IL-10-producing regulatory B cells (B10 cells) in autoimmune disease

B cell abnormalities contribute to the development and progress of autoimmune disease. Traditionally, the role of B cells in autoimmune disease was thought to be predominantly limited to the production of autoantibodies. Nevertheless, in addition to autoantibody production, B cells have other functions potentially relevant to autoimmunity. Such functions include antigen presentation to and activation of T cells, expression of co-stimulatory molecules and cytokine production. Recently, the ability of B cells to negatively regulate cellular immune responses and inflammation has been described and the concept of regulatory B cells has emerged. A variety of cytokines produced by regulatory B cell subsets have been reported, with IL-10 being the most studied. In this review, this specific IL-10-producing subset of regulatory B cells has been labeled B10 cells to highlight that the regulatory function of these rare B cells is mediated by IL-10, and to distinguish them from other B cell subsets that regulate immune responses through different mechanisms. B10 cells are a functionally defined subset currently identified only by their competency to produce and secrete IL-10 following appropriate stimulation. Although B10 cells share surface markers with other previously defined B cell subsets, currently there is no cell surface or intracellular phenotypic marker or set of markers unique to B10 cells. The recent discovery of an effective way to expand B10 cells ex vivo opens new horizons in the potential therapeutic applications of this rare B cell subset. This review highlights the current knowledge on B10 cells and discusses their potential as novel therapeutic agents in autoimmunity.


Introduction
Traditionally, B cells have been thought to contribute to the pathogenesis of autoimmune disease through antigen (Ag)-specifi c autoantibody production [1]. Nonetheless, the role of B cells in autoimmunity extends beyond the production of autoantibodies. B cells are now well established to have both positive and negative regulatory roles during immune responses.
B cells can positively regulate immune responses by producing Ag-specifi c antibody and inducing optimal T cell activation [2,3]. B cells can serve as professional Ag-presenting cells, capable of presenting Ag 10 3 -fold to 10 4 -fold more effi ciently than nonprofessional Agpresenting cells [4]. B cell Ag presentation is required for optimal Ag-specifi c CD4 + T cell expansion, memory formation, and cytokine production [5][6][7]. B cells may also positively regulate CD8 + T cell responses in mouse models of autoimmune disease [8,9]. Furthermore, costimulatory molecules (such as CD80, CD86, and OX40L) expressed on the surface of B cells are required for optimal T cell activation [10,11]. Th e positive regulatory roles of B cells extend to multiple immune system compo nents; the absence of B cells during mouse development results in signifi cant quantitative and qualitative abnormalities within the immune system, including a remarkable decrease in thymocyte numbers and diversity [12], signifi cant defects within spleen dendritic cell and T cell compartments [13][14][15], absence of Peyer's patch organogenesis and follicular dendritic cell networks [16,17], and absence of marginal zone and metallophilic macrophages with decreased chemokine expression [15,17]. B cells also positively regulate lymphoid tissue organization [18,19]. Finally, dendritic cell, macrophage, and T H cell development may all be infl uenced by B cells during the formation of immune responses [20].
B cells can also negatively regulate cellular immune responses through their production of immuno modulatory cytokines. B cell-negative regulation of immune responses has been demonstrated in a variety of mouse models of autoimmunity and infl ammation [21][22][23][24][25][26][27][28][29][30]. Although the identifi cation of B cell subsets with negative regulatory functions and the defi nition of their mechanisms of action are recent events, the important negative Abstract B cell abnormalities contribute to the development and progress of autoimmune disease. Traditionally, the role of B cells in autoimmune disease was thought to be predominantly limited to the production of autoantibodies. Nevertheless, in addition to autoantibody production, B cells have other functions potentially relevant to autoimmunity. Such functions include antigen presentation to and activation of T cells, expression of co-stimulatory molecules and cytokine production. Recently, the ability of B cells to negatively regulate cellular immune responses and infl ammation has been described and the concept of regulatory B cells has emerged. A variety of cytokines produced by regulatory B cell subsets have been reported, with IL-10 being the most studied. In this review, this specifi c IL-10-producing subset of regulatory B cells has been labeled B10 cells to highlight that the regulatory function of these rare B cells is mediated by IL-10, and to distinguish them from other B cell subsets that regulate immune responses through diff erent mechanisms. B10 cells are a functionally defi ned subset currently identifi ed only by their competency to produce and secrete IL-10 following appropriate stimulation. Although B10 cells share surface markers with other previously defi ned B cell subsets, currently there is no cell surface or intracellular phenotypic marker or set of markers unique to B10 cells. The recent discovery of an eff ective way to expand B10 cells ex vivo opens new horizons in the potential therapeutic applications of this rare B cell subset. This review highlights the current knowledge on B10 cells and discusses their potential as novel therapeutic agents in autoimmunity. regulatory roles of 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 B cells (B10 cells) are the most widely studied regulatory B cell subset [30,31,33]. Comprehensive reviews summar izing the variety of regulatory B cell subsets have been published during recent years [31,32]. Th e present review will therefore focus exclusively on the IL-10producing regulatory B cell subset. Th is specifi c subset of regulatory B cells has been labeled B10 cells to highlight that the regulatory function of these rare B cells is mediated by IL-10, and to distinguish them from other B cell subsets that regulate immune responses through diff erent mechanisms [34]. Th is functional subset of B cells is defi ned solely by its IL-10-dependent regulatory properties and extends beyond the concept of transcription factor-defi ned cell lineages. Th is review highlights 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 stimuli driving their develop ment. 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 be involved in the developmental process. Figure 1 illustrates our current understanding of B10 cell develop ment in vivo both in mice and humans, where their development shows multiple similarities.
B10 cells are a functionally defi ned B cell subset. Th ere are no unique phenotypic markers for B10 cells, and these cells are currently defi ned only by their competency to produce and secrete IL-10 following appropriate stimu lation. B10 cells share surface markers with other previously defi ned 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 identifi cation of B10 cells, intracellular cytoplasmic IL-10 staining is used, following ex vivo 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 (B10 PRO cells). B10 PRO cells develop into B10 cells after maturation through CD40 ligation or exposure to LPS or CpG. B10 PRO cells can be identifi ed indirectly following ex vivo stimulation with LPS or CpG in the presence of CD40 ligation for 48 hours with the addition of PMA and ionomycin for the last 5 hours. Th e IL-10 + B cells measured following this 48-hour stimulation include cells that would have been IL-10 + even with the shorter 5-hour stimulation (B10 cells), and thereby represent the sum of B10 plus B10 PRO cells (B10+B10 PRO ).

