- Open Access
Activation of human B cells negatively regulates TGF-β1 production
Journal of Neuroinflammation volume 14, Article number: 13 (2017)
Accumulating evidence indicate that B cells can exhibit pro- or anti-inflammatory activities. Similar to interleukin (IL)-10–competent B cells, we recently showed that transforming growth factor (TGF)-β1-producing regulatory B cells limit the induction of autoimmune neuroinflammation in mice, making them potentially important in maintaining peripheral immune tolerance in central nervous system inflammatory demyelinating disorders such as multiple sclerosis.
In this study, we compared B cell production of TGF-β1 and IL-10, the two most studied regulatory cytokines, and the pro-inflammatory B cell-derived IL-6 and tumor necrosis factor cytokines under basal conditions and following polyclonal stimulation with dual B cell receptor (BCR) cross-linking and Toll-like receptor (TLR)9 engagement.
We showed that resting TGF-β1–producing B cells fall within both the naïve (CD27−) and memory (CD27+) B cell compartments. We found no spontaneous B cell-derived IL-10, IL-6 or tumor necrosis factor (TNF) production. Human B cell activation with anti-Ig antibodies plus CPG-B leads to only modest IL-10 production by memory CD19+CD27+ B cells while expression levels of IL-6 and TNF by both naive and memory B cells were strongly induced. Remarkably, stimulated B cells showed significantly reduced capacity to produce TGF-β1.
These findings indicate that B cell activation may facilitate the development of excessive immune responses and autoimmunity by restricting B cell-derived TGF-β1 production by resting B cells and favoring in turns the proinflammatory actions of activated cytokine-producing B cells.
Recent years have seen a significant increase in the interest of the roles of B cells in autoimmune diseases such as multiple sclerosis (MS), not only as precursors of antibody-producing plasma cells [1, 2], but also as key regulators of T cell activation and differentiation through their antigen presentation function [3, 4] and cytokine production [3, 5, 6]. To date, the strongest evidence for B cells playing a crucial role in the immune pathology of MS arises from clinical trials uncovering the effect and potency of anti-CD20 B cell-depleting therapies (BCDT) [7–10]. In addition, a growing body of experimental studies suggests that activation of antigen-driven B cells can lead to central nervous system (CNS) autoimmune reactions [11, 12] and that B cells in MS may be inherently polarized towards a functional proinflammatory phenotype [6, 13–16]. Remarkably, some recent work has reported evidences of increased proinflammatory cytokine responses of B cells from MS patients upon non-CNS specific (i.e., CpG DNA) stimulation [17–19], suggesting that abnormal B cell effector cytokine responses in MS patients are not restricted to specific autoreactive B cells.
Recent research indicates that the activation status dictates the diverse roles a respective B cell may play in MS pathogenesis . Activated B cells, in particular memory B cells, are more pathogenic than naïve ones. While memory B cells are more easily activated to produce proinflammatory cytokines such as tumor necrosis factor (TNF), interleukin (IL)-6, and granulocyte macrophage-colony stimulating factor (GM-CSF), naïve B cells preferentially secrete the regulatory cytokine IL-10 [6, 17]. Although the current data support the conceptual idea of a prominent involvement of activated proinflammatory B cells in MS pathogenesis, accumulating experimental and clinical findings indicate that not all B cells play a pathogenic role in MS . Defects in regulatory B cell functions have been documented in MS patients [13, 17, 21–23], suggesting that a disrupted balance between proinflammatory and suppressive B cell properties may be particularly relevant to the regulation of CNS autoimmunity. While exacerbation of MS activity as a result of anti-CD20-mediated B cell depletion has not yet been documented, increased proinflammatory monocytic activity following B cell depletion has been reported in experimental autoimmune encephalomyelitis (EAE) , a model for MS, and more recently in some anti-CD20 monoclonal antibodies-treated MS patients . Evidence that BCDT also depletes potential protective B cell responses comes from clinical reports showing that infusion of the monoclonal anti-CD20 antibody Rituximab for autoimmunity has led to severe exacerbation of colitis and the spontaneous onset of colitis and psoriasis readily after the initiation of treatment [26–29]. Management of non-Hodgkin’s lymphoma with Rituximab has also been associated with the onset of autoimmunity . Interestingly, in MS, treatment with Atacicept, a decoy receptor for the B cell growth factors B-cell activating factor (BAFF) and a proliferation-inducing ligand (APRIL), was found to exacerbate MS, possibly by altering regulatory B cell functions . These cautionary data emphasize that pan depletion of B cells can be deleterious in some situations, and therefore supports further development of a therapeutic option for treating MS patients that spares regulatory B cell functions .
One of the first studies illustrating a decreased regulatory B cell function in patients with an autoimmune condition was performed in the context of MS . Decreased IL-10 production was seen upon B cell activation via CD40, or B cell receptor (BCR) in conjunction with CD40 , or Toll-like receptor (TLR)9 [21, 23], indicating a general alteration of B cell functions in MS rather than a defect in certain activation signaling pathways. Similar to IL-10-producing B cells [33–35], transforming growth factor (TGF)-β1-producing regulatory B cells have recently been shown by our group to restrain the initiation phase of experimental autoimmune neuroinflammation , making them potentially important in maintaining peripheral immune tolerance in organ-specific autoimmune disease such as MS. Consistent with this premise, recent evidence indicates that B cell subpopulations expressing TGF-β can control regulatory T cell induction, immune tolerance promotion, and/or innate and adaptive immune response suppression [37–50]. With regards to the importance of TGF-β1-producing B cells in the regulation of CNS autoimmunity, evaluating whether B cell activation governs TGF-β1 expression by human B cells may provide a better understanding of the contribution and mechanism of regulatory B cell functions in autoimmune manifestations.
