Cellular & Molecular Immunology (2013) 10, 122–132 ß 2013 CSI and USTC. All rights reserved 1672-7681/13 $32.00 www.nature.com/cmi

REVIEW

Regulatory B cells in autoimmune diseases

Min Yang1,3,KeRui2,3, Shengjun Wang2 and Liwei Lu1

B cells are generally considered to be positive regulators of the immune response because of their capability to produce , including autoantibodies. The production of antibodies facilitates optimal CD41 T-cell activation because B cells serve as antigen-presenting cells and exert other modulatory functions in immune responses. However, certain B cells can also negatively regulate the immune response by producing regulatory cytokines and directly interacting with pathogenic T cells via cell-to-cell contact. These types of B cells are defined as regulatory B (Breg) cells. The regulatory function of Breg cells has been demonstrated in mouse models of , cancer, transplantation, and particularly in autoimmunity. In this review, we focus on the recent advances that lead to the understanding of the development and function of Breg cells and the implications of B cells in human autoimmune diseases. Cellular & Molecular Immunology (2013) 10, 122–132; doi:10.1038/cmi.2012.60; published online 7 January 2013

Keywords: ; interleukin-10; regulatory B cells

INTRODUCTION we review the recent literature studying both the phenotypic and B-cell development in the bone marrow is a dynamic and com- functional characterization of Breg cells and the implications B plex process involving a delicate balance between cell proliferation cells have on the pathogenesis of autoimmune diseases. and apoptotic selection. This balance results in the generation of functional B cells that are responsible for eliciting humoral IDENTIFICATION OF BREG CELLS 1–3 immunity. The concept that suppressor B cells could regulate Despite the observations made in the 1970s that B cells with the immune response originated in 1974, when the ability of B suppressive functions possibly existed, the potential role of B cells to suppress delayed-type hypersensitivity responses in guinea cells with regulatory functions in inflammatory and auto- 4,5 pigs was described. However, the term ‘regulatory B cells’, immune diseases has only been recently appreciated. Janeway which defines B-cell subsets with regulatory properties, was first and colleagues first observed that B10.PL mice lacking B cells introduced by Mizoguchi and Bhan nearly 30 years later.6 Similar suffered an unusually severe and chronic form of experimental to regulatory T (Treg) cells, the regulatory function of B cells is autoimmune encephalomyelitis (EAE), indicating that B cells exerted via the production of regulatory cytokines, such as IL-10 have regulatory properties in a mouse model of EAE.12 and TGF-b, and the ability to express inhibitory molecules that Subsequently, it was found that B cells affected this auto- suppress pathogenic T cells and autoreactive B cells in a cell-to- immune disease by regulating IL-10.13 Mizoguchi and Bhan cell contact-dependent manner.7 Until recently, the exact origin were the first to introduce the term ‘regulatory B cells’ to and molecular identity of regulatory B (Breg) cells remained elu- describe these B-cell subsets with regulatory properties.6 sive. Accumulating evidence suggests that the Breg cell population While studying the putative pathogenic role of B cells in the is heterogeneous, meaning that this population can be derived development of colitis, the authors unexpectedly observed that from all B cells under the correct stimulatory context and time.8 It receptor alpha (TCRa)2/2 mice that were crossed with B has been postulated that Breg cells can exert their suppressive cell-deficient mice spontaneously developed an earlier onset of functions with different mechanisms in various mouse models colitis that was more severe compared to TCRa2/2 mice.14 of disease, including inflammation, cancer and autoimmunity.9 Moreover, Mizoguchi et al. further demonstrated that a certain Moreover, dynamic changes in Breg cells have been associated B-cell subset from gut-associated lymphoid tissues in a chronic with the progression of human autoimmune diseases.10,11 Here, inflammatory environment secreted IL-10, upregulated

1Department of Pathology and Center for Infection and Immunology, The University of Hong Kong, Hong Kong, China and 2Department of Immunology, School of Medical Science and Laboratory Medicine, Jiangsu University, Zhenjiang, China 3These authors contributed equally to this work. Correspondence: Dr LW Lu, Department of Pathology and Center for Infection and Immunology, The University of Hong Kong, Pokfulam Road, Hong Kong, China. Received: 14 October 2012; accepted: 6 November 2012 Regulatory B cells in autoimmune diseases M Yang et al 123 expression of CD1d and suppressed the progression of intest- characterized human Breg cells with the phenotype inal inflammation by downregulating inflammatory cascades CD24hiCD271, which is a phenotype related to memory B associated with IL-1 and signal transducer and activator of cells.11 transcription 3 (STAT3) activation.15 Although great progress has been made in the characteriza- Early studies have revealed that B-1 cells in the peritoneal tion of Breg cells, the cell surface markers and/or specific tran- cavity are a major source of -derived IL-10.16 Upon IL- scription factor(s) that are unique to Breg cells have not been 12 stimulation, B1a cells, but not B-1b cells, have the ability defined in mice and humans. Most of the currently identified to produce IL-10.17 Recently, the production of Th2 cyto- Breg cells exert their suppressive function at least partially kines by conventional B2 cells has been extensively investi- through the production of regulatory cytokines, such as IL- gated. The marginal-zone (MZ) B cells have been shown to 10 and TGF-b. This production of regulatory cytokines has regulate immunity by producing IL-10 in response to CpG been demonstrated by both in vitro functional assays and in stimulation in a mouse model of .18 Moreover, splenic vivo mouse studies. transitional 2-MZ precursor (T2-MZP) B cells that express high amounts of CD21, CD23, CD24, IgM and CD1d from BREG CELLS IN AUTOIMMUNE DISEASES both naive and arthritic mice are capable of producing IL- The regulatory functions of Breg cells have been extensively 10. Remarkably, the regulatory function of T2-MZP cells characterized in various animal models of inflammation, can- depends on IL-10 production because T2-MZP cells from cer and autoimmune diseases. B cells are generally considered IL-102/2 DBA mice show no protection against the develop- to play a pathogenic role in the development of autoimmune 19 diseases because B cells produce autoantibodies that cause tar- ment of arthritis. Tedder and colleagues have identified a 26 subset of IL-10-producing B10 cells that contain the unique get tissue damage. However, autoantibodies can also exert a hi 1 protective effect via the clearance of apoptotic cells and reduc- phenotype CD1d CD5 . These cells share certain pheno- 27 typic markers with B-1a, MZ B and T2-MZP cells.20 B10 tion of autoantigen load. Moreover, B cells also act as anti- gen-presenting cells, which are cells that contribute to the cells normally represent 1%–2% of splenocytes in wild-type activation and amplification of naive, activated and autoreac- mice and approximately 10% in hCD19 transgenic mice. tive T-cell responses.28–30 It has been reported that antigens Notably, IL-10 production has been found to be restricted presented by resting B cells can induce the differentiation of to this B10 cell subset. Interestingly, IL-10 production was tolerogenic CD41 T cells.31,32 Furthermore, B cells, similar to T decreased in CD192/2 mice but increased in hCD19 trans- cells, can be defined as B effector 1 and 2 cells. B effector 1 cells genic mice. Rafei et al. have reported a Breg cell subset produce Th1-associated pro-inflammatory cytokines, includ- induced by a granulocyte-macrophage colony-stimulating ing tumor-necrosis factor (TNF)-a, IFN-c and IL-12, whereas factor–IL-15 fusion protein known as GIFT15.21 These B effector 2 cells produce Th2-associated cytokines, including GIFT15-induced Breg cells possess a phenotype akin to IL-4 and IL-13.33 Notably, certain regulatory B cells that pro- B10 and T2-MZP Breg cells. A study by Ding et al.revealed duce IL-10 or TGF-b have recently been shown to possess that T-cell Ig domain and mucin domain protein 1 (TIM-1) 6 inhibitory functions in autoimmune diseases. Thus, current is expressed by a large majority of IL-10-producing Breg B studies on the functional implications of Breg cells in the cells, which consist of a heterogeneous population including hi 1 pathogenesis of autoimmune diseases can facilitate the transitional B, MZ B, FO B and CD1d CD5 B10 cells. development of combined therapies for autoimmune diseases. TIM-11 B cells express IL-4 and IL-10, promote Th2 res- 22 In the following sections, the role of Breg cells in mouse models ponse and directly transfer allograft tolerance. Recently, of various autoimmune diseases, including rheumatoid arth- Qian et al. have reported that regulatory dendritic cells can ritis, autoimmune diabetes, autoimmune encephalomyelitis program splenic T1, T2, MZ and B1 cells to differentiate into and lupus, will be discussed. a distinct regulatory B-cell subset with the phenotype hi hi CD19 FccIIb . This B-cell subset exerts potent regulatory Breg cells in experimental arthritis functions, such as the secretion of IL-10 both in vitro and in (RA) is a chronic inflammatory disease 23 vivo. that is characterized by inflammation in the synovium. This Aside from mouse Breg cells, the existence of human Breg inflammation is associated with the infiltration of activated T cells has recently been revealed. A number of studies have cells, B cells and macrophages, as well as the progressive 24,25 reported that certain B cells can produce IL-10. destruction of cartilage and bone structures, which eventually Remarkable progress in identifying the phenotype of human leads to joint destruction and deformity.34 RA is a common Breg cells has been achieved by the group led by Mauri. In an systemic autoimmune disease that has a prevalence of approxi- elegant study, Mauri and colleagues demonstrated that mately 0.5%–1% in the adult population.35 An animal model CD191CD24hiCD38hi B cells, which are cells that have a of human RA exists whereby collagen-induced arthritis (CIA) phenotype that has been previously associated with immature is induced in a susceptible strain of DBA/1J mice that are B cells, comprise the highest fraction of IL-10-producing B cells immunized with heterologous type II collagen emulsified in upon CD40 stimulation in human peripheral blood from complete Freund’s adjuvant.36 CIA is characterized by severe healthy individuals.10 Separately, Tedder and colleagues also swelling of the paws, extensive synovial hyperplasia, cartilage

