GHV that either contained or lacked a viral cyclin to test whether the viral cyclin lost importance when these inhibitory pathways were removed. These studies revealed that the viral cyclin was required for optimal function in normal mice but that it was no longer required following removal or reduced function of a single cellular inhibitor. These data define a very specific role for the viral cyclin in bypassing one cellular inhibitor and point to new methods to intervene with viral cyclins., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

B cell activating factor (BAFF), which belongs to the tumor necrosis factor (TNF) family, is testified to play a critical role in B cell survival, proliferation, maturation and immunoglobulin secretion. In the present study, the cDNA of open reading frame (ORF) in African ostrich (Struthio camelus) BAFF (designated OsBAFF) was cloned by reverse transcription-PCR (RT-PCR). The OsBAFF gene encodes a 288-amino acid protein containing a predicted transmembrane domain and a putative furin protease cleavage site like BAFFs from chicken (cBAFF), quail (qBAFF), duck (dBAFF), goose (gBAFF) and dove (doBAFF). RT-PCR analysis showed that the OsBAFF gene is strongly expressed in the bursa of Fabricius, thymus, spleen, and bone marrow. The soluble OsBAFF had been cloned into pET28a. SDS-PAGE and Western blotting analysis confirmed that the soluble fusion protein His-OsBAFF was efficiently expressed in Escherichia coli Rosset (DE3). In vitro, purified OsBAFF was not only able to promote the survival of African ostrich bursal lymphocytes, but also able to co-stimulate proliferation of mouse splenic B cells. The expression of OsBAFF in lymphocyte cells was higher than the control after LPS stimulation. These findings indicated that OsBAFF plays an important role in survival and proliferation of African ostrich bursal lymphocytes, which may provide valuable information for research into the immune system of African ostrich and OsBAFF may serve as a potential immunologic factor for enhancing immunological efficacy in African ostrich and any other birds., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Both tumor-associated neutrophils (TAN) and cancer-associated fibroblasts (CAFs) display specific phenotypic and functional features and contribute to tumor cell niche. However, their bidirectional crosstalk has been poorly studied, in particular in the context of hematological malignancies. Follicular lymphomas (FL) and diffuse large B-cell lymphomas (DLBCL) are two germinal center-derived lymphomas where various cell components of infiltrating microenvironment, including TAN and CAFs, have been demonstrated to favor directly and indirectly malignant B-cell survival, growth, and drug resistance. We show here that, besides a direct and contact-dependent supportive effect of neutrophils on DLBCL B-cell survival, mediated through the BAFF/APRIL pathway, neutrophils and stromal cells cooperate to sustain FL B-cell growth. This cooperation relies on an overexpression of IL-8 by lymphoma-infiltrating stromal cells that could thereafter efficiently promote neutrophil survival and prime them to neutrophil extracellular trap. Conversely, neutrophils are able to activate stromal cells in a NF-κB-dependent manner, inducing their commitment towards an inflammatory lymphoid stroma phenotype associated with an increased capacity to trigger malignant B-cell survival, and to recruit additional monocytes and neutrophils through the release of CCL2 and IL-8, respectively. Altogether, a better understanding of the lymphoma-supporting effects of neutrophils could be helpful to design new anti-tumor therapeutic strategies.

Background: B cell activating factor belonging to the TNF family (BAFF) is vital for B cell survival, proliferation and activation. Evidence indicates that BAFF is systemically or locally increased in glomerulonephritis (e.g. lupus nephritis, IgA nephropathy). However, the effect of BAFF on human mesangial cells is not known., Methods: The impact of BAFF on the proliferation of a human mesangial cell line in vitro was investigated. The expression of BAFF receptor (BAFF-R) and downstream signal transduction were explored. The influence of BAFF on the expression of related genes was also studied., Results: Our data indicated that BAFF had a proliferative effect on human mesangial cells, as supported by the results of cell proliferation assays and the inhibited expression of the pro-apoptotic gene Bim. BAFF-R was expressed on the cell membrane of human mesangial cells and blockade of BAFF/BAFF-R binding abrogated the proliferative effect of BAFF on human mesangial cells. BAFF stimulation led to rapid phosphorylation of NF-κBp65, Akt and MAPK p38 kinase in human mesangial cells, whereas it had no effect on the expression of NF-κB p100 and phosphorylation of Erk. The phosphorylation of Akt was very sensitive to blockade of BAFF/BAFF-R ligation, although activation of MAPK p38 and NF-κBp65 was not. BAFF treatment resulted in decreased expression of BAFF-R, which implied negative feedback regulation after its binding., Conclusions: BAFF promoted proliferation of human mesangial cells, which was mediated via BAFF-R. The BAFF/BAFF-R interaction triggered Akt, p65 and p38 activation, with Akt phosphorylation being tightly dependent on BAFF/BAFF-R interaction.

