Regulatory B10 Cells Differentiate Into Antibody-Secreting Cells After Transient IL-10 Production In Vivo

Abstract

Regulatory B cells that are functionally defined by their capacity to express IL-10 (B10 cells) downregulate inflammation and autoimmunity. In studies using well-defined IL-10-reporter mice, this rare B10 cell subset was also found to maintain a capacity for plasma cell differentiation. During a transient period of il10 transcription, the blimp1 and irf4 transcription factors were induced in B10 cells while pax5 and bcl6 were downregulated as a significant fraction of B10 cells completed the genetic and phenotypic program leading to antibody-secreting cell differentiation in vitro and in vivo. B10 cell-derived IgM reacted with both self and foreign Ags, whereas B10 cells generated Ag-specific IgG in response to immunizations. Moreover, B10 cells represented a significant source of serum IgM and IgG during adoptive transfer experiments, and produced Ag-specific, polyreactive and autoreactive antibody specificities that were consistent with their expression of a diverse Ag receptor repertoire. Thereby, B10 cells not only limit inflammation and immune responses by the transient production of IL-10, but may also facilitate clearance of their eliciting Ags through an inherent capacity to quickly generate polyreactive and/or Ag-specific antibodies during humoral immune responses.

Introduction

B lymphocytes mediate humoral immunity through their production of secreted antibody, but are also central regulators of CD4⁺ T cell activation by serving as APCs and providing co-stimulatory molecules and cytokines that regulate cellular immune responses during T cell expansion, memory formation, and cytokine production (1). However, B cells and specific B cell subsets can also negatively regulate immune responses (2). The absence or loss of these regulatory B cells exacerbates disease symptoms in diverse models of inflammation and autoimmunity, predominantly through the production of the regulatory cytokine, IL-10 (3–11).

A specific subset of regulatory B cells was recently found to inhibit inflammation, autoimmunity, and innate and adaptive immune responses through the production of IL-10 (8, 9, 12, 13), a potent and pleiotropic cytokine (14). We call these B cells “regulatory B10 cells” because IL-10 is required for their negative regulatory function (2) and additional B cell subsets with unique regulatory properties also exist. For example, IL-12-producing B cells regulate intestinal inflammation (15). In mice, regulatory B10 cells are functionally identified by cytoplasmic IL-10 expression following in vitro stimulation with LPS, PMA, and ionomycin (L+PI), with monensin (L+PIM) included in the cultures to block IL-10 secretion (8, 9). Spleen B10 cells are found at low frequencies (1–5%), where they are predominantly found within the phenotypically unique CD1d^hiCD5⁺CD19^hi B cell subpopulation (8–10). Regulatory B10 cells share overlapping cell surface markers with multiple other phenotypically-defined B cell subsets (B1a, marginal zone, and marginal zone precursor cells), potentially consistent with their localization within spleen follicles and marginal zones (16). B10 cells are presumed to be functionally mature since they are competent to express IL-10 after 5 h of ex vivo stimulation, and they proliferate rapidly following in vitro or in vivo activation (12, 17). Additional B cells within the CD1d^hiCD5⁺ B cell subpopulation acquire the ability to function like B10 cells during 48 h of in vitro stimulation with either agonistic CD40 mAb or LPS (17). These B10 progenitor (B10pro) cells are then able to express cytoplasmic IL-10 following L+PIM stimulation for 5 h. Regulatory B10 cell functions are Ag-restricted in vivo (8, 9), with B10pro and B10 cells requiring diverse Ag receptors (BCR) for their development (17). Spleen B10 cell numbers increase significantly during inflammation and autoimmunity, with the adoptive transfer of Ag-primed CD1d^hiCD5⁺ B cells suppressing inflammation and disease in mouse models (8, 9, 11, 17, 18). Human blood B10 and B10pro cells that parallel their mouse counterparts are equally rare, and represent a subset of the circulating CD24^hiCD27⁺ “memory” B cell subset (12). Thus, the capacity of human and mouse B10pro and B10 cells to express IL-10 is central to their regulatory function.

IL-10 reporter mice have been developed to examine regulatory T cell IL-10 expression and cell fates. In Tiger mice, an internal ribosomal entry site-GFP construct follows the genomic il10 coding sequence, resulting in cytoplasmic GFP expression during il10 transcription (19). Similarly, 10BiT mice express Thy1.1 under the control of il10 BAC-transgene regulatory elements, leading to cell surface Thy1.1 expression following IL-10 production (20). In the current studies, IL-10 reporter expression was used to track regulatory B10 cell induction and fates in Tiger and 10BiT mice, with the findings that regulatory B10 cells only transiently express IL-10 prior to their terminal differentiation into clonally diverse antibody-secreting plasmablasts and plasma cells that contribute significantly to the serum antibody pool. Thereby, regulatory B10 cells not only limit inflammation and immune responses by the production of IL-10, but also contribute to humoral immunity.

Material and Methods

Mice

C57BL/6 and Rag2^−/− mice were from NCI Frederick (Bethesda, MD). Tiger mice (19) were from The Jackson Laboratory (Bar Harbor, ME). A gene dose-dependent decrease in IL-10 production was not observed in homozygous Tiger mice, which occurs with T cells (19). Hemizygous 10BiT mice were as described (20). Mice were housed in a specific pathogen free barrier facility with end-point analyses carried out between 8–14 weeks of age. Mice were given (i.p.) sterile LPS in PBS (25 μg, E. coli, clone 0111:B4; Sigma, St. Louis, MO), CFA or IFA (200 μl of 1:1 emulsified mixture with PBS, Sigma, St. Louis, MO), Imject® Alum (200 μl of 1:1 emulsified mixture with PBS, Pierce, Rockford, IL), or alum with TNP₂₉KLH (50 μg/200 μl; Biosearch Technologies, Novato, CA). All studies and procedures were approved by the Duke University Animal Care and Use Committee.

B cell purification, cultures, and immunofluorescence analysis

B cells enriched (>95% CD19⁺) from single cell tissue suspensions by MACS selection using CD19-microbeads (Miltenyi Biotec Inc., Auburn, CA) were cultured in complete medium (RPMI 1640 medium containing 10% FBS, 1% HEPES, 1% L-Glutamine, 1% Pen/Strep, and 0.1% 2-ME). Sterile LPS (10 μg/ml), goat F(ab')₂ anti-mouse IgM antibody (5 μg/ml, Jackson ImmunoResearch, West Grove, PA), and CD40 mAb (2 μg/ml, clone HM40-3; BD Pharmingen, San Jose, CA) were added to cultures where indicated.

Single cell leukocyte suspensions were stained with pre-determined optimal antibody concentrations as described (21) with cytoplasmic IL-10 expression assessed as described (22). Antibodies included anti-mouse IL-10 (JES5-2A5), CD138 (281–2), CD43 (S7), CD38 (90) and GL7 (Ly-77) mAbs from BD Pharmingen; CD16/CD32 (FcBlock), FITC-, PE-, PE.Cy5-, PE.Cy7-, Biotin- or APC-conjugated anti-mouse B220 (clone RA3-6B2), CD19 (eBio1D3), CD1d (1B1), CD5 (53–7.3), Thy1.1 (HIS51), Thy1.1 (OX-7), CD21/35 (eBio8D9) and CD23 (B3B4) mAbs from eBioscience, Inc. (San Diego, CA); anti-mouse IL-10 (JES5-16E3), CD19 (6D5) and CD16/32 (TruStain) from BioLegend (San Diego, CA); and goat anti-mouse IgM antibody (Southern Biotech, Birmingham, AL). In some instances, Streptavidin conjugated to PE.Cy5 or PE.Cy7 (eBioscience) was used to reveal biotinylated antibody binding. Anti-mouse IgG1, IgG2a, IgG3 and IgA antibodies were from Southern Biotech. Anti-mouse Blimp-1 mAb (3H2-E8) was from Novus Biologicals (Littleton, CO). Data were collected on a FACSCantoII™ flow cytometer (BD Biosciences, Franklin Lakes, NJ) and analyzed using Flowjo Software (TreeStar, Inc., Ashland, OR).

Adoptive transfers of syngeneic spleen B cell populations were as described (22). For some experiments, purified spleen CD19⁺ B cells were first cultured overnight with LPS in complete medium, then washed twice and suspended in sterile PBS prior to i.v. injection through lateral tail veins.