Here, we measured B cell production of TGF-β1 and IL-10, the two most studied regulatory cytokines, and the proinflammatory B cell-derived IL-6 and TNF cytokines under basal conditions and following polyclonal stimulation with dual BCR cross-linking and TLR9 engagement. Spontaneous TGF-β1 production by resting B cells was observed in both the naïve (CD27−) and memory (CD27+) B-cell compartments. In contrast, we found that IL-10 production was negligible in B cells under basal culture conditions. Likewise, unstimulated B cells did not produce the proinflammatory cytokines IL-6 and TNF. Remarkably, B cells stimulated with combined anti-Ig antibodies + CpG-B showed decreased TGF-β1 production capacity. We also noted that once activated, a fraction of memory, but not naïve, B cells expressed low, but significant, IL-10 levels. Finally, B cell stimulation strongly enhanced production of IL-6 and TNF by B cells. These findings indicate that B cell activation may contribute to immunological abnormalities seen in autoimmune disorders such as MS by restricting the regulatory functions of resting TGF-β1-producing B cells and in turn favoring the proinflammatory effects of activated B cells.
Standard protocol approvals
Peripheral blood B cells were isolated from buffy coats of blood from healthy volunteers (five male and one female, median age 55 years, range 29–67 years) provided by the Geneva Hospital Blood Transfusion Center. In accordance with the ethical committee of the Geneva Hospital, the blood bank obtained informed consent from the donors that a part of their blood would be used for research purposes.
Cell preparation and B cell purification
Highly purified CD19+ B cells (<90% purities) were isolated from the mononuclear fraction using negative selection microbeads (Human B cell Enrichment Kit; EasySep 19054, STEMCELL Technologies).
AffiniPure F(ab')2 Fragment Goat Anti-Human IgA + IgG + IgM (H + L) (anti-Ig) was from Jackson ImmunoResearch. ODN 2006 (ODN 7909) Class B CpG oligonucleotide; a human TLR9 ligand, was purchased from Invivogen. X-Vivo 15 medium for culturing B cells was from Lonza.
Staining and immunofluorescence analysis
Cells (106 cells/mL) were suspended in complete medium (X-Vivo 15 medium containing 200 mg/mL penicillin, 200 U/mL streptomycin, 4 mM L-Glutamine and 1 mM Sodium Pyruvate, all from Gibco) in the presence of CpG-B 2006 (10 μg/mL, Tib Molbiol) and anti-Ig (10 μg/mL) in 24-well flat-bottom plates for 24 h, at 37 °C. During the last 5 h, cells were additionally stimulated with 50 ng/mL of phorbol myristate acetate (PMA), 1 nM ionomycin (Sigma-Aldrich), and 2 mM monensin (all from eBioscience). Viability was assessed using a UV Live/Dead Fixable dead cell staining kit (Invitrogen). Nonspecific antibody (Ab) interactions of Fc receptors (FcRs) on cells were limited using TruStain FcX reagent (BioLegend), as per the manufacturer’s instructions. Cells were stained with antibodies to the following cell surface markers: CD19–FITC, CD19–APC, CD27–FITC, and CD27–PE (all from BioLegend), isotype-matched controls were from Biolegend or BD Pharmingen. After washing, cells were fixed and permeabilized using a Fix and Perm kit (Invitrogen) and stained by intracellular staining (ICS) with IL-10–PE-Cy7, IL–6-PE, TNF–PerCP-Cy5.5 (all from BioLegend), and anti-human TGF-β1–APC mAb (R&D). Isotype controls were from BioLegend and R&D. After the cells were stained, they were washed twice and acquired within 1 h. For cell-surface LAP staining, B cells were stimulated as described above without the addition of monensin and stained after non-specific binding (TruStain FcX reagent) with anti-human LAP–PE-Cy7 (TGF-β1) (BioLegend) and appropriate PE-Cy7 mouse IgG1 κ isotype-matched control antibody (BD Biosciences). Samples were run through a FACS Cyan flow cytometer (Becton Dickinson) with standard equipment. Normalized median fluorescence intensity (MFI) was calculated by subtracting the MFI of isotype control antibody-stained cells from the MFI of specific antibody-stained cells. Gating strategy for B cell phenotyping is shown in Additional file 1: Figure S1A.
Analysis of cytokine production
For ELISA assays, B cells (4 × 105) were cultured in 250 μL of serum-free complete X-Vivo 15 medium with CpG-B 2006 (10 μg/mL) + anti-Ig (10 μg/mL) in 96-well flat-bottom plates. At 24 h, cell-free supernatants were analyzed for IL-6 and TNF cytokine content using commercially available Duoset ELISA kits (R&D Systems) according to the manufacturer’s instructions. The Quantikine Human LAP (TGF-β1) Immunoassay (R&D systems) was used to measure total TGF-β1 in acid activated cell culture supernatants. IL-10 cytokine content was analyzed using the commercially available Kit IL-10 Ready Set-Go (Ebioscience) according to the manufacturer’s instructions. The results are expressed as an average of triplicate wells ± standard error of the mean (SEM).
Highly purified CD19+ B cells (<90% purities) were left unstimulated or stimulated in the presence of CpG-B 2006 (10 μg/mL) + anti-Ig (10 μg/mL) in 24-well flat-bottom plates for 3 h at 37 °C. Total RNA was extracted with TRIzol reagent (Invitrogen), or using ReliaPrep RNA Cell Miniprep System (Promega) according to the manufacturer’s instructions, and reverse-transcribed into cDNA using iScript™ cDNA Synthesis Kit (Bio-Rad Laboratories). Real-time PCRs were performed using Power SYBR Green PCR Master Mix Reagent (Applied Biosystems) on a 7500 Real-time PCR system (Applied Biosystems). The human TGF-β1 forward and reverse primers were 5'-CCCAGCATCTGCAAAGCTC-3' and 5'-GTCAATGTACAGCTGCCGCA-3', alongside primers targeting the internal normalizer gene β-actin (Hs_ACTB_2_SG Quantitect Primer Assay, QT01680476; Qiagen). Relative expression levels were calculated for each gene using the ΔΔCt method.