The selection and maturation of B-cell clones are critically determined by tonic signals from activated B cell receptors (BCR) and survival signals from BAFF cytokine. These finely tuned and coordinated signals provide a net positive signal that can promote the selection, maturation, proliferation and differentiation of a developing B cell. Stimulation with an anti-IgD antibody can also activate BCR but can lead to depletion and an arrest of mature B-cell development in vivo. It is not known whether survival signals from excess BAFF can override the suppressive effects of treatment with anti-IgD on mature B cells in vivo. Herein, we examined the effects of co-treatment of BAFF and anti-IgD on the mature B-cell compartment and antibody production in vivo by treating mice with either 1mg/kg BAFF or anti-IgD alone or in combination for 3 consecutive days. We found that co-treatment with anti-IgD significantly abrogated these stimulatory effects of BAFF treatment on splenic CD19+ B cells as well as mature CD19+IgD(hi)IgM+ B cells in vivo. Anti-IgD down-regulated the expression of the BCR complex (mIgM, mIgD and CD19) and the BAFF receptor TACI without regard to the presence of BAFF. Anti-IgD treatment also significantly negated BAFF-induced IgM production in vivo. Both BAFF and anti-IgD could individually stimulate IL-10 synthesis in B cells but did not affect one another. Taken together, our data suggest that activation of BCR with an anti-IgD antibody can override the stimulatory effects from excess BAFF on B cell proliferation and antibody production by down-regulating the expression of BCR complex and BAFF receptors., (Copyright © 2014 Elsevier B.V. All rights reserved.)

B-cell activating factor-receptor (BAFF-R) is the primary BAFF receptor that is responsible for promoting B-cell development and survival. Malignant B-cells exploit the BAFF/BAFF-R system, and high serum BAFF levels or genetic alterations in BAFF receptors have been found in B-cell cancers. BAFF signaling impacts pro-survival pathways. However, other than nuclear factor-κB2 (NF-κB2), little is known about the specific pathways activated by individual BAFF receptors. Using a novel BAFF-R expression model we have demonstrated that activation of BAFF-R, independent of transmembrane activator and cytophilin ligand interactor (TACI) and B-cell maturation antigen (BCMA), can induce phosphorylation of Akt and glycogen synthase kinase 3β (GSK3β). Expression of an activated form of BAFF-R also enhanced a pro-survival gene expression pattern, including the novel BAFF-regulated gene Pin1, whose expression was phosphatidyl inositol 3-kinase (PI3K)-dependent. Additionally, we showed that TRAF6 is essential for mediating BAFF-R dependent activation of Akt. Together these data describe a novel role for TRAF6 in BAFF-R-specific activation of the PI3K pathway and provide evidence suggesting a new role for Pin1 in BAFF-R signaling.

B-cell activating factor (BAFF) is involved in not only the physiology of normal B cells, but also the pathophysiology of aggressive B cells related to malignant and autoimmune diseases. However, how excessive BAFF promotes aggressive B-cell proliferation and survival is not well understood. Here we show that excessive human soluble BAFF (hsBAFF) enhanced cell proliferation and survival in normal and B-lymphoid (Raji) cells, which was associated with suppression of PP2A, resulting in activation of Erk1/2. This is supported by the findings that pretreatment with U0126 or PD98059, expression of dominant negative MKK1, or overexpression of PP2A prevented hsBAFF-induced activation of Erk1/2 and cell proliferation/viability in the cells. It appears that hsBAFF-mediated PP2A-Erk1/2 pathway and B-cell proliferation/viability was Ca(2+)-dependent, as pretreatment with BAPTA/AM, EGTA or 2-APB significantly attenuated these events. Furthermore, we found that inhibiting CaMKII with KN93 or silencing CaMKII also attenuated hsBAFF-mediated PP2A-Erk1/2 signaling and B-cell proliferation/viability. The results indicate that BAFF activates Erk1/2, in part through Ca(2+)-CaMKII-dependent inhibition of PP2A, increasing cell proliferation/viability in normal and neoplastic B-lymphoid cells. Our data suggest that inhibitors of CaMKII and Erk1/2, activator of PP2A or manipulation of intracellular Ca(2+) may be exploited for prevention of excessive BAFF-induced aggressive B-cell malignancies and autoimmune diseases., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Introduction: B lymphocytes might play a pathogenic role in dermal fibrosis in systemic sclerosis (SSc). B-cell activating factor (BAFF), a key cytokine for B-cell activation, is increased in the serum and the skin of patients with SSc. However, the ability of B cells directly to stimulate dermal fibroblasts and the role of BAFF are not fully understood. We therefore investigated the involvement of B cells and BAFF in the expression of collagen and profibrotic markers by dermal fibroblasts., Methods: Cocultures of blood B cells from healthy blood donors and normal or SSc dermal fibroblasts stimulated with anti-IgM and BAFF were performed. Alpha-SMA, TIMP1, MMP9, COL1A1, COL1A2, and COL3A1 mRNA expression were determined by quantitative RT-PCR. Soluble collagen, BAFF, IL-6, IL-1β, TGF-β1, and CCL2 protein secretion were assessed., Results: Coculture of blood B cells and dermal fibroblasts isolated from SSc patients induced IL-6, TGF-β1, CCL2, and collagen secretion, as well as Alpha-SMA, TIMP1, and MMP9 expression in dermal fibroblasts. Transwell assays demonstrated that this induction was dependent on cell-cell contact. Addition of anti-IgM and BAFF to the coculture increased IL-6, CCL2, TGF-β1, and collagen secretion. B cell- and BAFF-induced collagen secretion was highly reduced by anti-TGF-β1 antibodies., Conclusions: Our results showed for the first time a direct role of B cells on the production of collagen by dermal fibroblasts, which is further enhanced by BAFF. Thus, these results demonstrate a new pathogenic role of B cells and BAFF in fibrosis and systemic sclerosis.