Transcript quantification

RNA extracted from enriched spleen B cells was used to generate cDNA, with relative transcript levels determined by reverse transcriptase quantitative real-time PCR of triplicate samples as described (9). Thy1.1 transcripts were amplified using forward (CGTTGGCGCACCAGGAGGAG) and reverse (TGGAGAGGGTGACGCGGGAG) primers. Other primers were as described: gapdh and il10 (9); xbp1 (23); bcl6 (24); blimp1, irf4, and pax5 (25). Cycle conditions were as follows: 1 denaturation step of 94° C for 2 minutes followed by 40 cycles of 94° C for 30 seconds, 60° C for 30 seconds, and 72° C for 1 minute. PCR products were controlled for purity by analyses of their melting curves. Expression threshold values (ΔCt) for each transcript were determined by normalizing to gapdh expression within each sample group.

ELISA and ELISPOT assays

Sera were collected weekly, with Ag-specific antibodies quantified by ELISA using DNP-BSA. Serum IgM and IgG levels, autoantibody levels, and TNP- or DNP-specific antibodies were quantified by ELISA as described (21, 26). ASC frequencies from cell sorter purified B10 and non-B10 cells were determined using ELISpot assays as described (27).

Ig sequences

Purified spleen B cells from three individual mice were stimulated with LPS (10 μg/ml), PMA (50 ng/ml), and ionomycin (1 μg/ml) for 5 h. IL-10-secreting cells were identified using the Mouse IL-10 Secretion Assay Kit (Miltenyi Biotech Inc., Auburn, CA). Individual IL-10⁺λ-CD19⁺ cells were sorted into single wells of 96-well PCR plates using a FACSAria II cell sorter (BD Biosciences). cDNA was synthesized with Ig H and L chain transcripts amplified using nested PCR primers as described (28). PCR products were purified (QIAquick PCR Purification Kit, Qiagen, Valencia, CA) and cloned (StrataClone PCR Cloning Kit, Agilent Technologies, La Jolla, CA) before sequencing (Duke University DNA Analysis Facility). Productive Ig rearrangements were compared against germline Ig sequences according to the Ig Basic Local Alignment Search Tool (IgBLAST) database (National Center for Biotechnology Information, Bethesda, MD) and analyzed using the Immunogenetics V-query and Standardization tool (29) to determine V(D)J gene family usage. Mutation frequencies were determined using germline V, D and J sequences from IgBLAST. When light chain sequences obtained from adjacent wells were identical, only one sequence was reported. V_H-D-J_H and V_K-J_K transcript alignments and phylogenetic trees based on average percent identity were constructed using ClustalW2 (30).

Statistical analysis

Data are shown as means (±SEM). The two-tailed Student's t test was used to identify significant differences between sample means.

Results

B cell GFP IL-10 reporter expression in Tiger mice

Spleen GFP⁺ or cytoplasmic IL-10⁺ B cells were not observed in Tiger mice at frequencies significantly above background levels in monensin-treated wild type mice and their IL-10^−/− littermates (Fig. 1A–B, not shown). However, GFP⁺ and cytoplasmic IL-10⁺ B cell frequencies increased significantly after ex vivo stimulation using L+PIM for 5 h. GFP⁺ or IL-10⁺ B cells represented between 2–3% of spleen B cells in both Tiger and wild type mice. Furthermore, 72±3% of IL-10⁺ B cells from Tiger mice expressed readily measurable GFP in these assays. Likewise, the majority of GFP⁺ B cells expressed IL-10 (Fig. 1C). In comparison with spleen, significantly fewer IL-10- or GFP-competent B10 cells were found within peripheral or mesenteric lymph nodes after L+PIM stimulation (Fig. 1D). Thus, GFP mimicked cytoplasmic IL-10 expression by most B10 cells during 5 h induction assays.

Figure 1. B cell GFP expression in Tiger mice parallels cytoplasmic IL-10 expression.

(A) B cell IL-10 production relative to GFP expression in Tiger mice. Splenocytes were cultured for 5 h with L+PIM before cell surface CD19 and cytoplasmic IL-10 immunofluorescence staining with flow cytometry analysis. Cells cultured with monensin alone served as negative controls for IL-10 staining, with results similar to isotype control mAb staining (not shown). Representative contour plots show the IL-10⁺, IL-10⁺GFP⁺ and GFP⁺ cell frequencies within the indicated gates for CD19⁺ B cells (n=5 mice). (B) Mean IL-10⁺ and GFP⁺ B cell frequencies (±SEM) in wild type and Tiger mice (n=5 mice/group) as identified in (A). (C) Representative IL-10 expression by GFP⁺ B cells in Tiger mice. GFP⁺ and GFP^– CD19⁺ B cells were assessed for IL-10 expression (thick lines) relative to control mAb staining (shaded histograms) after 5 h L+PIM stimulation (n=5 mice) as in (A). (D) Mean frequencies and numbers of IL-10⁺ and GFP⁺ B cells in tissues of Tiger mice among CD19⁺ B cells from spleen (SPL), peripheral lymph nodes (PLN, inguinal), or mesenteric lymph node (MLN) (≥3 mice) as in (A). (E) GFP expression by B10+B10pro cells from Tiger mice. Spleen CD19⁺ cells were cultured for 48 h in media alone or with agonistic CD40 mAb, LPS or anti-IgM antibody. Monensin, L+PIM or PIM were added during the final 5 h of culture, with IL-10⁺ or GFP⁺ B cells identified as in (A). Cultured spleen B cells from wild type mice served as background controls for GFP expression. Bar graphs show mean frequencies of GFP⁺ B cells after culture (n≥3 mice/group). (B, D, E) Significant differences between cultures with media alone or between values are indicated: *p<0.05, **p≤0.01. All experiments were performed ≥3 times.

Agonistic CD40 signals provided during 48 h in vitro cultures render B10pro cells competent to express IL-10 when subsequently stimulated with L+PIM. Under these conditions, similar frequencies of cytoplasmic IL-10⁺ (7.3±0.2%) and GFP⁺ (6.3±0.1%) B10+B10pro cells were enumerated (Fig. 1E). By contrast, LPS induces both B10pro cell maturation and B10 cell IL-10 secretion during 48 h assays (17). Under these conditions, the frequency of GFP⁺ B cells (9.3±0.1%) was consistently higher than the frequency of cytoplasmic IL-10⁺ B cells (7.8±0.5%), while BCR ligation did not induce B10pro maturation into GFP-competent B cells. Thus, GFP expression was more durable than IL-10 expression following prolonged (48 h) LPS stimulation due to IL-10 secretion and/or relative differences in protein turnover.

B cell Thy1.1 IL-10 reporter expression in 10BiT mice

A small fraction of spleen CD19⁺ B cells (0.16±0.02%) from 10BiT mice expressed cell surface Thy1.1⁺ ex vivo relative to background staining in wild type mice (Fig. 2A–B). However, significantly increased Thy1.1⁺ (0.9±0.1%, p<0.01) and IL-10⁺ (1.8±0.4%, p<0.05) B10 cell frequencies were found after 5 h L+PIM stimulation. Only 30±2% of IL-10⁺ B cells from 10BiT mice expressed measurable Thy1.1 in these assays, while 47±4% of the Thy1.1⁺ B cells expressed IL-10 (Fig. 2A–C). Mesenteric lymph nodes had the highest frequencies of Thy1.1⁺ B cells (2.1±0.2%) when observed directly ex vivo (not shown), as shown for T cells in mesenteric lymph nodes of 10BiT mice (20). Mesenteric lymph node Thy1.1⁺ B10 cell frequencies were also higher following 5 h L+PIM stimulation, but the highest numbers of Thy1.1⁺ B cells were in the spleen (Fig. 2D). To determine whether the il10 and thy1.1 genes were transcribed with similar kinetics in 10BiT spleen B cells, their transcripts were measured after in vitro LPS stimulation. Both transcript levels rose congruently in CD1d^hiCD5⁺ B cells and peaked at 24 h relative to CD1d^loCD5⁻ cells (Fig. 2E). Thus, the temporal delay in cell surface Thy1.1 expression relative to cytoplasmic IL-10 was likely due to Thy1.1 processing and cell surface transport during the 5 h assays.