Data are presented as mean ± SEM. Comparisons between sample means were performed by Student’s t test. Values of p < 0.05 were considered statistically significant.
Induction of IL-10 production by human blood memory B cells following CpG-B + anti-Ig in vitro stimulation
We assessed the ability of purified human blood B cells to produce IL-10 under both basal culture conditions and after stimulation with a combination of CpG-B, a ligand for human B cell TLR9, and anti-Ig antibodies. In the basal unstimulated state, IL-10–producing B cells were barely detected (Fig. 1a–c and Table 1). After stimulation, low but detectable frequencies of B cells expressing IL-10 were found (Fig. 1a–c and Table 1). However, the combination of CpG-B and anti-Ig did not statistically increase the percentages of CD19+CD27− B cells expressing IL-10 (Fig. 1a–c and Table 1). As previously reported , the frequency of IL-10+ B cells was four to six times higher in the CD19+CD27+ subpopulation than in the CD19+CD27− subset (Table 1). The frequency of IL-10–producing CD19+CD27+ B cells was comparable to that previously reported . Moreover, we noted that stimulating circulating B cells with the combination of activators statistically increased within CD19+CD27+ B cells the amount of IL-10 produced per cell (Fig. 1d, e and Table 2). While a similar pattern was observed for CD19+CD27− B cells expressing IL-10, the increased levels following B cell stimulation were not as important (Fig. 1d).
Increased IL-6 and TNF production in human B cells stimulated with CpG-B and anti-Ig
We next evaluated the capacity of purified circulating B cells to produce the proinflammatory cytokines IL-6 and TNF under basal and stimulated conditions. IL-6 production by B cells is a major pathogenicity factor for B cells in CNS autoimmunity [3, 5]. TNF is also considered as an important factor that is secreted by active B cells in MS . Analysis of frequencies of B cells under resting conditions revealed that B cells did not produce IL-6 (Fig. 2a) or TNF (Fig. 2b). Coordinated stimulation of BCR and TLR9 however drastically increased IL-6 and TNF levels in these cells. In contrast to IL-10 production, which was confined to stimulated CD19+CD27+ B cells, high expression levels of IL-6 and TNF were found in both CD19+CD27+ and CD19+CD27− subsets. These data suggest that polyclonal B cell stimulation shifts B cells towards a proinflammatory dominant phenotype. In line with these results, B cells from patients with MS were on average shown to produce more proinflammatory TNF and IL-6 and less regulatory IL-10 [5, 17].
BCR ligation together with TLR9 engagement inhibits TGF-β1 production by human blood B cells
We next assessed the ability of human blood B cells to express TGF-β1 under our experimental conditions. In the basal unstimulated state we could readily detect TGF-β1–producing B cells (Fig. 3a–c and Table 1). The frequencies of TGF-β1+ B cells were comparable between the CD19+CD27+ and CD19+CD27− subpopulations. Strikingly, TGF-β1-producing B cell proportions significantly decreased after the combined CpG and anti-Ig stimulation (Fig. 3a–c and Table 1). A comparable degree of reduction in TGF-β1 expression was observed in both naïve and memory B cells after stimulation. Furthermore, we noted that stimulating circulating B cells with the combination of activators statistically reduced the expression levels of TGF-β1 per cell, measured as mean florescence intensity (MFI) (Fig. 3d, e and Table 2). Taken together these data indicate that dual BCR ligation and TLR9 engagement induces substantial decreases in both the frequency of B cells that express TGF-β1 and the absolute levels of TGF-β1 expression by B cells.
Analysis of cytokines secreted by human blood B cells after CpG-B and anti-Ig stimulation
We next measured the effect of combined CpG and anti-Ig stimulation on pro- and anti-inflammatory cytokine secretion by purified B cells. As expected, high levels of IL-6 (Fig. 4a) and TNF (Fig. 4b) were detected in supernatants from B cells cultures following stimulation. Likewise, stimulation of B cells also increased IL-10 secretion, although to a much lesser extent (Fig. 4c). As TGF-β1 is secreted in a latent form, linked to Latency Associated Protein (LAP) , latent TGF-β1 was analyzed by enzyme-linked immunosorbent assay (ELISA) after dissociation of TGF-β1 from LAP by acidification of supernatant samples. This method measures total TGF-β1, equivalent to dissociated latent TGF-β1 plus any free TGF-β1 present prior to acidification. Compared to control serum-free conditions, low concentrations of total TGF-β1 were detected in cell-free supernatants of resting B cells (Fig. 4d). Under these conditions, levels of total TGF-β1 secreted by resting B cells was not inferior to those measured by stimulated B cells (Fig. 4d).
Reduced TGF-β1 expression in human circulating B cells following activation
As TGF-β1 is produced in a latent form, linked LAP, and is predominantly expressed on the surface of TGF-β1-producing cells , we next evaluated the cell-surface expression of LAP–TGF-β1 on B cells by flow cytometry. Comparable frequencies of LAP–TGF-β1+ B cells were seen in unstimulated CD19+CD27+ and CD19+CD27− subpopulations (Fig. 5a and Table 1). Remarkably, B cell stimulation significantly reduced the percentage of CD19+CD27+ and CD19+CD27− B cells bearing LAP–TGF-β1 (Fig. 5a–c and Table 1). Moreover, we observed a substantial decreased density (MFI) of cell-surface expression of LAP–TGF-β1 on naïve CD19+CD27− B cells (Fig. 5d, e and Table 2), which were significantly more abundant in peripheral blood than memory B cells (Additional file 1: Figures S1A-B). A similar trend, with poorer correlation, was observed for expression levels of LAP–TGF-β1 per cell within the CD19+CD27+ B cell subpopulation (Fig. 5d, e and Table 2). Consistent with these data, B cell stimulation significantly reduced the level of TGF-β1 messenger RNA (mRNA) by whole CD19+ B cells as measured by quantitative reverse transcription real-time polymerase chain reaction (PCR) (Additional file 1: Figure S1C). Altogether, our results indicate that activation of human circulating B cells through BCR/TLR9 co-engagement, a remarkably potent mechanism of activation of autoreactive B cells , shifts B cells from a regulatory/suppressive phenotype associated with TGF-β1 expression to a proinflammatory state characterized by low expression levels of TGF-β1.