Objective: To determine the necessity for any individual BAFF receptor in the development of systemic lupus erythematosus (SLE)., Methods: Bcma-, Taci-, and Br3-null mutations were introgressed into NZM 2328 mice. NZM.Bcma-/-, NZM.Taci-/-, and NZM.Br3-/- mice were evaluated for lymphocyte phenotype and BAFF receptor expression by flow cytometry; for B cell responsiveness to BAFF by in vitro culture; for serum levels of BAFF and total IgG and IgG anti-double-stranded DNA (anti-dsDNA) by enzyme-linked immunosorbent assay; for renal immunopathology by immunofluorescence and histopathology; and for clinical disease., Results: BCMA, TACI, and B lymphocyte stimulator receptor 3 (BR3) were not surface-expressed in NZM.Bcma-/-, NZM.Taci-/-, and NZM.Br3-/- mice, respectively. Transitional and follicular B cells from NZM.Br3-/- mice were much less responsive to BAFF than were the corresponding cells from wild-type, NZM.Bcma-/-, or NZM.Taci-/- mice. In comparison with wild-type mice, NZM.Bcma-/- and NZM.Taci-/- mice harbored an increased number of spleen B cells, T cells, and plasma cells, whereas serum levels of total IgG and IgG anti-dsDNA were similar to those in wild-type mice. Despite their paucity of B cells, NZM.Br3-/- mice had an increased number of T cells, and the numbers of plasma cells and levels of IgG anti-dsDNA were similar to those in wild-type mice. Serum levels of BAFF were increased in NZM.Taci-/- and NZM.Br3-/- mice but were decreased in NZM.Bcma-/- mice. Despite their phenotypic differences, NZM.Bcma-/-, NZM.Taci-/-, and NZM.Br3-/- mice had renal immunopathology and clinical disease that were at least as severe as that in wild-type mice., Conclusion: Any single BAFF receptor, including BR3, is dispensable for the development of SLE in NZM mice. Development of disease in NZM.Br3-/- mice demonstrates that BAFF-BCMA and/or BAFF-TACI interactions contribute to SLE, and that a profound, life-long reduction in the numbers of B cells does not guarantee protection against SLE., (Copyright © 2013 by the American College of Rheumatology.)