Figure 2. Divergent IL-10 and Thy1.1 expression by 10BiT B cells.

(A) B cell IL-10 production relative to cell surface Thy1.1 expression in 10BiT mice. Splenocytes were stimulated for 5 h before IL-10 and CD19 staining as in figure 1A. Representative contour plots show the IL-10⁺, IL-10⁺Thy1.1⁺ and Thy1.1⁺ cell frequencies within the indicated gates for CD19⁺ B cells. (B) Mean IL-10⁺ and Thy1.1⁺ B cell frequencies in wild type and 10BiT mice (n=5 mice/group) as in (A). (C) Representative IL-10 expression by Thy1.1⁺ B cells in 10BiT mice. Thy1.1⁺ and Thy1.1⁻ CD19⁺ B cells were assessed for IL-10 expression (thick lines) relative to control mAb staining (shaded histograms) after 5 h cultures with L+PIM (n=5 mice) as in (A). (D) Mean frequencies and numbers of tissue IL-10⁺ or Thy1.1⁺ B cells in spleen (SPL), lymph nodes (PLN), or mesenteric lymph node (MLN) of 10BiT mice (n≥3 mice) as in (A). (E) Relative il10 and thy1.1 transcript expression by B cells from 10BiT mice. Purified CD1d^hiCD5⁺ (black boxes) and CD1d^loCD5⁻ (empty boxes) CD19⁺ B cells were cultured alone or with LPS for 5, 24 and 48 h prior to RNA isolation and reverse transcriptase quantitative real-time PCR analysis. Values were normalized to the CD1d^loCD5⁻ population at each time point, with relative values shown as mean frequencies from 3 experiments. (F) Thy1.1 expression by B10+B10pro cells from 10BiT mice. Contour plots and bar graphs (representative of two experiments) show mean frequencies of Thy1.1⁺ spleen CD19⁺ B cells from wild type (background controls) and 10BiT mice (n≥3 mice/group) after 48 h cultures with the indicated stimuli as in figure 1E. (B, D, F) Significant differences between cultures with media alone or between the indicated values are indicated: **p≤0.01. Unless indicated, all experiments were performed ≥3 times.

CD40-induced B10pro cell maturation did not induce nascent cell surface Thy1.1 expression or change the kinetics of Thy1.1 expression induced by PIM stimulation. A normal portion of B cells cultured with CD40 mAb for 48 h expressed cytoplasmic IL-10 after L+PIM stimulation for 5 h, while Thy 1.1 expression was only modestly induced (Fig. 2F). However, a higher fraction of 10BiT B cells expressed Thy1.1 than expressed IL-10 after 48 h cultures with LPS plus 5 h PIM stimulation. Thus, cell surface Thy1.1 expression served as a more durable marker than IL-10 induction, with a large portion of the B10 cells having terminated IL-10 expression during the 48 h LPS cultures.

LPS drives B10 cell expansion in vivo

To evaluate B10 cell expansion in vivo, wild type mice were given complete and incomplete Freund's adjuvants, alum, or low-dose LPS, with spleen B10 cell numbers enumerated 3 days later by IL-10 staining after 5 h monensin or L+PIM treatment. Freund's adjuvants did not drive B10 cell expansion, while B10 cell numbers increased 2- to 3-fold after alum and LPS treatments (Fig. 3A). When Tiger mice were given LPS, ex vivo IL-10⁺ or GFP⁺ B10 cell frequencies and numbers remained low, but expanded 2- to 4-fold relative to their frequencies in littermates given only PBS (monensin treatment, Fig. 3B). Following 5 h of in vitro L+PIM stimulation, there were 2- to 3-fold increases in IL-10⁺ or GFP⁺ B10 cell frequencies and numbers relative to control mice, with most B10 cells expressing both IL-10 and GFP. Thus, GFP served as a reliable reporter for IL-10 expression in Tiger mice.

Figure 3. B10 cells expand after in vivo LPS treatment.

(A) Alum and LPS drive B10 cell expansion in vivo. Spleen B10 cell numbers were quantified as in figure 1, three days after PBS, CFA, IFA, alum or LPS treatment. Values represent mean frequencies or numbers of IL-10⁺ CD19⁺ B cells from one of two experiments with similar results (n≥3 mice/group/experiment). (B) LPS drives GFP⁺ B10 cell expansion in Tiger mice. Representative contour plots show IL-10 and GFP expression by spleen CD19⁺ B cells 3 days after PBS or LPS treatment. B cells were cultured with monensin alone or L+PIM for 5 h before IL-10 and GFP analysis as in figure 1A. Bar graphs show mean frequencies or numbers of IL-10⁺ or GFP⁺ B cells from PBS- (d 3) or LPS-treated (days 1–3) mice (≥3 mice/group). (C) LPS treatment drives Thy1.1⁺ B10 cell expansion in 10BiT mice. Representative contour plots and bar graphs indicate frequencies and total numbers of IL-10⁺ or Thy1.1⁺ B cells from 10BiT mice (3–4 mice per group) as assessed in (B). (A–C) Means significantly different from PBS-treated control mice are indicated: *p≤0.05, **p≤0.01. Data presented in Fig. 3B–C were pooled from 3 independent experiments. (D) Ex vivo cell surface phenotype of B cells from wild type, Tiger or 10BiT mice. Spleen B cells were isolated 3 days after LPS treatment, with subsequent L+PIM stimulation for 5 h before cell surface staining. Open histograms (thick lines) represent the IL-10⁺, GFP⁺ or Thy1.1⁺ B cell subsets, while shaded histograms represent IL-10⁻, GFP^- or Thy1.1⁻ B cells, as indicated. Similar results were obtained in 2 experiments.

After 3 days of LPS-treatment in vivo, Thy1.1⁺ and IL-10⁺ B cell frequencies and numbers in 10BiT mice increased by 4- and 2-fold, respectively (Fig. 3C). However, the higher frequencies and numbers of Thy1.1⁺ B cells relative to IL-10⁺ cells demonstrated that Thy1.1 expression served as a more durable B cell marker than IL-10 expression since half of the cells had already lost the capacity to express IL-10 following in vitro L+PIM stimulation. Thus, ongoing and terminated IL-10 production in vivo was reported by B cell Thy1.1 expression in 10BiT mice.

B10 cells differentiate into ASCs following IL-10 production in vivo

After in vivo low-dose LPS treatment for 3 days, the phenotype of spleen IL-10⁺, GFP⁺ or Thy1.1⁺ B cells remained predominantly IgM^hiCD1d^hiCD5⁺CD19^hiCD23^lowCD38^hiB220^hi (Fig. 3D), consistent with the ex vivo phenotype of B10 cells from untreated wild type mice (8, 10). However, variable frequencies of LPS-induced B10 cells also expressed the CD43 and GL7 activation markers (31), suggesting that LPS drives a subset of the reporter-positive B10 cells towards an antibody-secreting cell (ASC) phenotype.

Spleen ASCs are predominantly found within the rare CD138^hiB220^int/lo B cell subset (27). However, CD138 staining is lost under the conditions used to visualize cytoplasmic IL-10⁺ cells. Therefore, Tiger and 10BiT mice were used to determine whether in vivo LPS treatment induced B10 cells to differentiate into ASCs. In Tiger mice, GFP⁺ B cells expanded in vivo after LPS treatment, but predominantly remained CD138^low (Fig. 4A). Rare GFP⁺ B cells (<2%) were found within the CD138^hiB220^int/lo B cell subset in untreated Tiger mice, with LPS inducing significant numbers of GFP⁺ B cells (16%, p<0.01) that peaked 1 day after LPS treatment and subsequently declined (Fig. 4B). By contrast, a significant portion of Thy1.1⁺ B cells (17–40%) in 10BiT mice expressed CD138 after 2–3 days of LPS treatment (Fig. 4A). Before receiving LPS, 14% of CD138^hiB220^lo B cells expressed Thy1.1, with almost half of the CD138^hiB220^lo B cells expressing Thy1.1 2 days after LPS treatment (Fig. 4C). Thus, Thy1.1⁺ B cells contributed significantly to the ASC pool following LPS treatment.

Figure 4. B10 cells differentiate into ASC in vivo.

