Introduction

Traditionally, B cells have been thought to contribute to the pathogenesis of autoimmune diseasethrough antigen (Ag)-specfic autoantibody production [1]. Nonetheless, the role of B cells in autoimmunity extends beyond the production ofautoantibodies. B cells are now well established to have both positive and negative regulatory rolesduring immune responses.

B cells can positively regulate immune responses by producing Ag-specfic antibody and inducingoptimal T cell activation [2, 3]. B cells can serve as professional Ag-presenting cells, capable of presenting Ag103-fold to 104-fold more efficiently than nonprofessional Ag-presenting cells [4]. B cell Ag presentation is required for optimal Ag-specific CD4+ T cellexpansion, memory formation, and cytokine production [5–7]. B cells may also positively regulate CD8+ T cell responses in mouse models ofautoimmune disease [8, 9]. Furthermore, costimulatory molecules (such as CD80, CD86, and OX40L) expressed on thesurface of B cells are required for optimal T cell activation [10, 11]. The positive regulatory roles of B cells extend to multiple immune system components;the absence of B cells during mouse development results in significant quantitative and qualitativeabnormalities within the immune system, including a remarkable decrease in thymocyte numbers anddiversity [12], significant defects within spleen dendritic cell and T cell compartments [13–15], absence of Peyer's patch organogenesis and follicular dendritic cell networks [16, 17], and absence of marginal zone and metallophilic macrophages with decreased chemokineexpression [15, 17]. B cells also positively regulate lymphoid tissue organization [18, 19]. Finally, dendritic cell, macrophage, and TH cell development may all beinfluenced by B cells during the formation of immune responses [20].

B cells can also negatively regulate cellular immune responses through their production ofimmunomodulatory cytokines. B cell-negative regulation of immune responses has been demonstrated ina variety of mouse models of autoimmunity and inflammation [21–30]. Although the identification of B cell subsets with negative regulatory functions and thedefinition of their mechanisms of action are recent events, the important negative regulatory rolesof B cells in immune responses are now broadly recognized [31, 32]. A variety of regulatory B cell subsets have been described; IL-10-producing regulatory Bcells (B10 cells) are the most widely studied regulatory B cell subset [30, 31, 33]. Comprehensive reviews summarizing the variety of regulatory B cell subsets have beenpublished during recent years [31, 32]. The present review will therefore focus exclusively on the IL-10 producing regulatory Bcell subset. This specific subset of regulatory B cells has been labeled B10 cells to highlight thatthe regulatory function of these rare B cells is mediated by IL-10, and to distinguish them fromother B cell subsets that regulate immune responses through different mechanisms [34]. This functional subset of B cells is defined solely by its IL-10-dependent regulatoryproperties and extends beyond the concept of transcription factor-defined cell lineages. This reviewhighlights our current knowledge on B10 cells, with emphasis on their roles in autoimmune disease,and discusses their potential as a novel therapeutic approach in the treatment of autoimmunity.

Biology of B10 cells

One of the most fundamental basic biology questions about B10 cells relates to the stimulidriving their development. Ag and B cell receptor (BCR) signaling are critical in early development,although additional stimuli such as CD40 ligation and Toll-like receptor (TLR) ligands appear to beinvolved in the developmental process. Figure 1 illustrates our currentunderstanding of B10 cell development in vivo both in mice and humans, where theirdevelopment shows multiple similarities.

Figure 1
figure 1

Linear differentiation model of B10 cell development in vivo in mice and humans.B10 cells originate from a progenitor population (B10PRO). In mice, B10PROcells are found in the CD1d−CD5− adult blood and lymph nodeB cell subsets and within the CD1d−CD5+ neonatal spleen and adultperitoneal cavity B cell subsets. CD40 stimulation induces B10PRO cells to becomecompetent for IL-10 expression, while lipopolysaccharide (LPS) induces B10PRO cells tobecome competent for IL-10 expression and induces B10 cells to produce and secrete IL-10.CD1dhiCD5+ IL-10-competent B10 cells in the adult spleen are induced toexpress IL-10 following stimulation with phorbol esters (phorbol-12-myristate-13-acetate (PMA)) andionomycin or LPS plus PMA and ionomycin for 5 hours. Following a transient period of IL-10expression, a small subset of B10 cells can differentiate into antibody-secreting plasma cells (PC).B10 cells also possibly differentiate into memory B10 cells (B10M). B10 cell developmentin humans appears to follow the differentiation scheme observed in mice. B10 cells and B10PROcells have been identified in human newborn and adult blood. B10+B10PRO cells inadult human blood express CD27 and CD24. Whether human B10 cells further differentiate into PCs orB10M remains to be determined. Solid arrows, known associations; dashed arrows,speculated associations. MHC-II, major histocompatibility complex class II.