Follicular B cell survival requires signaling from BAFFR, a receptor for BAFF and the B cell antigen receptor (BCR). This "tonic" BCR survival signal is distinct from that induced by antigen binding and may be ligand-independent. We show that inducible inactivation of the Syk tyrosine kinase, a key signal transducer from the BCR following antigen binding, resulted in the death of most follicular B cells because Syk-deficient cells were unable to survive in response to BAFF. Genetic rescue studies demonstrated that Syk transduces BAFFR survival signals via ERK and PI3 kinase. Surprisingly, BAFFR signaling directly induced phosphorylation of both Syk and the BCR-associated Igα signaling subunit, and this Syk phosphorylation required the BCR. We conclude that the BCR and Igα may be required for B cell survival because they function as adaptor proteins in a BAFFR signaling pathway leading to activation of Syk, demonstrating previously unrecognized crosstalk between the two receptors., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Chronic inflammation is an important contributor to the development and progression of metabolic syndrome. Recent evidence indicates that, in addition to innate immune cells, adaptive immune cells have an important role in this process. We previously showed that the serum level of B-cell-activating factor (BAFF) was increased in patients with nonalcoholic steatohepatitis. However, it is currently unknown whether BAFF and BAFF-R (BAFF-R) have a role in lipid metabolism in the liver. To address this issue, the role played by BAFF and BAFF-R signaling in the development of insulin resistance and hepatic steatosis was examined in BAFF-R(-/-) mice fed a high-fat diet (HFD). Furthermore, the effect of BAFF on lipid metabolism in hepatocytes was analyzed in vitro. BAFF-R(-/-) mice showed improvements in HFD-induced obesity and insulin resistance. In addition, the number of B cells, levels of serum IgG, and inflammation of visceral fat were reduced in these mice. However, the expression of steatogenic genes and fatty acid deposition in the liver was higher in these mice than in control mice. BAFF was also found to downregulate the expression of steatogenesis genes and enhance steatosis in hepatocytes through BAFF-R. Collectively, these data indicated that, in addition to its known functions in inflammation and glucose metabolism, BAFF has a protective role in hepatic steatosis by regulating lipid metabolism in the liver.

3T3-L1 adipocytes express the B-cell-activating factor (BAFF) and three different BAFF receptors (BAFF-Rs). Furthermore, BAFF expression is regulated by inflammatory modulators, such as tumor necrosis factor-α and rosiglitazone. Here we investigated the function of BAFF in 3T3-L1 adipocytes and RAW 264.7 macrophages. We examined adipokine expression in 3T3-L1 adipocytes treated with 10 ng ml(-1) BAFF. We also examined inflammatory molecule expression in RAW 264.7 macrophages treated with 10 or 100 ng ml(-1) BAFF. We examined BAFF expression in the coculture of 3T3-L1 adipocytes and RAW 264.7 macrophages, as well as in white adipose tissue (WAT) of diet-induced obese (DIO) mice. We found that BAFF decreases leptin and adiponectin expression, but increases the expression of proinflammatory adipokines monocyte chemotactic protein-1, interleukin-6 (IL-6), cyclooxygenase-2 (COX-2) and haptoglobin. Coculturing the two cell types resulted in increased BAFF mRNA and protein expression, as well as modulation of BAFF-R mRNA expression in both cell types. These data indicate that BAFF might mediate adipocyte and macrophage interaction. When RAW 264.7 macrophages were treated with BAFF, BAFF-R expression was modulated as in coculture, and nitric oxide synthase and IL-6 expression increased. BAFF expression also increased in WAT of DIO mice. We propose that BAFF can regulate adipokine expression and possibly mediate adipocyte and macrophage interaction.

We aim to investigate the additive value of B cell-activating factor (BAFF) when added to oligodeoxynucleotides (ODN)-activated B cells with respect to TLR-9, CD69, MHC-II expression, IL-6 and IL-10 secretion and B cell cycling. Therefore, B cells from healthy individuals were incubated under the following conditions: (1) B cells with medium, (2) B cells with ODN 0.5 μm, (3) B cells with BAFF 20 μm and (4) B cells with both ODN 0.5 μm and BAFF 20 μm. We found that addition of BAFF did not enhance the expression of TLR-9, CD69 and MHC-II in ODN-activated B cells. Incubation of B cells with BAFF and ODN together leads to a marked elevation of IL-6 and IL-10 levels compared to ODN alone. Synthesis and mitosis were higher in B cells stimulated by BAFF than in B cells stimulated by ODN. These findings suggest that both BAFF and TLR-9 contribute independently to B cell function., (© 2012 The Authors. Scandinavian Journal of Immunology © 2012 Blackwell Publishing Ltd.)

The TNF ligand family member "B cell-activating factor belonging to the TNF family" (BAFF, also called BLyS, TALL-1, zTNF-4, and THANK) is an important survival factor for B and T cells. In this study, we show that BAFF is able to induce CD4(+) spleen T cell proliferation when co-stimulated with anti-CD3. Expression of phosphorylated FOXO3A was notably down-regulated and cyclins D2 and D3 were up-regulated and higher in the CD4(+) T cells when treated with BAFF and anti-CD3, as assessed by Western blotting. Furthermore, after FOXO3A was knocked down, expression of cyclin D1 was unchanged, compared with control group levels, but the expression of cyclins D2 and D3 increased, compared with the control group. In conclusion, our results suggest that BAFF induced CD4(+) spleen T cell proliferation by down-regulating the phosphorylation of FOXO3A and then activating cyclin D2 and D3 expression, leading to CD4(+) T cell proliferation., (Copyright © 2012 Elsevier Inc. All rights reserved.)