Mouse B10 cell development
BCR specifi city, affi nity and signaling are the most important currently identifi ed factors in B10 cell development. B10 cell regulation of infl ammation and autoimmunity is Ag specifi c [23,30,36]. Th e importance of BCR diversity is demonstrated by the fact that B10+B10 PRO cells are reduced by approximately 90% in transgenic mice with a fi xed BCR [37]. Signaling through the BCR appears critical during early development in vivo. CD19-defi cient mice (where BCR signaling is decreased) have a 70 to 80% decrease in B10+B10 PRO cells [30]. In contrast, B10 cells are expanded in human CD19 transgenic mice (where the overexpression of CD19 augments BCR signaling). Th e absence of CD22, which normally dampens CD19 and BCR 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 also ectopically express CD40L show dramatically enhanced numbers of CD1d hi CD5 + B cells and B10 cells [40]. Th e induction of IL-10 + B cells with regulatory activity by T cell immunoglobulin domain and mucin domain protein 1 (TIM-1) ligation [41] further highlights the importance of BCR signaling in B10 cell development. BCR signaling and 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 antibody production both in vitro and in vivo [43]. Th e importance of BCR-related signals is further highlighted by the observation that the stromal 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 produce IL-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 B10 cell development.
Despite the requirement for BCR expression and function during mouse B10 cell development, B cell stimulation with mitogenic anti-IgM antibody alone does not induce cytoplasmic IL-10 expression. Th e combination of anti-IgM stimulation with CD40 ligation and LPS or CpG signifi cantly reduces IL-10 competence [37]. BCR-generated signals thus inhibit the abilities of LPS or CpG and CD40 ligation to induce cytoplasmic IL-10 production. Whether BCR stimulation inhibits the induc tion of IL-10 competence by inducing B cells to mature or diff erentiate down a divergent pathway or diverts intracellular signaling is unknown. Another possibility is that the signals generated by mitogenic anti-IgM BCR cross-linking are too intense and that low-affi nity Ag-BCR interactions drive B10 PRO 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 purifi ed spleen CD19 + B cells with IL-21 induced 2.7-fold to 3.2-fold higher B10 cell frequencies, and 4.4-fold to 5.3-fold more IL-10 secretion compared with stimulation with media alone. Remarkably, IL-21 induced B10 cells to produce IL-10 without the need for stimulation with phorbol esters and ionomycin. Interestingly, IL-21 induced a three fold increase in IL-10 + B cells within the splenic CD1d hi CD5 + B cell subset, but did not induce IL-10 + B cells within the CD1d lo CD5 -B cell subset. Both B10 cells and non-B10 cells expressed IL-21R at similar levels, and ex vivo B10, B10pro and CD1d hi CD5 + B cell numbers were similar among IL-21R-defi cient (IL-21R -/-), MHC-IIdefi cient (MHC-II -/-) and CD40-defi cient (CD40 -/-) mice. Nevertheless, IL-21R, MHC-II and CD40 appear to be required for B10 cell eff ector functions, at least in experimental auto immune encephalomyelitis (EAE) [36]. Regulatory B10 cell function therefore requires IL-21R signaling, as well as CD40 and MHC-II interactions, potentially explaining Ag-specifi c B10 cell eff ector function [37].
Although cognate interactions with CD4 + T cells are important for B10 cell eff ector functions [36], T cells do not appear to be required for B10 cell development. In mice, B10 PRO cells are found in the CD1d − CD5 − adult blood and lymph node B cell subsets and within the CD1d − CD5 + neonatal spleen and adult peritoneal cavity B cell subsets. CD40 stimulation induces B10 PRO cells to become competent for IL-10 expression, while lipopolysaccharide (LPS) induces B10 PRO cells to become competent for IL-10 expression and induces B10 cells to produce and secrete IL-10. CD1d hi CD5 + IL-10-competent B10 cells in the adult spleen are induced to express IL-10 following stimulation with phorbol esters (phorbol-12-myristate-13-acetate (PMA)) and ionomycin or LPS plus PMA and ionomycin for 5 hours. Following a transient period of IL-10 expression, a small subset of B10 cells can diff erentiate into antibody-secreting plasma cells (PC). B10 cells also possibly diff erentiate into memory B10 cells (B10 M ). B10 cell development in humans appears to follow the diff erentiation scheme observed in mice. B10 cells and B10 PRO cells have been identifi ed in human newborn and adult blood. B10+B10 PRO cells in adult human blood express CD27 and CD24. Whether human B10 cells further diff erentiate into PCs or B10 M remains to be determined. Solid arrows, known associations; dashed arrows, speculated associations. MHC-II, major histocompatibility complex class II. B10 cells are present in T cell-defi cient nude mice, and their frequencies and numbers are approximately fi vefold higher when compared with wildtype mice. Th is observation is strengthened by the fact that MHC class I and MHC-II molecules and CD1d expression are not required for B10 cell development [37]. Th e presence or absence of T cells in vitro also does not aff ect the frequency of B10 cells. Although increased B10 cell frequencies in T celldefi cient mice suggest that T cells might actually inhibit B10 cell development, it is equally possible that the immunodefi cient state of these mice allows subclinical infl ammation that induces B10 cell generation. Th e role of T cells in B10 cell development in vivo is thereby complex and, although T cells are not required for B10 cell development, cognate interactions between CD4 + T cells and B10 cells are required for B10 cell eff ector function. B10 cells can be driven to produce IL-10 by TLR4 (LPS) or TLR9 (CpG oligonucleotides) ligands. Mouse B10 PRO 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 agonistic CD40 mAb [32]. TLR4 and TLR9 signaling through myeloid diff er entiation primary response gene 88 (MyD88) is necessary for the optimal maturation and IL-10 induction of B10pro and B10 cells following LPS stimulation and LPS or CpG stimulation, respectively [37]. Nevertheless, MyD88 expression is not an absolute requirement for B10 cell development in vivo, since B10 cells develop normally in MyD88 -/mice [37]. Specifi cally, numbers of B cells with the capacity to produce IL-10 are equivalent in wildtype and MyD88 -/mice when their maturation or IL-10 production are measured following CD40 ligation or PMA plus ionomycin stimulation, respectively, demon strating that B10 PRO and B10 cells are present at normal frequencies in MyD88 -/mice. Th ereby, while TLR signaling is not required for B10 cell development, MyD88 expression is required for LPS to induce optimal B cell IL-10 expression and secretion in vitro.