Data indicate that depending on the signals B cells receive, proinflammatory or immunoregulatory cytokines can be produced, and the shift towards an inflammatory or a protective/suppressive immune response will occur . The goal of this investigation was to evaluate the ability of human blood B cells to produce the classical pro- and anti-inflammatory cytokines IL-6, TNF, IL-10, and TGF-β1, respectively, under both basal and stimulated conditions. We found that a combination of adaptive signals by BCR cross-linking and innate signals through TLR9 engagement strongly induced IL-6 and TNF production by human B cells in vitro. Although modest, the signals induced by CpG-DNA and BCR ligation also caused induction of IL-10 production by memory B cells. Strikingly, stimulation of B cells by engagement of BCR and TLR9 decreased TGF-β1 production. In regards to the implication of TGF-β1 expression in regulatory B cell functions in immune tolerance [36, 38, 55, 56], the present findings imply that loss of B cell-derived TGF-β1 production following B cell activation may confer increased susceptibility to autoimmune responses.
Accumulating evidence from both mouse and human studies confirms the existence of regulatory B cells, and is beginning to define their mechanisms of action. In EAE, a CD4+ T cell-mediated autoimmune disease and a well-established model of MS, data indicate that separate B cell subsets regulate the induction and recovery of autoimmune neuroinflammation [35, 57–61]. While initially all regulatory functions of suppresive B cells were attributed to their capabilities to produce IL-10, recent studies have highlighted additional mechanisms by which Bregs suppress immunity. In addition to the recently reported regulatory role of IL-35-producing B cells during disease recovery , data from our group have established that TGF-β1-producing B cells restrain the initiation phase of EAE and the development of Th17 cells . Consistent with a regulatory role of B cell-derived of TGF-β1 production in CNS autoimmunity, B cells were previously found to have a central function in induction of oral tolerance and amelioration of EAE via the up-regulation of TGF-β in gut associated lymphoid tissue (GALT) [44, 62]. Moreover, other related work has reported that TGF-β1 negatively regulates the encephalitogenic capacity of Th17 cells  and EAE [64–66].
Although data indicate that TGF-β1-producing B cells exert a regulatory function in EAE, their relevance to MS is yet to be determined. Due to its prominent role in controlling the immune system , TGF-β1 has been suggested to be one of the central regulatory cytokines in MS. Interestingly, data obtained from MS patients have reported that TGF-β expression is increased in peripheral blood mononuclear cells (PBMCs) from MS patients with slight or no disability  or stable disease . In a longitudinal study, TGF-β expression was further shown to decline in relapse and to return to baseline values four to eight weeks following the MS relapse . Of note, myelin-stimulated peripheral blood T cell clones from MS patients failed to secrete TGF-β during acute attacks, while TGF-β expression was restored during disease remission . While no data to date have investigated TGF-β production by B cells in MS, our findings, despite being derived from healthy people, indicate that activation of human blood B cells reduced their capacity to produce TGF-β1. Considering the importance of TGF-β1 in modulating immunity and the abnormal proinflammatory polarization of B cells of MS patients [5, 6, 13, 17], inefficient production of TGF-β1 by B cells may be associated with an increased risk of developing MS or driving new relapsing MS disease activity. It is also tempting to postulate that exacerbation of symptoms, or onset of new pathologies following B cell depletion with Rituximab in a few patients with immune-mediated diseases [26–29], could be associated with the depletion of unactivated TGF-β1-producing B cells. Future studies aimed at evaluating TGF-β1 production by B cells, regardless of their antigenic specificity, from MS subjects and healthy controls in response to modes of activation relevant to B-cell–T-cell interaction and MS [13, 17, 72] will allow for a better understanding of the contribution of B cell-derived TGF-β1 in this disease.
The phenotypic characterization of human IL-10-producing B cells has been integral in accelerating assessment of their function and relevance in immune-mediated diseases . B cells isolated according to the cell surface expression of receptors classically used to separate known B cell subsets have revealed that CD19+CD27− naïve-like B cells produced more IL-10 than CD19+CD27+ B cells after stimulation via BCR and CD40 . In a separate study, it was shown that the proportion of IL-10-producing B cells was higher in the CD19+CD27+ subpopulation than in the CD19+CD27− subset after stimulation with CpG-DNA and BCR ligation . These different results may stem from the use of distinct stimulatory conditions. Here, our data confirm that IL-10-producing B cells activated via BCR and TLR9 fall within the CD19+CD27+ subpopulation . Using CD27 as a surrogate marker of human memory B cells, our data demonstrate that TGF-β1-producing B cells do not belong to a unique B cell subset. The characterization of human TGF-β1-producing B cells may provide a better understanding of their function and development. Interestingly, the quantification of TGF-β1 expression by B cells in EAE mice  indicate that murine TGF-β1-producing B cells do not belong to the CD1dhiCD5+ B10 subset  or CD138+ plasma cells, which have been shown to be the main source of IL-10 and IL-35 during disease development [61, 75].