Cellular & Molecular Immunology Regulatory B cells in autoimmune diseases M Yang et al 124

damage, bone erosion and joint ankylosis.36–39 Collagen decrease joint pathology in CIA mice.45,46 Adoptive transfer immunization induces chronic inflammatory arthritis, which of in vitro expanded B10 cells in mice on the day of the second is the result of infiltration of CD41 T cells into the synovial collagen immunization resulted in a marked delay in the onset membrane and the production of collagen-specific IgG auto- of arthritis and a reduced severity of both clinical symptoms antibodies by B cells.40 Many types of immune cells, including and joint damage. This delay of onset was accompanied by a natural killer cells, have been shown to possess a regulatory substantial reduction in the number of pathogenic IL-17-pro- 1 function in the development of autoimmune arthritis.41 In ducing CD4 T cells. Thus, Breg cells expanded in vitro can be a CIA mice, B-cell depletion using the CD20 monoclonal anti- potential treatment for autoimmune arthritis. body (mAb) significantly ameliorates disease severity.42 Interestingly, B-cell depletion before collagen immunization Breg cells in autoimmune diabetes delays disease onset and autoantibody production and mark- Type 1 diabetes (T1D) is characterized by the destruction of edly diminishes the severity of arthritis as indicated by both insulin-producing pancreatic b cells. This destruction is prim- 1 1 47 clinical symptoms and histological changes in joint tissue. arily mediated by CD4 and CD8 T cells. In non-obese However, B-cell depletion after collagen immunization does diabetic (NOD) mice, which is a spontaneous model of human not show any significant effect on arthritis progression or dis- T1D, the onset of diabetes is initiated at the age of 13–15 weeks, and approximately 80% of female mice and 20% of male mice ease severity. These observations suggest that B cells may play a 48 more prominent regulatory role during the initiation of dis- develop diabetes by the age of 30 weeks. There is increasing ease. In CIA mice, IL-10-producing B-cell subsets with varying evidence that B cells play a pathogenic role in the initiation of phenotypes and origins have also been identified during arth- T1D. B cells are among the earliest cells to infiltrate the pan- ritis development. Mauri et al. have performed a comprehens- creatic islets in NOD mice, which is where they organize with T cells into lymphoid structures within germinal centers that ive study that examines whether adoptive transfer of activated promotes the selection of autoreactive B cells.49,50 These B cells from arthritic mice has an inhibitory effect on CIA.43 ectopic lymphoid structures, which consist of a central zone Mauri and colleagues found that the in vitro activation of of T cells surrounded by B cells, begin to generate at the early splenic arthritogenic B cells with collagen and a CD40 mAb stage of peri-insulitis.51 Because B cell-deficient NOD mice fail resulted in IL-10 production. B cells that were injected intra- to develop diabetes, targeting B cells may be a potential peritoneally into recipient DBA/1-T-cell Ag receptor b-trans- approach to treating b-cell mediated autoimmune diabetes.52 genic mice that were concurrently immunized with collagen In 5-week-old female NOD mice treated with CD20 mAbs for a significantly reduced the incidence and severity of arthritis and short amount of time, approximately 95% of B cells were markedly inhibited Th1 cell differentiation. Moreover, in vitro- depleted, which subsequently led to reduced insulitis. activated arthritogenic B cells were also effective in ameliorat- Moreover, diabetes was prevented in more than 60% of the ing the disease. Further studies have demonstrated that IL-10 is littermates. However, treating 15-week-old NOD female mice essential for the regulatory function of this subset of B cells with a CD20 mAb substantially delays the onset of diabetes, but because B cells isolated from IL-10 knockout mice failed to does not prevent the disease.53 Recent studies conducted by mediate protective functions. Consistently, B cells isolated Grey and colleagues have shown that B-cell depletion delays from arthritogenic splenocytes treated with anti-IL-10/anti- and reduces diabetes by increasing the number of IL-10R antibodies in vitro were unable to protect recipient mice CD251Foxp31CD41 Treg T cells, thereby enforcing long-term from developing arthritis. When sorted MZ B, FO B or T2- tolerance.54 Moreover, studies by Smith and Tedder have sug- MZP arthritic B cells were transferred into DBA/1J mice on the gested that Breg cells, such as B10 cells, may represent a signifi- day of the second immunization, only T2-MZP B cells signifi- cant component of the reconstituted B-cell pool after B-cell cantly delayed the development of arthritis, and approximately depletion.55 These findings suggest an involvement of Breg cells 40% of the mice that received T2-MZP cells developed arth- in the development of diabetes. ritis. Thus, T2-MZP B cells and cells within this phenotypically There is compelling evidence that activated B cells can main- 19 defined subset can inhibit CIA progression. Administration tain immune tolerance because the transfer of activated B cells of apoptotic to mice up to 1 month before the protects NOD mice from diabetes.56,57 Repeated intravenous clinical onset of CIA has also been shown to have protective transfer of BCR-activated B cells into 5- to 6-week-old NOD 44 effects on joint inflammation and bone destruction. mice delays the onset and reduces the incidence of diabetes, Activated splenic B cells respond directly to apoptotic cell treat- while treatment starting at 9 weeks of age only delays diabetes ment by increasing the secretion of IL-10, which is important onset. The therapeutic effect from transfusing activated B cells for inducing T cell-derived IL-10. Moreover, the passive trans- from NOD mice correlates with the polarization of CD41 T fer of B cells from apoptotic cell-treated mice provides signifi- cells toward a Th2 phenotype in recepient NOD mice.58 cant protection against developing arthritis. These findings Notably, B cell-derived IL-10 is required for protection against suggest that certain subsets of IL-10-producing B cells gene- T1D because the transfusion of activated NOD-IL-102/2 B rated in vivo can suppress autoimmune pathogenesis. This cells neither confers protection against diabetes nor reduces notion is supported by our recent findings that in vitro induced the severity of insulitis. Tian et al. have reported that B10 cells can suppress the development of arthritis and LPS-activated B cells expressed Fas ligand and secreted TGF-