PMA + Ionomycin + Brefeldin
Th e involvement of TLR signals in B10-cell IL-10 production was recently demonstrated [45]. IL-10 produc tion by B cells, stimulated by contact with apoptotic cells, results from the engagement of TLR9 within the B cell after recognition of DNA-containing complexes on the surface of apoptotic cells by the BCR. An earlier study also highlights the eff ects of apoptotic cells on B cell IL-10 production, where apoptotic cells protected mice from developing collagen-induced arthritis (CIA) by the induction of IL-10-producing regulatory B cells [46]. Cell death products may therefore represent one of the physio logic triggers for B10 cell development 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 be determined.
Although certain transcription factors are involved at some point in B10 cell development, it is important 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 the blimp1 and irf4 transcription factors along with decreased expression of pax5 and bcl6, a signifi cant but small fraction of B10 cells can diff erentiate into antibodysecreting cells pro duc ing IgM and IgG polyreactive antibodies that are enriched for autoreactivity to singlestranded 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
B10 PRO cells and B10 cells have been recently identifi ed in humans [48] and their responses to LPS, CpG and CD40 ligation appear to follow the general scheme of mouse B10 cell development ( Figure 1). One notable diff erence in mouse versus human B10 cell development is the lack of response of mouse B10 PRO cells to CpG compared with their human counterparts. Human B10 PRO cells can be driven to develop ex vivo into B10 cells with LPS or CpG stimulation, or CD40 ligation. Interestingly, BCR ligation augmented human B cell IL-10 responses to CpG in one study [49]. Th is fi nding is in discordance with our fi ndings in both humans [48] and mice [37], where BCRgenerated signals inhibit the abilities of LPS or CpG and CD40 ligation to induce cytoplasmic IL-10 production. Whether human B10 cells develop into antibodysecreting cells or enter the memory pool (memory B10 cells, B10 M ) remains to be determined.