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B10 cells are a functionally defined B cell subset. There are no unique phenotypic markers forB10 cells, and these cells are currently defined only by their competency to produce and secreteIL-10 following appropriate stimulation. B10 cells share surface markers with other previouslydefined B cell subsets both in mice and humans, such as marginal zone B cells, transitional B cells,B1a B cells, and memory B cells. However, no one marker or set of markers is unique to B10 cells.For identification of B10 cells, intracellular cytoplasmic IL-10 staining is used, following exvivo stimulation with lipopolysaccharide (LPS) or CpG oligonucleotides, phorbol esters(phorbol-12-myristate-13-acetate (PMA)) and ionomycin for 5 hours [35]. B10 cells originate from a progenitor population (B10PRO cells). B10PROcells develop into B10 cells after maturation through CD40 ligation or exposure to LPS or CpG.B10PRO cells can be identified indirectly following ex vivo stimulation with LPSor CpG in the presence of CD40 ligation for 48 hours with the addition of PMA and ionomycin for thelast 5 hours. The IL-10+ B cells measured following this 48-hour stimulation includecells that would have been IL-10+ even with the shorter 5-hour stimulation (B10 cells),and thereby represent the sum of B10 plus B10PRO cells (B10+ B10PRO).

Mouse B10 cell development

BCR specificity, affinity and signaling are the most important currently identified factors inB10 cell development. B10 cell regulation of inflammation and autoimmunity is Ag specific [23, 30, 36]. The importance of BCR diversity is demonstrated by the fact that B10+ B10PROcells are reduced by approximately 90% in transgenic mice with a fixed BCR [37]. Signaling through the BCR appears critical during early development in vivo.CD19-deficient mice (where BCR signaling is decreased) have a 70 to 80% decrease in B10+B10PROcells [30]. In contrast, B10 cells are expanded in human CD19 transgenic mice (where theoverexpression of CD19 augments BCR signaling). The absence of CD22, which normally dampens CD19 andBCR signaling [38], also results in increased B10 cell numbers. Ectopic B cell expression of CD40L (CD154)in transgenic mice, which induces increased CD40 signaling [39], also increases B10 cell numbers. CD22−/−mice that alsoectopically express CD40L show dramatically enhanced numbers of CD1dhiCD5+ Bcells and B10 cells [40]. The induction of IL-10+ B cells with regulatory activity by T cellimmunoglobulin domain and mucin domain protein 1 (TIM-1) ligation [41] further highlights the importance of BCR signaling in B10 cell development. BCR signalingand TIM-1 are closely related. BCR ligation induces TIM-1 expression on B cells [41, 42], and TIM-1 ligation appears to enhance BCR signaling since it increases antibodyproduction both in vitro and in vivo [43]. The importance of BCR-related signals is further highlighted by the observation that thestromal interaction molecules 1 (STIM1) and 2 (STIM2) are required for B cell IL-10 production [44]. Remarkably, B cells lacking both stromal interaction molecule proteins failed to produceIL-10 after BCR stimulation in the presence of PMA and ionomycin for 5 hours [44]. All of the above indicate that BCR-related signals are particularly important in B10cell development.

Despite the requirement for BCR expression and function during mouse B10 cell development, B cellstimulation with mitogenic anti-IgM antibody alone does not induce cytoplasmic IL-10 expression. Thecombination of anti-IgM stimulation with CD40 ligation and LPS or CpG significantly reduces IL-10competence [37]. BCR-generated signals thus inhibit the abilities of LPS or CpG and CD40 ligation toinduce cytoplasmic IL-10 production. Whether BCR stimulation inhibits the induction of IL-10competence by inducing B cells to mature or differentiate down a divergent pathway or divertsintracellular signaling is unknown. Another possibility is that the signals generated by mitogenicanti-IgM BCR cross-linking are too intense and that low-affinity Ag--BCR interactions driveB10PRO cell development in vivo.

A recent study revealed the importance of IL-21, major histocompatibility complex class II(MHC-II) and CD40 during cognate interactions with CD4+ T cells in B10 cell development [36]. Ex vivo stimulation of purified spleen CD19+ B cells with IL-21induced 2.7-fold to 3.2-fold higher B10 cell frequencies, and 4.4-fold to 5.3-fold more IL-10secretion compared with stimulation with media alone. Remarkably, IL-21 induced B10 cells to produceIL-10 without the need for stimulation with phorbol esters and ionomycin. Interestingly, IL-21induced a threefold increase in IL-10+ B cells within the splenicCD1dhiCD5+ B cell subset, but did not induce IL-10+ B cells withinthe CD1dloCD5-- B cell subset. Both B10 cells and non-B10 cells expressedIL-21R at similar levels, and ex vivo B10, B10pro and CD1dhiCD5+ Bcell numbers were similar among IL-21R-deficient (IL-21R--/--), MHC-II-deficient(MHC-II--/--) and CD40-deficient (CD40--/--) mice. Nevertheless, IL-21R,MHC-II and CD40 appear to be required for B10 cell effector functions, at least in experimentalautoimmune encephalomyelitis (EAE) [36]. Regulatory B10 cell function therefore requires IL-21R signaling, as well as CD40 andMHC-II interactions, potentially explaining Ag-specific B10 cell effector function [37].