In summary, our data demonstrate that B cell activation by BCR/TLR9 co-engagement differentially regulates the production of pro- and anti-inflammatory cytokines by human B cells. B cell stimulation with combined CpG-B + anti-Ig induced strong IL-6 and TNF production. IL-10 was modestly elevated after B cell activation. Remarkably, B cells stimulated in this way had a significantly reduced capacity to produce TGF-β1. These findings imply that activation of human B cells may amplify autoimmune responses by restricting the regulatory functions of resting TGF-β1-producing B cells and favoring the proinflammatory activities of B cells. These observations further support the consensus that the action of naïve B cells is fundamentally regulatory and that activated B cells preferentially produce proinflammatory cytokines [6, 17].
A proliferation-inducing ligand
B cell-depleting therapies
B cell receptor
Central nervous system
Experimental autoimmune encephalomyelitis
Enzyme-linked immunosorbent assay
Granulocyte-macrophage colony-stimulating factor
Latency associated protein
Mean florescence intensity
Polymerase chain reaction
Phorbol 12-myristate 13-acetate
Transforming growth factor
Tumor necrosis factor
Lalive PH. Autoantibodies in inflammatory demyelinating diseases of the central nervous system. Swiss Med Wkly. 2008;138:692–707.
Menge T, Lalive PH, von Budingen HC, Genain CP. Conformational epitopes of myelin oligodendrocyte glycoprotein are targets of potentially pathogenic antibody responses in multiple sclerosis. J Neuroinflammation. 2011;8:161.
Molnarfi N, Schulze-Topphoff U, Weber MS, Patarroyo JC, Prod'homme T, Varrin-Doyer M, Shetty A, Linington C, Slavin AJ, Hidalgo J, et al. MHC class II-dependent B cell APC function is required for induction of CNS autoimmunity independent of myelin-specific antibodies. J Exp Med. 2013;210:2921–37.
Harp CT, Ireland S, Davis LS, Remington G, Cassidy B, Cravens PD, Stuve O, Lovett-Racke AE, Eagar TN, Greenberg BM, et al. Memory B cells from a subset of treatment-naive relapsing-remitting multiple sclerosis patients elicit CD4(+) T-cell proliferation and IFN-gamma production in response to myelin basic protein and myelin oligodendrocyte glycoprotein. Eur J Immunol. 2010;40:2942–56.
Barr TA, Shen P, Brown S, Lampropoulou V, Roch T, Lawrie S, Fan B, O'Connor RA, Anderton SM, Bar-Or A, et al. B cell depletion therapy ameliorates autoimmune disease through ablation of IL-6-producing B cells. J Exp Med. 2012;209:1001–10.
Li R, Rezk A, Miyazaki Y, Hilgenberg E, Touil H, Shen P, Moore CS, Michel L, Althekair F, Rajasekharan S, et al. Proinflammatory GM-CSF-producing B cells in multiple sclerosis and B cell depletion therapy. Sci Transl Med. 2015;7:310ra166.
Hauser SL, Waubant E, Arnold DL, Vollmer T, Antel J, Fox RJ, Bar-Or A, Panzara M, Sarkar N, Agarwal S, et al. B-cell depletion with rituximab in relapsing-remitting multiple sclerosis. N Engl J Med. 2008;358:676–88.
Kappos L, Li D, Calabresi PA, O'Connor P, Bar-Or A, Barkhof F, Yin M, Leppert D, Glanzman R, Tinbergen J, Hauser SL. Ocrelizumab in relapsing-remitting multiple sclerosis: a phase 2, randomised, placebo-controlled, multicentre trial. Lancet. 2011;378:1779–87.
Bar-Or A, Calabresi PA, Arnold D, Markowitz C, Shafer S, Kasper LH, Waubant E, Gazda S, Fox RJ, Panzara M, et al. Rituximab in relapsing-remitting multiple sclerosis: a 72-week, open-label, phase I trial. Ann Neurol. 2008;63:395–400.
Sorensen PS, Lisby S, Grove R, Derosier F, Shackelford S, Havrdova E, Drulovic J, Filippi M. Safety and efficacy of ofatumumab in relapsing-remitting multiple sclerosis: a phase 2 study. Neurology. 2014;82:573–81.
Palanichamy A, Apeltsin L, Kuo TC, Sirota M, Wang S, Pitts SJ, Sundar PD, Telman D, Zhao LZ, Derstine M, et al. Immunoglobulin class-switched B cells form an active immune axis between CNS and periphery in multiple sclerosis. Sci Transl Med. 2014;6:248ra106.
Stern JN, Yaari G, Vander Heiden JA, Church G, Donahue WF, Hintzen RQ, Huttner AJ, Laman JD, Nagra RM, Nylander A, et al. B cells populating the multiple sclerosis brain mature in the draining cervical lymph nodes. Sci Transl Med. 2014;6:248ra107.
Bar-Or A, Fawaz L, Fan B, Darlington PJ, Rieger A, Ghorayeb C, Calabresi PA, Waubant E, Hauser SL, Zhang J, Smith CH. Abnormal B-cell cytokine responses a trigger of T-cell-mediated disease in MS? Ann Neurol. 2010;67:452–61.
Aung LL, Balashov KE. Decreased Dicer expression is linked to increased expression of co-stimulatory molecule CD80 on B cells in multiple sclerosis. Mult Scler. 2015;21:1131–8.
Genc K, Dona DL, Reder AT. Increased CD80(+) B cells in active multiple sclerosis and reversal by interferon beta-1b therapy. J Clin Invest. 1997;99:2664–71.
Ireland SJ, Blazek M, Harp CT, Greenberg B, Frohman EM, Davis LS, Monson NL. Antibody-independent B cell effector functions in relapsing remitting multiple sclerosis: clues to increased inflammatory and reduced regulatory B cell capacity. Autoimmunity. 2012;45:400–14.
