The development and function of regulatory B cells expressing IL-10 (B10 cells) requires antigen receptor diversity and TLR signals.
Journal: 2009/June - Journal of Immunology
ISSN: 1550-6606
Abstract:
Autoimmunity and inflammation are controlled in part by regulatory B cells, including a recently identified IL-10-competent CD1d(high)CD5(+) B cell subset termed B10 cells that represents 1-3% of adult mouse spleen B cells. In this study, pathways that influence B10 cell generation and IL-10 production were identified and compared with previously described regulatory B cells. IL-10-competent B cells were predominantly CD1d(high)CD5(+) in adult spleen and were the prevalent source of IL-10, but not other cytokines. B10 cell development and/or maturation in vivo required Ag receptor diversity and intact signaling pathways, but not T cells, gut-associated flora, or environmental pathogens. Spleen B10 cell frequencies were significantly expanded in aged mice and mice predisposed to autoimmunity, but were significantly decreased in mouse strains that are susceptible to exogenous autoantigen-induced autoimmunity. LPS, PMA, plus ionomycin stimulation in vitro for 5 h induced B10 cells to express cytoplasmic IL-10. However, prolonged LPS or CD40 stimulation (48 h) induced additional adult spleen CD1d(high)CD5(+) B cells to express IL-10 following PMA plus ionomycin stimulation. Prolonged LPS or CD40 stimulation of newborn spleen and adult blood or lymph node CD1d(low) and/or CD5(-) B cells also induced cytoplasmic IL-10 competence in rare B cells, with CD40 ligation uniformly inducing CD5 expression. IL-10 secretion was induced by LPS signaling through MyD88-dependent pathways, but not following CD40 ligation. LPS stimulation also induced rapid B10 cell clonal expansion when compared with other spleen B cells. Thereby, both adaptive and innate signals regulate B10 cell development, maturation, CD5 expression, and competence for IL-10 production.
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J Immunol 182(12): 7459-7472

The Development and Function of Regulatory B Cells Expressing IL-10 (B10 cells) Requires Antigen Receptor Diversity and TLR Signals<sup><sup><a href="#FN3" rid="FN3" class=" fn">1</a></sup></sup>

Introduction

Immunological tolerance exemplifies the capacity of the immune system to down-modulate immune responses. B cells are generally considered to positively regulate immune responses by producing Ag-specific Ab and helping induce optimal CD4 T cell activation (1). However, B cells and specific B cell subsets can also negatively regulate immune responses in mice, validating the existence of regulatory B cells (28). The absence or loss of negative regulatory B cells exacerbates disease symptoms in contact hypersensitivity (CHS)4, experimental autoimmune encephalomyelitis (EAE), chronic colitis, and collagen-induced arthritis (CIA) models of autoimmunity and inflammation (918). Furthermore, IL-10-producing B cells down regulate autoimmune disease initiation, onset, or severity in EAE (13, 19), CIA (15), CHS (18), and inflammatory bowel disease (14). Although IL-10 can augment immune responses, it can also suppress both Th1 and Th2 polarization and inhibit Ag presentation and proinflammatory cytokine production by monocytes and macrophages (20). IL-10 production and/or regulatory B cell activities have been variably attributed to all B cells, CD5 B-1a cells, or cells with CD21CD23 marginal zone (MZ) or CD1dCD21CD23 T2-MZ precursor B cell phenotypes (17, 21, 22). B cells can also contribute to immunoregulation through the production of IL-4, IL-6, IFN-γ, and TGF-β (23, 24). Altogether, these studies have established that phenotypically-diverse regulatory B cells significantly influence immune responses.

A potent subset of regulatory B cells was recently found to regulate T cell-dependent CHS and EAE responses in an IL-10-dependent manner (18, 19). This phenotypically unique CD1dCD5CD19 subset of regulatory B cells shares overlapping cell surface markers with the CD5 B-1a, CD21CD23 MZ, and CD1dCD21CD23 T2-MZ precursor B cell subsets (17, 18, 21, 22), and is found within the spleens of naïve wild type mice at frequencies of 1–2% (18). These CD1dCD5 B cells are induced to express cytoplasmic IL-10 following 5 h in vitro stimulation with LPS, PMA, ionomycin, plus monensin (L+PIM), and are called B10 cells to distinguish them from other regulatory B cell subsets that may also exist and to identify them as the predominant source of B cell IL-10 production (25). B10 cell regulatory functions are Ag-restricted in vivo (18, 19), implying a requirement for Ag-specific BCR signaling. The adoptive transfer of Ag-primed B10 cells reduces inflammation during CHS responses and reduces EAE severity during disease onset (18, 19), and may also regulate other autoimmune diseases (25).

Regulatory B cells are commonly stimulated in vitro to induce their capacity to inhibit immune responses in mice with autoimmune disease. For example, the activation of arthritogenic splenocytes with collagen alone (17) or collagen plus agonistic CD40 mAb in vitro gives rise to B cells that produce high IL-10 levels and prevent arthritis (15). Transfusions of BCR-activated B cells also protect NOD mice from type 1 diabetes in an IL-10-dependent manner (26). LPS-activated B cells are also reported to prevent diabetes in NOD mice (24). Mouse B cells express toll-like receptor (TLR)-4 and RP-105 that bind LPS (27) and B10 cells produce IL-10 in response to LPS stimulation in vitro (18). BCR and CD40 engagement also appear to be required for IL-10-dependent regulatory B cell functions in CIA, CHS, and EAE models (13, 1719). Spleen B cells with a CD1dCD21CD23 “MZ” phenotype can also produce IL-10 in response to CpG stimulation in mice with lupus-like autoimmune disease (22). Based on these findings, Mizoguchi et al. have postulated that distinct regulatory B cell subsets might arise from separate follicular and MZ B cells through “innate type” (polyclonal stimulus-induced IL-10 production) or “acquired type” (Ag-specific IL-10 production) developmental pathways, respectively, or from B1 cells (2).

Identification of B10 cells as a phenotypically distinct, IL-10-producing B cell subset (18, 19) facilitates their characterization as either “innate type” or “acquired type” regulatory B cells. Whether B10 cells share the physiologic triggers that lead to their expansion and function as described for other regulatory B cells is unknown. It is also unknown whether B10 cells produce cytokines other than IL-10. Therefore, these issues and the factors that influence B10 cell generation in vivo and in vitro were examined in the current study with the finding that both adaptive and innate signals facilitate B10 cell generation, maturation, and optimal IL-10 production.

Material and Methods

Mice

Wild type C57BL/6 (B6), IL-10 (B6.129P2-Il10tmlCgn/J), NOD (NOD/Lt), DBA/1J, SJL/J, NZB/W F1 (NZBWF1/J), CD40 (B6.129P2-CD40tm1Kik/J), MRL/lpr (MRL/MpJ-Faslpr/J), MD4 (C57BL/6-Tg(TghelMD4)4Ccg/J) that express IgM and IgD specific for HEL (28), and nude (C57BL/6-Hfh11nu) mice were from the Jackson Laboratory (Bar Harbor, ME). MHC-I/II (B6.129-H2-Ab1B2mtmJaeN17 from Taconic Farms, Inc., Hudson, NY) mice were as described (29) and were provided by Y. Zhuang (Duke University, Durham, NC). MyD88 mice (30) were provided by Y. Yang (Duke University) with the permission of S. Akira (Osaka University, Osaka Japan). CD22, CD21, CD19, and hCD19Tg (h19-1 line) mice on a B6 genetic background were as described (3134). CD40L/BTg mice with B cells expressing cell surface CD40L were as described (35). CD40L/BTg/CD22 double mutant mice were generated by crossing CD40L/BTg mice with CD22 mice. B6 neonates were 3 to 10 days old. All mice were housed in a specific pathogen-free barrier facility and used at 12–16 wk of age, unless otherwise specified. All studies were approved by the Duke University Animal Care and Use Committee. Tissues from 6 mo-old gnotobiotic and specific-pathogen-free 129S6/SvEv mice were generously provided by Dr. Scott Plevy and the Univ. of North Carolina at Chapel Hill Center for Gastrointestinal Biology &amp; Disease Gnotobiotic Core.