Although cognate interactions with CD4+ T cells are important for B10 cell effectorfunctions [36], T cells do not appear to be required for B10 cell development. B10 cells are present inT cell-deficient nude mice, and their frequencies and numbers are approximately fivefold higher whencompared with wildtype mice. This observation is strengthened by the fact that MHC class I andMHC-II molecules and CD1d expression are not required for B10 cell development [37]. The presence or absence of T cells in vitro also does not affect the frequencyof B10 cells. Although increased B10 cell frequencies in T cell-deficient mice suggest that T cellsmight actually inhibit B10 cell development, it is equally possible that the immunodeficient stateof these mice allows subclinical inflammation that induces B10 cell generation. The role of T cellsin B10 cell development in vivo is thereby complex and, although T cells are not requiredfor B10 cell development, cognate interactions between CD4+ T cells and B10 cells arerequired for B10 cell effector function.

B10 cells can be driven to produce IL-10 by TLR4 (LPS) or TLR9 (CpG oligonucleotides) ligands.Mouse B10PRO cells acquire the ability to function like B10 cells after in vitro maturation following stimulation with LPS, but not CpG, in the presence or absence of agonisticCD40 mAb [32]. TLR4 and TLR9 signaling through myeloid differentiation primary response gene 88 (MyD88)is necessary for the optimal maturation and IL-10 induction of B10pro and B10 cells following LPSstimulation and LPS or CpG stimulation, respectively [37]. Nevertheless, MyD88 expression is not an absolute requirement for B10 cell developmentin vivo, since B10 cells develop normally in MyD88--/-- mice [37]. Specifically, numbers of B cells with the capacity to produce IL-10 are equivalent inwildtype and MyD88--/-- mice when their maturation or IL-10 production are measuredfollowing CD40 ligation or PMA plus ionomycin stimulation, respectively, demonstrating that B10PRO and B10 cells are present at normal frequencies in MyD88--/-- mice.Thereby, while TLR signaling is not required for B10 cell development, MyD88 expression is requiredfor LPS to induce optimal B cell IL-10 expression and secretion in vitro.

The involvement of TLR signals in B10-cell IL-10 production was recently demonstrated [45]. IL-10 production by B cells, stimulated by contact with apoptotic cells, results fromthe engagement of TLR9 within the B cell after recognition of DNA-containing complexes on thesurface of apoptotic cells by the BCR. An earlier study also highlights the effects of apoptoticcells on B cell IL-10 production, where apoptotic cells protected mice from developingcollagen-induced arthritis (CIA) by the induction of IL-10-producing regulatory B cells [46]. Cell death products may therefore represent one of the physiologic triggers for B10 celldevelopment by providing a combination of BCR and TLR signals. Additional non-TLR/non-BCR signals(such as alarmins) released from dying cells may be also involved but their identities remain to bedetermined.

Although certain transcription factors are involved at some point in B10 cell development, it isimportant to stress that there is no known transcription factor signature unique to B10 cells.Following a transient period of IL-10 transcription characterized by increased expression of theblimp1 and irf4 transcription factors along with decreased expression of pax5 and bcl6, a significant but small fraction of B10 cells can differentiate intoantibody-secreting cells producing IgM and IgG polyreactive antibodies that are enriched forautoreactivity to single-stranded or double-stranded DNA and histones [47]. Whether B10 cells can produce and secrete IL-10 repeatedly remains to be determined.

Human B10 cell development

B10PRO cells and B10 cells have been recently identified in humans [48] and their responses to LPS, CpG and CD40 ligation appear to follow the general scheme ofmouse B10 cell development (Figure 1). One notable difference in mouse versushuman B10 cell development is the lack of response of mouse B10PRO cells to CpG comparedwith their human counterparts. Human B10PRO cells can be driven to develop ex vivo into B10 cells with LPS or CpG stimulation, or CD40 ligation. Interestingly, BCR ligationaugmented human B cell IL-10 responses to CpG in one study [49]. This finding is in discordance with our findings in both humans [48] and mice [37], where BCR-generated signals inhibit the abilities of LPS or CpG and CD40 ligation toinduce cytoplasmic IL-10 production. Whether human B10 cells develop into antibody-secreting cellsor enter the memory pool (memory B10 cells, B10M) remains to be determined.

Unsolved questions on B10 cell development

The most critical unsolved issue relates to the nature of antigenic stimuli driving B10 celldevelopment. The identification of B10-cell BCR specificity is imperative since it will provide newinsights into their early development. The autoreactive nature of mouse B10-cell BCRs [47] suggests that autoantigens may be driving early B10 cell development and that B10 cellsmay represent one of the ways enabling the immune system to peripherally tolerate autoantigens. Bcells responding to autoantigens in an IL-10-dependent regulatory way can potentially limitinflammatory responses and limit autoimmune phenomena (see later section on B10 cell regulatoryeffects and Figure 2). Cell death products, by providing simultaneously bothantigenic and nonantigenic stimuli, may represent one of the physiologic triggers for B10 celldevelopment. The clearance of antigenic products of dying cells by noncomplement-fixing IgMpolyreactive/autoreactive antibodies (such as those made by mouse B10 cells) in an IL-10-richenvironment would be beneficial since it could potentially limit inflammatory responses to self-Ags.Additional unidentified antigenic and nonantigenic stimuli are probably involved in B10 celldevelopment. The identification of such stimuli will provide additional insights ito B celldevelopment that may prove invaluable for the future manipulation of B10 cells for treatingautoimmune disease. Another important question is whether B10 cells enter the B cell memory poolduring their development. This question is suggested by human studies demonstrating that B10PROcells and B10 cells share phenotypic features with memory B cells (see later section on HumanB10 cell phenotype).