Duddy M, Niino M, Adatia F, Hebert S, Freedman M, Atkins H, Kim HJ, Bar-Or A. Distinct effector cytokine profiles of memory and naive human B cell subsets and implication in multiple sclerosis. J Immunol. 2007;178:6092–9.
Miyazaki Y, Li R, Rezk A, Misirliyan H, Moore C, Farooqi N, Solis M, Goiry LG, de Faria JO, Dang VD, et al. A novel microRNA-132-sirtuin-1 axis underlies aberrant B-cell cytokine regulation in patients with relapsing-remitting multiple sclerosis [corrected]. PLoS One. 2014;9:e105421.
Panaretou B, Jones GW. Yeast models for amyloid disease. Essays Biochem. 2014;56:85–97.
Claes N, Fraussen J, Stinissen P, Hupperts R, Somers V. B cells are multifunctional players in multiple sclerosis pathogenesis: insights from therapeutic interventions. Front Immunol. 2015;6:642.
Knippenberg S, Peelen E, Smolders J, Thewissen M, Menheere P, Cohen Tervaert JW, Hupperts R, Damoiseaux J. Reduction in IL-10 producing B cells (Breg) in multiple sclerosis is accompanied by a reduced naive/memory Breg ratio during a relapse but not in remission. J Neuroimmunol. 2011;239:80–6.
Correale J, Farez M, Razzitte G. Helminth infections associated with multiple sclerosis induce regulatory B cells. Ann Neurol. 2008;64:187–99.
Hirotani M, Niino M, Fukazawa T, Kikuchi S, Yabe I, Hamada S, Tajima Y, Sasaki H. Decreased IL-10 production mediated by Toll-like receptor 9 in B cells in multiple sclerosis. J Neuroimmunol. 2010;221:95–100.
Weber MS, Prod'homme T, Patarroyo JC, Molnarfi N, Karnezis T, Lehmann-Horn K, Danilenko DM, Eastham-Anderson J, Slavin AJ, Linington C, et al. B-cell activation influences T-cell polarization and outcome of anti-CD20 B-cell depletion in central nervous system autoimmunity. Ann Neurol. 2010;68:369–83.
Lehmann-Horn K, Schleich E, Hertzenberg D, Hapfelmeier A, Kumpfel T, von Bubnoff N, Hohlfeld R, Berthele A, Hemmer B, Weber MS. Anti-CD20 B-cell depletion enhances monocyte reactivity in neuroimmunological disorders. J Neuroinflammation. 2011;8:146.
Ray A, Mann MK, Basu S, Dittel BN. A case for regulatory B cells in controlling the severity of autoimmune-mediated inflammation in experimental autoimmune encephalomyelitis and multiple sclerosis. J Neuroimmunol. 2011;230:1–9.
Dass S, Vital EM, Emery P. Development of psoriasis after B cell depletion with rituximab. Arthritis Rheum. 2007;56:2715–8.
Goetz M, Atreya R, Ghalibafian M, Galle PR, Neurath MF. Exacerbation of ulcerative colitis after rituximab salvage therapy. Inflamm Bowel Dis. 2007;13:1365–8.
El Fassi D, Nielsen CH, Kjeldsen J, Clemmensen O, Hegedus L. Ulcerative colitis following B lymphocyte depletion with rituximab in a patient with Graves’ disease. Gut. 2008;57:714–5.
Mielke F, Schneider-Obermeyer J, Dorner T. Onset of psoriasis with psoriatic arthropathy during rituximab treatment of non-Hodgkin lymphoma. Ann Rheum Dis. 2008;67:1056–7.
Kappos L, Hartung HP, Freedman MS, Boyko A, Radu EW, Mikol DD, Lamarine M, Hyvert Y, Freudensprung U, Plitz T, et al. Atacicept in multiple sclerosis (ATAMS): a randomised, placebo-controlled, double-blind, phase 2 trial. Lancet Neurol. 2014;13:353–63.
Lehmann-Horn K, Kronsbein HC, Weber MS. Targeting B cells in the treatment of multiple sclerosis: recent advances and remaining challenges. Ther Adv Neurol Disord. 2013;6:161–73.
Fillatreau S, Gray D, Anderton SM. Not always the bad guys: B cells as regulators of autoimmune pathology. Nat Rev Immunol. 2008;8:391–7.
Fillatreau S. Novel regulatory functions for Toll-like receptor-activated B cells during intracellular bacterial infection. Immunol Rev. 2011;240:52–71.
Lehmann-Horn K, Sagan SA, Winger RC, Spencer CM, Bernard CC, Sobel RA, Zamvil SS. CNS accumulation of regulatory B cells is VLA-4-dependent. Neurol Neuroimmunol Neuroinflamm. 2016;3:e212.
Bjarnadottir K, Benkhoucha M, Merkler D, Weber MS, Payne NL, Bernard CC, Molnarfi N, Lalive PH. B cell-derived transforming growth factor-beta1 expression limits the induction phase of autoimmune neuroinflammation. Sci Rep. 2016;6:34594.
Tian J, Zekzer D, Hanssen L, Lu Y, Olcott A, Kaufman DL. Lipopolysaccharide-activated B cells down-regulate Th1 immunity and prevent autoimmune diabetes in nonobese diabetic mice. J Immunol. 2001;167:1081–9.
Parekh VV, Prasad DV, Banerjee PP, Joshi BN, Kumar A, Mishra GC. B cells activated by lipopolysaccharide, but not by anti-Ig and anti-CD40 antibody, induce anergy in CD8+ T cells: role of TGF-beta 1. J Immunol. 2003;170:5897–911.
Singh A, Carson WF, Secor Jr ER, Guernsey LA, Flavell RA, Clark RB, Thrall RS, Schramm CM. Regulatory role of B cells in a murine model of allergic airway disease. J Immunol. 2008;180:7318–26.