Cellular & Molecular Immunology Regulatory B cells in autoimmune diseases M Yang et al 125 b.57 Transfer of the activated B cells into prediabetic NOD mice in the control of disease initiation, whereas Treg cells exert their inhibited spontaneous Th1 immune responses and delayed the regulatory function during the late-phase of disease. onset of diabetes. Cotransfer of activated B cells and diabeto- genic splenic T cells into NOD/scid mice prevented the Breg cells in murine lupus development of diabetes; however, cotransfer of control cells Systemic lupus erythematous (SLE) is a systemic autoimmune with diabetogenic T cells showed no protective effect. These disease that is characterized by high autoantibody production, results suggest that activated B cells may downregulate patho- increased immune complex deposition, and multiple organ genic Th1 immunity by triggering the apoptosis of Th1 cells damage. Both T and B cells contribute to the pathogenesis of and/or inducing the secretion of the regulatory cytokine TGF- human SLE.66,67 NZB/NZW (NZB/W) F1 hybrid mice develop b. Together, these findings indicate that Breg cells play an a spontaneous lupus-like disease, which is characterized by indispensable role in the initiation of T1D, but have little effect immune complex-mediated glomerulonephritis that is assoc- on disease progression in NOD mice. iated with IgG autoantibody production against nuclear Ags, including dsDNA, RNA, chromatin and histones.68 The prom- Breg cells in EAE inent characteristics of NZB/W F1 mice include the expansion Multiple sclerosis (MS) is a prototypic T cell-mediated auto- of B-1a and MZ B cells, activation of polyclonal B cells, and immune disease that results in the demyelination of cells in the high serum levels of IgM and IgG.69–72 MRL/lpr mice also central nervous system (CNS). This demyelination is mediated spontaneously develop a similar lupus-like disease.73 by CD41 T cells specific for myelin oligodendrocyte glycopro- Therefore, both NZB/W F1 and MRL/lpr mouse models have tein and other autoantigens in the central nervous system.59 been extensively used for studying human SLE. When 12- to EAE is a mouse model of human MS. Accumulating evidence 28-week-old NZB/W F1 mice receive treatment with a low dose has suggested that B cells also play a pathogenic role in of the CD20 mAb, spontaneous disease progression is signifi- EAE.12,60,61 However, it has been reported that B cell-deficient cantly delayed. In contrast, B-cell depletion initiated in 4-week- mice develop a more severe and non-remitting form of old mice promotes disease onset, most likely due to the deple- EAE.12,60 Moreover, CD201 B cell depletion after EAE develop- tion of IL-10-producing Breg cells.74 It has been shown that ment dramatically suppresses the disease symptoms.62 there is significant expansion of B10 cells in young NZB/W F1 Although B-cell depletion ameliorates ongoing EAE, B-cell mice.65 Therefore, different B-cell populations can play either depletion occurring before EAE induction exacerbates the dis- protective or pathogenic roles during disease pathogenesis ease, suggesting that the Breg cells that negatively regulate because the timing of B-cell depletion has significant effects inflammatory reactions are possibly depleted.63 Furthermore, on disease progression in NZB/W F1 mice. To examine the the regulatory functions of B cells during EAE have been linked roles of B cells in disease pathogenesis in NZB/W F1 mice, to the production of IL-10 because adoptive transfer of wild- CD192/2 NZB/W mice were generated. The production of type B cells, rather than IL-102/2 B cells from mMT mice, anti-nuclear Abs was remarkably delayed in CD192/2 NZB/ decreases the severity of EAE. B cells from recovered mice W mice compared with wild-type NZB/W mice. However, produce IL-10, which is important for attenuating pro-inflam- CD192/2 NZB/W mice developed nephritis significantly earl- matory Th1 responses. Importantly, in the absence of IL-10- ier and had a substantially reduced survival rate. These results producing B cells, mice are not able to recover from EAE.60,64 In emphasized that B cells play both pathogenic and protective a recent study, fusokine GIFT15-induced Breg cells are shown roles in lupus pathogenesis.75 B10 cells were increased in wild- to secrete IL-10 and express MHC I, MHC II, and surface IgM type NZB/W mice during disease progression, whereas and IgD. Moreover, mice with EAE undergo complete CD192/2 NZB/W mice lacked B10 cells, which is similar to remission after the intravenous transfer of GIFT15-Breg cells. the findings in a previous report.20 Moreover, transfer of These cells function by suppressing neuro-inflammation.21 splenic B10 cells from wild-type NZB/W mice into CD192/2 Therefore, IL-10-producing B cells have been identified as NZB/W recipients significantly prolonged the survival rate of important regulators in controlling EAE. Recently, Tedder NZB/W mice. This increased survival was accompanied by and colleagues characterized the overlapping and differential expansion of Treg cells, which suggests that regulatory B10 cells roles of Breg and Treg cells in shaping the course of EAE immu- play a protective role in lupus pathogenesis.75 Moreover, Blair nopathogenesis.65 Adoptively transferred B10 cells can directly et al. have demonstrated that the transfer of in vitro anti-CD40- influence EAE pathogenesis by producing IL-10. Interestingly, induced T2 Breg cells significantly improved the severity of the number of B10 cells expands quickly in the spleen but not in renal disease and survival rate in MPL/lpr mice in an IL-10- the CNS, which is consistent with the regulatory function of dependent manner.76 Thus, both B10 cells and T2-MZP B cells B10 cells involved in disease initiation. Furthermore, transfer of can effectively protect mice from developing lupus. antigen-sensitized B10 cells into wild-type mice dramatically reduces EAE initiation, but B10 cells could not inhibit ongoing Breg cells in human autoimmune diseases EAE progression. However, the number of Treg cells expanded Extensive studies in mice have demonstrated that Breg cells markedly within the CNS during disease progression. This play important roles in the suppression of autoimmune dis- expansion negatively regulated the late phase of EAE. Thus, eases, but relatively little is known about human Breg cells in these findings suggest that Breg cells play a predominant role healthy individuals and patients. A remarkable study by Mauri

Cellular & Molecular Immunology Regulatory B cells in autoimmune diseases M Yang et al 126

and colleagues has identified a specific subset of human Breg which are both Th1-mediated autoimmune conditions.88,89 cells with a phenotype of CD191CD24hiCD38hi in the peri- These intriguing findings may indicate the existence of Breg cells pheral blood of healthy individuals.10 This phenotype has been that modulate T cell-mediated inflammatory responses in vivo. previously associated with immature transitional B cells.77,78 Taken together, B cells, autoantibodies and T cells are all However, these B cells were able to produce IL-10 in response involved in the development of autoimmune diseases and have to CD40 stimulation. However, CD191CD24hiCD38hi B cells unique functions in each autoimmune disease. B cells not only isolated from the peripheral blood of patients with SLE lacked produce autoantibodies and act as antigen-presenting cells for the suppressive capacity possessed by their counterparts in CD41 T-cell activation, but also serve as negative regulators healthy individuals. Moreover, comparisons between B cells that dampen harmful immune responses. Therefore, the time from SLE patients and healthy controls indicate that the defect window for depleting B cells or transferring Breg cells is in IL-10-production in response to CD40 stimulation in B cells important because the changes in immunological balance from SLE patients is possibly due to altered activation of may result in either exacerbation or amelioration of disease STAT3.10 Interestingly, SLE patients who received rituximab progression. treatment had an increased ratio of CD191CD24hiCD38hi B cells to memory B cells, which supports the notion that B cell MICRO-ENVIRONMENTAL SIGNALING IN MODULATING depletion may result in an increased generation of tolerogenic BREG CELL GENERATION 77,79 B cells. No common surface markers or specific transcription factor(s) Tedder and colleagues have identified a subset of human B10 have been identified yet that define both human and mouse 1 cells with a phenotype of CD24hiCD27 ; approximately 60% of Breg cells. Moreover, Breg cells can be generated with the these B10 cells express CD38.11 CD27 is a well-characterized appropriate stimulation both in vitro and in vivo, which rein- 1 marker for human memory B cells. Moreover, CD27 B cells forces the notion that factors present in the microenvironment can expand during autoimmune diseases and act as a biomar- may play a crucial role in the induction of Breg cells.8 Certain ker for disease activity.80,81 B10 cells in the blood have a higher TLR agonists have been demonstrated to be potent inducers of proliferative capacity than other B cells in response to mitogen B cells with suppressive functions. LPS from Gram-negative stimulation, which indicates that these B cells have not recently bacteria and CpG-containing oligonucleotides that mimic bac- emigrated from the bone marrow.11 The frequency of terial DNA have been shown to induce IL-10-producing B cells CD24hiCD271 B10 cells in human blood was even higher in and inhibit disease progression in a mouse model of EAE.90 the autoimmune diseases including SLE, RA, autoimmune skin Mice containing B-cell deletions of Tlr2, Tlr4 or the TLR disease and MS. Recently, Bouaziz et al. have demonstrated that adaptor myeloid differentiation primary-response gene 88 human B cells stimulated with anti-Ig and CpG produced IL-10 (MyD88) could not recover from EAE, suggesting that TLRs and enriched both CD271 memory and CD38hi transitional B are directly involved in modulating the regulatory function of B cell compartments.82 Although current findings cannot recon- cells.90 cile the different phenotypes of human Breg cells, it is clear that TLR-signaling has been shown to initiate IL-10 production in Breg cells exist in human blood and lymphoid organs. These naive B cells. However, B cells also require CD40 and BCR cells have regulatory functions that are partially dependent on ligation to enable further IL-10-production.60 Accumulating IL-10 production. data support a two-step model for the establishment of B-cell- Similar to findings in animal models, the relative contribution mediated suppression. During the initial stage, TLR stimulation of different immune components to the pathogenesis of human induces only a few IL-10-producing B cells. During the second autoimmune disease differs from one disease to another. B cells phase, BCR and CD40 ligation, which are classically involved in may play either a crucial role in the initiation of the disease or B-cell survival and expansion, further amplifies the population contribute to autoimmune pathogenesis after disease onset.40 of IL-10-producing B cells, which results in sufficient IL-10 Recently, B-cell depletion strategies have been used to treat production for effective suppression.90 Yanaba et al. have patients with autoimmune diseases. In RA patients, B-cell deple- demonstrated that splenic B cells treated with LPS, PMA and tion using rituximab significantly diminished ongoing joint ionomycin in vitro for 5 h results in optimal IL-10 production.91 inflammation, but the recrudescence of disease activity was often Moreover, LPS or LPS plus CD40 stimulation for 48 h induces accompanied by B-cell recovery.83–85 In human MS, B-cell additional splenic CD1dhiCD51 B10 cells to express IL-10 fol- depletion appears to be more effective after the onset of disease. lowing PMA plus ionomycin stimulation. Human B cells express B-cell depletion after the onset of the symptoms can ameliorate TLR9, which is a receptor for CpG, but not TLR4. Bouaziz et al. disease progression.86 Clinical studies have shown that B-cell have shown that TLR9 is a potent inducer of IL-10 production. depletion with rituximab improves the clinical manifestations Moreover, the optimal stimulus for human B cells found in the of SLE.87 These clinical studies have suggested that B-cell deple- blood is the combination of CpG and anti-Ig, which can act tion is an effective therapy for treating autoimmune diseases. synergistically to induce human B cells to produce IL-10.82 However, B-cell depletion may exacerbate disease in some auto- Interestingly, CpG and anti-Ig stimuli can effectively induce immune conditions. For example, B-cell depletion has been memory B cells (CD271), CD51 B cells and immature trans- shown to exacerbate ulcerative colitis and trigger psoriasis, itional B cells (CD38hiCD24hi) to produce IL-10.10,11,92 These