Abs

Anti-mouse mAbs included: B220 mAb RA3-6B2 (provided by Dr. Robert Coffman, DNAX Corp., Palo, Alto, CA); and CD19 (1D3), CD5 (53–7.3), CD1d (1B1), CD40 (HM40-3), CD21/35 (7G6), CD23 (B3B4), CD24 (M1/69), CD43 (S7), and CD93 (AA4.1) mAbs from BD PharMingen (San Diego, CA). Anti-mouse IgM Ab was from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA). Phycoerythrin-conjugated anti-mouse IL-10 mAb (JES5-16E3) was from eBioscience (San Diego, CA).

B cell isolation, immunofluorescence analysis and cell sorting

Blood mononuclear cells were isolated from heparinized blood after centrifugation over a discontinuous Lymphoprep (Axis-Shield PoC As, Oslo, Norway) gradient. Single cell splenocyte suspensions were generated by gentle dissection with >90% cell viability as determined by trypan blue exclusion. Cell numbers were quantified using a hemocytometer, with relative lymphocyte percentages among viable cells (based on scatter properties) determined by flow cytometry analysis. B220- or CD19-mAb coated microbeads (Miltenyi Biotech) were used to purify spleen B cells by positive selection following the manufacturer’s instructions. When necessary, the cells were enriched a second time using a fresh MACS column to obtain >99% purities.

Single cell leukocyte suspensions were stained on ice using predetermined optimal concentrations of each Ab for 20–60 min, and fixed as described (32). Cells with the light scatter properties of lymphocytes were analyzed by 2–4 color immunofluorescence staining and FACScan or FACSCalibur flow cytometers (Becton Dickinson, San Jose, CA). Dead cells were excluded from the analysis based on their forward- and side-light scatter properties and the use of LIVE/DEAD Fixable Dead Cell Stain Kits (Invitrogen-Molecular Probes, Carlsbad, CA). All histograms are shown on a 4 decade logarithmic scale, with gates shown to indicate background isotype-matched control mAb staining set with <2% of the cells being positive. Background staining was determined using unreactive isotype-matched control mAbs (Caltag Laboratories, San Francisco, CA) with gates positioned to exclude ≥98% of unreactive cells. Spleen CD1dCD5, CD1dCD5, CD1dCD5 B cells were isolated using a FACSVantage SE flow cytometer (Becton Dickinson, San Jose, CA) with ~75%–95% purities.

Analysis of IL-10 production

Intracellular IL-10 analysis by flow cytometry was as described (18). Briefly, isolated leukocytes or purified cells were resuspended (2 × 10 cells/ml) in complete medium [RPMI 1640 media containing 10% FCS, 200 μg/ml penicillin, 200 U/ml streptomycin, 4 mM L-Glutamine, and 5 × 10 M 2-mercaptoethanol (all from Gibco, Carlsbad, CA)] with LPS (10 μg/ml, Escherichia coli serotype 0111: B4, Sigma), PMA (50 ng/ml; Sigma), ionomycin (500 ng/ml; Sigma), and monensin (2 μM; eBioscience) for 5 h, in 24-well flat-bottom plates. In some experiments, the cells were incubated for 48 h with LPS (10 μg/ml) and/or anti-mouse CD40 mAb (1 μg/ml), and/or anti-mouse IgM Ab (10 μg/ml, Jackson ImmunoResearch Laboratories, Inc, West Grove, PA). For analysis of cell proliferation, leukocytes were stained with CFSE Vybrant CFDA SE fluorescent dye (0.1 μM; CFSE; Invitrogen-Molecular Probes) according to the manufacturer’s instructions. For IL-10 detection, Fc receptors were blocked with mouse Fc receptor mAb (2.4G2; BD PharMingen) with dead cells detected by using a LIVE/DEAD® Fixable Green Dead Cell Stain Kit (Invitrogen-Molecular Probes) before cell surface staining. Stained cells were fixed and permeabilized using a Cytofix/Cytoperm kit (BD PharMingen) according to the manufacturer’s instructions and stained with phycoerythrin-conjugated mouse anti-IL-10 mAb. Leukocytes from IL-10 mice served as negative controls to demonstrate specificity and to establish background IL-10 staining levels.

Secreted IL-10 was quantified by ELISA. Purified B cells (4 × 10) were cultured in 0.2 ml of complete medium in a 96-well flat-bottom tissue culture plates. Culture supernatant fluid IL-10 concentrations were quantified using IL-10 OptEIA ELISA kits (BD PharMingen) following the manufacturer’s protocols. All assays were carried out using triplicate samples.

B cell cytokine transcript expression analysis

Purified spleen B cells were cultured for 5 h with LPS+PMA+ionomycin (L+PI). IL-10-secreting spleen B cells were identified using an IL-10 secretion detection kit (Miltenyi Biotech, Auburn, CA) with subsequent staining for CD19 expression before cell sorting into IL-10CD19 and IL-10CD19 populations. Total RNA was extracted from the purified B cells using TRIzol (Invitrogen-Molecular Probes), with relative cytokine transcripts quantified by GeneChip analysis (Affymetrix Mouse Genome 430 2.0 GeneChips; Affymetrix, Santa Clara, CA). All quality parameters for the arrays were confirmed to be in the range recommended by the manufacturer.

Statistical analysis

All data are shown as means (±SEM). Significant differences between sample means were determined using the Student’s t test.

Mice

Wild type C57BL/6 (B6), IL-10 (B6.129P2-Il10tmlCgn/J), NOD (NOD/Lt), DBA/1J, SJL/J, NZB/W F1 (NZBWF1/J), CD40 (B6.129P2-CD40tm1Kik/J), MRL/lpr (MRL/MpJ-Faslpr/J), MD4 (C57BL/6-Tg(TghelMD4)4Ccg/J) that express IgM and IgD specific for HEL (28), and nude (C57BL/6-Hfh11nu) mice were from the Jackson Laboratory (Bar Harbor, ME). MHC-I/II (B6.129-H2-Ab1B2mtmJaeN17 from Taconic Farms, Inc., Hudson, NY) mice were as described (29) and were provided by Y. Zhuang (Duke University, Durham, NC). MyD88 mice (30) were provided by Y. Yang (Duke University) with the permission of S. Akira (Osaka University, Osaka Japan). CD22, CD21, CD19, and hCD19Tg (h19-1 line) mice on a B6 genetic background were as described (3134). CD40L/BTg mice with B cells expressing cell surface CD40L were as described (35). CD40L/BTg/CD22 double mutant mice were generated by crossing CD40L/BTg mice with CD22 mice. B6 neonates were 3 to 10 days old. All mice were housed in a specific pathogen-free barrier facility and used at 12–16 wk of age, unless otherwise specified. All studies were approved by the Duke University Animal Care and Use Committee. Tissues from 6 mo-old gnotobiotic and specific-pathogen-free 129S6/SvEv mice were generously provided by Dr. Scott Plevy and the Univ. of North Carolina at Chapel Hill Center for Gastrointestinal Biology &amp; Disease Gnotobiotic Core.