Figure 2
figure 2

B10 cell regulatory effects in autoimmune disease. In this model, unidentifiedautoantigens (auto-Ags) drive early development of B10PRO cells. Following exposure toCD40 ligation and/or Toll-like receptor (TLR) ligands (lipopolysaccharide (LPS), CpG), B10PROcells mature into B10 cells that can actively secrete IL-10 and regulate both innate andadaptive immune responses. IL-21R signaling along with major histocompatibility complex class II(MHC-II) and CD40 cognate interactions with CD4+ T cells, although not needed for B10cell development, are necessary for B10 cell effector functions and result in antigen-specificresponses. B10 cells regulate macrophage function by decreasing their activation, phagocytosis andcytokine and nitric oxide (NO) production. In antigen-presenting cells (APCs), B10-cell-negativeregulation of antigen presentation, expression of co-stimulatory molecules (such as CD86) andproinflammatory cytokine production limits T cell activation. In CD4+ T helper(TH) cells, B10 cells skew responses towards a TH2 phenotype and away fromTH1 and TH17 responses. The negative regulatory effects of B10 cells therebylimit inflammatory responses and subsequent tissue damage. Arrows with solid outline, knownassociations; arrows with dashed outline, speculated associations.

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Mouse B10 cell phenotype

Although a variety of cell surface markers have been proposed [31, 32], there is no known surface phenotype unique to B10 cells and, currently, the only way toidentify these cells is functionally by intracellular IL-10 staining [35]. Only a small portion of B cells (that is, ~1 to 3% of splenic B cells in wildtypeC57BL/6 mice) produce IL-10 following PMA and ionomycin stimulation, implying that not all B cellsare competent to produce IL-10. Intracellular cytokine staining combined with flow cytometricphenotyping shows that mouse spleen B10 cells are enriched within the smallCD1dhiCD5+ B cell subset, where they represent 15 to 20% of the cells inC57BL/6 mice. This phenotypically unique CD1dhiCD5+ subset shares overlappingcell surface markers with a variety of phenotypically defined B cell subsets such as CD5+B-1a B cells, CD1dhiCD23−IgMhiCD1dhimarginal zone B cells, and CD1dhiCD23+IgMhiCD1dhiT2 marginal zone precursor B cells, which all undoubtedly contain both B10PRO cellsand B10 cells [23, 26, 30, 50]. Mouse B10 cells are predominantly IgDlowIgMhi, and <10%co-express IgG or IgA, but they can differentiate into antibody-secreting cells secretingpolyreactive or Ag-specific IgM and IgG [47]. IL-10+ B cells were recently shown to be enriched in the TIM-1+ compartmentand TIM-1+ B cells are enriched in the CD1dhiCD5+ compartment [41]. However, IL-10+ B cells are also present in the TIM-1--compartment and TIM-1+ B cells are present in the non-CD1dhiCD5+compartment. Intracellular cytoplasmic IL-10 staining thereby remains the only current way tovisualize the entire subset of IL-10-competent B cells. Nonetheless, the isolation ofCD1dhiCD5+ B cells or other phenotypically defined B cell subsets where B10cells are enriched currently provides the best current means for isolating a viable B cellpopulation that is significantly enriched for B10 cells and can be used for adoptive transferexperiments and functional studies in mice.

Human B10 cell phenotype

The IL-10-producing B cell subset characterized in humans normally represents <1% ofperipheral blood B cells [48]. Peripheral blood B10 cells and B10PRO cells are highly enriched in theCD24hiCD27+ B cell subset, with approximately 60% also expressing CD38.Similar total numbers of IL-10+ B cells have been described in theCD24hiCD38hi and CD24intCD38int B cell subsets [51]. A separate study showed that B10 cells did not fall within any of the previously definedB cell subsets, but they were enriched in the CD27+ and the CD38hicompartments [49]. Human B10 cells also highly express CD48 and CD148 [48]. CD48 is a B cell activation marker [52] and CD148 is considered a marker for human memory B cells [53]. CD27 expression is another well-characterized marker for memory B cells, although somememory B cells may be CD27-- [54–56]. The CD27+ B cell subset can also expand during the course of autoimmunity andhas been proposed as a marker for disease activity [54, 56]. The CD24hiCD148+ phenotype of B10 cells and B10PROcells may thereby indicate their selection into the memory B cell pool during development, orthey may represent a distinct B cell subset that shares common cell surface markers with memory Bcells. Consistent with a memory phenotype, the proliferative capacity of human blood B10 cells inresponse to mitogen stimulation is higher than that for other B cells [48], as is seen for mouse B10 cells [37]. Human transitional B cells are rare (2 to 3% of B cells) in adult human blood and aregenerally CD10+CD24hiCD38hi cells that are also CD27-negative [55, 56]; since CD10 expression is a well-accepted marker for most cells within the transitional Bcell pool [57], its absence on B10 cells suggests that these cells are not recent emigrants from thebone marrow. In summary, human B10 cells share phenotypic characteristics with other previouslydefined B cell subsets, and, currently, there is no known surface phenotype unique to B10 cells.