Cellular & Molecular Immunology Regulatory B cells in autoimmune diseases M Yang et al 127 findings suggest that TLR signaling plays an important role in the spleen.98 Interestingly, studies by Rafei et al. have shown the induction of Breg cells. that GIFT15 can induce Breg cells to have a phenotype that is a CD40 engagement has been found to be required for the hybrid between CD1dhiCD51 B10 and T2-MZP Breg cells and suppression of both EAE and CIA, suggesting that an inter- plasma cells expressing CD138.21 Recently, Qian et al. have action between B cells and CD40L-expressing CD41 T helper reported that regulatory dendritic cells (DCs) can induce cells is necessary for B cell-mediated suppression.43,60,76 Mauri splenic B cells to differentiate into IL-10-producing B cells that hi hi et al. have demonstrated that stimulation of splenocytes from have the phenotype CD19 FccIIb through IFN-b and 23 CIA mice during remission with anti-CD40 mAb induces the CD40L. These results suggest that Breg cells can be generated differentiation of IL-10-producing B cells. Furthermore, B cells in the appropriate temporal and spatial microenvironment. from normal mice that have recovered from EAE produced IL- Future studies identifying more signals involved in the differ- 10 upon the ligation of CD40.43 In contrast, mice with chimeric entiation of B cells into Breg cells are anticipated. bone marrow that contained B cells lacking CD40 expression failed to recover from EAE. However, the transfer of B cells MECHANISMS UNDERLYING BREG CELL FUNCTION from mice that had recovered from EAE into these chimeric There are several direct and indirect mechanisms by which mice compensated for their inability to recover from EAE.60 In Breg cells exert their regulatory functions during the humans, B cells from the blood that were treated with CD40L immune response (Figure 1). In mice, there are currently effectively induced CD4hiCD251Foxp31 Treg cells, which sup- two well-characterized subsets of IL-10-producing B cells. 1 2 93 The first is a B10 cell subset with the phenotype press CD4 CD25 T cells. Blair et al. have reported that CD40L hi hi 1 1 hi hi CD19 CD1d CD5 , and the second is a T2-MZP cell sub- stimulation induces CD19 CD24 CD38 B-cell expansion and 1 1 1 hi 20,99 1 hi set with the phenotype CD19 CD23 CD21 CD1d . suppresses Th1 cell differentiation. However, CD19 CD24 hi Although the frequency of naturally existing IL-10-pro- CD38 B cells from the peripheral blood of SLE patients were ducing regulatory B cells is extremely low, Breg cells can insensitive to stimulation by CD40L and produced a reduced be expanded in vitro. This expansion allows for the enrich- amount of IL-10. Consequently, these B cells were unable to ment of Breg cells and permits a more comprehensive study suppress CD41CD252 T cells from healthy donors.10 These of the mechanisms by which Breg cells mediate immune results suggest that anti-CD40 ligation is critically involved in suppression. These two Breg subsets produce IL-10 and sup- Breg cell activation. press both the proliferation of T cells and cytokine produc- B-cell activating factor (BAFF), which is a member of the tion (IFN-c and TNF-a)byTh1cells.20,46,99 Moreover, TNF family, acts as a key regulator of B-cell maturation and transfer of a relatively low number of in vitro expanded survival. Analyses of BAFF-deficient mice reveal a fundamental Breg cells maintains long-term protection against several role of BAFF in promoting the maturation of T1 B cells to T2 B 94 autoimmune diseases in animal models, which suggests that cells. In addition to the crucial role BAFF plays in the main- Breg cells can either further proliferate in vivo or initiate an tenance of the peripheral B cell pool, BAFF has been found to be 95 efficient immunosuppressive cascade with other immune essential for MZ B-cell development. Moreover, new evid- suppressive cells.100 Breg cells can not only suppress Th1- ence from BAFF-transgenic mice indicates that BAFF induces 1 1 mediated immune responses but also convert effector T cells CD4 Foxp3 T cells to suppress T-cell responses in an indir- into regulatory Tr1 cells.76,97,101 Gray et al. have clearly ect, B cell-dependent manner, which suggests a regulatory role shown that ACs induce B and T cells to produce IL-10. of BAFF in vivo.96 Recently, we have shown that low dosages of hi 1 Moreover, B cell-derived IL-10 has been shown to be essen- BAFF can induce B cells of the phenotype CD1d CD5 to tial in the induction of T cells to secrete IL-10 in vitro.97 induce IL-10, which is similar to B10 cells. BAFF stimulation Furthermore, Mauri et al. have observed that there is a can selectively induce the expansion of IL-10-producing B cells longer contact time between CD41CD252 T cells and IL- after 3-days in culture. Moreover, BAFF treatment in vivo 10-producing B cells than IL-10-deficient B cells. This longer increased the number of IL-10-producing B cells in the mar- contact time enables IL-101 B cells to convert effector T cells 46 ginal zone regions. These findings reveal a previously unap- into Tr1 cells, which is mediated by IL-10 that is produced preciated function of BAFF, which is to induce B cells with by B cells.101 B cells can also promote DCs to not only regulatory function. secrete IL-4 but also downregulate IL-12, which affects the In addition, other signals have been reported to be important Th1/Th2 balance.102 In addition to IL-10-producing Breg in the generation of Breg cells. For example, apoptotic cells cells, TGF-b1-producing Breg cells have been identified in (ACs) have been shown to act as endogenous signals that response to LPS stimulation in vitro.103,104 These B cells can trigger IL-10 production, leading to the amelioration of trigger pathogenic Th1 cells to undergo apoptosis through CIA.97 ACs are able to induce splenic B cells to secrete IL-10, Fas–FasL interactions and/or the inhibition of antigen-pre- which further enhances antigen-specific T cells to secrete IL-10 senting cell activity via the secretion of TGF-b1.103 and exert immunosuppressive functions. Moreover, ACs can In addition to regulating the Th1/Th2 balance, Breg cells preferentially induce MZ B cells, rather than FO B cells, to have been shown to affect the balance between Foxp31 and secrete IL-10, which is most likely related to the fact that MZ IL-17–producing T cells.101,105 Worm-induced Breg cells have B cells reside on the border between the red and white pulp in been shown to suppress allergic airway inflammation by