Unsolved questions on B10 cell development
Th e most critical unsolved issue relates to the nature of antigenic stimuli driving B10 cell development. Th e identifi cation of B10-cell BCR specifi city is imperative since it will provide new insights into their early development. Th e autoreactive nature of mouse B10-cell BCRs [47] suggests that autoantigens may be driving early B10 cell development and that B10 cells may represent one of the ways enabling the immune system to peripherally tolerate autoantigens. B cells responding to autoantigens in an IL-10-dependent regulatory way can potentially limit infl ammatory responses and limit autoimmune phenomena (see later section on B10 cell regulatory eff ects and Figure 2). Cell death products, by providing simultaneously both antigenic and nonantigenic stimuli, may represent one of the physio logic triggers for B10 cell development. Th e clearance of antigenic products of dying cells by non complement-fi xing IgM polyreactive/ autoreactive anti bodies (such as those made by mouse B10 cells) in an IL-10-rich environment would be benefi cial since it could potentially limit infl ammatory responses to self-Ags. Additional unidentifi ed antigenic and nonantigenic stimuli are probably involved in B10 cell development. Th e identifi cation of such stimuli will provide additional insights ito B cell development that may prove invaluable for the future manipulation of B10 cells for treating autoimmune disease. Another important question is whether B10 cells enter the B cell memory pool during their development. Th is question is suggested by human studies demonstrating that B10 PRO cells and B10 cells share phenotypic features with memory B cells (see later section on Human B10 cell phenotype).

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 to identify 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 wildtype C57BL/6 mice) produce IL-10 following PMA and ionomycin stimulation, implying that not all B cells are competent to produce IL-10. Intracellular cytokine staining combined with fl ow cytometric phenotyping shows that mouse spleen B10 cells are enriched within the small CD1d hi CD5 + B cell subset, where they represent 15 to 20% of the cells in C57BL/6 mice. Th is phenotypically unique CD1d hi CD5 + subset shares overlapping cell surface markers with a variety of phenotypically defi ned B cell subsets such as CD5 + B-1a B cells, CD1d hi CD23 − IgM hi CD1d hi marginal zone B cells, and CD1d hi CD23 + IgM hi CD1d hi T2 marginal zone precursor B cells, which all undoubtedly contain both B10 PRO cells and B10 cells [23,26,30,50]. Mouse B10 cells are predominantly IgD low IgM hi , and <10% co-express IgG or IgA, but they can diff erentiate into antibody-secreting cells secreting polyreactive or Ag-specifi c IgM and IgG [47]. IL-10 + B cells were recently shown to be enriched in the TIM-1 + compartment and TIM-1 + B cells are enriched in the CD1d hi CD5 + compartment [41]. However, IL-10 + B cells are also present in the TIM-1compartment and TIM-1 + B cells are present in the non-CD1d hi CD5 + compartment. Intracellular cytoplasmic IL-10 staining thereby remains the only current way to visualize the entire subset of IL-10-competent B cells. Nonetheless, the isolation of CD1d hi CD5 + B cells or other phenotypically defi ned B cell subsets where B10 cells are enriched currently provides the best current means for isolating a viable B cell population that is signifi cantly enriched for B10 cells and can be used for adoptive transfer experiments and functional studies in mice.

Human B10 cell phenotype
Th e IL-10-producing B cell subset characterized in humans normally represents <1% of peripheral blood B cells [48]. Peripheral blood B10 cells and B10 PRO cells are highly enriched in the CD24 hi CD27 + B cell subset, with approximately 60% also expressing CD38. Similar total numbers of IL-10 + B cells have been described in the CD24 hi CD38 hi and CD24 int CD38 int B cell subsets [51]. A separate study showed that B10 cells did not fall within any of the previously defi ned B cell subsets, but they were enriched in the CD27 + and the CD38 hi compart ments [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-charac ter ized marker for memory B cells, although some memory B cells may be CD27 - [54][55][56]. Th e CD27 + B cell subset can also expand during the course of autoimmunity and has been proposed as a marker for disease activity [54,56]. Th e CD24 hi CD148 + phenotype of B10 cells and B10 PRO cells may thereby indicate their selection into the memory B cell pool during development, or they may represent a distinct B cell subset that shares common cell surface markers with memory B cells. Consistent with a memory phenotype, the proliferative capacity of human blood B10 cells in response 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 are generally CD10 + CD24 hi CD38 hi cells that are also CD27-negative [55,56]; since CD10 expression is a well-accepted marker for most cells within the transitional B cell pool [57], its absence on B10 cells suggests that these cells are not recent emigrants from the bone marrow. In summary, human B10 cells share phenotypic characteristics with other previously defi ned B cell subsets, and, currently, there is no known surface phenotype unique to B10 cells.