B10 cell regulatory effects

B10 cells exert a variety of IL-10-dependent regulatory effects potentially involved inautoimmune disease. The anti-inflammatory effects of IL-10 are mediated by multiple mechanismsinvolving both the innate and adaptive arms of the immune system. In innate cells, these mechanismsinclude downregulation of proinflammatory cytokine production [58] and decreased expression of MHC-II and co-stimulatory molecules [59] resulting in decreased T cell activation. B10 cells negatively regulate the ability ofdendritic cells to present Ag [60]. In CD4+ T cells, IL-10 suppresses TH1 [50] and enhances TH2 polarization [41, 59]. B10 cells suppress IFNγ and TNFα responses in vitro [60] and INFγ responses in vivo [36] by Ag-specific CD4+ T cells. Co-culture of mouse CD1dhiCD5+B cells with CFSE-labeled naive CD4+ T cells suppresses TH17 celldifferentiation [61] and IL-10 is known to suppress TH17 responses [62]. The suppression of TH17 responses by B10 cells in vivo wasdemonstrated recently [36]. IL-10 production by human B10 cells inhibits Ag-specific CD4+CD25--T cell proliferation [49] and regulates monocyte activation and cytokine production [48]in vitro.

A number of studies suggest that IL-10-producing B cells are important for the generation and/ormaintenance of the regulatory T cell (TREG) pool [46, 63–72]. However, a recent study [73] and our previously published data [23] do not support this view. The reason for this discrepancy is unclear but may be relatedto the different models of inflammation and conditions used to study the relationship of B10 cellsand TREGS. These two studies suggesting that B10 cells are not involved in the generationand maintenance of the TREG pool are both in models of EAE [23, 73]. In contrast, only one study suggests that B10 cells are important for the generationand/or maintenance of the TREG pool specifically in EAE [63]. The results of a different study clarify the picture in EAE further by showing that asubset of regulatory B cells control TREG numbers through IL-10-independent mechanisms [34]. Human B10 cell IL-10 production will therefore probably also have pleiotropic regulatoryeffects on the immune system, as occurs in mice. The potential regulatory effects of B10 cells inautoimmune disease limiting inflammatory responses and subsequent tissue damage are summarized inFigure 2.

B10 cells in human autoimmune disease

Studies of B10 cells and human autoimmune disease are limited but of outmost importance sincethey provide valuable insights relevant to the potential future therapeutic application of B10 cellsin humans. Peripheral blood B10 cells and B10PRO cells are present in patients withautoimmune diseases, including rheumatoid arthritis, systemic lupus erythematosus, primarySjögren's syndrome, autoimmune bullous diseases, and multiple sclerosis. Interestingly,B10+B10PRO cell frequencies are expanded in some but not all cases, while mean B10+B10PRO cell frequencies are significantly higher in patients with autoimmune diseasecompared with age-matched healthy controls [48]. A different study examined cytoplasmic IL-10 production by B cells from systemic lupuserythematosus patients and normal controls [74]. Blood mononuclear cells were cultured for 24 hours in the presence or absence of PMA,ionomycin, or LPS; significantly more systemic lupus erythematosus CD5+ B cells producedcytoplasmic IL-10 than did controls. A different study also demonstrated spontaneous B cell IL-10production that is higher in untreated rheumatoid arthritis, systemic sclerosis, and systemic lupuserythematosus patients than in controls [75].

By contrast, the concept of functional impairment of B10 cells in autoimmune disease was recentlyintroduced by demonstrating functional impairment of CD24hiCD38hi regulatory Bcells in human systemic lupus erythematosus [51]. Cultures of peripheral blood mononuclear cells were stimulated with plate-bound anti-CD3mAb for 72 hours, followed by the measurement of IFNγ and TNFα CD4+ T cellresponses. When CD24hiCD38hi B cells were removed from the culture, higherfrequencies of CD4+IFNγ+ and CD4+TNFα+ T cellswere noted in healthy individuals but not in systemic lupus erythematosus patients; this effect waspartially IL-10 dependent. In addition, CD24hiCD38hi B cells isolated from theperipheral blood of systemic lupus erythematosus patients were refractory to CD40 ligation andproduced less IL-10 compared with their healthy counterparts. The results of this study are ratherintriguing but these findings need to be validated in view of the complexity of the culture systemused and the non-uniformity of the CD24hiCD38hi B cell subset with regards toits IL-10-dependent regulatory properties. In conclusion, B10 cells are present in the peripheralblood of autoimmune disease patients, where they appear to be expanded, whereas the functionalcapacity of human B10 cells in autoimmunity needs to be further defined.