Cellular & Molecular Immunology Regulatory B cells in autoimmune diseases M Yang et al 128

DC

Treg TH17

b IL-10

IL-10,TLR IL-10,TGF-

CD80 CD28 CD86 CTLA-4 Breg Teff FasL Fas IL-10 CD1d

TH2 NKT IL-4,IL-10 c IL-13 INF- TH1 AC TNF-a

Figure 1 Mechanisms of action for Breg cells in immune responses. The possible mechanisms by which Breg cells modulate immune responses may include the following: Breg cells restore the Th1/Th2 balance by producing IL-10; Breg cells inhibit Th1 and Th17 cell differentiation, but promote Treg cell expansion. These effects are mediated not only through the release of soluble factors such as IL-10 and TGF-b, but also via cell- to-cell contact involving CD80, CD86 and FasL, etc. Interactions between Breg and Teffs) can result in the induction of ACs as well as the induction of both Foxp31 Treg cells and IL-10-producing Tr1 cells. Breg cells can dampen the activation of DCs and macrophages. Moreover, Breg cells express CD1d, which may activate iNKT cells with regulatory functions. AC, apoptotic cell; Breg, regulatory B; DC, dendritic cell; iNKT, invariant natural killer T; Teff, effector T cell; TNF, tumor-necrosis factor; Treg, regulatory T.

promoting pulmonary infiltration of CD41CD251Foxp31 Treg roles in autoimmune development. Interestingly, EAE is exa- cells, which is a IL-10-dependent but TGF-b-independent mech- cerbated in CD1d2/2 mice, which lack NKT cells.110 Recently, anism.105,106 Carter et al. have elegantly demonstrated that endo- human transitional B cells (CD191CD24hiCD38hi) have been genous IL-10-producing B cell-deficient mice develop an shown to play an essential role in iNKT cell expansion and exacerbated case of arthritis and exhibit an increased frequency activation in healthy individuals, but not in SLE patients of Th1/Th17 pro-inflammatory cells, but a decreased frequency of because transitional B cells from SLE patients have defects in Treg cells.101,107 Consistent with these findings, we have also CD1d recycling.111 Thus, CD1d-expressing Breg cells can exert demonstrated that B10 cells induced in vitro can suppress Th17 their regulatory functions by activating NKT cells. cell differentiation by decreasing the phosphorylation levels of The mechanisms for regulating the immune response are Stat3, which subsequently reduces the levels of RORct, and par- mediated by either the release of suppressive soluble cytokines, tially inhibits the Th17 cell population in an IL-10-dependent including IL-10 and TGF-b, by regulatory cells or promotion of manner.45 Based on these current findings, it is likely that Breg activation-induced cell death (or apoptosis), which is mediated cells play an important role in T-cell plasticity. by death-inducing ligands, including FasL, TNF-related apopto- Apart from cytokine-mediated suppression, B cells can also sis-inducing ligand, and programmed death ligands 1 and 2 (PD- exert their regulatory effects by cellular interactions. Both B10 L1 and PD-L2), etc.112–114 B cells can express FasL and other and T2-MZP Breg cells share the phenotype CD1dhi, which is a death-inducing ligands under many circumstances. Both FasL MZ B cell marker. CD1d-expressing MZ B cells have been and IL-10 are highly expressed in the CD51 B-cell population, shown to activate invariant natural killer T (iNKT) cells in which indicates that CD51 B cells may exert regulatory effects by the presence of DCs and aid in the establishment of peripheral their killing ability.7 Interestingly, a recent study by Ray et al.has tolerance by the induction of Tr1 cells, which is a process that is suggested that B cells can induce the proliferation of Treg cells in dependent on the activation of iNKT cells via CD1d.108,109 theCNSduringthedevelopmentofEAEvia the expression of Moreover, CD1dhi MZ B cells are capable of presenting glyco- glucocorticoid-induced TNF receptor ligand rather than IL- lipids through CD1d. These glycolipids are recognized by NKT 10.115 In addition, costimulatory molecules are also involved in cells, which are cells that have been shown to play important Breg-mediated suppression. The synergistic effects of IL-10,

Cellular & Molecular Immunology Regulatory B cells in autoimmune diseases M Yang et al 129

CD80 and CD86 interactions have been demonstrated in both functional features of Breg cells in vivo will provide a more mouse and humans.10,116 Thus, signaling through CD80 and complete understanding of the roles Breg cells play in auto- CD86 is an additional effector mechanism for immune suppres- immune pathogenesis. The knowledge gained is essential to sion. facilitate the development of Breg cells as a potential cellular In summary, Breg cells can exert their suppressive effects by therapy for human autoimmune diseases. secreting anti-inflammatory cytokines, such as IL-10 and TGF- b, and engaging in cell-to-cell contact via activating cell death ACKNOWLEDGEMENTS markers or costimulatory molecules. Moreover, Breg cells can The authors dedicate this review manuscript to Dr Dennis G Osmond not only regulate the balance of T helper cells, but also induce at McGill University for his mentorship. Dr Lu is a Croucher Senior tolerogenic DC or invariant NKT cells to further influence T Research Fellow supported by Hong Kong Croucher Foundation. This helper cell plasticity. work was supported by grants from the National Basic Research Program of China (Grant No. 2010 CB 529100) and Research Grants CONCLUDING REMARKS: BREG CELLS FROM BENCH Council of Hong Kong. The authors apologize to those researchers TO BEDSIDE whose work could not be cited due to space limitations. The pathogenic roles of B cells in autoimmune diseases have been extensively characterized. These roles have been further confirmed by the efficacy of B-cell depletion in treating auto- 1 Osmond DG. B cell development in the bone marrow. Semin Immunol immune diseases in both humans and mice. Although B cell- 1990; 2: 173–180. targeted therapies are very promising, long-term B-cell deple- 2 Cooper MD. Exploring differentiation pathways. Immunol tion may lead to the development of immunopathology. CD20 Rev 2002; 185: 175–185. 3 Welner RS, Pelayo R, Kincade PW. Evolving views on the genealogy of is expressed on B cells ranging from the pre-B to mature stages, B cells. Nat Rev Immunol 2008; 8: 95–106. but not on plasma cells, which suggests that long-lived plasma 4 Katz SI, Parker D, Turk JL. B-cell suppression of delayed hypersensitivity cells that produce autoantibodies may not be affected by anti- reactions. Nature 1974; 251: 550–551. CD20 mAb treatment. Moreover, current approaches to target 5 Neta R, Salvin SB. Specific suppression of delayed hypersensitivity: the possible presence of a suppressor B cell in the regulation of B cells cannot distinguish between pathogenic B and Breg cells. delayed hypersensitivity. J Immunol 1974; 113: 1716–1725. Recently, the anti-BAFF mAb has been shown to be effective in 6 Mizoguchi A, Bhan AK. A case for regulatory B cells. J Immunol 2006; treating autoimmune diseases such as SLE. Although blocking 176: 705–710. BAFF can inhibit the survival and maturation of B cells that 7 Lundy SK. Killer B : the evidence and the potential. contribute to autoimmune pathogenesis, this approach may Inflamm Res 2009; 58: 345–357. 8 Gray D, Gray M. What are regulatory B cells? Eur J Immunol 2010; 40: also reduce the number of IL-10-producing Breg cells. 2677–2679. Because low dosages of BAFF induce the generation of Breg 9 DiLillo DJ, Matsushita T, Tedder TF. B10 cells and regulatory B cells cells, the timing and choice of suitable antibodies for B cell balance immune responses during inflammation, autoimmunity, and depletion is critical depending on the pathogenic features of cancer. Ann NY Acad Sci 2010; 1183: 38–57. 10 Blair PA, Norena LY, Flores-Borja F, Rawlings DJ, Isenberg DA, each autoimmune disease. Increasing evidence indicates that Ehrenstein MR et al. CD191CD24hiCD38hi B cells exhibit regulatory B-cell depletion results in long-term remission, which is most capacity in healthy individuals but are functionally impaired in likely due to the expansion of Treg and Breg cells. In particular, systemic Lupus Erythematosus patients. Immunity 2010; 32: 129– recent studies from various mouse models have suggested that 140. 11 Iwata Y, Matsushita T, Horikawa M, Dilillo DJ, Yanaba K, Venturi GM et B-cell depletion leads to an increased Breg cell subset in the al. Characterization of a rare IL-10-competent B-cell subset in reconstituted B-cell population. Thus, it would be of interest to humans that parallels mouse regulatory B10 cells. Blood 2011; determine the effects of the adoptive transfer of Breg cells either 117: 530–541. alone or in combination with B-cell depletion for the treatment 12 Wolf SD, Dittel BN, Hardardottir F, Janeway CA Jr. Experimental autoimmune encephalomyelitis induction in genetically B cell- of autoimmune diseases. deficient mice. J Exp Med 1996; 184: 2271–2278. The adoptive transfer of Breg cells is a potential therapeutic 13 Fillatreau S, Sweenie CH, McGeachy MJ, Gray D, Anderton SM. B cells strategy. IL-10-producing Breg cells can continually secrete IL- regulate autoimmunity by provision of IL-10. Nat Immunol 2002; 3: 10, whereas the direct administration of IL-10 has a restricted 944–950. 14 Mizoguchi A, Mizoguchi E, Smith RN, Preffer FI, Bhan AK. therapeutic effect due to its short half-life. There is also com- Suppressive role of B cells in chronic colitis of T cell receptor alpha pelling evidence that transferred Breg cells can migrate to local mutant mice. J Exp Med 1997; 186: 1749–1756. inflammatory sites and reside in joint tissue for more than 3 15 Mizoguchi A, Mizoguchi E, Takedatsu H, Blumberg RS, Bhan AK. weeks in CIA mice. Hence, Breg cells may exert regulatory Chronic intestinal inflammatory condition generates IL-10- producing regulatory B cell subset characterized by CD1d functions in local sites depending on their homing capacity upregulation. Immunity 2002; 16: 219–230. and the survival signals present in the local environment. 16 O’Garra A, Chang R, Go N, Hastings R, Haughton G, Howard M. Ly-1 B However, there are also some critical questions that need to (B-1) cells are the main source of B cell-derived . Eur J be addressed before the clinical application of Breg cells can be Immunol 1992; 22: 711–717. 17 Spencer NF, Daynes RA. IL-12 directly stimulates expression of IL-10 considered. Similar to the therapeutic application of Treg cells, by CD51 B cells and IL-6 by both CD51 and CD52 B cells: possible one of the major challenges is the functional stability of trans- involvement in age-associated cytokine dysregulation. Int Immunol ferred Breg cells in vivo. Thus, further characterization of the 1997; 9: 745–754.