Abs

Anti-mouse mAbs included: B220 mAb RA3-6B2 (provided by Dr. Robert Coffman, DNAX Corp., Palo, Alto, CA); and CD19 (1D3), CD5 (53–7.3), CD1d (1B1), CD40 (HM40-3), CD21/35 (7G6), CD23 (B3B4), CD24 (M1/69), CD43 (S7), and CD93 (AA4.1) mAbs from BD PharMingen (San Diego, CA). Anti-mouse IgM Ab was from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA). Phycoerythrin-conjugated anti-mouse IL-10 mAb (JES5-16E3) was from eBioscience (San Diego, CA).

B cell isolation, immunofluorescence analysis and cell sorting

Blood mononuclear cells were isolated from heparinized blood after centrifugation over a discontinuous Lymphoprep (Axis-Shield PoC As, Oslo, Norway) gradient. Single cell splenocyte suspensions were generated by gentle dissection with >90% cell viability as determined by trypan blue exclusion. Cell numbers were quantified using a hemocytometer, with relative lymphocyte percentages among viable cells (based on scatter properties) determined by flow cytometry analysis. B220- or CD19-mAb coated microbeads (Miltenyi Biotech) were used to purify spleen B cells by positive selection following the manufacturer’s instructions. When necessary, the cells were enriched a second time using a fresh MACS column to obtain >99% purities.

Single cell leukocyte suspensions were stained on ice using predetermined optimal concentrations of each Ab for 20–60 min, and fixed as described (32). Cells with the light scatter properties of lymphocytes were analyzed by 2–4 color immunofluorescence staining and FACScan or FACSCalibur flow cytometers (Becton Dickinson, San Jose, CA). Dead cells were excluded from the analysis based on their forward- and side-light scatter properties and the use of LIVE/DEAD Fixable Dead Cell Stain Kits (Invitrogen-Molecular Probes, Carlsbad, CA). All histograms are shown on a 4 decade logarithmic scale, with gates shown to indicate background isotype-matched control mAb staining set with <2% of the cells being positive. Background staining was determined using unreactive isotype-matched control mAbs (Caltag Laboratories, San Francisco, CA) with gates positioned to exclude ≥98% of unreactive cells. Spleen CD1dCD5, CD1dCD5, CD1dCD5 B cells were isolated using a FACSVantage SE flow cytometer (Becton Dickinson, San Jose, CA) with ~75%–95% purities.

Analysis of IL-10 production

Intracellular IL-10 analysis by flow cytometry was as described (18). Briefly, isolated leukocytes or purified cells were resuspended (2 × 10 cells/ml) in complete medium [RPMI 1640 media containing 10% FCS, 200 μg/ml penicillin, 200 U/ml streptomycin, 4 mM L-Glutamine, and 5 × 10 M 2-mercaptoethanol (all from Gibco, Carlsbad, CA)] with LPS (10 μg/ml, Escherichia coli serotype 0111: B4, Sigma), PMA (50 ng/ml; Sigma), ionomycin (500 ng/ml; Sigma), and monensin (2 μM; eBioscience) for 5 h, in 24-well flat-bottom plates. In some experiments, the cells were incubated for 48 h with LPS (10 μg/ml) and/or anti-mouse CD40 mAb (1 μg/ml), and/or anti-mouse IgM Ab (10 μg/ml, Jackson ImmunoResearch Laboratories, Inc, West Grove, PA). For analysis of cell proliferation, leukocytes were stained with CFSE Vybrant CFDA SE fluorescent dye (0.1 μM; CFSE; Invitrogen-Molecular Probes) according to the manufacturer’s instructions. For IL-10 detection, Fc receptors were blocked with mouse Fc receptor mAb (2.4G2; BD PharMingen) with dead cells detected by using a LIVE/DEAD® Fixable Green Dead Cell Stain Kit (Invitrogen-Molecular Probes) before cell surface staining. Stained cells were fixed and permeabilized using a Cytofix/Cytoperm kit (BD PharMingen) according to the manufacturer’s instructions and stained with phycoerythrin-conjugated mouse anti-IL-10 mAb. Leukocytes from IL-10 mice served as negative controls to demonstrate specificity and to establish background IL-10 staining levels.

Secreted IL-10 was quantified by ELISA. Purified B cells (4 × 10) were cultured in 0.2 ml of complete medium in a 96-well flat-bottom tissue culture plates. Culture supernatant fluid IL-10 concentrations were quantified using IL-10 OptEIA ELISA kits (BD PharMingen) following the manufacturer’s protocols. All assays were carried out using triplicate samples.

B cell cytokine transcript expression analysis

Purified spleen B cells were cultured for 5 h with LPS+PMA+ionomycin (L+PI). IL-10-secreting spleen B cells were identified using an IL-10 secretion detection kit (Miltenyi Biotech, Auburn, CA) with subsequent staining for CD19 expression before cell sorting into IL-10CD19 and IL-10CD19 populations. Total RNA was extracted from the purified B cells using TRIzol (Invitrogen-Molecular Probes), with relative cytokine transcripts quantified by GeneChip analysis (Affymetrix Mouse Genome 430 2.0 GeneChips; Affymetrix, Santa Clara, CA). All quality parameters for the arrays were confirmed to be in the range recommended by the manufacturer.

Statistical analysis

All data are shown as means (±SEM). Significant differences between sample means were determined using the Student’s t test.

Results

IL-10-producing B cells preferentially secrete IL-10

Spleen B cells that are competent to express cytoplasmic IL-10 following 5 h L+PIM stimulation were predominantly found within the CD1dCD5 CD19 subset in wild type B6 mice (Fig. 1A), as described (18, 19). By contrast, IL-10 expressing B cells were significantly less common within the CD1dCD5, CD1dCD5, or CD1dCD5 B cell subsets (p<0.01), with B cells from IL-10 mice used as negative controls for background IL-10 staining. We have previously shown that 5 h L+PIM stimulation does not influence the phenotype of these B cell subsets (18). IL-10CD19 B10 cells were predominantly CD21, CD23, CD24, CD43, and CD93 (AA4.1) (Fig. 1B). Thereby, spleen B10 cells are relatively rare and share some overlapping phenotypic markers with the B-1a, MZ, and T2-MZ precursor B cell subsets, but are nonetheless phenotypically distinct, (18, 19).

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B10 cells preferentially secret IL-10. (A) IL-10-producing B cells were predominantly found within the CD1dCD5CD19 B cell subset. Splenocytes from wild type and IL-10 mice were cultured with L+PIM for 5 h, then stained with CD1d, CD5, and CD19 mAb before permeabilization and staining using IL-10 mAb. Percentages and bar graphs indicate mean (±SEM) B cell subset frequencies and numbers among CD19 splenocytes or IL-10 cell frequencies among the indicated B cell subsets (a, CD1dCD5; b, CD1dCD5; c, CD1dCD5; d, CD1dCD5) from 3 mice as determined by flow cytometry analysis. Values significantly different from background frequencies or numbers for IL-10 mice are indicated: *, p<0.05; **, p<0.01. (B) CD21, CD23, CD24, CD43, and CD93 expression by IL-10-producing (thick line) and IL-10 (thin line) CD19 spleen B cells from wild type mice cultured with L+PIM for 5 h, then stained for cell surface antigens before permeabilization and cytoplasmic IL-10 staining. Gray histograms represent isotype-matched control mAb staining. Results are representative of those obtained with B cells from ≥3 mice as determined by flow cytometry analysis. (C) IL-10-producing B cells from hCD19Tg mice are predominantly found within the CD1dCD5CD19 B cell subset. Staining and analysis was as described in (A). (D) Representative isolation of IL-10-secreting B cells. Splenic B220 cells purified from three hCD19Tg mice were pooled and cultured with L+PI for 5 h before staining for CD19 and secreted IL-10 capture (left panel). Cytoplasmic IL-10 and IL-10 B cells were isolated by cell sorting using the indicated gates and subsequently reassessed for IL-10 secretion and CD19 expression (right panels). (E) Cytokine gene expression by IL-10-secreting and non-secreting B cells purified as in (D). Mean fold-differences (±SEM) in cytokine transcript levels (IL-10/IL-10 cells) from 3 independent experiments are shown. Values of 1 (dashed line) indicate no difference in cytokine expression between the IL-10 and IL-10 B cells, with significant differences indicated: **, p<0.005.