To investigate the role of PI3K/Akt/mTOR signaling mediated by B cell-activating factor belonging to the TNF family (BAFF) involved in anti-apoptosis of B lymphocytes in rats with collagen-induced arthritis (CIA) and the regulation of epigallo-catechin-3-gallate (EGCG). Sprague-Dawley rats were immunized to induce CIA. CIA rats were randomly separated into different groups and treated with EGCG (40, 80 mg/kg), Paeoniflorin (100mg/kg) from day 18 to day 38 after immunization. The effects of EGCG on B lymphocytes were evaluated by the levels of BAFF, anti-CII antibody, IgA, IgG and IgM, and the expressions of BAFF receptor, P110δ, p-Akt, mTORC1, Bcl-xL and Bim. B lymphocyte proliferations were analyzed by MTT assay. Apoptosis of B lymphocyte were assayed by flow cytometry. Results showed that, in CIA rats, the levels of BAFF, anti-CII antibody, IgA, IgG and IgM enhanced. BAFF receptor, P110δ, p-AKT, mTORC1 and Bcl-xL were expressed highly, while Bim expression decreased. EGCG (40, 80 mg/kg) and Paeoniflorin decreased the levels of BAFF, anti-CII antibody, IgA, IgG, IgM and the expressions of BAFF receptor, P110δ, p-AKT, mTORC1, Bcl-xL in CIA rats, and increased Bim expression. Further studies showed that EGCG could reduce the expression of P110δ and mTORC1 in vitro. EGCG inhibited B lymphocyte proliferation and induced B lymphocyte apoptosis. In conclusion, BAFF/BAFF receptor might regulate B cell anti-apoptosis through PI3K/Akt/mTOR pathway. EGCG had therapeutic effects on CIA rats, which might be relative to the inhibition effects of EGCG on BAFF and PI3K/Akt/mTOR signaling, and then the apoptosis of B lymphocytes was promoted further., (Copyright © 2012 Elsevier B.V. All rights reserved.)

B cell activating factor (BAFF), a member of the TNF family, is a critical cytokine for the survival, proliferation, maturation, and differentiation of B cells. In the present study, Père David's deer BAFF (miBAFF) was amplified from Elaphurus davidianus using RT-PCR. This is the first BAFF cloned from a member of Cervidae family. The open reading frame (ORF) of the miBAFF cDNA consists of 843 bases that encode a 280-amino acid protein bearing typical TNF homology domain. Sequence alignment shows that miBAFF shares 39.3%-97% sequence homology with the BAFF sequences of other mammals. Comparative protein modeling predicted that the 3D structure of the soluble mature portion of miBAFF (misBAFF) is very similar to that of human BAFF (hsBAFF). Recombinant misBAFF fused to a SUMO-tag was efficiently expressed in Escherichia coli BL21 (DE3) cells. The protein molecular weight of ~36 KDa was determined using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. In vitro, purified misBAFF was shown to promote the survival and proliferation of Père David's deer peripheral blood lymphocytes and mouse B cells. These results indicate that miBAFF plays an important role in the survival/proliferation of mouse B cells and, shows highly conserved evolutionarily, leading to functional cross-reactivity that exists between mouse and Père David's deer BAFF., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Neutralizing Abs provide the protective effect of the majority of existing human vaccines. For a prophylactic vaccine against HIV-1, broadly neutralizing Abs targeting conserved epitopes of the viral envelope glycoproteins (Env) are likely required, because the pool of circulating HIV-1 variants is extremely diverse. The failure to efficiently induce broadly neutralizing Abs by vaccination may be due to the use of suboptimal immunogens or immunization regimens, or it may indicate that B cells specific for broadly neutralizing Env determinants are selected against during peripheral checkpoints, either before or after Ag encounter. To investigate whether perturbation of B cell subsets prior to immunization with recombinant Env protein affects the vaccine-induced Ab response in mice, we used B lymphocyte stimulator (BLyS), a cytokine that regulates survival and selection of peripheral B cells. We show that the transient BLyS treatment used in this study substantially affected naive B cell populations; in particular, it resulted in more B cells surviving counter-selection at the transitional stages. We also observed more mature naive B cells, especially marginal zone B cells, in BLyS-treated mice. Intriguingly, provision of excess BLyS prior to immunization led to a consistent improvement in the frequency and potency of HIV-1 Env vaccine-induced neutralizing Ab responses, without increasing the number of Env-specific Ab-secreting cells or the Ab-binding titers measured after boosting. The results presented in this article suggest that an increased understanding of BLyS-regulated processes may help the design of vaccine regimens aimed at eliciting improved neutralizing Ab responses against HIV-1.