B10 cell regulatory eff ects
B10 cells exert a variety of IL-10-dependent regulatory eff ects potentially involved in autoimmune disease. Th e anti-infl ammatory eff ects of IL-10 are mediated by multiple mechanisms involving both the innate and adaptive arms of the immune system. In innate cells, these mechanisms include downregulation of proinfl ammatory cytokine production [58] and decreased expression of MHC-II and co-stimulatory molecules [59] result ing in decreased T cell activation. B10 cells negatively regulate the ability of dendritic cells to present Ag [60]. In CD4 + T cells, IL-10 suppresses T H1 [50] and enhances T H2 polarization [41,59]. B10 cells suppress IFNγ and TNFα responses in vitro [60] and INFγ responses in vivo [36] by Ag-specifi c CD4 + T cells. Co-culture of mouse CD1d hi CD5 + B cells with CFSE-labeled naive CD4 + T cells suppresses T H17 cell diff erentiation [61] and IL-10 is known to suppress T H17 responses [62]. Th e suppression of T H17 responses by B10 cells in vivo was demonstrated recently [36]. IL-10 produc tion by human B10 cells inhibits Ag-specifi c CD4 + CD25 -T cell proliferation [49] and regulates mono cyte activation and cytokine production [48] in vitro.
A number of studies suggest that IL-10-producing B cells are important for the generation and/or maintenance of the regulatory T cell (T REG ) pool [46,[63][64][65][66][67][68][69][70][71][72]. However, a recent study [73] and our previously published data [23] do not support this view. Th e reason for this discrepancy is unclear but may be related to the diff erent models of infl ammation and conditions used to study the relationship of B10 cells and T REGS . Th ese two studies suggesting that B10 cells are not involved in the generation and maintenance of the T REG pool are both in models of EAE [23,73]. In contrast, only one study suggests that B10 cells are important for the generation and/or maintenance of the T REG pool specifi cally in EAE [63]. Th e results of a diff erent study clarify the picture in EAE further by showing that a subset of regulatory B cells control T REG numbers through IL-10-independent mecha nisms [34]. Human B10 cell IL-10 production will there fore probably also have pleiotropic regulatory eff ects on the immune system, as occurs in mice. Th e potential regulatory eff ects of B10 cells in autoimmune disease limiting infl ammatory responses and subsequent tissue damage are summarized in Figure 2.

B10 cells in human autoimmune disease
Studies of B10 cells and human autoimmune disease are limited but of outmost importance since they provide valuable insights relevant to the potential future therapeutic application of B10 cells in humans. Peripheral blood B10 cells and B10 PRO cells are present in patients with autoimmune diseases, including rheumatoid arthritis, systemic lupus erythematosus, primary Sjögren's syndrome, autoimmune bullous diseases, and multiple sclerosis. Interestingly, B10+B10 PRO cell frequencies are expanded in some but not all cases, while mean B10+B10 PRO cell frequencies are signifi cantly higher in patients with autoimmune disease compared with agematched healthy controls [48]. A diff erent study examined cytoplasmic IL-10 production by B cells from systemic lupus erythematosus patients and normal controls [74]. Blood mononuclear cells were cultured for 24 hours in the presence or absence of PMA, ionomycin, or LPS; signifi cantly more systemic lupus erythematosus CD5 + B cells produced cytoplasmic IL-10 than did controls. A diff erent study also demonstrated spontaneous B cell IL-10 production that is higher in untreated rheumatoid arthritis, systemic sclerosis, and systemic lupus erythematosus patients than in controls [75].
By contrast, the concept of functional impairment of B10 cells in autoimmune disease was recently introduced by demonstrating functional impairment of CD24 hi CD38 hi regulatory B cells in human systemic lupus erythematosus [51]. Cultures of peripheral blood mononuclear cells were stimulated with plate-bound anti-CD3 mAb for 72 hours, followed by the measurement of IFNγ and TNFα CD4 + T cell responses. When CD24 hi CD38 hi B cells were removed from the culture, higher frequencies of CD4 + IFNγ + and CD4 + TNFα + T cells were noted in healthy individuals but not in systemic lupus erythe matosus patients; this eff ect was partially IL-10 dependent. In addition, CD24 hi CD38 hi B cells isolated from the peripheral blood of systemic lupus erythematosus patients were refractory to CD40 ligation and produced less IL-10 compared with their healthy counterparts. Th e results of this study are rather intriguing but these fi ndings need to be validated in view of the complexity of the culture system used and the non-uniformity of the CD24 hi CD38 hi B cell subset with regards to its IL-10dependent regulatory properties. In conclusion, B10 cells are present in the peripheral blood of autoimmune disease patients, where they appear to be expanded, whereas the functional capacity of human B10 cells in autoimmunity needs to be further defi ned.