Shah S, Qiao L. Resting B cells expand a CD4 + CD25 + Foxp3+ Treg population via TGF-beta3. Eur J Immunol. 2008;38:2488–98.
Kessel A, Haj T, Peri R, Snir A, Melamed D, Sabo E, Toubi E. Human CD19(+)CD25(high) B regulatory cells suppress proliferation of CD4(+) T cells and enhance Foxp3 and CTLA-4 expression in T-regulatory cells. Autoimmun Rev. 2012;11:670–7.
Natarajan P, Singh A, McNamara JT, Secor Jr ER, Guernsey LA, Thrall RS, Schramm CM. Regulatory B cells from hilar lymph nodes of tolerant mice in a murine model of allergic airway disease are CD5+, express TGF-beta, and co-localize with CD4 + Foxp3+ T cells. Mucosal Immunol. 2012;5:691–701.
Liu ZQ, Wu Y, Song JP, Liu X, Liu Z, Zheng PY, Yang PC. Tolerogenic CX3CR1+ B cells suppress food allergy-induced intestinal inflammation in mice. Allergy. 2013;68:1241–8.
Gonnella PA, Chen YH, Waldner H, Weiner HL. Induction of oral tolerization in CD86 deficient mice: a role for CD86 and B cells in the up-regulation of TGF-beta. J Autoimmun. 2006;26:73–81.
Lee JH, Noh J, Noh G, Choi WS, Cho S, Lee SS. Allergen-specific transforming growth factor-beta-producing CD19(+)CD5(+) regulatory B-Cell (Br3) responses in human late eczematous allergic reactions to cow’s milk. J Interferon Cytokine Res. 2011;31(5):441–9.
Mishima Y, Ishihara S, Aziz MM, Oka A, Kusunoki R, Otani A, Tada Y, Li YY, Moriyama I, Oshima N, et al. Decreased production of interleukin-10 and transforming growth factor-beta in Toll-like receptor-activated intestinal B cells in SAMP1/Yit mice. Immunology. 2010;131:473–87.
Toubi E, Nussbaum S, Staun-Ram E, Snir A, Melamed D, Hayardeny L, Miller A. Laquinimod modulates B cells and their regulatory effects on T cells in multiple sclerosis. J Neuroimmunol. 2012;251:45–54.
Nouel A, Pochard P, Simon Q, Segalen I, Le Meur Y, Pers JO, Hillion S. B-Cells induce regulatory T cells through TGF-beta/IDO production in A CTLA-4 dependent manner. J Autoimmun. 2015;59:53–60.
Joly MS, Martin RP, Mitra-Kaushik S, Phillips L, D'Angona A, Richards SM, Joseph AM. Transient low-dose methotrexate generates B regulatory cells that mediate antigen-specific tolerance to alglucosidase alfa. J Immunol. 2014;193:3947–58.
Guo Y, Zhang X, Qin M, Wang X. Changes in peripheral CD19(+)Foxp3(+) and CD19(+)TGFbeta(+) regulatory B cell populations in rheumatoid arthritis patients with interstitial lung disease. J Thorac Dis. 2015;7:471–7.
Bouaziz JD, Calbo S, Maho-Vaillant M, Saussine A, Bagot M, Bensussan A, Musette P. IL-10 produced by activated human B cells regulates CD4(+) T-cell activation in vitro. Eur J Immunol. 2010;40:2686–91.
Arestrom I, Zuber B, Bengtsson T, Ahlborg N. Measurement of human latent transforming growth factor-beta1 using a latency associated protein-reactive ELISA. J Immunol Methods. 2012;379:23–9.
Viglianti GA, Lau CM, Hanley TM, Miko BA, Shlomchik MJ, Marshak-Rothstein A. Activation of autoreactive B cells by CpG dsDNA. Immunity. 2003;19:837–47.
Vadasz Z, Haj T, Kessel A, Toubi E. B-regulatory cells in autoimmunity and immune mediated inflammation. FEBS Lett. 2013;587:2074–8.
Caver TE, O'Sullivan FX, Gold LI, Gresham HD. Intracellular demonstration of active TGFbeta1 in B cells and plasma cells of autoimmune mice. IgG-bound TGFbeta1 suppresses neutrophil function and host defense against Staphylococcus aureus infection. J Clin Invest. 1996;98:2496–506.
Lee KM, Stott RT, Zhao G, SooHoo J, Xiong W, Lian MM, Fitzgerald L, Shi S, Akrawi E, Lei J, et al. TGF-beta-producing regulatory B cells induce regulatory T cells and promote transplantation tolerance. Eur J Immunol. 2014;44:1728–36.
Mann MK, Maresz K, Shriver LP, Tan Y, Dittel BN. B cell regulation of CD4 + CD25+ T regulatory cells and IL-10 via B7 is essential for recovery from experimental autoimmune encephalomyelitis. J Immunol. 2007;178:3447–56.
Matsushita T, Yanaba K, Bouaziz JD, Fujimoto M, Tedder TF. Regulatory B cells inhibit EAE initiation in mice while other B cells promote disease progression. J Clin Invest. 2008;118:3420–30.
Ray A, Basu S, Williams CB, Salzman NH, Dittel BN. A novel IL-10-independent regulatory role for B Cells in suppressing autoimmunity by maintenance of regulatory T cells via GITR Ligand. J Immunol. 2012;188:3188–98.
Fillatreau S, Sweenie CH, McGeachy MJ, Gray D, Anderton SM. B cells regulate autoimmunity by provision of IL-10. Nat Immunol. 2002;3:944–50.