B10 cells in mouse models of autoimmune disease

The important regulatory effects of B10 cells in vivo and their therapeutic potential inautoimmunity have been demonstrated in a variety of mouse models of human autoimmune disease.

Experimental autoimmune encephalomyelitis

EAE is an established model of multiple sclerosis induced by immunization with myelin peptides(such as myelin oligodendrocyte glycoprotein) leading to demyelination mediated by auto-Ag-specificCD4+ T cells [76, 77]. B cells were shown over a decade ago to have regulatory properties during the inductionof EAE, with genetically B cell-deficient mice developing a severe nonremitting form of the disease [21]. However, these B cell regulatory effects were recently shown not to be IL-10 dependent [34]. Nonetheless, other studies highlight the importance of B cell-derived IL-10 in EAE.Specifically, EAE severity during the late phase of disease increases in B cell-deficient μMTmice that do not fully recover from their disease when compared with wildtype mice, and the adoptivetransfer of wildtype B cells but not IL-10--/-- B cells normalizes EAE severity inμMT mice [22]. Disease recovery is dependent on the presence of autoantigen-reactive B cells, and Bcells isolated from mice with disease produced IL-10 in response to autoantigen stimulation. In theabsence of Ag-specific B cell IL-10 production, the proinflammatory TH1-mediated immuneresponses persist and mice do not recover from the disease.

The EAE model demonstrates the complexity of regulatory mechanisms mediated by different cellsubsets during different stages of the disease. When B cells from wildtype mice are depleted by CD20mAb treatment 7 days before EAE induction, there is an increased influx or expansion ofencephalitogenic T cells within the central nervous system and exacerbation of disease symptoms [23]. This effect is related to B10 cell depletion since similar effects are observed withselective B10 depletion by means of CD22 mAb [60]. The adoptive transfer of Ag-specific (myelin oligodendrocyte glycoprotein-sensitized)B10 cells into wildtype mice also reduces EAE initiation dramatically. The protective effect isIL-10 dependent since the adoptive transfer of CD1dhiCD5+ B cells purifiedfrom IL-10−/−mice does not affect EAE severity. B10 cell effectorfunctions in EAE require IL-21 along with cognate interactions with CD4+ T cells sincethe adoptive transfer of CD1dhiCD5+ B cells into CD19--/-- micefrom IL-21R--/--, MHC-II--/-- or CD40--/-- mice prior to theinduction of EAE does not alter disease course [36]. Once disease is established, adoptive transfer of B10 cells does not suppress ongoingEAE. B10 cells thereby appear to normally regulate acute autoimmune responses in EAE. In contrast tothe role of B10 cells in early disease, TREG depletion enhances late-phase disease.Therefore, in EAE, depending on the stage of the disease, different regulatory mechanisms areinvolved in limiting inflammatory responses, with B10 cells regulating disease initiation andTREGS being involved predominantly in the regulation of late-phase disease.

Inflammatory bowel disease

IL-10-producing B cells regulate intestinal inflammation in inflammatory bowel disease [26]. Early studies showed that B cells and their autoantibody products suppress colitis in Tcell receptor alpha chain-deficient mice that spontaneously develop chronic colitis, while B cellsare not required for disease initiation [78]. B cells with upregulated CD1d expression in the gut-associated lymphoid tissues of micewith intestinal inflammation were subsequently demonstrated to be regulatory [25]. This IL-10-producing B cell subset appears during chronic inflammation in T cellreceptor alpha chain-deficient mice and suppresses the progression of intestinal inflammation bydownregulating inflammatory cascades associated with IL-1 upregulation and signal transducer andactivator of transcription 3 (stat3) activation rather than by altering polarized THcell responses. The adoptive transfer of these mesenteric lymph node B cells also suppressesinflammatory bowel disease through a mechanism that correlates with an increase in TREGsubsets [67]. Oral administration of dextran sulfate sodium solution to mice is widely used as a modelof human ulcerative colitis. Dextran sulfate sodium-induced intestinal injury is more severe inCD19--/-- mice (where B10 cells are absent) than in wildtype mice [79], and these inflammatory responses are negatively regulated by CD1dhiCD5+B cells producing IL-10. B10 cells therefore emerge during chronic inflammation in mousemodels of inflammatory bowel disease, where they suppress the progression of inflammatory responsesand ameliorate disease manifestations.

Collagen-induced arthritis

CIA is a model for human rheumatoid arthritis that develops in susceptible mouse strainsimmunized with heterologous type II collagen emulsified in complete Freund's adjuvant [80, 81]. CIA and rheumatoid arthritis share in common an association with a limited number ofMHC-II haplotypes that determine disease susceptibility [82, 83]. B cells are important for initiating inflammation and arthritis since mature B celldepletion significantly reduces disease severity prior to CIA induction but does not inhibitestablished disease [84]. Several studies on CIA demonstrate the negative regulatory effects and therapeuticpotential of B10 cells.