B10 cells in mouse models of autoimmune disease
Th e important regulatory eff ects of B10 cells in vivo and their therapeutic potential in autoimmunity 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-specifi c CD4 + T cells [76,77]. B cells were shown over a decade ago to have regulatory properties during the induction of EAE, with genetically B cell-defi cient mice developing a severe nonremitting form of the disease [21]. However, these B cell regulatory eff ects were recently shown not to be IL-10 dependent [34]. Nonetheless, other studies highlight the importance of B cell-derived IL-10 in EAE. Specifi cally, EAE severity during the late phase of disease increases in B celldefi cient μMT mice that do not fully recover from their disease when compared with wildtype mice, and the adoptive transfer of wildtype B cells but not IL-10 -/-B cells normalizes EAE severity in μMT mice [22]. Disease recovery is dependent on the presence of auto antigen-reactive B cells, and B cells isolated from mice with disease produced IL-10 in response to autoantigen stimulation. In the absence of Ag-specifi c B cell IL-10 production, the proinfl ammatory T H1mediated immune responses persist and mice do not recover from the disease. Th e EAE model demonstrates the complexity of regulatory mechanisms mediated by diff erent cell subsets during diff erent stages of the disease. When B cells from wildtype mice are depleted by CD20 mAb treatment 7 days before EAE induction, there is an increased infl ux or expansion of encephalitogenic T cells within the central nervous system and exacerbation of disease symptoms [23]. Th is eff ect is related to B10 cell depletion since similar eff ects are observed with selective B10 depletion by means of CD22 mAb [60]. Th e adoptive transfer of Ag-specifi c (myelin oligodendrocyte glycoprotein-sensitized) B10 cells into wildtype mice also reduces EAE initiation dramatically. Th e protective eff ect is IL-10 dependent since the adoptive transfer of CD1d hi CD5 + B cells purifi ed from IL-10 −/− mice does not aff ect EAE severity. B10 cell eff ector functions in EAE require IL-21 along with cognate interactions with CD4 + T cells since the adoptive transfer of CD1d hi CD5 + B cells into CD19 -/mice from IL-21R -/-, MHC-II -/or CD40 -/mice prior to the induction of EAE does not alter disease course [36]. Once disease is established, adoptive transfer of B10 cells does not suppress ongoing EAE. B10 cells thereby appear to normally regulate acute autoimmune responses in EAE. In contrast to the role of B10 cells in early disease, T REG depletion enhances late-phase disease. Th erefore, in EAE, depending on the stage of the disease, diff erent regulatory mechanisms are involved in limiting infl ammatory responses, with B10 cells regulating disease initiation and T REGS being involved predominantly in the regulation of late-phase disease.

Infl ammatory bowel disease
IL-10-producing B cells regulate intestinal infl ammation in infl ammatory bowel disease [26]. Early studies showed that B cells and their autoantibody products suppress colitis in T cell receptor alpha chain-defi cient mice that spontaneously develop chronic colitis, while B cells are not required for disease initiation [78]. B cells with upregulated CD1d expression in the gut-associated lymphoid tissues of mice with intestinal infl ammation were subsequently demonstrated to be regulatory [25]. Th is IL-10-producing B cell subset appears during chronic infl ammation in T cell receptor alpha chain-defi cient mice and suppresses the progression of intestinal infl ammation by down regulating infl ammatory cascades associated with IL-1 upregulation and signal transducer and activator of transcription 3 (stat3) activation rather than by altering polarized T H cell responses. Th e adoptive transfer of these mesenteric lymph node B cells also suppresses infl ammatory bowel disease through a mechanism that correlates with an increase in T REG subsets [67]. Oral administration of dextran sulfate sodium solution to mice is widely used as a model of human ulcerative colitis. Dextran sulfate sodium-induced intestinal injury is more severe in CD19 -/mice (where B10 cells are absent) than in wildtype mice [79], and these infl ammatory responses are negatively regulated by CD1d hi CD5 + B cells producing IL-10. B10 cells therefore emerge during chronic infl am mation in mouse models of infl amma tory bowel disease, where they suppress the progression of infl am matory responses and ameliorate disease manifestations.