Shen P, Roch T, Lampropoulou V, O'Connor RA, Stervbo U, Hilgenberg E, Ries S, Dang VD, Jaimes Y, Daridon C, et al. IL-35-producing B cells are critical regulators of immunity during autoimmune and infectious diseases. Nature. 2014;507:366–70.
Gonnella PA, Waldner HP, Weiner HL. B cell-deficient (mu MT) mice have alterations in the cytokine microenvironment of the gut-associated lymphoid tissue (GALT) and a defect in the low dose mechanism of oral tolerance. J Immunol. 2001;166:4456–64.
Lee PW, Yang Y, Racke MK, Lovett-Racke AE. Analysis of TGF-beta1 and TGF-beta3 as regulators of encephalitogenic Th17 cells: Implications for multiple sclerosis. Brain Behav Immun. 2015;46:44–9.
Racke MK, Dhib-Jalbut S, Cannella B, Albert PS, Raine CS, McFarlin DE. Prevention and treatment of chronic relapsing experimental allergic encephalomyelitis by transforming growth factor-beta 1. J Immunol. 1991;146:3012–7.
Racke MK, Cannella B, Albert P, Sporn M, Raine CS, McFarlin DE. Evidence of endogenous regulatory function of transforming growth factor-beta 1 in experimental allergic encephalomyelitis. Int Immunol. 1992;4:615–20.
Johns LD, Sriram S. Experimental allergic encephalomyelitis: neutralizing antibody to TGF beta 1 enhances the clinical severity of the disease. J Neuroimmunol. 1993;47:1–7.
Prud'homme GJ, Piccirillo CA. The inhibitory effects of transforming growth factor-beta-1 (TGF-beta1) in autoimmune diseases. J Autoimmun. 2000;14:23–42.
Link J, Soderstrom M, Olsson T, Hojeberg B, Ljungdahl A, Link H. Increased transforming growth factor-beta, interleukin-4, and interferon-gamma in multiple sclerosis. Ann Neurol. 1994;36:379–86.
Rieckmann P, Albrecht M, Kitze B, Weber T, Tumani H, Broocks A, Luer W, Poser S. Cytokine mRNA levels in mononuclear blood cells from patients with multiple sclerosis. Neurology. 1994;44:1523–6.
Rieckmann P, Albrecht M, Kitze B, Weber T, Tumani H, Broocks A, Luer W, Helwig A, Poser S. Tumor necrosis factor-alpha messenger RNA expression in patients with relapsing-remitting multiple sclerosis is associated with disease activity. Ann Neurol. 1995;37:82–8.
Correale J, Gilmore W, McMillan M, Li S, McCarthy K, Le T, Weiner LP. Patterns of cytokine secretion by autoreactive proteolipid protein-specific T cell clones during the course of multiple sclerosis. J Immunol. 1995;154:2959–68.
Duddy ME, Alter A, Bar-Or A. Distinct profiles of human B cell effector cytokines: a role in immune regulation? J Immunol. 2004;172:3422–7.
Iwata Y, Matsushita T, Horikawa M, Dilillo DJ, Yanaba K, Venturi GM, Szabolcs PM, Bernstein SH, Magro CM, Williams AD, et al. Characterization of a rare IL-10-competent B-cell subset in humans that parallels mouse regulatory B10 cells. Blood. 2011;117:530–41.
Yanaba K, Bouaziz JD, Haas KM, Poe JC, Fujimoto M, Tedder TF. A regulatory B cell subset with a unique CD1dhiCD5+ phenotype controls T cell-dependent inflammatory responses. Immunity. 2008;28:639–50.
Matsumoto M, Baba A, Yokota T, Nishikawa H, Ohkawa Y, Kayama H, Kallies A, Nutt SL, Sakaguchi S, Takeda K, et al. Interleukin-10-producing plasmablasts exert regulatory function in autoimmune inflammation. Immunity. 2014;41(6):1040–51.
NM is supported by grants from the Swiss MS Society (SMSS) and is a recipient of an advanced researcher exchange 2011 fellowship from the European Committee for Treatment and Research in Multiple Sclerosis (ECTRIMS) Foundation. This work was supported by grants to PHL from the Swiss National Science Foundation (No.310030_153164) and the SMSS.
Availability of data and materials
All data supporting the conclusions of this manuscript are shown in the text and figures.
NM, CJ, and PHL designed research, analyzed data, and wrote the article. CJ performed the experiments. MB and KB gave conceptual advice. NM and PHL supervised the study. All authors read, commented on, and approved the final manuscript.
The authors declare that they have no competing interests.
Consent for publication
Ethics approval and consent to participate
In accordance with the ethical committee of the Geneva Hospital, the blood bank obtained informed consent from the donors, who are thus informed that part of their blood will be used for research purposes.
B cell activation reduced TGF-β1 mRNA expression. a A representative gating of naïve and memory B cells in PBMCs is shown. Cells were first gated for singlets (FSC-H vs. FSC-A) and lymphocytes (SSC-A vs. FSC-A), followed by a live/dead gate. Naïve B cells (CD27–) and resting and activated memory B cells (CD27+) were distinguished by CD19 versus CD27 gating. FSC-H, forward scatter height; FSC-A, forward scatter area; SSC-A, side scatter area. b Scatter plot indicates mean (± SEM) percentages of naïve (CD27–) versus memory (CD27+) B cell subpopulations (n = 6). c Quantitative RT-PCR analysis of human B cells for the expression of TGF-β1. Expression levels are normalized using the ΔΔCt method, relative to β-actin. Results show mean of 6 independent sets of samples. Errors bars represent SEM. (DOCX 286 kb)
About this article
Cite this article
Molnarfi, N., Bjarnadóttir, K., Benkhoucha, M. et al. Activation of human B cells negatively regulates TGF-β1 production. J Neuroinflammation 14, 13 (2017) doi:10.1186/s12974-017-0798-5
- B cells
- Multiple sclerosis