Cellular & Molecular Immunology Regulatory B cells in autoimmune diseases M Yang et al 130

18 Lenert P, Brummel R, Field EH, Ashman RF. TLR-9 activation of 40 Yanaba K, Bouaziz JD, Matsushita T, Magro CM, St Clair EW, Tedder marginal zone B cells in lupus mice regulates immunity through TF. B-lymphocyte contributions to human autoimmune disease. increased IL-10 production. J Clin Immunol 2005; 25: 29–40. Immunol Rev 2008; 223: 284–299. 19 Evans JG, Chavez-Rueda KA, Eddaoudi A, Meyer-Bahlburg A, 41 Lo CK, Lam QL, Sun L, Wang S, Ko KH, Xu H et al. Natural killer Rawlings DJ, Ehrenstein MR et al. Novel suppressive function of cell degeneration exacerbates experimental arthritis in mice via transitional 2 B cells in experimental arthritis. JImmunol2007; enhanced interleukin-17 production. Arthritis Rheum 2008; 58: 178: 7868–7878. 2700–2711. 20 Yanaba K, Bouaziz JD, Haas KM, Poe JC, Fujimoto M, Tedder TF. A 42 Yanaba K, Hamaguchi Y, Venturi GM, Steeber DA, St Clair EW, Tedder regulatory B cell subset with a unique CD1dhiCD51 phenotype controls TF. B cell depletion delays collagen-induced arthritis in mice: arthritis T cell-dependent inflammatory responses. Immunity 2008; 28: 639– induction requires synergy between humoral and cell-mediated 650. immunity. J Immunol 2007; 179: 1369–1380. 21 Rafei M, Hsieh J, Zehntner S, Li M, Forner K, Birman E et al.A 43 Mauri C, Gray D, Mushtaq N, Londei M. Prevention of arthritis by granulocyte-macrophage colony-stimulating factor and interleukin- interleukin 10-producing B cells. J Exp Med 2003; 197 : 489–501. 15 fusokine induces a regulatory B cell population with immune 44 Gray M, Miles K, Salter D, Gray D, Savill J. Apoptotic cells protect mice suppressive properties. Nat Med 2009; 15: 1038–1045. from autoimmune inflammation by the induction of regulatory B cells. 22 Ding Q, Yeung M, Camirand G, Zeng Q, Akiba H, Yagita H et al. Proc Natl Acad Sci USA 2007; 104: 14080–14085. Regulatory B cells are identified by expression of TIM-1 and can be 45 Yang M, Deng J, Liu Y, Ko KH, Wang X, Jiao Z et al. IL-10-producing induced through TIM-1 ligation to promote tolerance in mice. J Clin regulatory B10 cells ameliorate collagen-induced arthritis via Invest 2011; 121: 3645–3656. suppressing Th17 cell generation. Am J Pathol 2012; 180: 2375– 23 Qian L, Qian C, Chen Y, Bai Y, Bao Y, Lu L et al. Regulatory dendritic 2385. cells program B cells to differentiate into CD19hiFcgammaIIbhi 46 Yang M, Sun L, Wang S, Ko KH, Xu H, Zheng BJ et al. Novel function of regulatory B cells through IFN-beta and CD40L. Blood 2012; 120: B cell-activating factor in the induction of IL-10-producing regulatory 581–591. B cells. J Immunol 2010; 184: 3321–3325. 24 Duddy M, Niino M, Adatia F, Hebert S, Freedman M, Atkins H et al. 47 Anderson MS, Bluestone JA. The NOD mouse: a model of immune Distinct effector cytokine profiles of memory and naive human B cell dysregulation. Annu Rev Immunol 2005; 23: 447–485. subsets and implication in multiple sclerosis. J Immunol 2007; 178: 48 Silveira PA, Grey ST. B cells in the spotlight: innocent bystanders or 6092–6099. major players in the pathogenesis of type 1 diabetes. Trends 25 Duddy ME, Alter A, Bar-Or A. Distinct profiles of human B cell effector Endocrinol Metab 2006; 17: 128–135. cytokines: a role in immune regulation? J Immunol 2004; 172: 3422– 49 Fox CJ, Danska JS. Independent genetic regulation of T-cell and 3427. antigen-presenting cell participation in autoimmune islet 26 Edwards JC, Cambridge G, Abrahams VM. Do self-perpetuating B inflammation. Diabetes 1998; 47: 331–338. lymphocytes drive human autoimmune disease? Immunology 1999; 50 Kendall PL, Yu G, Woodward EJ, Thomas JW. Tertiary lymphoid 97: 188–196. structures in the pancreas promote selection of B lymphocytes in 27 Shimomura Y, Mizoguchi E, Sugimoto K, Kibe R, Benno Y, Mizoguchi autoimmune diabetes. J Immunol 2007; 178: 5643–5651. A et al. Regulatory role of B-1 B cells in chronic colitis. Int Immunol 51 Henry RA, Kendall PL. CXCL13 blockade disrupts B lymphocyte 2008; 20: 729–737. organization in tertiary lymphoid structures without altering B cell 28 Shlomchik MJ, Craft JE, Mamula MJ. From T to B and back again: receptor bias or preventing diabetes in nonobese diabetic mice. J positive feedback in systemic autoimmune disease. Nat Rev Immunol Immunol 2010; 185: 1460–1465. 2001; 1: 147–153. 52 Yanaba K, Bouaziz JD, Matsushita T, Magro CM, St Clair EW, Tedder 29 Rodriguez-Pinto D, Moreno J. B cells can prime naive CD41 T cells in TF. B-lymphocyte contributions to human autoimmune disease. vivo in the absence of other professional antigen-presenting cells in a Immunol Rev 2008; 223: 284–299. CD1542CD40-dependent manner. Eur J Immunol 2005; 35: 1097– 53 Xiu Y, Wong CP, Bouaziz JD, Hamaguchi Y, Wang Y, Pop SM et al.B 1105. lymphocyte depletion by CD20 monoclonal prevents 30 Yan J, Harvey BP, Gee RJ, Shlomchik MJ, Mamula MJ. B cells drive diabetes in nonobese diabetic mice despite isotype-specific early T cell autoimmunity in vivo prior to dendritic cell-mediated differences in Fc gamma R effector functions. J Immunol 2008; autoantigen presentation. J Immunol 2006; 177: 4481–4487. 180: 2863–2875. 31 Fuchs EJ, Matzinger P. B cells turn off virgin but not memory T cells. 54 Marino E, Villanueva J, Walters S, Liuwantara D, Mackay F, Grey ST. Science 1992; 258: 1156–1159. CD41CD251 T-cells control autoimmunity in the absence of B-cells. 32 Eynon EE, Parker DC. Small B cells as antigen-presenting cells in the Diabetes 2009; 58: 1568–1577. induction of tolerance to soluble protein antigens. J Exp Med 1992; 55 Smith SH, Tedder TF. Targeting B-cells mitigates autoimmune 175: 131–138. diabetes in NOD mice: what is plan B? Diabetes 2009; 58: 1479– 33 Harris DP, Haynes L, Sayles PC, Duso DK, Eaton SM, Lepak NM et al. 1481. Reciprocal regulation of polarized cytokine production by effector B 56 Hussain S, Delovitch TL. Intravenous transfusion of BCR-activated B and T cells. Nat Immunol 2000; 1: 475–482. cells protects NOD mice from type 1 diabetes in an IL-10-dependent 34 Feldmann M, Brennan FM, Maini RN. Rheumatoid arthritis. Cell manner. J Immunol 2007; 179: 7225–7232. 1996; 85: 307–310. 57 Tian J, Zekzer D, Hanssen L, Lu Y, Olcott A, Kaufman DL. 35 Scott DL, Wolfe F, Huizinga TW. Rheumatoid arthritis. Lancet 2010; -activated B cells down-regulate Th1 immunity 376: 1094–1108. and prevent autoimmune diabetes in nonobese diabetic mice. J 36 Trentham DE, Townes AS, Kang AH. Autoimmunity to type II collagen Immunol 2001; 167: 1081–1089. an experimental model of arthritis. J Exp Med 1977; 146: 857–868. 58 Hussain S, Delovitch TL. Intravenous transfusion of BCR-activated B 37 Lu L, Osmond DG. Apoptosis and its modulation during B cells protects NOD mice from type 1 diabetes in an IL-10-dependent in mouse bone marrow. Immunol Rev 2000; 175: manner. J Immunol 2007; 179: 7225–7232. 158–174. 59 Williams KC, Ulvestad E, Hickey WF. Immunology of multiple 38 Zhang M, Srivastava G, Lu L. The pre-B cell receptor and its function sclerosis. Clin Neurosci 1994; 2: 229–245. during B cell development. Cell Mol Immunol 2004; 1: 89–94. 60 Fillatreau S, Sweenie CH, McGeachy MJ, Gray D, Anderton SM. B cells 39 Zhang M, Ko KH, Lam QL, Lo CK, Srivastava G, Zheng B et al. regulate autoimmunity by provision of IL-10. Nat Immunol 2002; 3: Expression and function of TNF family member B cell-activating 944–950. factor in the development of autoimmune arthritis. Int Immunol 61 Cross AH, Trotter JL, Lyons J. B cells and antibodies in CNS 2005; 17: 1081–1092. demyelinating disease. J Neuroimmunol 2001; 112: 1–14.