Calcium-modulating cyclophilin ligand (CAML) is a ubiquitously expressed protein that is important during thymopoiesis. However, whether it serves a function in mature lymphocytes is unknown. In this article, we show that CAML is essential for survival of peripheral follicular (Fo) B cells. Conditional deletion of CAML in CD19-Cre transgenic mice caused a significant reduction in Fo cell numbers and increased rates of homeostatic proliferation. CAML-deficient Fo cells showed increased cellular turnover and normal proliferative ability. Although CAML-deficient Fo cells responded to AgR stimulation and to B cell activating factor, they displayed decreased survival and increased apoptosis following stimulation with LPS and IL-4 in vitro. Failure to survive was not due to aberrant B cell development in the absence of CAML, because induced deletion of the gene in mature cells resulted in a similar phenotype. These data establish an essential and ongoing role for CAML in the long-term survival of mature B cells.

We previously reported that CD21(+) B cells purified from bovine blood do not respond to CpG-ODN stimulation unless either CD14(+) monocytes or B-cell Activating Factor (BAFF), a cytokine produced by activated monocytes, are present. In this report, we present evidence that CD14(+) monocytes are critical for CpG-specific lymphocyte proliferation within the peripheral blood mononuclear cell (PBMC) population but that this response is not mediated by soluble factors produced by CpG-activated monocytes. We further determine that bovine monocytes stimulated with IFN-γ induce expression of the BAFF gene and that recombinant IFN-γ and BAFF induced robust B cell activation when cultured in the absence of CpG ODN. These data suggest that CpG-stimulated monocytes may indirectly promote B cell activation by promoting release of cytokines and/or other soluble factors from accessory cells which in turn act on CpG-stimulated B cells to promote antigen-independent and T cell independent B cell activation. Understanding the T cell independent signals that induce B cell activation has important implications for understanding B cell development in locations where T cells are limited and in understanding polyclonal B cell activation that may contribute to autoimmune diseases., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Members of the tumor necrosis factor (TNF) family play key roles in the regulation of inflammation, immune responses and tissue homeostasis. Here we describe the identification of the Japanese sea perch (Lateolabrax japonicus) homologue of mammalian B cell activating factor of the TNF family (BAFF/BLyS) (designated LjBAFF). The cDNA contains an open reading frame (ORF) of 783 nucleotides that are translated into a predicted 260 amino acid protein. Like most known BAFFs, Japanese sea perch BAFF contains three cysteine residues (Cys(123), Cys(218) and Cys(232)) which are conserved in the aligned BAFF sequences and a furin protease cleavage site (R-K-K-R). Real-time quantitative PCR (qPCR) analysis revealed that LjBAFF could be detected in various tissues and predominantly expressed in lymphoid tissue spleen. The soluble BAFF (LjsBAFF) had been cloned into a pET28a vector to express the recombinant protein. The His-LjsBAFF was efficiently expressed in Escherichia coli BL21 (DE3) and its expression was confirmed by SDS-PAGE and Western blotting analysis. After purification, MTT assays and flow cytometric analysis revealed that LjsBAFF could promote the survival/proliferation of splenic B cells in vitro. Furthermore, bacterially expressed LjsBAFF induced the selective expansion of B cells in the spleen when administered to young mice. Our results suggest that like its mammalian counterparts, LjsBAFF plays an important role in the survival and/or proliferation of B cells., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Rheumatoid arthritis (RA) is a chronic and systemic autoimmune disease, it's pathogenic mechanism is unknown and there is a lack of safe and effective therapeutic drug on RA. Because of important function of B cells in RA pathogenic mechanism, many therapeutic drugs targeted on different positions of B cells are emerging in RA therapy. These B cell-targeted therapeutic biologic agents include anti-CD20 monoclonal antibody (such as rituximab, ocrelizumab and ofatumumab), biologic agents which targeted B lymphocyte stimulator (BLyS) and their receptors (such as belimumab and atacicept), anti-CD22 monoclonal antibody and CD40/CD40L co-stimulation reaction blocker. Efficacy of above-mentioned targeting agents has confirmed in studies of RA and it's animal models, suggesting that it is a promising therapeutic strategy that B cells are regarded as targeting position in RA therapy.