Activation of arthritogenic splenocytes with Ag and agonistic anti-CD40 mAb induces a B cellpopulation that produces high levels of IL-10 and low levels of IFNγ [85]. The adoptive transfer of these B cells into DBA/1--T cell receptor--β-Tg mice,immunized with bovine collagen (type II collagen) emulsified in complete Freund's adjuvant, inhibitsTH1 responses, prevents arthritis development, and is effective in amelioratingestablished disease. The adoptive transfer of CD21hiCD23+IgM+ Bcells from DBA/1 mice in the remission phase prevents CIA and reduces disease severity through IL-10secretion [86]; a significant but less dramatic therapeutic effect on CIA progression is seen when cellsfrom naïve mice are adoptively transferred. In addition, the adoptive transfer of ex vivo expanded CD1dhiCD5+ B cells in collagen-immunized mice delays arthritisonset and reduces disease severity, accompanied by a substantial reduction in the number ofTH17 cells [61]. Co-culture of CD1dhiCD5+ B cells with naive CD4+ Tcells suppresses TH17 cell differentiation in vitro, and co-culture ofCD1dhiCD5+ B cells with TH17 cells results in decreasedproliferation responses in vitro. Furthermore, the adoptive transfer of TH17cells triggers CIA in IL-17--/-- DBA mice; however, when TH17 cells areco-transferred with CD1dhiCD5+ B cells, the onset of CIA is significantlydelayed. Finally, in a different study, administration of apoptotic thymocytes along with ovalbuminpeptide and complete Freund's adjuvant to mice carrying an ovalbumin-specific rearranged T cellreceptor transgene (DO11.10 mice) up to 1 month before the onset of CIA resulted in an increase inovalbumin-specific IL-10 secretion and is protective for severe joint inflammation and bonedestruction [46]. Activated spleen B cells responded directly to apoptotic cell treatment in vitro by increasing secretion of IL-10, and inhibition of IL-10 in vivo reversed thebeneficial effects of apoptotic cell treatment [46].

Systemic lupus erythematosus

B cell-negative regulatory effects are important in NZB/W mice, a spontaneous lupus model, sincemature B cell depletion initiated in 4-week-old NZB/W F1 mice hastens disease onset, which parallelsdepletion of B10 cells [87]. B10 cells are phenotypically similar in NZB/W F1 and C57BL/6 mice, but are expandedsignificantly in young NZB/W F1 mice [87]. In wildtype NZB/W mice, the CD1dhiCD5+B220+ B cellsubset, which is enriched in B10 cells, is increased 2.5-fold during the disease course, whereasCD19--/-- NZB/W mice lack this CD1dhiCD5+ regulatory B cell subset [88]. Finally, the potential therapeutic effect of B10 cells in lupus is highlighted by theprolonged survival of CD19--/-- NZB/W recipients following the adoptive transfer ofsplenic CD1dhiCD5+ B cells from wildtype NZB/W mice [88]. Studies in the NZB/W spontaneous lupus model therefore suggest that B10 cells haveprotective and potentially therapeutic effects.

In the MRL.Fas(lpr) mouse lupus model, B cell-derived IL-10 does not regulate spontaneousautoimmunity [89]. B cell-specific deletion of IL-10 in MRL.Fas(lpr) mice indicates that B cell-derivedIL-10 is ineffective in suppressing the spontaneous activation of self-reactive B cells and T cellsduring lupus. The severity of organ disease and survival rates in mice harboring IL-10-deficient Bcells were unaltered. MRL.Fas(lpr) IL-10 reporter mice illustrate that B cells comprise only a smallfraction of the pool of IL-10-competent cells. In contrast to previously published studies from ourlaboratory and elsewhere, putative regulatory B cell phenotypic subsets, such asCD1dhiCD5+ and CD21hiCD23hi B cells, were not enrichedin IL-10 transcription. This observation suggests fundamental differences in the pathogenesis andimmune dysregulation in the NZB/W lupus model compared with the MRL.Fas(lpr) model.

Type 1 diabetes

Studies on B10 cells and mouse models of diabetes are limited to the nonobese diabetic (NOD)mouse, a spontaneous model of type 1 diabetes in which autoimmune destruction of theinsulin-producing pancreatic β cells is primarily T cell mediated [90]. Although B cells clearly have a pathogenic role in disease initiation [91], B cells activated in vitro can maintain tolerance and transfer protection fromtype 1 diabetes in NOD mice [92, 93]. The adoptive transfer of BCR-stimulated B cells into NOD mice starting at 5 to 6 weeksof age both delays the onset and reduces the incidence of type 1 diabetes, while treatment at 9weeks of age delays disease onset. Protection from type 1 diabetes requires B cell IL-10 productionsince the adoptive transfer-activated NOD-IL-10--/-- B cells do not confer protectionfrom type 1 diabetes or the severe insulitis in NOD recipients. The therapeutic effect of adoptivelytransferred activated NOD B cells correlates with TH2 polarization. The limited dataabove suggest that B10 cells may be protective in preventing establishment of type 1 diabetes in NODmice.