(A) Representative spleen GFP⁺ or Thy1.1⁺ cell frequencies versus CD138 expression among B220^hi/int B cells in Tiger (left) and 10BiT (right) mice before (day 0) or 1–3 days following LPS treatment. Numbers within quadrants indicate means (n=3–5 mice). (B) Spleen CD138^hiB220^int/lo B cells in Tiger mice express GFP after LPS treatment in vivo. Representative contour plots show CD138^hiB220^lo B cell frequencies in Tiger mice before (day 0) or 1–3 days following LPS treatment. Representative histograms indicate GFP expression by CD138^hiB220^int/lo B cells at the same time points (heavy lines, lower panels) relative to CD138^hiB220^int/lo B cells from wild type mice as negative controls (shaded lines). Mean CD138^hiB220^int/lo B cell frequencies or percentages of reporter-positive cells within the indicated gates are shown with backgrounds subtracted (n=3–5 mice). (C) CD138^hiB220^int/lo B cells in 10BiT mice express Thy1.1 before and after LPS treatment in vivo. Representative contour plots and histograms are shown as in (B). (D) Thy1.1⁺ B10 cells secrete IgM in vitro. Purified spleen B cells from 10BiT mice given LPS 3 days earlier were sorted into Thy1.1⁺ or Thy1.1⁻ CD19⁺ cell fractions and cultured on ELISpot plates overnight to enumerate IgM-secreting cells from 3–8 individual mice. (A–D) Data are pooled from 3 independent experiments. (E) Thy1.1⁺ B10 cells express transcription factors associated with plasma cell differentiation. Spleen Thy1.1⁺ or Thy1.1⁻ CD19⁺ B cells were purified from 10BiT mice given LPS 3 days earlier, with relative transcription factor expression measured by reverse transcriptase quantitative real-time PCR. Bars indicate mean fold differences between Thy1.1⁺ B cells normalized to Thy1.1⁻ B cells from 3 experiments (n=5 mice/experiment). (F) B10 cells from wild type mice express blimp1 and irf4. Purified spleen CD1d^hiCD5⁺ and CD1d^loCD5⁻ B cells were stimulated with L+PI for 5 h (B10 cells) or were cultured with CD40 mAb for 48 h with L+PI added during the final 5 h (B10+B10pro cells). Values indicate mean fold differences between CD1d^hiCD5⁺ and CD1d^loCD5⁻ B cells (n=3 mice). (G) IL-10⁺ B10 cells from wild type mice express blimp1. B cells were stimulated with L+PI for 5 h before IL-10⁺ and IL-10⁻ CD19⁺ B cells were purified. Values indicate mean fold differences between IL-10⁺ and IL-10⁻ B cells (n=3 mice). (F–G) il10, irf4 and blimp1 transcripts were quantified as in (E). (H) Intracellular Blimp-1 expression by spleen IL-10⁺, IL-10⁻ or monensin only-treated B cells following 5 h of L+PIM stimulation. (I) Intracellular Blimp-1 levels in IL-10⁺, IL-10⁻ or monensin only treated cells following 24 h LPS stimulation with PIM added during the final 5 h. (H–I) Mean MFI values for the indicated populations are shown (n=3 mice). (D–I) Significant differences between means are indicated: *p≤0.05, **p≤0.01.

Since some pre-B cells, immature B cells and plasma cells express CD43, GL-7, and CD138 (32), an association between B10 cells and ASCs was more rigorously tested. Thy1.1⁺ B10 cells purified from LPS-treated 10BiT mice spontaneously secreted IgM in ELISpot assays at 5.5-fold higher frequencies than Thy1.1⁻ B cells (Fig. 4D). IgG-secreting cells were not detectable within the Thy1.1⁺ or Thy1.1⁻ B cell subsets under these conditions (not shown). Furthermore, Thy1.1⁺ B cells from LPS-treated 10BiT mice expressed transcripts for the plasma cell-associated transcription factors blimp1 (also known as prdm1), xbp1 and irf4 at 2- to 6-fold higher levels than Thy1.1⁻ B cells (Fig. 4E). Likewise, pax5 and bcl6 transcripts were markedly reduced in Thy1.1⁺ B cells relative to Thy1.1⁻ B cells, suggesting that reporter-positive B10 cells adopt an ASC or plasma cell fate.

B10 cell Blimp-1 expression was also measured during IL-10 induction. CD1d^hiCD5⁺ B cells (B10 cell-enriched) from wild type mice expressed significantly higher il10 and blimp1 transcript levels relative to CD1d^loCD5^- B cells after 5 h of L+PI stimulation (Fig. 4F). Similarly, CD1d^hiCD5⁺ B cells cultured with CD40 mAb for 48 h expressed significant il10, blimp1 and irf4 transcripts relative to CD1d^loCD5⁻ B cells following 5 h of L+PI stimulation. Independently, blimp1 transcripts were significantly increased in purified IL-10⁺ B10 cells when compared with IL-10⁻ B cells after 5 h of L+PI stimulation (Fig. 4G). Measurable B10 cell intracellular Blimp-1 protein expression was confirmed by immunofluorescence staining in comparison with non-B10 cells (Fig. 4H) using described methods (33). Intracellular Blimp-1 expression increased when purified B cells were cultured in the presence of LPS for 24 h, with ~2-fold higher Blimp-1 levels in IL-10⁺ B cells than in IL-10⁻ B cells (Fig. 4I). Thus, B10 cells expressed Blimp-1 before initiating the ASC differentiation program.

IL-10 is not required for B10 cell ASC differentiation

IL-10 induces human plasma cell differentiation in vitro (34–36). To determine whether autocrine IL-10 drives mouse B10 cell development or differentiation, the 10BiT transgene was bred into an IL-10^−/− background to create 10BiT.IL-10^−/− mice. Spleen Thy1.1⁺ B cell frequencies were identical in both 10BiT and 10BiT.IL-10^−/− mice after in vitro stimulation with agonistic CD40 mAb or LPS for 48 h (Fig. 5A). Identical frequencies of IgM ASCs were also found within the spleen Thy1.1⁺ subsets of 10BiT and 10BiT.IL-10^−/− mice following in vivo LPS treatment (Fig. 5B). ASC frequencies within the spleen CD1d^hiCD5⁺ subset were also equivalent in LPS-treated IL-10^−/− and wild type mice, with the B10 cell-enriched CD1d^hiCD5⁺ B cells containing a higher frequency of ASCs when compared with CD1d^loCD5⁻ B cells. Thus, autocrine IL-10 was not required for either B10 cell development or ASC differentiation.

Figure 5. B10 cells produce Ag-specific antibody and autoantibodies.

(A) IL-10 is not required for B10+B10pro cell development in 10BiT mice. Splenocytes from 10BiT or IL-10^−/−10BiT mice were cultured for 48 h with media alone, CD40 mAb, or LPS, with the frequency of Thy1.1⁺ B10+B10pro cells determined as in figure 1E. Representative contour plots show CD19⁺ B cells from LPS-stimulated cultures. Bar graphs indicate relative mean frequencies of Thy1.1⁺ cells among CD19⁺ B cells (n=3 mice/group). (B) IL-10 expression is not required for B10 cell differentiation into ASCs. 10BiT or wild type mice (open bars) and IL-10^−/−10BiT or IL-10^−/− mice (filled bars) were given LPS 3 days before relative ASC frequencies were determined among Thy1.1⁺ or Thy1.1⁻ subsets from 10BiT mice and CD1d^hiCD5⁺ or CD1d^loCD5⁻ subsets from wild type mice as in fig. 4D (n=3 mice/group, data represent 2 experiments). (C) B10 cell expression of cell surface IgG and IgA. Spleen B cells from wild type mice were stimulated with L+PIM for 5 h before staining for IL-10 and cell surface IgG and IgA. Bar graphs show mean frequencies of B cells expressing each isotype (n=8 mice/group) from 2 experiments. (D) B10 cells from Tiger mice can secrete IgM. Purified spleen CD19⁺ B cells from Tiger mice were stimulated for 5 h with L+PI before GFP⁺ and GFP⁻ B cells were isolated by cell sorting. After 18 h of culture with LPS, the cells were cultured on ELISpot plates for 5 h. Bar graphs show mean IgM ASC frequencies (n=3 mice/group). (E) B10 cells can secrete Ag-specific IgM and IgG. Tiger mice were immunized with TNP-KLH plus alum, or PBS plus alum. Spleen TNP-specific IgM and IgG ASCs were quantified 7 days later using ELISpot assays as in (B). Bar graphs indicate mean ASC frequencies from 2 PBS- and 3 TNP-immunized mice in 2 experiments. (F) B10 cells contribute to serum antibody titers in vivo. In 2 experiments, purified spleen B cells from 4 or 8 Tiger mice were pooled and cultured overnight (18 h) with LPS, followed by 5 h stimulation with L+PI to induce GFP expression. Cell sorter purified GFP⁺ (closed squares) and GFP⁻ (open squares) B cells were then transferred into 5 and 6 Rag2^−/− recipients, respectively. Serum was collected at the indicated times, with antibody levels quantified by ELISA. Background IgM and IgG levels were determined using serum from untreated Rag2^−/− mice (dashed lines). (G) Reactivity of antibodies produced by B10 cells. Serum from Rag2^−/− mice given GFP⁺ (closed squares) or GFP⁻ (open squares) B cells 10 days earlier (as in D) was analyzed for reactivity with the indicated Ags by ELISA. Positive and negative controls included pooled sera from two-month-old wild type mice before (closed triangles) and 7 days after (diamonds) TNP-KLH-immunization, 10-month-old CD22^−/− mice (open circles), and a 6-month-old female MRL^lpr mouse (open triangles). Values indicate results from individual mice. (A–G) Means significantly different between groups are indicated: *p≤0.05, **p≤0.01.