Determining whether spleen B10 cells purified from wild type mice produce only Il-10 was problematic due to the inherent technical difficulties when purifying such low-frequency cells and the predominantly low level induction of most cytokines by B cells (data not shown). However, spleen B10 cell frequencies and numbers are expanded in mice expressing a human CD19 transgene (hCD19Tg, Fig. 1C) (18). Within the CD1dCD5 B cell subset in hCD19Tg mice, 58% of the cells were induced to express cytoplasmic IL-10 following L+PIM stimulation for 5 h, but were significantly less common within the CD1dCD5, CD1dCD5, or CD1dCD5 B cell subsets (p<0.01). Whether IL-10-competent B cells represent a heterogeneous population capable of producing other cytokines was therefore examined by purifying IL-10-secreting CD19 B cells from hCD19Tg mice (Fig. 1D). IL-10 transcripts were expressed at ~6-fold higher frequencies in IL-10-secreting B cells when compared with B cells that did not secrete detectable IL-10 (Fig. 1E). Furthermore, IL-10 B cells did not produce transcripts for 31 additional cytokines at levels higher than IL-10 B cells under these culture conditions. Thus, the IL-10-secreting CD1dCD5 B10 cell subset was phenotypically and functionally unique.

B10 cell numbers during development

To characterize B10 cell development, the frequencies and numbers of spleen CD1dCD5 B cells and IL-10-producing B cells were assessed in neonatal, 2-mo-old, and 6-mo-old wild type B6 mice. CD1dCD5 B cells were virtually absent in neonatal spleen, with 5-fold lower frequencies than in 2-mo-old mice (Fig. 2A). Remarkably, neonatal spleen had 6.8-fold higher frequencies of IL-10-producing B cells than the 1–2% frequency induced in 2-mo-old wild type spleen B cells following 5 h L+PIM stimulation (Fig. 2B). Nonetheless, the majority of IL-10 B cells in neonates had a CD1dCD5 phenotype, with 6.5-fold higher CD5 expression levels than IL-10 B cells (p<0.001, Fig. 2C). Conversely, the frequencies and numbers of CD1dCD5 B cells were 1.4- and 1.8-fold higher in 6-mo-old mice than in 2-mo-old mice. IL-10 B cell frequencies and numbers were also 1.8- and 1.6-fold higher, respectively, in 6-mo-old mice compared with 2-mo-old mice (p<0.05). Spleen IL-10 B cells from 2- and 6-mo-old mice were predominantly CD1dCD5. Thus, neonatal IL-10-producing CD1dCD5 B cells were present at relatively high frequencies and numbers, while CD1dCD5 B10 cells expanded with age in the spleens of adult mice.

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B10 cell development in neonatal and 2- or 6-mo-old wild type B6 mice. (A) Representative CD1d and CD5 expression by CD19 B cells. Splenocytes were stained with CD1d, CD5, and CD19 mAbs with flow cytometry analysis of cells. Results represent one mouse indicating the frequency of CD1dCD5 B cells among total B cells within the indicated gates. Bar graphs indicate mean (±SEM) percentages and numbers of CD1dCD5 B cells in one of two independent experiments with three mice in each group. (B) IL-10 production by B cells. Splenocytes were cultured with L+PIM for 5 h, then stained with CD19 mAb to identify B cells, permeabilized, and stained using IL-10 mAb with flow cytometry analysis. Representative results demonstrate the frequency of IL-10-producing cells among total CD19 B cells within the indicated gates. Bar graphs indicate mean (±SEM) percentages and numbers of B cells that produced IL-10 in one of two independent experiments with three mice in each group. (A, B) Significant differences between sample means are indicated: *p<0.05, **, p<0.01. (C) Representative CD1d and CD5 expression by IL-10 or IL-10 B cells from neonatal mice. Horizontal and vertical gates delineate background staining using unreactive isotype-matched control mAbs.

B10 cell development is T cell and pathogen independent

To identify factors that influence B10 cell development, CD1dCD5 and IL-10-producing B cells were assessed in T cell-deficient nude mice and in gnotobiotic mice. CD1dCD5 B cell frequencies and numbers were ~5-fold higher in adult nude mice than in age-matched wild type mice (p<0.05; Fig. 3A). Cytoplasmic IL-10 B cell frequencies and numbers were also ~4.5-fold higher in L+PIM-stimulated splenocytes from nude mice when compared with wild type mice (p<0.05; Fig. 3B). The majority of IL-10 B cells in nude and wild type mice had a CD1dCD5 phenotype, while IL-10 B cells were CD1dCD5 (Fig. 3C). Whether B cell IL-10 production in vitro was influenced by the presence of T cells was also assessed by culturing whole splenocytes or purified B cells alone with L+PIM for 5 h. The frequency of B cells that expressed cytoplasmic IL-10 among all B cells was comparable in both cultures (Fig. 3D). Thus, spleen B10 cell development does not require the presence of T cells in nude mice.

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B10 cell development in T cell-deficient and gnotobiotic mice. (A) CD1d and CD5 expression by spleen CD19 B cells from 2 mo-old wild type and nude mice. Results represent one mouse indicating the frequency of CD1dCD5 B cells within the indicated gates among total B cells. Bar graphs indicate mean (±SEM) percentages and numbers of CD1dCD5 B cells in one of two independent experiments with three mice in each group. (B) IL-10 production by B cells from wild type and nude mice. Splenocytes were cultured with L+PIM for 5 h, stained with CD19 mAb, permeabilized, and stained using IL-10 mAb with flow cytometry analysis. Representative results demonstrate the frequency of IL-10-producing cells within the indicated gates among total CD19 B cells. Bar graphs indicate mean (±SEM) percentages and numbers of B cells that produced IL-10 in one of two independent experiments with three mice in each group. (C) CD1d and CD5 expression by IL-10 or IL-10 B cells from wild type and nude mice. Data are representative of 2 independent experiments with three mice in each group. Horizontal and vertical gates delineate background staining using unreactive isotype-matched control mAbs. (D) The presence of T cells during in vitro cultures does not influence B cell IL-10 production. Wild type splenocytes or purified B220 B cells were cultured with L+PIM for 5 h, then stained with CD19 mAb, permeabilized, and stained using IL-10 mAb with flow cytometry analysis. Representative results demonstrate the frequency of IL-10-producing cells within the indicated gates among total CD19 B cells. Bar graphs indicate mean (±SEM) percentages and numbers of B cells that produced IL-10 in one of two independent experiments with three mice in each group. (E) CD1d and CD5 expression by spleen CD19 B cells from specific pathogen free (SPF) and gnotobiotic mice. Bar graphs indicate mean (±SEM) percentages and numbers of CD1dCD5 B cells in three mice. (F) IL-10 production by B cells from specific-pathogen-free (SPF) and gnotobiotic mice cultured as in (B). Bar graphs indicate mean (±SEM) percentages and numbers of B cells that produced IL-10 in three mice. (A, B, D–F) Significant differences between sample means are indicated: *p<0.05, **, p<0.01.

To determine whether environmental factors influence B10 cell development, germ-free mice were assessed. CD1dCD5 B cell frequencies and numbers were similar, if not identical, in age-matched mice reared in gnotobiotic and specific pathogen-free colonies (Fig. 3E). Cytoplasmic IL-10 B cell frequencies and numbers were also similar (Fig. 3F) and the majority of IL-10 B cells had a CD1dCD5 phenotype (data not shown). Thus, environmental flora and gut-associated bacteria are not required for spleen B10 cell development.