Therapeutic potential of B10 cells

Harvesting the anti-inflammatory properties of B10 cells can provide a new approach to thetreatment of autoimmunity. Manipulation of this subset for treating autoimmune disease is possibleby either selective depletion of mature B cells while sparing B10/B10PRO cells or theselective expansion of B10 cells. Since there are no identified surface molecules specific fornon-B10/B10PRO cells, it is currently impossible to selectively target and deplete matureB cells while sparing B10/B10PRO cells. B10 cell expansion appears to be a more viableapproach since some of the stimuli driving their development have been identified. B10 cells can beexpanded for therapeutic purposes either in vivo or ex vivo. Expansion of B10cells in vivo by means of agonistic CD40 antibody has shown benefit in CIA [85]. However, expanding B10 cells in vivo carries additional risks since thecurrently identified stimuli driving B10 cell development are rather nonspecific and, ifadministered systemically, will trigger responses in a variety of immune cells. For example, thesystemic administration of agonistic CD40 antibodies in humans has been associated with seriousadverse effects such as cytokine release syndrome [94]. In summary, selective depletion of mature B cells while sparing B10/B10PROcells is not currently possible, and in vivo B10 cell expansion by nonspecific agentssuch as agonistic CD40 antibody is potentially associated with serious off-target effects.

Expanding B10 cells ex vivo appears more preferable than in vivo B10 cellexpansion by nonspecific agents because it offers a potential therapy without the risk ofundesirable nonspecific off-target effects. However, ex vivo B10 cell expansion introducesnew challenges related to the method of expansion, to the magnitude of expansion and to the time ittakes to generate B10 numbers that will be sufficient for therapeutic use. The method of ex vivo B10 cell expansion can be the source of safety concerns when it comes to human applications.Large numbers of regulatory B cells have been successfully generated in mice by means of geneticmanipulation of immature B cells through lentiviral transfection [95]. These cells were effective in treating EAE. However, although this method canefficiently generate large numbers of regulatory B cells ex vivo, concerns remain aboutadministering infusions of lentivirus-infected B cells to humans (with retroviral and infectiouspotential). Safety concerns thereby limit the use of infectious agents in manipulating human cells,which could render this approach inappropriate for use in humans.

The magnitude of ex vivo B10 cell expansion is very important since the number of cellsinfused during adoptive transfer experiments is critical. In humans, the most convenient potentialsource of B10/B10PRO cells prior to ex vivo expansion is obviously peripheralblood. Since B10/B10PRO cells are rare in peripheral blood and there are limitations onthe volume that can be drawn at any given time, a method of expanding B10 cells by severalmillion-fold is needed. Furthermore, since this method will be used for treatment of active disease,the time it will take to expand these cells ex vivo is also of great significance; ideally,this process should not take more than 1 or 2 weeks. There is accumulating hope that such anapproach will soon be available for human cells since mouse B10 cell ex vivo expansion canbe accomplished within 9 days by means of combined CD154, B-lymphocyte stimulator, IL-4 and IL-21stimulation [36]. After the 9-day culture period, B10 cell numbers are increased 4,000,000-fold, with 38%of the B cells actively producing IL-10. Fluorescence-activated cell sorting based on CD5 expressionincreases the B10 cell purity to75%, thus providing not only large numbers of B10 cells but also a Bcell population predominantly consisting of B10 cells. These ex vivo expanded B10 cells arevery effective in limiting inflammatory responses in EAE. This approach appears promising since itprovides an effective way of generating large numbers of B10 cells without the use of infectiousagents. The development of a similar system for expanding human B10 cells is of outmostimportance.

Conclusion

The phenotypic and functional characterization of B10 cells is an important advance for theregulatory B cell field. Numerous additional functionally defined subsets of regulatory B cells willprobably be identified in the future. B10 cells share phenotypic markers with a variety ofpreviously defined subsets, but their only unique phenotypic marker is intracellular IL-10production. Although certain transcription factors are involved at different points in B10 celldevelopment, there is currently no transcription factor signature unique to B10 cells. BCR-relatedsignals are most critical in B10 cell development and the finding of B10-cell BCR auto-reactivitysuggests that autoantigens may be of particular importance. The recent discovery of an in vitro method to efficiently expand mouse B10 cells provides an invaluable tool for studying the basicbiology of B10 cells as well as manipulating them for therapeutic purposes. The development of asimilar method for human cells will open new opportunities for studying the basic biology of humanB10 cells and a promising novel approach in treating human autoimmune disease, potentially withoutundesirable off-target effects.

Declarations

This article has been published as part of Arthritis Research & Therapy Volume 15Supplement 1, 2013: B cells in autoimmune diseases: Part 2. The supplement was proposed by thejournal and content was developed in consultation with the Editors-in-Chief. Articles have beenindependently prepared by the authors and have undergone the journal's standard peer review process.Publication of the supplement was supported by Medimmune.