Cellular & Molecular Immunology Regulatory B cells in autoimmune diseases M Yang et al 131

62 Bouaziz JD, Yanaba K, Tedder TF. Regulatory B cells as inhibitors of 82 Bouaziz JD, Calbo S, Maho-Vaillant M, Saussine A, Bagot M, immune responses and inflammation. Immunol Rev 2008; 224: 201– Bensussan A et al. IL-10 produced by activated human B cells 214. regulates CD41 T-cell activation in vitro. Eur J Immunol 2010; 40: 63 Matsushita T, Yanaba K, Bouaziz JD, Fujimoto M, Tedder TF. 2686–2691. Regulatory B cells inhibit EAE initiation in mice while other B cells 83 Edwards JC, Cambridge G. Sustained improvement in rheumatoid promote disease progression. J Clin Invest 2008; 118: 3420–3430. arthritis following a protocol designed to deplete B lymphocytes. 64 Ray A, Mann MK, Basu S, Dittel BN. A case for regulatory B cells in Rheumatology (Oxford) 2001; 40: 205–211. controlling the severity of autoimmune-mediated inflammation in 84 Edwards JC, Szczepanski L, Szechinski J, Filipowicz-Sosnowska A, experimental autoimmune encephalomyelitis and multiple sclerosis. Emery P, Close DR et al. Efficacy of B-cell-targeted therapy with J Neuroimmunol 2011; 230: 1–9. rituximab in patients with rheumatoid arthritis. NEnglJMed 65 Matsushita T, Horikawa M, Iwata Y, Tedder TF. Regulatory B cells 2004; 350: 2572–2581. (B10 cells) and regulatory T cells have independent roles in 85 Leandro MJ, Cambridge G, Ehrenstein MR, Edwards JC. Recon- controlling experimental autoimmune encephalomyelitis initiation stitution of peripheral blood B cells after depletion with rituximab and late-phase immunopathogenesis. JImmunol2010; 185: in patients with rheumatoid arthritis. Arthritis Rheum 2006; 54: 2240–2252. 613–620. 66 Lipsky PE. Systemic lupus erythematosus: an autoimmune disease of 86 Hauser SL, Waubant E, Arnold DL, Vollmer T, Antel J, Fox RJ et al. B cell hyperactivity. Nat Immunol 2001; 2: 764–766. B-cell depletion with rituximab in relapsing-remitting multiple 67 Grammer AC, Slota R, Fischer R, Gur H, Girschick H, Yarboro C et al. sclerosis. N Engl J Med 2008; 358: 676–688. Abnormal germinal center reactions in systemic lupus erythematosus 87 Looney RJ, Anolik JH, Campbell D, Felgar RE, Young F, Arend LJ et al. demonstrated by blockade of CD154–CD40 interactions. J Clin Invest B cell depletion as a novel treatment for systemic lupus erythe- 2003; 112: 1506–1520. matosus: a phase I/II dose-escalation trial of rituximab. Arthritis 68 Mountz J. Animal models of systemic lupus erythematosus and Rheum 2004; 50: 2580–2589. Sjogren’s syndrome. Curr Opin Rheumatol 1990; 2: 740–748. 88 Goetz M, Atreya R, Ghalibafian M, Galle PR, Neurath MF. Exa- 69 Wither JE, Roy V, Brennan LA. Activated B cells express increased cerbation of ulcerative colitis after rituximab salvage therapy. levels of costimulatory molecules in young autoimmune NZB and Inflamm Bowel Dis 2007; 13: 1365–1368. (NZB3NZW)F mice. Clin Immunol 2000; 94: 51–63. 89 Dass S, Vital EM, Emery P. Development of psoriasis after B cell 1 depletion with rituximab. Arthritis Rheum 2007; 56: 2715–2718. 70 Schuster H, Martin T, Marcellin L, Garaud JC, Pasquali JL, Korganow AS. Expansion of marginal zone B cells is not sufficient for the 90 Lampropoulou V, Hoehlig K, Roch T, Neves P, Calderon Gomez E, Sweenie CH et al. TLR-activated B cells suppress T cell-mediated development of renal disease in NZB3NZW F mice. Lupus 2002; 1 autoimmunity. J Immunol 2008; 180: 4763–4773. 11: 277–286. 91 Yanaba K, Bouaziz JD, Matsushita T, Tsubata T, Tedder TF. The 71 Atencio S, Amano H, Izui S, Kotzin BL. Separation of the New Zealand development and function of regulatory B cells expressing IL-10 Black genetic contribution to lupus from New Zealand Black (B10 cells) requires antigen receptor diversity and TLR signals. determined expansions of marginal zone B and B1a cells. J J Immunol 2009; 182: 7459–7472. Immunol 2004; 172: 4159–4166. 92 Gary-Gouy H, Harriague J, Bismuth G, Platzer C, Schmitt C, Dalloul 72 Vyse TJ, Halterman RK, Rozzo SJ, Izui S, Kotzin BL. Control of AH. Human CD5 promotes B-cell survival through stimulation of separate pathogenic autoantibody responses marks MHC gene autocrine IL-10 production. Blood 2002; 100: 4537–4543. contributions to murine lupus. Proc Natl Acad Sci USA 1999; 96: 93 Tu W, Lau YL, Zheng J, Liu Y, Chan PL, Mao H et al.Efficient 8098–8103. generation of human alloantigen-specific CD41 regulatory T cells 73 Chan OT, Madaio MP, Shlomchik MJ. The central and multiple roles of from naive precursors by CD40-activated B cells. Blood 2008; B cells in lupus pathogenesis. Immunol Rev 1999; 169: 107–121. 112: 2554–2562. 74 Haas KM, Watanabe R, Matsushita T, Nakashima H, Ishiura N, Okochi 94 Schiemann B, Gommerman JL, Vora K, Cachero TG, Shulga- H et al. Protective and pathogenic roles for B cells during systemic Morskaya S, Dobles M et al. An essential role for BAFF in the autoimmunity in NZB/W F1 mice. JImmunol2010; 184: 4789– normal development of B cells through a BCMA-independent 4800. pathway. Science 2001; 293: 2111–2114. 