Collagen-induced arthritis
CIA is a model for human rheumatoid arthritis that develops in susceptible mouse strains immunized with heterologous type II collagen emulsifi ed in complete Freund's adjuvant [80,81]. CIA and rheumatoid arthritis share in common an association with a limited number of MHC-II haplotypes that determine disease susceptibility [82,83]. B cells are important for initiating infl ammation and arthritis since mature B cell depletion significantly reduces disease severity prior to CIA induction but does not inhibit established disease [84]. Several studies on CIA demonstrate the negative regulatory eff ects and therapeutic potential of B10 cells.
Activation of arthritogenic splenocytes with Ag and agonistic anti-CD40 mAb induces a B cell population that produces high levels of IL-10 and low levels of IFNγ [85]. Th e adoptive transfer of these B cells into DBA/1-T cell receptor-β-Tg mice, immunized with bovine collagen (type II collagen) emulsifi ed in complete Freund's adjuvant, inhibits T H1 responses, prevents arthritis develop ment, and is eff ective in ameliorating established disease. Th e adoptive transfer of CD21 hi CD23 + IgM + B cells from DBA/1 mice in the remission phase prevents CIA and reduces disease severity through IL-10 secretion [86]; a signifi cant but less dramatic therapeutic eff ect on CIA progression is seen when cells from naïve mice are adoptively transferred. In addition, the adoptive transfer of ex vivo expanded CD1d hi CD5 + B cells in collagenimmunized mice delays arthritis onset and reduces disease severity, accompanied by a substantial reduction in the number of T H17 cells [61]. Co-culture of CD1d hi CD5 + B cells with naive CD4 + T cells suppresses T H17 cell diff erentiation in vitro, and co-culture of CD1d hi CD5 + B cells with T H17 cells results in decreased proliferation responses in vitro. Furthermore, the adoptive transfer of T H17 cells triggers CIA in IL-17 -/-DBA mice; however, when T H17 cells are co-transferred with CD1d hi CD5 + B cells, the onset of CIA is signifi cantly delayed. Finally, in a diff erent study, administration of apoptotic thymocytes along with ovalbumin peptide and complete Freund's adjuvant to mice carrying an ovalbumin-specifi c rearranged T cell receptor transgene (DO11.10 mice) up to 1 month before the onset of CIA resulted in an increase in ovalbumin-specifi c IL-10 secretion and is protective for severe joint infl ammation and bone destruction [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 the benefi cial eff ects of apoptotic cell treatment [46].

Systemic lupus erythematosus
B cell-negative regulatory eff ects are important in NZB/ W mice, a spontaneous lupus model, since mature B cell depletion initiated in 4-week-old NZB/W F1 mice hastens disease onset, which parallels depletion of B10 cells [87]. B10 cells are phenotypically similar in NZB/W F1 and C57BL/6 mice, but are expanded signifi cantly in young NZB/W F1 mice [87]. In wildtype NZB/W mice, the CD1d hi CD5 + B220 + B cell subset, which is enriched in B10 cells, is increased 2.5-fold during the disease course, whereas CD19 -/-NZB/W mice lack this CD1d hi CD5 + regulatory B cell subset [88]. Finally, the potential therapeutic eff ect of B10 cells in lupus is highlighted by the prolonged survival of CD19 -/-NZB/W recipients following the adoptive transfer of splenic CD1d hi CD5 + B cells from wildtype NZB/W mice [88]. Studies in the NZB/W spontaneous lupus model therefore suggest that B10 cells have protective and potentially therapeutic eff ects.
In the MRL.Fas(lpr) mouse lupus model, B cell-derived IL-10 does not regulate spontaneous autoimmunity [89]. B cell-specifi c deletion of IL-10 in MRL.Fas(lpr) mice indicates that B cell-derived IL-10 is ineff ective in suppressing the spontaneous activation of self-reactive B cells and T cells during lupus. Th e severity of organ disease and survival rates in mice harboring IL-10defi cient B cells were unaltered. MRL.Fas(lpr) IL-10 reporter mice illustrate that B cells comprise only a small fraction of the pool of IL-10-competent cells. In contrast to previously published studies from our laboratory and elsewhere, putative regulatory B cell phenotypic subsets, such as CD1d hi CD5 + and CD21 hi CD23 hi B cells, were not enriched in IL-10 transcription. Th is observation suggests fundamental diff erences in the pathogenesis and immune 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 auto immune destruction of the insulin-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 from type 1 diabetes in NOD mice [92,93]. Th e adoptive transfer of BCR-stimulated B cells into NOD mice starting at 5 to 6 weeks of age both delays the onset and reduces the incidence of type 1 diabetes, while treatment at 9 weeks of age delays disease onset. Protection from type 1 diabetes requires B cell IL-10 production since the adoptive transfer-activated NOD-IL-10 -/-B cells do not confer protection from type 1 diabetes or the severe insulitis in NOD recipients. Th e therapeutic eff ect of adoptively transferred activated NOD B cells correlates with T H2 polarization. Th e limited data above suggest that B10 cells may be protective in preventing establishment of type 1 diabetes in NOD mice.