B10 cells differentiate into IgM and IgG ASCs

Although spleen B10 cells are predominantly cell surface IgM^hi (Fig. 3D), B10 cells co-expressing IgG2c, IgG3 and IgA were over-represented in the B10 cell subset relative to non-B10 cells (Fig. 5C). The relative contribution of B10 cells to the ASC pool was therefore assessed using GFP⁺ B10 cells purified from Tiger mice. Spleen B cells were stimulated for 5 h with L+PI to induce GFP expression, sorted into GFP⁺ and GFP⁻ fractions, and cultured overnight with LPS prior to ELISPOT analysis. Consistent with the B10 cell ASC potential demonstrated in 10BiT mice (Fig. 4D), GFP⁺ B10 cells were also a major source of IgM ASCs (Fig. 5D). Thus, a large portion of B cells in both Tiger and 10BiT mice produced IL-10 prior to ASC differentiation.

To determine whether B10 cells produce Ag-specific antibody, Tiger mice were immunized with the T cell-dependent Ag 2,4,6-trinitrophenol-conjugated keyhole limpet hemocyanin (TNPKLH) in alum. Seven days later, spleen B cells were stimulated for 5 h with L+PI to induce GFP expression, with purified GFP⁺ and GFP⁻ cells assessed for anti-TNP IgM and IgG ASC potential. GFP⁺ B cells from both unimmunized and TNP-immunized Tiger mice produced TNP-reactive IgM, indicating that some reactivity was attributable to polyreactive or natural antibodies (Fig. 5E). TNP-reactive IgG was only produced by GFP⁺ B cells from immunized mice. Thereby, B10 cells produced both polyreactive IgM and Ag-specific IgM and IgG.

B10 cells contribute to serum antibody levels

To determine whether B10 cells contribute to serum Ig, equal numbers of spleen GFP⁺ B10 cells or GFP⁻ non-B10 cells were transferred from unimmunized Tiger mice into Rag2^−/− hosts. Serum IgM and IgG were first detected in mice given GFP⁺ cells after 1 and 4 days, respectively, and increased thereafter (Fig. 5F). In mice receiving non-B10 cells, IgM and IgG were detected after 4 and 6 days, respectively. At day 10 post-transfer, serum IgM levels from Rag2^−/− mice that had received GFP⁺ B10 cells were significantly higher than those of untreated Rag2^−/− mice controls or Rag2^−/− mice given non-B10 cells. Serum IgG levels in Rag2^−/− recipients given either B10 or non-B10 cells were below the levels found in wild type mice (Fig. 5G). Rag2^−/− recipients given B10 cells produced IgM but not IgG antibodies reactive with TNP, further confirming that B10 cells produce polyreactive IgM. Serum IgM from these mice also reacted with nuclear Ags, including single- and double-stranded DNA and histone proteins. IgM or IgG autoantibodies were not detected in sera from Rag2^−/− mice given non-B10 cells. Thus, B10 cells contributed to the serum IgM and IgG pools, including IgM antibodies with autoreactive/polyreactive specificities.

B10 cells express diverse Ag receptors

PCR methods were used to obtain an unbiased representation of the IgH and IgL repertoires of single IL-10⁺λ⁻ CD19⁺ cells from wild type mice. Both H and L chain transcripts revealed the utilization of diverse V_H and V_K family members (Fig. 6, Tables I–II). V_H1 (J558) was the most frequently observed V_H family, reflecting the predominance of this family within the Ig locus. Germline sequences without mutations encoded 84% of 50 representative V_H-D-J_H sequences and 91% of 69 representative V_K-J_K sequences. Thereby, B10 cells express diverse BCRs that were predominantly germline-encoded.

Figure 6. B10 cells utilize diverse V genes that are largely unmutated.

(A) V_H family gene usage by 50 representative IL-10⁺ B cells from 3 individual mice. Mutation frequencies within the V_H-D-J_H gene sequences are shown on the right. (B) V_K gene family usage by 69 representative IL-10⁺ B cells. V_K-J_K mutation frequencies are shown on the right. (C) Phylogenetic tree showing relationships between the V_H-D-J_H amino acid sequences of individual B cells from mice named A–C with numbers indicating different B cells. Branches indicate the average distance between two sequences based on percent identity. (D) Phylogenetic tree showing the relationship between the V_K-J_K amino acid sequences of individual B cells.

Table II.

B10 cell V_K-J_K sequences.