B cell activating factor belonging to the TNF family (BAFF, also called BLyS, TALL-1, THANK, or zTNF4) is an important survival factor for B cells, and is able to regulate T-cell activation. Recently, we have demonstrated that treatment of mice with human soluble BAFF (hsBAFF) causes a significant increase of percentages of splenic CD4(+) T lymphocytes dose-dependently, but the CD8(+) T lymphocyte percentages maintained unchanged. Here, we show that hsBAFF significantly enhanced CD4(+) T lymphocyte response of cultured mouse splenic cells, and hsBAFF induced the proliferation and IL-2/IFN-γ secretion of purified CD4(+) T lymphocytes suboptimally stimulated through anti-CD3. Of importance, we observed that IL-2 or IFN-γ cytokine has additive effect on the proliferation and activity of hsBAFF-stimulated CD4(+) T lymphocytes. Using Flow cytometry with fluorescent probe, Fluo-3/AM, we found that hsBAFF elicited [Ca(2+)](i) elevation contributing to CD4(+) T cell proliferation. This is evidenced by our finding that pretreatment with BAPTA/AM, an intracellular Ca(2+) chelator, significantly attenuated the proliferation of hsBAFF-stimulated CD4(+) T lymphocytes. Subsequently, we revealed that hsBAFF-stimulated CD4(+) T cell proliferation was markedly suppressed after pretreatment with EGTA, an extracellular Ca(2+) chelator, or with 2-APB, an inhibitor of Ca(2+) influx through CRAC channels, respectively, suggesting that extracellular Ca(2+) influx due to hsBAFF is closely associated with [Ca(2+)](i) elevation contributing to CD4(+) T cell proliferation. In addition, we noticed that hsBAFF-treated cells conferred partial resistance to decrease of cellular viability induced by thapsigargin (Tg), an endoplasmic reticulum (ER) Ca(2+)-ATPase inhibitor. Taken together, our data indicate that hsBAFF may promote CD4(+) T cell proliferation and response by upregulation of [Ca(2+)](i) homeostasis., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

It is controversial whether naïve B cells are directly activated in response to TLR9 ligand, CpG ODN. Although bovine blood-derived CD21(+) B cells express TLR9 and proliferate in response to CpG in mixed-cell populations, purified bovine B cells do not proliferate significantly in response to CpG ODN, even when the B cell receptor is engaged. When co-cultured with CD14(+) myeloid cells and/or B-cell activating factor (BAFF), a cytokine produced by activated myeloid cells, there was a significant increase in CpG-specific B cell proliferation, and the number of large B cells in general or positive for CD25, all of which are markers for B cell activation. These data suggest that activated myeloid cells and BAFF prime B cells for significant CpG-specific activation. Understanding the signals required to mediate efficient CpG-induced, antigen-independent and T-cell independent activation of B cells has implications for polyclonal B cell activation and the development of autoimmune diseases., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Aberrant signal transducer and activator of transcription (STAT)3 signaling participates in the development and progress of human cancers. We previously generated a highly cytotoxic fusion toxin designated rGel/BLyS for receptor-mediated delivery of the rGel toxin to malignant B-cells. In this study, we examined this fusion toxin for its ability to impact STAT3 signaling in diffuse large B-cell lymphoma (DLBCL). The activated B cell-like DLBCL lines were found to express higher levels of interleukin-6 receptor (IL-6R) and STAT3 than did the germinal center B cell-like DLBCL lines. Treatment of DLBCL cells with rGel/BLyS resulted in down-regulation of IL-6R and inhibited STAT3 phosphorylation, STAT3-DNA binding activity, and IL-6-inducible STAT3 reporter gene activity. In agreement with these results, we additionally found that rGel/BLyS down-regulated levels of several STAT3 targets (c-Myc, p21, Mcl-1, and Bcl-x(L)) and p-SYK, a positive regulator of STAT3. Inhibition of IL-6R-mediated STAT3 signaling by rGel/BLyS led to growth inhibition, triggered accumulation of cells in the sub-G(1) phase of the cell cycle, and induced apoptosis. Our results indicate that rGel/BLyS is an excellent candidate for the treatment of aggressive DLBCL which is resistant to conventional chemotherapeutic regimens and STAT3 signaling pathway may be an attractive therapeutic target for non-Hodgkin's lymphoma., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Survival of mature B cells is regulated by B cell receptor and BAFFR-dependent signals. We show that B cells from mice lacking the G(alphaq) subunit of trimeric G proteins (Gnaq(-/-) mice) have an intrinsic survival advantage over normal B cells, even in the absence of BAFF. Gnaq(-/-) B cells develop normally in the bone marrow but inappropriately survive peripheral tolerance checkpoints, leading to the accumulation of transitional, marginal zone, and follicular B cells, many of which are autoreactive. Gnaq(-/-) chimeric mice rapidly develop arthritis as well as other manifestations of systemic autoimmune disease. Importantly, we demonstrate that the development of the autoreactive B cell compartment is the result of an intrinsic defect in Gnaq(-/-) B cells, resulting in the aberrant activation of the prosurvival factor Akt. Together, these data show for the first time that signaling through trimeric G proteins is critically important for maintaining control of peripheral B cell tolerance induction and repressing autoimmunity.