Autoimmunity promotes B10 cell development

The influence of autoimmunity on B10 cell development was assessed in the NOD, NZB/W F1, MRL/lpr, DBA/1, and SJL mouse strains. NOD mice are a spontaneous model of type 1 diabetes (36). DBA/1 mice develop CIA after collagen immunization (37). SJL mice are susceptible to EAE after myelin proteolipid protein immunization (38). MRL/lpr and NZB/W mice spontaneously develop lupus-like disease (39). Most B cells in NOD (85±2%, n>3), MRL/lpr (80±12%, n=3), and SJL (94±1% n=3) mice expressed cell surface CD5 at levels that were significantly higher than background control mAb staining in comparison with B cells from B6 (25±2%, n>3), NZB/W (28±1%, n=3), and DBA/1 (14±1%, n=3) mice in side-by-side comparisons (Fig. 4A). Nonetheless, the frequency of CD1dCD5 B cells was limited, but 3- to 9-fold higher in NZB/W, MRL/lpr, NOD, and SJL mice than in 2-mo-old B6 mice. CD1dCD5 B cell numbers were also 3.8- to 5.9-fold increased in NZB/W, MRL/lpr, and NOD mice. Thus, the CD1dCD5 B cell subset increased in frequency in mice predisposed to autoimmunity.

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Autoimmunity promotes B10 cell expansion. (A) CD1d and CD5 expression by spleen B cells from 2 mo-old wild type B6, NZB/W F1, MRL/lpr, NOD, DBA/1, and SJL/J mice. Representative results demonstrate the frequency of CD1dCD5 B cells within the indicated gates among total CD19 B cells. Horizontal and vertical gates are set to delineate the CD1dCD5 B cell subset. Bar graphs indicate mean (±SEM) percentages and numbers of CD1dCD5 B cells in one of two independent experiments with 3 mice in each group. (B) IL-10 production by B cells. Splenocytes were cultured with L+PIM for 5 h, then stained with CD19 mAb, permeabilized, and stained using IL-10 mAb with flow cytometry analysis. Representative results demonstrate the frequency of IL-10-producing cells within the indicated gates among total B cells. Bar graphs indicate mean (±SEM) percentages and numbers of B cells that produced IL-10 in one of two independent experiments with 3 mice in each group. (A, B) Significant differences between sample means are indicated: *p<0.05, **, p<0.01. (C) CD1d and CD5 expression by IL-10 or IL-10 B cells. Horizontal and vertical gates are set to delineate the CD1dCD5 B cell subset as in (A). Data are representative of 2 independent experiments with 3 mice in each group.

The numbers of cytoplasmic IL-10 B cells were 2- to 4-fold higher in NZB/W, MRL/lpr, and NOD mice than in B6 wild type mice after L+PIM-stimulation (Fig. 4B). By contrast, IL-10-producing B cell numbers were 49% and 55% lower in DBA/1 and SJL mice, respectively, relative to wild type mice (p<0.01). In all cases, the majority of cytoplasmic IL-10 B cells also retained a CD1dCD5 phenotype (Fig. 4C). Thus, B10 cell numbers were significantly higher in diabetes- and lupus-prone mice, but significantly below wild type levels in DBA/1 and SJL mice that are susceptible to exogenous autoantigen-induced autoimmune disease.

Receptors that regulate B10 cell development in vivo

B cell development is regulated through the BCR and other molecules that inform B cells of their extracellular microenvironment, including CD19, CD21, CD22, and CD40 (40). Whether cell surface signals influence B10 cell development was determined by assessing CD1dCD5 and IL-10 producing B cell development in IL-10, MD4, CD19, CD21, CD40, MHC-I/II, hCD19Tg, CD22, CD40L/BTg, and CD40L/BTg/CD22 mice. MD4 transgenic mice have a fixed BCR specific for hen egg lysozyme (28). MHC-I/II mice are deficient in cell surface MHC class II, and most MHC class I and CD1 molecules due to combined disruption of the H2-Ab1 and β2-microglobulin genes (29, 41). B cells from CD40L/BTg mice express ectopic cell surface CD40L constitutively, with some mice developing lupus-like disease (35).

CD1dCD5 B cells were present at similar frequencies and numbers in IL-10, wild type, and MD4 mice (Fig. 5A). However, both the frequencies (65% decrease, p<0.01) and numbers (90% decrease, p<0.01) of L+PIM-induced cytoplasmic IL-10 B cells were reduced in MD4 mice when compared with wild type mice (Fig. 5B). In CD19 mice, the frequency and number of CD1dCD5 B cells was 87–92% lower than in wild type littermates, while L+PIM-induced IL-10 B cell frequencies and numbers were 73% and 89% lower, respectively (p<0.01). By contrast, CD21- or CD40-deficiencies did not affect the frequencies or numbers of CD1dCD5 or IL-10 producing B cells. CD1dCD5 B cell frequencies could not be assessed in MHCI/II mice that do not express CD1d, but IL-10 producing spleen B cell frequencies and numbers were normal. Spleen CD1dCD5 B cell frequencies and numbers were normal in MyD88 mice (Fig. 5A), while L+PIM-induced cytoplasmic IL-10 B cell frequencies and numbers were reduced by 40% and 46%, respectively, in 5 h assays (Fig. 5B). Thus, BCR diversity, and CD19- and MyD88-generated signals were critical for normal IL-10 producing CD1dCD5 B10 cell development and/or peripheral expansion in vivo, or visualization in vitro.

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Cell surface molecules that regulate B10 cell development in vivo. (A) CD1d and CD5 expression by spleen B cells from wild type, IL-10, MD4, CD19, CD21, CD40, MHC-I/II, MyD88, hCD19Tg, CD22, CD40L/BTg, and CD40L/BTg/CD22 mice. Splenocytes were stained with CD1d, CD5, and CD19 or CD20 mAbs (for CD19 mice). Representative results demonstrate the frequency of CD1dCD5 B cells within the indicated gates among total CD19 or CD20 B cells. Bar graphs indicate mean (±SEM) percentages and numbers of CD1dCD5 B cells in one of two independent experiments with 3 mice in each group. The horizontal dashed line is provided for reference to wild type mice. (B) IL-10 production by B cells. Splenocytes were cultured with L+PIM for 5 h, stained with CD19 or CD20 mAb, permeabilized, and stained using IL-10 mAb with flow cytometry analysis. Representative frequencies of IL-10-producing cells within the indicated gates among total CD19 or CD20 B cells. Bar graphs indicate mean (±SEM) percentages and numbers of B cells that produced IL-10 in one of two independent experiments with 3 mice in each group. The horizontal dashed line is for reference.

The frequencies and numbers of CD1dCD5 B cells were 5.8- and 1.5-fold higher in hCD19Tg mice than in wild type littermates, respectively (Fig. 5A). IL-10-producing B cell frequencies and numbers were 7.9- and 2.1-fold higher in hCD19Tg mice, respectively (Fig. 5B). Similarly, the frequency and number of CD1dCD5 B cells was 2.7- and 1.9-fold higher in CD22 mice than in wild type mice, while the frequency and number of IL-10-producing B cells was 4.1- and 2.8-fold higher, respectively. The frequency and number of CD1dCD5 B cells was 1.4- and 3.9-fold higher in CD40L/BTg mice than in wild type mice, while the frequency and number of IL-10-producing B cells was 1.4- and 3.7-fold higher, respectively. Thus, CD19 overexpression, CD22-deficiency, and ectopic CD40L expression on B cells significantly enhanced B10 cell numbers in vivo.