Cell	V Gene	J Gene	V End	P	N	P	J End	CDR3 Translation	Mutation Locations
A2	bt20	JK4	TGT TTGCAAAGTGATAACTTGCCT		C		TCACG	LQSDNLPLT
A3	cr1	JK2	TGC TTTCAAGGTTCACATGTTCC	G			TACACG	FQGSHVPYT
A5	cp9	JK2	TGT CAGCAGTATAGTAAGCTTCC	G			TACACG	QQYSKLPYT
A7	kn4	JK5	TGC CATCAGCGGAGTAGTTA(C)				ACG	HQRSSYT
A13	bt20	JK5	TGT TTGCAAAGTGATAACTTGCCT				CTCACG	LQSDNLPLT
A15	21–12	JK5	TGT CAGCACAGTAGGGAGCTTCC	G			CTCACG	QHSRELPLT
A17	8–34	JK2	TGT CAGCAGTCCTACAGCGCTCC	G			TACACG	QQSYSAPYT
A22	kn4	JK5	TGC CATCAGCGGAGTAGTTA(C)				ACG	HQRSSYT
A23	bt20	JK2	TGT TTGCAAAGTGATAACTTGCC	G			TACACG	LQSDNLPYT
A24	21–10	JK5	TGT CAGCAAAATAATGAGGATCC	G			CTCACG	QQNNEDPLT
A25	8–34	JK1	TGT CAGCAATATTATAGCTATCC				GACG	QQYYSYPT	T88C, FWR1
A26	fl12	JK2	TGT CAAAATGTGTTAAGTACTCCT				TACACG	QNVLSTPYT	G313A, J region
A27	fl12	JK1	TGT CAAAATGTGTTAAGTACTCC	G			TGGACG	QNVLSTPWT
A28	ba9	JK2	TGT CTACAGTATGATGAGTTTCC	G			TACACG	LQYDEFPYT
A29	at4	JK5	TGC CAGCAGTGGAGTAGTTACCC	G			CTCACG	QQWSSYPLT
A30	21–2	JK1	TGT CAGCAAAGTAAGGAGGTTCC	G			TGGACG	QQSKEVPWT
A31	21–12	JK5	TGT CAGCACAGTAGGGAGCTTCC	G			CTCACG	QHSRELPLT
A32	8–28	JK2	TGT CAGAATGATCATAGTTATCC	G			TACACG	QNDHSYPYT
A33	cf9	JK2	TGT GTACAGTATGCTCAGTTTCC	G			TACACG	VQYAQFPYT
A34	cf9	JK2	TGT GTACAGTATGCTCAGTTTCC	G			TACACG	VQYAQFPYT
A35	cr1	JK1	TGC TTTCAAGGTTCACATGTTCC		TC		GGACG	FQGSHVPRT
A36	cr1	JK1	TGC TTTCAAGGTTCACATGTTCC	G			TGGACG	FQGSHVPWT
A37	gn33	JK2	TGT CAACAGTATTGGAGTACTCC	G			TACACG	QQYWSTPYT
A38	19–32	JK1	TGT CAGCAGGATTATAGCTCTCC	G			TGGACG	QQDYSSPWT
A39	bb1	JK2	TGC TCTCAAAGTACACATGTTCC	G			TACACG	SQSTHVPYT
A40	bd2	JK1	TGC TGGCAAGGTACACATTT		G		TGGACG	WQGTHLWT
A41	bb1	JK2	TGC TCTCAAAGTACACATGTTCC	G			TACACG	SQSTHVPYT
A42	ce9	JK1	TGC CAACAGGGTAATACGCTGCTTCCT			C	GGACG	QQGNTLPRT
A43	bd2	JK1	TGC TGGCAAGGTACACATTTTCCT		CA		GACG	WQGTHFPQT
A44	n12–46	JK2	TGT CAACATTTTTGGGGTACTCC	G			TACACG	QHFWGTPYT
A45	fl12	JK2	TGT CAAAATGTGTTAAGTACTCCT		CCG		TACACG	QNVLSTPPYT
A46	ba9	JK2	TGT CTACAGTATGATGAGTTTCC	G			TACACG	LQYDEFPYT
A47	21–5	JK2	TGT CAGCAAAGTAATGAGGATCC	G			TACACG	QQSNEDPYT
A48	ap4	JK4	TGC CAGCAAAGGAGTAGTTACCCA				TTCACG	QQRSSYPFT
A49	19–15	JK4	TGT CAGCAATATAACAGCTATCC			A	TTCACG	QQYNSYPFT
A51	bb1	JK2	TGC TCTCAAAGTACACATGTTCC	G			TACACG	SQSTHVPYT
A52	23–43	JK4	TGT CAACAGAGTAACAGCTGGCC			A	TTCACG	QQSNSWPFT
A53	ce9	JK1	TGC CAACAGGGTAATACGCTTCCT		C	C	GACG	QQGNTLPPT
A54	bd2	JK2	TGC TGGCAAGGTACACATTTTCC	G			TACACG	WQGTHFPYT
A56	bd2	JK1	TGC TGGCAAGGTACACATTTTCC	G			TGGACG	WQGTHFPWT
B7	cw9	JK1	TGT CTACAATATGCTAGTTATCCT		C	C	GACG	LQYASYPPT
B8	bl1	JK2	TGC CTCCAAGTTACACATGTCCC	G			TACACG	LQVTHVPYT
B13	23–39	JK5	TGT CAAAATGGTCACAGCTTTCC	G			CTCACG	QNGHSFPLT
B14	19–32	JK4	TGT CAGCAGGATTATAGCTCTCC				CACG	QQDYSSPT
B15	RF	JK1	TGT CAACAGCATAATGAAT ACCCG(T)				GGACG	QQHNEYPWT
B16	8–24	JK1	TGT CAGCAACATTATAGCACTCC	G			TGGACG	QQHYSTPWT
B23	bb1	JK5	TGC TCTCAAAGTACACATGTTCC	G			CTCACG	SQSTHVPLT
B25	bb1	JK1	TGC TCTCAAAGTACACATGTTCCT		C	C	GACG	SQSTHVPPT
B26	gm33	JK1	TGT CAACAGTATTGGAGTACTCCT			C	GGACG	QQYWSTPRT
B28	ae4	JK4	TGC CATCAGTGGAGTAGTTACCCA				TTCACG	HQWSYPFT
B29	kh4	JK4	TGT CAACAGTGGAGTAGTTACCCATT(C)				ACG	QQWSSYPFT	C289A, CDR3
B30	cr1	JK1	TGC TTTCAAGGTTCACATGTTCC	G			TGGACG	FQGSHVPWT
B31	8–30	JK1	TGT CAGCAATATTATAGCTATCCT			C	GGACG	QQYYSYPRT	G151A, FWR3
B32	bd2	JK1	TGC TGGCAAGGTACACATTTTCCT			C	GGACG	WQGTHFPRT
B33	21–4	JK2	TGT CAGCAAAGTAATGAGGATCC	G			TACACG	QQSNEDPYT
B34	aa4	JK1	TGC CAGCAGTATCATAGTTACCCAC				GGACG	QQYHSYPRT
B35	bb1	JK1	TGC TCTCAAAGTACACATGT		G		TGGACG	SQSTHVWT
B37	23–43	JK5	TGT CAACAGAGTAACAGCTGGCCT		GC	G	CTCACG	QQSNSWPALT
B38	n8–30	JK1	TGT CAGCAATATTATAGCTATCCT			C	GGACG	QQYYSYPRT	C224T, FWR3; A322G, J region
B39	23–39	JK5	TGT CAAAATGGTCACAGCTTTCCT		CC		CACG	QNGHSFPPT
B40	cr1	JK2	TGC TTTCAAGGTTCACATGTTCC	G			TACACG	FQGSHVPYT
B41	bt20	JK2	TGT TTGCAAAGTGATAACTTGCC	G			TACACG	LQSDNLPYT
B43	19–32	JK1	TGT CAGCAGGATTATAGCTCTCCT		C	C	GACG	QQDYSSPPT
B44	bb1	JK2	TGC TCTCAAAGTACACATGTTCC	G			TACACG	SQSTHVPYT
B45	n12–46	JK1	TGT CAACATTTTTGGGGTACTCC	G			TGGACG	QHFWGTPWT
B47	cr1	JK1	TGC TTTCAAGGTTCACATGTTCC		T	C	GGACG	FQGSHVPRT
B48	bd2	JK1	TGC TGGCAAGGTACACATTTTCCT		CA		GACG	WQGTHFPQT	T66C, FWR1; G217A, FWR3
B49	bd2	JK1	TGC TGGCAAGGTACACATTTTCC	G			TGGACG	WQGTHFPWT
B50	bd2	JK1	TGC TGGCAAGGTACACATTTTCC	G			TGGACG	WQGTHFPWT

V_K-J_K genes from single IL-10⁺ B cells were identified as in figure 6D–F. P, P nucleotide addition; N, N nucleotide addition; FWR, framework region.

Discussion

These results demonstrate that the B10 cell subset not only regulates inflammatory immune responses through the production of IL-10, but also maintains a capacity for plasma cell differentiation. Following a transient period of IL-10 production, a significant fraction of B10 cells initiated the genetic and phenotypic program leading to ASC differentiation in vitro and in vivo (Figs. 4 and 5). B10 cells not only produced Ag-specific antibodies and represented a significant source of serum IgM and IgG (Figs. 5D–F), but also contributed polyreactive and autoreactive antibody specificities (Fig. 5G), consistent with the broad diversity of their expressed BCRs (Fig. 6). Hence, B10 cells do not define a distinct B cell lineage committed exclusively to IL-10-dependent immunoregulation. Instead, Ag-specific in vivo signals select B10pro cells, which develop into IL-10-competent B10 cells that secrete IL-10 in response to Ag exposure and/or TLR signaling before plasma cell differentiation (Fig. 7). Thus, B10 cells not only regulate acute inflammation and immune responses by the transient production of IL-10, but may also have the capacity to clear their inducing Ags by producing polyreactive and/or Ag-specific antibody.

Figure 7. B10 cells regulate antibody production in vivo.

Model for B10 cell maturation and antibody production. Transient B10 cell IL-10 production parallels GFP expression in IL-10 reporter mice, while cell surface Thy1.1 expression is observed later and accumulates over time. Although other B10 cell fates are possible, some spleen B10 cells differentiate into ASC cells that predominantly produce IgM. Antibody production by B10-derived B cells may constitute a second wave of humoral regulation during immune responses.