Assessment of immune responses induced by mucosal vaccines is to a large extent based on measurement of IgA levels in mucosal secretions and detection of short-lived effector IgA-secreting cells circulating in peripheral blood. Since these immunological parameters poorly reflect long-term IgA-mediated responses, we sought to investigate novel approaches that would enable detection of vaccine specific IgA memory B cells. We demonstrate that stimulation of human peripheral blood mononuclear cells in vitro with immunostimulatory DNA in combination with B cell-activating factor (BAFF) and IL-15 promotes differentiation of IgA memory B cells to IgA-secreting cells. By using the inactivated oral cholera vaccine Dukoral we demonstrate that vaccine specific IgA memory B cells are induced by oral immunization and are circulating for at least 9 months after vaccination. We also show that stimulated IgA memory B cells do not secrete IgA unless they reencounter the specific antigen., (Copyright 2010 Elsevier Ltd. All rights reserved.)

B cells are typically characterized as positive regulators of the immune response, primarily by producing antibodies. However, recent studies indicate that various subsets of B cells can perform regulatory functions mainly through IL-10 secretion. Here we discovered that peritoneal B-1 (B-1P) cells produce high levels of IL-10 upon stimulation with several Toll-like receptor (TLR) ligands. High levels of IL-10 suppressed B-1P cell proliferation and differentiation response to all TLR ligands studied in an autocrine manner in vitro and in vivo. IL-10 that accumulated in cultures inhibited B-1P cells at second and subsequent cell divisions mainly at the G1/S interphase. IL-10 inhibits TLR induced B-1P cell activation by blocking the classical NF-kappaB pathway. Co-stimulation with CD40 or BAFF abrogated the IL-10 inhibitory effect on B-1P cells during TLR stimulation. Finally, B-1P cells adoptively transferred from the peritoneal cavity of IL-10(-/-) mice showed better clearance of Borrelia hermsii than wild-type B-1P cells. This study described a novel autoregulatory property of B-1P cells mediated by B-1P cell derived IL-10, which may affect the function of B-1P cells in infection and autoimmunity.

B-lineage acute lymphoblastic leukemia (ALL) arises by transformation of a progenitor (pre-B) cell. Cure rates in adults remain low and treatment is complicated by support provided by the microenvironment to the leukemic cells, indicating an urgent need to better understand the factors that promote their survival. B-cell-activating factor (BAFF) and its receptor BAFF-R are important for survival and growth of mature normal and malignant B cells but are not expressed on pre-B cells. Unexpectedly, all cells in the primary Philadelphia chromosome (Ph)-positive and Ph-negative ALL samples tested were positive for high BAFF-R cell surface expression. BAFF-R was fully competent to bind BAFF, and stimulation of the receptor activated both the classic and the noncanonical NF-kappaB pathways. Recombinant BAFF supported survival of the ALL cells in the absence of stroma, and it significantly attenuated the rate of apoptosis caused by exposure to nilotinib, a drug used therapeutically to treat Ph-positive ALLs. Surprisingly, BAFF mRNA and protein were also expressed in the same cells but BAFF was not shed into the medium. Our report is the first showing universal expression of BAFF-R by pre-B ALL cells and opens the possibility of blocking its function as an adjuvant therapeutic strategy., (Copyright 2010 AACR.)

The capacity of immature B cells of the spleen and bone marrow to differentiate in vitro into cells representing mature end stage cells was investigated using B-cell activating factor belonging to the TNF family (BAFF) and Notch pathway activators. Immature splenic and bone marrow B cells were found, in the presence of both of these activators, to mature into cells with follicular mature (FM) and marginal zone (MZ) cell phenotypes. Such cells were functionally responsive to B-cell-specific activation. The derivation in vitro of cells with an MZ phenotype was more robust from CD23(-) populations than CD23(+) immature/transitional B cells, suggesting a direct immature/T1 B cell to MZ cell differentiation pathway. Transcript analysis of the in vitro-derived B-cell populations demonstrated expression profiles similar to maturing B cells in vivo. FACS-purified populations of B220(+)CD19(+)CD21(-)CD23(-) cells from bone marrow of 2-wk-old mice gave rise to populations of CD21(+)CD23(-) cells with MZ cell phenotypes as well as CD21(+)CD23(+) cells with FM cell phenotypes in percentages similar to those found in vivo. These data suggest that the commitment to an MZ and FM B cell phenotype is set prior to immature B-cell release from the marrow.