Combined CD22-deficiency and CD40L expression dramatically expanded the B10 cell subset in CD40L/BTg/CD22 mice (Fig. 5). The frequency and number of CD1dCD5 B cells was 7.0- and 16-fold higher in CD40L/BTg/CD22 mice, while the frequency and number of IL-10-producing B cells was 11- and 26-fold higher in CD40L/BTg/CD22 mice than in wild type mice, respectively (p<0.01). Thus, the absence of CD22 regulation combined with CD40L expression by B cells dramatically increased B10 cell numbers in vivo. In all mouse lines except MHC-I/II mice, L+PIM-induced IL-10 B cells maintained a CD1dCD5 phenotype when present (data not shown). Thus, spleen B10 cell development or expansion in vivo is not intrinsic, but depends in part on transmembrane signals.

LPS and CD40 stimulation induce B cell cytoplasmic IL-10 production in vitro

Signals that regulate B cell IL-10 production were assessed by culturing wild type spleen B cells with LPS, agonistic CD40 mAb, or mitogenic anti-IgM Ab at predetermined optimal concentrations (data not shown). PMA, ionomycin, and monensin (PIM)-stimulation for 5 h induced cytoplasmic IL-10 expression by 0.5–2% of B cells, which was 8 to 13-fold higher than for media alone and >5-fold higher than for LPS alone (Fig. 6A). The addition of CD40 mAb or anti-IgM Ab to PIM-stimulated cultures did not significantly increase IL-10 B cell frequencies. However, L+PIM-stimulation for 5 h induced >2-fold higher frequencies of IL-10 B cells than PIM, or CD40 mAb plus PIM, or anti-IgM Ab plus PIM (p<0.01). Thus, L+PIM stimulation induced optimal B cell cytoplasmic IL-10 expression in 5 h assays.

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In vitro B cell stimulation induces IL-10 production and secretion. CD19 splenocytes were purified from (A, B) wild type mice, or (C, D) wild type (filled bars) and MyD88 (open bars) littermates. Purified B cells were cultured with media alone, LPS, L+PIM, agonistic CD40 mAb, mitogenic anti-IgM Ab, or various combinations of these stimuli for the times indicated. For cytoplasmic IL-10 staining, PIM was added as indicated during the last 5 h of all cultures before the cells were isolated, stained with CD19 mAb, permeabilized, and stained with IL-10 mAb for flow cytometry analysis. (A) Values within representative histograms indicate the percentage of IL-10-producing cells within the gates shown among total B cells. Monensin was added for 5 h to media-only and LPS-only cultures. (B, D) For measuring secreted IL-10, culture supernatant fluid was harvested from cultured cells at the times indicated, with IL-10 concentrations determined by ELISA. Bar graphs indicate mean (±SEM) percentages or mean IL-10 (±SEM) concentrations from (A, B) one of 3 independent experiments with 3 mice in each group, or (C, D) one experiment with 3 mice in each group. (A–D) Significant differences between sample means are indicated: *p<0.05, **, p<0.01.

Culturing B cells with LPS or CD40 mAb for 48 h with PIM added during the last 5 h of culture induced significantly higher frequencies of cytoplasmic IL-10 B cells than anti-IgM Ab with PIM added during the last 5 h of culture (Fig. 6A). LPS stimulation was also significantly more robust than CD40 mAb stimulation. Unexpectedly however, the combination of LPS plus CD40 mAb for 48 h, or anti-IgM Ab plus either LPS or CD40 mAb, or all three together with PIM stimulation during the last 5 h did not increase IL-10 B cell frequencies significantly beyond what was normally observed with 5 h PIM stimulation alone. Thus, culturing B cells with LPS or CD40 mAb for 48 h before PIM stimulation induced the highest numbers of B cells with cytoplasmic IL-10 expression.

Spleen B cells stimulated with CD40 mAb for 48 h plus L+PIM for 5 h did not induce significantly higher numbers of cytoplasmic IL-10 B cells than LPS for 48 h plus PIM for 5 h (Fig. 6A). However, this sequential combination of stimuli induced the most robust levels of cytoplasmic IL-10 expression when compared with independent LPS or CD40 mAb stimulation. By contrast, adding L+PIM during the last 5 h of anti-IgM Ab, or CD40 mAb plus anti-IgM Ab cultures only induced ~2-fold higher numbers of IL-10 B cells than anti-IgM Ab or CD40 mAb alone. Thus, CD40 ligation with subsequent 5 h L+PIM stimulation was the most potent strategy for inducing the highest numbers of cytoplasmic IL-10 B cells with the highest levels of cytoplasmic IL-10.

LPS but not BCR or CD40 ligation induces B cell IL-10 secretion in vitro

Signals that regulate B cell IL-10 secretion were assessed by culturing spleen B cells with LPS, agonistic CD40 mAb, or mitogenic anti-IgM Ab, with culture supernatant fluid IL-10 levels determined by ELISA. LPS stimulation of spleen B cells for 24 h induced 3.5- to 3.8-fold more IL-10 than unstimulated cells, or cells cultured with CD40 mAb or anti-IgM Ab (p<0.01; Fig. 6B). LPS stimulation alone for 72 h induced significant B cell IL-10 secretion in contrast to CD40 mAb, anti-IgM Ab, or CD40 mAb plus anti-IgM Ab (p<0.01). In fact, simultaneous CD40 mAb or anti-IgM Ab treatment reduced LPS-induced IL-10 secretion by >68%. Furthermore, B cells cultured with CD40 mAb, anti-IgM Ab, and CD40 mAb plus anti-IgM Ab did not secrete significantly more IL-10 when LPS was added during the last 24 h of culture. Thus, LPS was the most potent stimulus for inducing both IL-10 production and secretion, while CD40-generated signals promoted cytoplasmic IL-10 generation but inhibited its secretion.

Normal B10 cell development in MyD88 mice

L+PIM-induced cytoplasmic IL-10 B cell frequencies and numbers were reduced in MyD88 mice (Fig. 5B). Whether this represented a developmental defect in vivo or reflected the absence of LPS-induced IL-10 production was therefore assessed in vitro. The frequency of cytoplasmic IL-10 MyD88 spleen B cells was also significantly reduced after 48 h of LPS stimulation relative to wild type B cells (Fig. 6C). By contrast, the frequency of CD40 mAb-induced cytoplasmic IL-10 B cells was equivalent in MyD88 and wild type littermates. Adding LPS to MyD88 B cell cultures during the last 5 h did not increase the frequency of CD40 mAb-induced cytoplasmic IL-10 B cells. IL-10 secretion was also significantly reduced in LPS-stimulated cultures of MyD88 B cells (Fig. 6D). Therefore, MyD88 expression was not required for normal B10 cell development and/or expansion in vivo, but MyD88 was required for optimal IL-10 production and secretion following LPS stimulation.

LPS and CD40 stimulation promotes B cell competence for cytoplasmic IL-10 production

Although CD5 B cells predominate in the spleens of neonatal wild type mice (Fig. 2), IL-10 production was not constitutive since culturing neonatal spleen B cells with monensin alone did not result in detectable cytoplasmic IL-10 staining (data not shown). Nonetheless, relatively high frequencies of IL-10-producing B cells were generated after 5 h of L+PIM stimulation (Fig. 2 and and7A).7A). Whether additional neonatal B cells could be induced to produce IL-10 was therefore assessed by culturing spleen B cells with LPS or agonistic CD40 mAb for 48 h. IL-10 B cells were 40% more frequent after prolonged LPS stimulation (p<0.05) despite lower level cytoplasmic IL-10 staining (Fig. 7A). Culturing neonatal splenocytes with CD40 mAb induced significantly fewer IL-10 B cells (p<0.05). The combination of CD40 mAb for 48 h with L+PIM stimulation during the last 5 h of culture generated similar numbers of IL-10 B cells as in the 48 h LPS cultures, but the overall intensity of cytoplasmic IL-10 staining was highest. Therefore, the majority of CD5 neonatal B cells were already competent for L+PIM-induced IL-10 production, with additional in vitro stimulation increasing B10 cell numbers significantly.