The BCR repertoire of spleen B10 cells was remarkably diverse, involving a wide spectrum of V_H, D and J_H elements, normal frequencies of noncoded nucleotide (N) insertions, as well as considerable complementarity-determining region 3 diversity (Fig. 6, Tables I–II). Regulatory B10 cell BCRs were predominantly germline-encoded with no somatic mutations in most clones. Thereby, spleen B10 cell V_H utilization was similar to that observed for conventional spleen B cells (37) and did not exhibit the skewed pattern associated with peritoneal cavity B-1a cells (38, 39). While different selective and/or developmental forces may ultimately shape the regulatory B10 cell BCR repertoire, the current findings demonstrate that IL-10 competent B cells are generated in response to diverse foreign and self Ags, including a T cell-dependent Ag. Some B10 cells also produced “natural” IgM antibody that was characteristically polyreactive (Fig. 5E and G). Consistent with their IgM^hiIgD^lo phenotype (Fig. 3D) and ability to clonally expand rapidly in vitro (12, 17), it is likely that B10 cells contribute substantially to the short-lived plasma cell pool that develops rapidly following Ag encounter. Regulatory B10 cells also develop at normal frequencies in T cell-deficient mice (17), suggesting that many respond to T cell-independent Ags and are unlikely products of germinal center reactions. Germinal center-independent B cell isotype switching may apply to B10 cells as described (40, 41), although it remains possible that some B10 cells are recruited into germinal centers. Whether B10 cells re-enter the memory B cell pool after IL-10 production is also unknown since methods are not currently available to track B10 cells after they lose Thy1.1 expression. Regardless, B10 cell production of diverse antibody products following transient IL-10 production highlights their functional plasticity.

There were significant changes in B10 cell expression of the blimp1, xbp1, irf4, pax5 and bcl6 transcription factors following activation in vivo, which paralleled ASC differentiation (Fig. 4E). Upregulated B10 cell expression of blimp1 and irf4 (Fig. 4F–I) may be of considerable functional significance since these transcription factors cooperatively induce regulatory T cell differentiation and il10 gene expression (42). The Blimp-1 transcriptional repressor is well known for its role in promoting plasma cell differentiation (43), with IRF4 required for blimp1 expression (44). Blimp-1 may also exert its normal function as a transcriptional repressor and stop IL-10 expression during B10 cell differentiation into ASCs. Identifying the overlapping upregulation of il10, blimp1, and irf4 by B10 cells highlights the potential importance of these transcription factors for regulatory B10 cell function, although other B cells also upregulate blimp1 and irf4 as they differentiate.

Based on their unique phenotypes and ability to proliferate rapidly following mitogenic stimulation, it is likely that mouse and human regulatory B10 cells represent subsets of Ag-experienced B cells (12, 17). Despite high IgM expression by most B10 cells (Fig. 3D), some B10 cells have undergone isotype switching (Fig. 5C). Furthermore, B10 cells do not develop in transgenic mice with fixed Ag receptors and genetic alterations that regulate BCR signaling significantly influence B10 cell numbers (17, 45–47). Since only a small subset of B cells have the capacity to produce IL-10 in vivo or in vitro (Figs. 1–2) and not all ASC expressed IL-10 before differentiation (Fig. 4A–C), specific in vivo signals must be required to induce IL-10 competence. This may explain why potent BCR ligation alone does not induce B10pro cells to mature into B10 cells in vitro, but may instead drive these cells towards different functional programs (Figs. 1E and 2F) (17). Since neither CD40 nor MyD88 expression are absolutely required for B10 cell development in vivo (17), it is likely that these signals and polyclonal mitogens such as LPS expand B10pro and B10 cells subsequent to Ag encounter. Consistent with this, murine cytomegalovirus infection leads to the development of IL-10-expressing CD138^hi B cells by 7 days (48). Salmonella infection also results in the rapid development of IL-10-expressing CD138^hi B cells, which is maximal at day 1 post infection (49). Thereby, pathways that modify intrinsic BCR signals will drive IL-10 competence and B10 cell differentiation (16).

B10 cell antibody production in vitro and in vivo suggests that B10 cells contribute significantly to the serum IgM and IgG antibody pool after transient IL-10 secretion. The spleen marginal zone and B1a cell subsets also contribute significantly to antibody responses. In fact, spleen marginal zone B cells, by virtue of their preactivated state and topographical location, join B1 B cells to generate a wave of IgM producing plasmablasts during early responses to blood-borne antigens (50, 51). B10 cells also proliferate rapidly following in vitro or in vivo activation (12, 17) and rapidly convert to plasmablasts (Figs. 4–5). Since the regulatory B10 cell, B1a and marginal zone B cell subsets share overlapping cell surface markers, it is not currently possible to ascertain whether individual members of any one of these functionally- or phenotypically-defined subsets are the primary source of natural, polyreactive, autoreactive or antigen-specific antibody. Furthermore, B10pro cells cannot be identified apart from the CD1d^hiCD5⁺ subset of B cells, so it is not possible to remove B cells that have the functional capacity to become IL-10 competent from either the CD5⁺ B1a or the CD1d^hi marginal zone subsets for functional studies. Thus, B1a, marginal zone and B10 B cells share the capacity to produce antibodies in vivo and contribute to early innate and subsequent adaptive immune responses.

B10 cell antibody secretion may also contribute to their immunosuppressive functions in vivo. Soluble antibodies can quickly reduce Ag load and promote Ag clearance by opsonization or complement-mediated phagocytosis. In addition, bound antibody can directly interfere with Ag recognition by other cell types, effectively reducing the availability of activation signals via Ag neutralization. Autoantibodies can also be important negative regulators of intestinal inflammation and suppress colitis (52, 53). B10 cells may thus exhibit two waves of protection that are first IL-10- and subsequently antibody-dependent. For example, B10 cell IL-10 production inhibits the initial pathology associated with experimental autoimmune encephalomyelitis induction (9, 18), while others have defined a subsequent wave of B cell-mediated immunosuppression in this model that is both Ag-specific and enhanced by CD40 signals (4, 54). Since B10 cells can produce autoantibodies (Fig. 5G), it is possible that their antibody products reduce inflammation and disease through a second wave of Ag clearance. Also, B10 cells primarily produced germline-encoded IgM antibodies that are likely to be of low affinity and non-pathogenic, which may be optimally suited to neutralize self-Ags, preempt pathogenic IgG production, and contribute to the suppression of autoimmunity (Fig. 7). Consistent with this, treatment of MRL^lpr mice with unmutated IgM autoantibodies confers protection against lupus nephritis (55). Further characterization of the B10 cell repertoire will be important for understanding both B10 cell development and expansion, particularly during autoimmune disease. Defining the BCR ligands and other signals important for B10 cell expansion and subsequent antibody production may also lead to new therapies for treating both inflammatory and autoimmune conditions.

Table I.

B10 cell V_H-D_H-J_H sequences.