75 Watanabe R, Ishiura N, Nakashima H, Kuwano Y, Okochi H, Tamaki 95 Schneider P, Takatsuka H, Wilson A, Mackay F, Tardivel A, Lens S K et al. Regulatory B cells (B10 cells) have a suppressive role in et al. Maturation of marginal zone and follicular B cells requires B murine lupus: CD19 and B10 cell deficiency exacerbates systemic cell activating factor of the tumor necrosis factor family and is autoimmunity. J Immunol 2010; 184: 4801–4809. independent of B cell maturation antigen. J Exp Med 2001; 194: 76 Blair PA, Chavez-Rueda KA, Evans JG, Shlomchik MJ, Eddaoudi A, 1691–1697. Isenberg DA et al. Selective targeting of B cells with agonistic anti- 96 Walters S, Webster KE, Sutherland A, Gardam S, Groom J, Liuwantara CD40 is an efficacious strategy for the generation of induced D et al. Increased CD41Foxp31 T cells in BAFF-transgenic mice regulatory T2-like B cells and for the suppression of lupus in MRL/ suppress T cell effector responses. J Immunol 2009; 182: 793–801. lpr mice. J Immunol 2009; 182: 3492–3502. 97 Gray M, Miles K, Salter D, Gray D, Savill J. Apoptotic cells protect 77 Palanichamy A, Barnard J, Zheng B, Owen T, Quach T, Wei C et al. mice from autoimmune inflammation by the induction of regulatory Novel human populations revealed by B cell B cells. Proc Natl Acad Sci USA 2007; 104: 14080–14085. depletion therapy. J Immunol 2009; 182: 5982–5993. 98 Lopes-Carvalho T, Kearney JF. Development and selection of 78 Plebani A, Lougaris V, Soresina A, Meini A, Zunino F, Losi CG et al.A marginal zone B cells. Immunol Rev 2004; 197: 192–205. novel immunodeficiency characterized by the exclusive presence of 99 Evans JG, Chavez-Rueda KA, Eddaoudi A, Meyer-Bahlburg A, Rawlings transitional B cells unresponsive to CpG. Immunology 2007; 121: DJ, Ehrenstein MR et al. Novel suppressive function of transitional 183–188. 2 B cells in experimental arthritis. J Immunol 2007; 178: 7868–7878. 79 Anolik JH, Barnard J, Owen T, Zheng B, Kemshetti S, Looney RJ et al. 100 Mauri C. Regulation of immunity and autoimmunity by B cells. Curr Delayed recovery in peripheral blood and lymphoid Opin Immunol 2010; 22: 761–767. tissue in systemic lupus erythematosus after B cell depletion 101 Carter NA, Vasconcellos R, Rosser EC, Tulone C, Munoz-Suano A, therapy. Arthritis Rheum 2007; 56: 3044–3056. Kamanaka M et al. Mice lacking endogenous IL-10-producing 80 Sanz I, Wei C, Lee FE, Anolik J. Phenotypic and functional regulatory B cells develop exacerbated disease and present with an heterogeneity of human memory B cells. Semin Immunol 2008; 20: increased frequency of Th1/Th17 but a decrease in regulatory T cells. 67–82. J Immunol 2011; 186: 5569–5579. 81 Agematsu K, Hokibara S, Nagumo H, Komiyama A. CD27: a memory 102 Moulin V, Andris F, Thielemans K, Maliszewski C, Urbain J, Moser M. B-cell marker. Immunol Today 2000; 21: 204–206. B lymphocytes regulate dendritic cell (DC) function in vivo: increased

Cellular & Molecular Immunology Regulatory B cells in autoimmune diseases M Yang et al 132

interleukin 12 production by DCs from B cell-deficient mice 109 Sonoda KH, Stein-Streilein J. CD1d on antigen-transporting APC and results in type 1 deviation. J Exp Med 2000; 192: splenic marginal zone B cells promotes NKT cell-dependent 475–482. tolerance. Eur J Immunol 2002; 32: 848–857. 103 Tian J, Zekzer D, Hanssen L, Lu Y, Olcott A, Kaufman DL. 110 Croxford JL, Miyake S, Huang YY, Shimamura M, Yamamura T. Lipopolysaccharide-activated B cells down-regulate Th1 immunity Invariant Valpha19i T cells regulate autoimmune inflammation. Nat and prevent autoimmune diabetes in nonobese diabetic mice. Immunol 2006; 7: 987–994. J Immunol 2001; 167: 1081–1089. 111 Bosma A, Abdel-Gadir A, Isenberg DA, Jury EC, Mauri C. Lipid-antigen 104 Parekh VV, Prasad DV, Banerjee PP, Joshi BN, Kumar A, Mishra GC. presentation by CD1d1 B cells is essential for the maintenance of B cells activated by lipopolysaccharide, but not by anti-Ig and anti- invariant natural killer T cells. Immunity 2012; 36: 477–490. CD40 antibody, induce anergy in CD81 T cells: role of TGF-beta 1. 112 Sharpe AH, Wherry EJ, Ahmed R, Freeman GJ. The function of J Immunol 2003; 170: 5897–5911. programmed cell death 1 and its ligands in regulating autoimmunity 105 Tedder TF, Matsushit, T. Regulatory B cells that produce IL-10: a and infection. Nat Immunol 2007; 8: 239–245. breath of fresh air in allergic airway disease. J Allergy Clin Immunol 113 Alderson MR, Lynch DH. Receptors and ligands that mediate 2010; 125: 1125–1127. activation-induced death of T cells. Springer Semin Immunopathol 106 Amu S, Saunders SP, Kronenberg M, Mangan NE, Atzberger A, 1998; 19: 289–300. Fallon PG. Regulatory B cells prevent and reverse allergic airway 114 Anel A, Bosque A, Naval J, Pineiro A, Larrad L, Alava MA et al. Apo2L/ inflammation via FoxP3-positive T regulatory cells in a murine TRAIL and immune regulation. Front Biosci 2007; 12: 2074–2084. model. J Allergy Clin Immunol 2010; 125: 1114–1124.e8. 115 Ray A, Basu S, Williams CB, Salzman NH, Dittel BN. A novel IL-10- 107 Carter NA, Rosser EC, Mauri C. IL-10 produced by B cells is crucial independent regulatory role for B cells in suppressing autoimmunity for the suppression of Th17/Th1 responses, induction of Tr1 cells by maintenance of regulatory T cells via GITR ligand. J Immunol and reduction of collagen-induced arthritis. Arthritis Res Ther 2012; 2012; 188: 3188–3198. 14: R32. 116 Mann MK, Maresz K, Shriver LP, Tan Y, Dittel BN. B cell regulation 108 Bialecki E, Paget C, Fontaine J, Capron M, Trottein F, Faveeuw C. Role of CD41CD251 T regulatory cells and IL-10 via B7 is essential of marginal zone B lymphocytes in invariant NKT cell activation. for recovery from experimental autoimmune encephalomyelitis. J Immunol 2009; 182: 6105–6113. J Immunol 2007; 178: 3447–3456.

Cellular & Molecular Immunology