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LPS and CD40 signals induce the maturation of B10 progenitor cells. LPS and CD40 mAb induce IL-10 production by (A) neonatal spleen or (B) adult blood B cells from wild type mice. (A–B) Cells were cultured with LPS, agonistic CD40 mAb, or both for the times indicated, with PIM added during the last 5 h of each culture. The cultured cells were isolated, stained with CD19 mAb, permeabilized, and stained using IL-10 mAb with flow cytometry analysis. Values within representative histograms indicate the percentage of IL-10-producing cells among CD19 B cells within the gates shown. Bar graphs indicate mean (±SEM) percentages of IL-10 producing B cells in one of two independent experiments with 3 mice in each group. Significant differences between sample means are indicated: *p<0.05, **, p<0.01. (C) CD40 stimulation induces B cell CD5 expression. Cell surface CD1d and CD5 expression by wild type CD19 cells was determined by immunofluorescence staining. Neonatal splenocytes, or adult blood and spleen B cells were freshly isolated, or cultured for 48 h with LPS or agonistic CD40 mAb (plus or minus LPS for the last 5 h of culture). Values indicate the percentage of CD1dCD5 B cells among total B cells within the indicated gates. Single color histograms are representative of two independent experiments with 3 mice in each group.

CD1dCD5 or IL-10-competent B cells are not commonly observed in the blood or peripheral lymph nodes of naïve wild type mice, even after 5 h of L+PIM stimulation in vitro (Fig. 7B, ref. 18). Whether prolonged LPS or CD40 stimulation could induce B cell competence for IL-10 production was therefore examined. LPS or agonistic CD40 mAb stimulation induced 6–9-fold higher frequencies of cytoplasmic IL-10 B cells in 48 h cultures than in 5 h L+PIM cultures (p<0.01; Fig. 7B). The combination of CD40 mAb for 48 h with L+PIM stimulation during the last 5 h of culture also generated high numbers of IL-10 B cells with the highest intensity of cytoplasmic IL-10 staining. Similar results were obtained using peripheral lymph node B cells (data not shown). These results suggest that prolonged LPS or CD40 stimulation can promote the maturation of CD5 progenitor B10 cells into an IL-10 competent state.

Whether LPS or CD40 generated signals induce B cells to express a CD1dCD5 phenotype was therefore assessed. Neonatal spleen, and adult blood and spleen B cells were cultured with LPS or agonistic CD40 mAb for 48 h and examined for CD1d and CD5 expression by immunofluorescence staining. CD40 mAb but not LPS stimulation induced markedly higher CD5 expression on most B cells (Fig. 7C). By contrast, B cell CD1d expression was not induced or changed by LPS or CD40 mAb stimulation or the combination of both treatments for 48 h. Thus, CD5 was an induced marker for CD40-stimulated B10 cells.

IL-10 production by adult spleen B cells is restricted to the CD1dCD5 B cell subset

Splenic B10 cells that express cytoplasmic IL-10 after L+PIM stimulation localize primarily within the CD1dCD5 subset (Fig. 1A). It was therefore determined whether the increased frequency of IL-10 B cells in LPS or CD40 stimulated cultures results from the maturation of B10 cell progenitor cells within the CD1dCD5 subset or other B cell populations. Spleen CD1dCD5 or non-CD1dCD5 B cells from wild type mice were purified and cultured with LPS for 48 h, or with agonistic CD40 mAb for 48 h with LPS added during the last 5 h of culture. The CD1dCD5 B cell subset from B6 mice normally contains ~9–18% IL-10 B cells after 5 h of L+PIM stimulation (Fig. 1A, ref. 18). However, 33–43% of the CD1dCD5 B cells expressed cytoplasmic IL-10 after 48 h LPS or CD40 mAb stimulation, whereas <3% of CD1dCD5 B cells produced IL-10 (Fig. 8A). Thus, splenic B cells capable of producing IL-10 after prolonged LPS or CD40 mAb stimulation predominantly derive from the CD1dCD5 subset.

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Effect of LPS or CD40 ligation on IL-10 production, proliferation, and the phenotype of CD1dCD5 B cells. (A) LPS and CD40 mAb-induced cytoplasmic IL-10 production are restricted to CD1dCD5 B cells. CD1dCD5 or CD1dCD5 B220 B cells were purified from pooled splenocytes of three wild type mice by cell sorting and reassessed for CD1d and CD5 expression (middle panels). The purified B cell subsets were cultured with LPS or CD40 mAb for 48 h, with L+PIM added for the last 5 h of culture before permeabilization, staining for IL-10, and flow cytometry analysis (right panels). The frequencies of IL-10 cells among the sorted CD1dCD5 or CD1dCD5 B cell subsets are shown for one of two independent experiments. (B) Clonal expansion of IL-10-producing B cells after LPS but not CD40 stimulation in vitro for 48 h. Wild type CD19 splenocytes were labeled with CFSE and cultured with LPS or CD40 mAb for 48 h, with L+PIM added for the last 5 h of culture. Histograms (right) represent CFSE expression by the IL-10 or IL-10 B cell subsets. Dashed lines represent CFSE staining of unstimulated B cells. (A–B) Data are representative of 2 independent experiments. (C) Potential B10 developmental pathway leading to the generation of the IL-10 secreting B10 cell subset. Dashed arrows and question marks represent potential maturation steps based on CD5 and CD1d expression patterns.

To determine whether the increased frequency of IL-10 B cells after LPS or CD40 stimulation results from the clonal expansion of existing IL-10-competent B cells or maturation of progenitor B10 cells, IL-10 B cell proliferation was assessed by labeling purified spleen B cells with CFSE before LPS or CD40 mAb stimulation in vitro. LPS stimulation for 48 h induced IL-10 and IL-10 B cell proliferation, although IL-10 B cells proliferated more than IL-10 B cells as measured by reduced CFSE staining (Fig. 8B). By contrast, CD40 mAb stimulation for 48 h plus LPS treatment for the last 5 h of culture only induced modest IL-10 or IL-10 B cell proliferation during these 48 h cultures. CD40 mAb stimulation predominantly induces B cell clonal expansion between 72–96 h as described (31, 42). Thus, LPS stimulation induces and expands the IL-10 B cell subset during 48 h cultures, while CD40 ligation induces B cell competence for cytoplasmic IL-10 production (Fig. 8C).

IL-10-producing B cells preferentially secrete IL-10

Spleen B cells that are competent to express cytoplasmic IL-10 following 5 h L+PIM stimulation were predominantly found within the CD1dCD5 CD19 subset in wild type B6 mice (Fig. 1A), as described (18, 19). By contrast, IL-10 expressing B cells were significantly less common within the CD1dCD5, CD1dCD5, or CD1dCD5 B cell subsets (p<0.01), with B cells from IL-10 mice used as negative controls for background IL-10 staining. We have previously shown that 5 h L+PIM stimulation does not influence the phenotype of these B cell subsets (18). IL-10CD19 B10 cells were predominantly CD21, CD23, CD24, CD43, and CD93 (AA4.1) (Fig. 1B). Thereby, spleen B10 cells are relatively rare and share some overlapping phenotypic markers with the B-1a, MZ, and T2-MZ precursor B cell subsets, but are nonetheless phenotypically distinct, (18, 19).