Cell	V Gene	VH	D	JH	V End	P	N	P	D	P	N	P	J End	CDR3 Translation	Mutation Locations
A1	7183.20.37	5	DFL16.1	JH2	TGT GCAAGG				TATTACTACGGTAGTAGCTAC		A		TTGACTAC	ARYYYGSSYIDY
A2	36–60.6.70	3	DSP2.x	JH2	TGT GCAAGAGA		TTCCC		ATAGTAACTAC	CCTTT			CTAC	ARDSHSNYPFY
A3	J558.39.129	1	DSP2.9	JH1	TGT GCAA(GA)				TGGTTAC		CGGT		CTACTGGTACTTCGATGTC	ARWLPVYWYFDV
A4	7183.a7.10	5	DST4.3	JH4	TGT GCAAGAC		CGGACGTGACGA		GG(GCTA)				TGGACTAC	ARPDVTRAMDY
A5	7183.20.37	5	DST4.3	JH2	TGT GCAAGG	GGAA	G	G	CAGCTCAGGCTAC		C		CTTTGACTAC	ARGRQLRLPFDY
A6	Q52.8.22	2	DSP2.2	JH3	TGT GCCAGACA		AGGG		GATTACGAC	T	CTC		CCTGGTTTGCTTAC	ARQGDYDSPWFAY
A7	J558.50.143	1	DFL16.1	JH3	TGT GCAAGA	G			ACTACGGTAGTAGCTAC		GATACTTC		C	ARDYGSSYDTS
A8	J558.53.146	1	DSP2.2	JH4	TGT GCAAGA		ACCCT		CTATGATTACG		GCC		CTATGGACTAC	ARTLYDYGPMDY
A9	J558.55.149	1	DSP2.x	JH4	TGT GCA				(A)GTAA		AG		GCTATGGACTAC	AVKAMDY
A10	36–60.8.74	3	DFL16.1	JH1	TGT GCAAGA		AATTTT	G	ATTACTACGGTAGTAG	TC	CT		TACTGGTACTTCGATGTC	ARNFDYYGSSPYWYFDV
A11	J558.67.166	1	DSP2.2	JH4	TGT GCAAGA	T	ATGT		CTATGATTACGAC	G	GACG		TGCTATGGACTAC	ARYVYDYDGRAMDY	T40C, FWR1
A12	7183.4.6	5	DSP2.2	JH2	TGT GCAAGA		GATGAGG		GATTACGAC		CT		TTTGACTAC	ARDEGLRPFDY	A78G, FWR1; G89A, CDR1
A13	J558.75.177	1	DSP2.2	JH3	TGT GCAAGA		G		ATGATTACG		GTC	G	CTGGTTTGCTTAC	ARDDYGRWFAY	G102A, C103A, A104C, FWR2
A14	J558.53.146	1	DSP2.8	JH3	TGT GCAAGA		GGG		GGTAACTAC	GTA	GT	A	TGGTTTGCTTAC	ARGGNYVWFAY	C40A, FWR1
A15	J606.1.79	6	DSP2.7	JH3	TGC ACAG		AAAGGAC		CTATGGTAAC		C		CCTGGTTTGCTTAC	TERTYGNPWFAY	T263A, FWR3
A16	7183.7.10	5	DSP2.9	JH1	TGT GCAAGA		CAGGCG		TCTATGATGGTTA		AGA		GGTACTTCGATGTC	ARQASMMVKRYFDV
A17	Q52.3.8	2	DSP2.9	JH3	TGT GCCAAACC	G			GATGGTTACTA(C)				TGGTTTGCTTAC	AKPDGYYWFAY
A18	VGAM3.8-1-57	9	DQ52	JH2	TGT GTAAG		GAG	G	CTAACTGGGA				ACT AC	VRRLTGNY
A19	J558.53.146	1	none	JH3	TGT GCAAGA		GA		none				TGCTTAC	ARDAY	C58T, FWR1
A20	VGAM3.8-3-61	9	DSP2.13	JH2	TGC GC(A)				CTAC		AGGGGCT		CTTTGACTAC	ALQGLFDY	T288C, FWR3
A21	J606.4.82	6	DFL16.1	JH2	TGT ACC(A)				CTACGGTAGTAGCT		GGGGAAGAC		TACTTTGACTAC	TTTVVAGEDYFDY
B1	J558.53.146	1	DSP2.9	JH2	TGT GCAAGA		GGG		GATGGTTACTAC		CCCCTCTAC		TACTTTGACTAC	ARGDGYYPLYYFDY
B2	J558.80.186	1	DST4	JH2	TGT GCAAGA		GACGAC		CAGGC				CTTTGACTAC	ARDDQAFDY
B3	J558.53.146	1	DSP2.9	JH2	TGT GCAAGA		GGG		GATGGTTACTAC		CCCCTCTAC		TACTTTGACTAC	ARGDGYYPLYYFDY
B4	VH10.3.91	10	DSP2.6	JH4	TGT GTGAGAGA	T	GATAGG		GGTTACGAC	G	GTGGA	T	ATTACTAT	VRDDRGYDGGYYY
B5	J558.53.146	1	DFL16.1	JH3	TGT GCAAGA		GGGG		ACTACGGTAGTAG	TC	TC		TTTGCTTAC	ARGDYGSSLFAY
B6	J558.80.186	1	DST4	JH2	TGT GCAAGA		GACGAC		CAGGC				CTTTGACTAC	ARDDQAFDY
B7	J606.1.79	6	DFL16.2	JH4	TGC ACAGG		A	A	ATTACT		C		C	TGNYS
B8	VH10.1.86	10	DSP2.8	JH2	TGT GT			C	GGTAA	T	G		TTGACTAC	VGNVDY
B9	VH11.2.53	11	DSP2.x	JH1	TGT ATGAGA(TA)				TAGTAA(CTAC)				TGGTACTTCGATGTC	MRYSNYWYFDV
B10	J558.55.149	1	DQ52	JH2	TGT GCAAGA		GGGGG		TAACTGGG		TCCT		CTTTGACTAC	ARGGNWVLFDY
B11	J558.50.143	1	DFL16.1	JH3	TGT GCAAGA		G		ACTACGGTAGTAGCTAC	G	ATACTTC		C	ARDYGSSYDTS
B12	VH11.2.53	11	DSP2.x	JH1	TGT ATGAGA(TA)				TAGTAA(CTAC)				TGGTACTTCGATGTC	MRYSNYWYFDV
B13	VH10.3.91	10	DSP2.9	JH2	TGT GTGAGAG		GGG		TCTATGATGGTTACTAC		C		TTGACTAC	VRGVYDGYYLDY
B14	SM7.3.54	14	DSP2.8	JH2	TGT G(CTAG)				G		GGGG		TTGACTAC	ARGVDY
B15	J558.77.180	1	DSP2.6	JH2	TGT GCAATA		G		AC(GAC)				TAC	AIDDY
B16	J558.72.173	1	DFL16.1	JH2	TGT GCAAGA		C		TACTACGGT		T		CTTTGACTAC	ARLLRFFDY
B17	VGAM3.8-3-61	9	DSP2.9	JH2	TGT GCAAGA	TCT	GTCGT		TTAC(TAC)				TTTGACTAC	ARSWYYFDY
B18	VH10.3.91	10	DQ52	JH2	TGT GTGAG		G		ACTGG			A	TTTGACTAC	VRTGFDYW
B19	Vh10.2b	10	DSP2.5	JH4	TGT GTGAGAC		T		TCTACTATGG		G	G	CTATGCTATGGACTAC	VRLLLWGYAMDY
B20	SM7.3.54	14	DFL16.1	JH4	TGT GCTAGA		AC		CGGTAGTAGC		CCCC		ATTACTATGCTATGGACTAC	ARTGSSPHYYAMDY
B21	S107.3.62	7	DQ52	JH1	TGT GCAAGA	T	TTCTCA		AACTGG		T		CTACTGGTACTTCGATGTC	ARFLKLVYWYFDV
B22	7183.4.6	5	DFL16.1	JH4	TGT GCAAGA		G	A	TATTACT		TTAGGG		GCTATGGACTAC	ARDITLGAMDY
B23	J558.53.146	1	DFL16.1	JH2	TGT GCAAGA	T	TTGGG		TACTACGGTAGTA	T			CTTTGACTAC	ARFGYYGSIFDY
B24	J558.50.143	1	DFL16.1	JH3	TGT GCAAGA	G			ACTACGGTAGTAG		TACGATAACTTC		C	ARDYGSSTITS
B25	7183.14.25	5	DQ52	JH4	TGT AC(AA)				CTGGGA(C)				TATGGACTAC	TTGTMDY
C1	VH11.2.53	11	DFL16.1	JH1	TGT AT		CCT		CTACGGTAGTAG(CTAC)				TGGTACTTCGATGTC	ILYGSSYWYFDV
C2	J606.1.79	6	DFL16.1	JH4	TGC ACAGG	C		A	TACTACGGT		CG		TATGCTATGGACTAC	TGILRSYAMDY
C3	J558.72.173	1	DFL16.1	JH4	TGT GCAAGA	TC			TTACTACGG		GACCCCC		TACTATGCTATGGACTAC	ARSYYGTPYYAMDY	T212G, FWR3
C4	VH10.3.91	10	DSP2.9	JH4	TGT GTGAGA(GA)				TGGTTACTA	T	TCCTT		CTATGCTATGGACTAC	VRDGYYSFYAMDY

V_H-D_H-J_H genes from single IL-10⁺ B cells were identified as in figure 6A–C. P, P nucleotide addition; N, N nucleotide addition; FWR, framework region.