Therapeutic potential of B10 cells
Harvesting the anti-infl ammatory properties of B10 cells can provide a new approach to the treatment of autoimmunity. Manipulation of this subset for treating autoimmune disease is possible by either selective depletion of mature B cells while sparing B10/B10 PRO cells or the selective expansion of B10 cells. Since there are no identifi ed surface molecules specifi c for non-B10/B10 PRO cells, it is currently impossible to selectively target and deplete mature B cells while sparing B10/B10 PRO cells. B10 cell expansion appears to be a more viable approach since some of the stimuli driving their development have been identifi ed. B10 cells can be expanded for therapeutic purposes either in vivo or ex vivo. Expansion of B10 cells in vivo by means of agonistic CD40 antibody has shown benefi t in CIA [85]. However, expanding B10 cells in vivo carries additional risks since the currently identifi ed stimuli driving B10 cell development are rather nonspecifi c and, if administered systemically, will trigger responses in a variety of immune cells. For example, the systemic administration of agonistic CD40 antibodies in humans has been associated with serious adverse eff ects such as cytokine release syndrome [94]. In summary, selective depletion of mature B cells while sparing B10/ B10 PRO cells is not currently possible, and in vivo B10 cell expansion by nonspecifi c agents such as agonistic CD40 antibody is potentially associated with serious off -target eff ects.
Expanding B10 cells ex vivo appears more preferable than in vivo B10 cell expansion by nonspecifi c agents because it off ers a potential therapy without the risk of undesirable nonspecifi c off -target eff ects. However, ex vivo B10 cell expansion introduces new challenges related to the method of expansion, to the magnitude of expansion and to the time it takes to generate B10 numbers that will be suffi cient for therapeutic use. Th e 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 genetic manipulation of immature B cells through lentiviral transfection [95]. Th ese cells were eff ective in treating EAE. However, although this method can effi ciently generate large numbers of regulatory B cells ex vivo, concerns remain about administering infusions of lentivirus-infected B cells to humans (with retroviral and infectious potential). Safety concerns thereby limit the use of infectious agents in manipulating human cells, which could render this approach inappropriate for use in humans.
Th e magnitude of ex vivo B10 cell expansion is very important since the number of cells infused during adoptive transfer experiments is critical. In humans, the most convenient potential source of B10/B10 PRO cells prior to ex vivo expansion is obviously peripheral blood. Since B10/B10 PRO cells are rare in peripheral blood and there are limitations on the volume that can be drawn at any given time, a method of expanding B10 cells by several million-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 signifi cance; ideally, this process should not take more than 1 or 2 weeks. Th ere is accumulating hope that such an approach will soon be available for human cells since mouse B10 cell ex vivo expansion can be accomplished within 9 days by means of combined CD154, Blymphocyte stimulator, IL-4 and IL-21 stimulation [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 expression increases the B10 cell purity to 75%, thus providing not only large numbers of B10 cells but also a B cell population predominantly consisting of B10 cells. Th ese ex vivo expanded B10 cells are very eff ective in limiting infl ammatory responses in EAE. Th is approach appears promising since it provides an eff ective way of generating large numbers of B10 cells without the use of infectious agents. Th e development of a similar system for expanding human B10 cells is of outmost importance.

Conclusion
Th e phenotypic and functional characterization of B10 cells is an important advance for the regulatory B cell fi eld. Numerous additional functionally defi ned subsets of regulatory B cells will probably be identifi ed in the future. B10 cells share phenotypic markers with a variety of previously defi ned subsets, but their only unique phenotypic marker is intracellular IL-10 production. Although certain transcription factors are involved at diff erent points in B10 cell development, there is currently no transcription factor signature unique to B10 cells. BCR-related signals are most critical in B10 cell development and the fi nding of B10-cell BCR autoreactivity suggests that autoantigens may be of particular importance. Th e recent discovery of an in vitro method to effi ciently expand mouse B10 cells provides an invaluable tool for studying the basic biology of B10 cells as well as manipulating them for therapeutic purposes. Th e development of a similar method for human cells will open new opportunities for studying the basic biology of human B10 cells and a promising novel approach in treating human autoimmune disease, potentially without undesirable off -target eff ects.