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B10 cells preferentially secret IL-10. (A) IL-10-producing B cells were predominantly found within the CD1dCD5CD19 B cell subset. Splenocytes from wild type and IL-10 mice were cultured with L+PIM for 5 h, then stained with CD1d, CD5, and CD19 mAb before permeabilization and staining using IL-10 mAb. Percentages and bar graphs indicate mean (±SEM) B cell subset frequencies and numbers among CD19 splenocytes or IL-10 cell frequencies among the indicated B cell subsets (a, CD1dCD5; b, CD1dCD5; c, CD1dCD5; d, CD1dCD5) from 3 mice as determined by flow cytometry analysis. Values significantly different from background frequencies or numbers for IL-10 mice are indicated: *, p<0.05; **, p<0.01. (B) CD21, CD23, CD24, CD43, and CD93 expression by IL-10-producing (thick line) and IL-10 (thin line) CD19 spleen B cells from wild type mice cultured with L+PIM for 5 h, then stained for cell surface antigens before permeabilization and cytoplasmic IL-10 staining. Gray histograms represent isotype-matched control mAb staining. Results are representative of those obtained with B cells from ≥3 mice as determined by flow cytometry analysis. (C) IL-10-producing B cells from hCD19Tg mice are predominantly found within the CD1dCD5CD19 B cell subset. Staining and analysis was as described in (A). (D) Representative isolation of IL-10-secreting B cells. Splenic B220 cells purified from three hCD19Tg mice were pooled and cultured with L+PI for 5 h before staining for CD19 and secreted IL-10 capture (left panel). Cytoplasmic IL-10 and IL-10 B cells were isolated by cell sorting using the indicated gates and subsequently reassessed for IL-10 secretion and CD19 expression (right panels). (E) Cytokine gene expression by IL-10-secreting and non-secreting B cells purified as in (D). Mean fold-differences (±SEM) in cytokine transcript levels (IL-10/IL-10 cells) from 3 independent experiments are shown. Values of 1 (dashed line) indicate no difference in cytokine expression between the IL-10 and IL-10 B cells, with significant differences indicated: **, p<0.005.

Determining whether spleen B10 cells purified from wild type mice produce only Il-10 was problematic due to the inherent technical difficulties when purifying such low-frequency cells and the predominantly low level induction of most cytokines by B cells (data not shown). However, spleen B10 cell frequencies and numbers are expanded in mice expressing a human CD19 transgene (hCD19Tg, Fig. 1C) (18). Within the CD1dCD5 B cell subset in hCD19Tg mice, 58% of the cells were induced to express cytoplasmic IL-10 following L+PIM stimulation for 5 h, but were significantly less common within the CD1dCD5, CD1dCD5, or CD1dCD5 B cell subsets (p<0.01). Whether IL-10-competent B cells represent a heterogeneous population capable of producing other cytokines was therefore examined by purifying IL-10-secreting CD19 B cells from hCD19Tg mice (Fig. 1D). IL-10 transcripts were expressed at ~6-fold higher frequencies in IL-10-secreting B cells when compared with B cells that did not secrete detectable IL-10 (Fig. 1E). Furthermore, IL-10 B cells did not produce transcripts for 31 additional cytokines at levels higher than IL-10 B cells under these culture conditions. Thus, the IL-10-secreting CD1dCD5 B10 cell subset was phenotypically and functionally unique.

B10 cell numbers during development

To characterize B10 cell development, the frequencies and numbers of spleen CD1dCD5 B cells and IL-10-producing B cells were assessed in neonatal, 2-mo-old, and 6-mo-old wild type B6 mice. CD1dCD5 B cells were virtually absent in neonatal spleen, with 5-fold lower frequencies than in 2-mo-old mice (Fig. 2A). Remarkably, neonatal spleen had 6.8-fold higher frequencies of IL-10-producing B cells than the 1–2% frequency induced in 2-mo-old wild type spleen B cells following 5 h L+PIM stimulation (Fig. 2B). Nonetheless, the majority of IL-10 B cells in neonates had a CD1dCD5 phenotype, with 6.5-fold higher CD5 expression levels than IL-10 B cells (p<0.001, Fig. 2C). Conversely, the frequencies and numbers of CD1dCD5 B cells were 1.4- and 1.8-fold higher in 6-mo-old mice than in 2-mo-old mice. IL-10 B cell frequencies and numbers were also 1.8- and 1.6-fold higher, respectively, in 6-mo-old mice compared with 2-mo-old mice (p<0.05). Spleen IL-10 B cells from 2- and 6-mo-old mice were predominantly CD1dCD5. Thus, neonatal IL-10-producing CD1dCD5 B cells were present at relatively high frequencies and numbers, while CD1dCD5 B10 cells expanded with age in the spleens of adult mice.

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B10 cell development in neonatal and 2- or 6-mo-old wild type B6 mice. (A) Representative CD1d and CD5 expression by CD19 B cells. Splenocytes were stained with CD1d, CD5, and CD19 mAbs with flow cytometry analysis of cells. Results represent one mouse indicating the frequency of CD1dCD5 B cells among total B cells within the indicated gates. Bar graphs indicate mean (±SEM) percentages and numbers of CD1dCD5 B cells in one of two independent experiments with three mice in each group. (B) IL-10 production by B cells. Splenocytes were cultured with L+PIM for 5 h, then stained with CD19 mAb to identify B cells, permeabilized, and stained using IL-10 mAb with flow cytometry analysis. Representative results demonstrate the frequency of IL-10-producing cells among total CD19 B cells within the indicated gates. Bar graphs indicate mean (±SEM) percentages and numbers of B cells that produced IL-10 in one of two independent experiments with three mice in each group. (A, B) Significant differences between sample means are indicated: *p<0.05, **, p<0.01. (C) Representative CD1d and CD5 expression by IL-10 or IL-10 B cells from neonatal mice. Horizontal and vertical gates delineate background staining using unreactive isotype-matched control mAbs.

B10 cell development is T cell and pathogen independent

To identify factors that influence B10 cell development, CD1dCD5 and IL-10-producing B cells were assessed in T cell-deficient nude mice and in gnotobiotic mice. CD1dCD5 B cell frequencies and numbers were ~5-fold higher in adult nude mice than in age-matched wild type mice (p<0.05; Fig. 3A). Cytoplasmic IL-10 B cell frequencies and numbers were also ~4.5-fold higher in L+PIM-stimulated splenocytes from nude mice when compared with wild type mice (p<0.05; Fig. 3B). The majority of IL-10 B cells in nude and wild type mice had a CD1dCD5 phenotype, while IL-10 B cells were CD1dCD5 (Fig. 3C). Whether B cell IL-10 production in vitro was influenced by the presence of T cells was also assessed by culturing whole splenocytes or purified B cells alone with L+PIM for 5 h. The frequency of B cells that expressed cytoplasmic IL-10 among all B cells was comparable in both cultures (Fig. 3D). Thus, spleen B10 cell development does not require the presence of T cells in nude mice.

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B10 cell development in T cell-deficient and gnotobiotic mice. (A) CD1d and CD5 expression by spleen CD19 B cells from 2 mo-old wild type and nude mice. Results represent one mouse indicating the frequency of CD1dCD5 B cells within the indicated gates among total B cells. Bar graphs indicate mean (±SEM) percentages and numbers of CD1dCD5 B cells in one of two independent experiments with three mice in each group. (B) IL-10 production by B cells from wild type and nude mice. Splenocytes were cultured with L+PIM for 5 h, stained with CD19 mAb, permeabilized, and stained using IL-10 mAb with flow cytometry analysis. Representative results demonstrate the frequency of IL-10-producing cells within the indicated gates among total CD19 B cells. Bar graphs indicate mean (±SEM) percentages and numbers of B cells that produced IL-10 in one of two independent experiments with three mice in each group. (C) CD1d and CD5 expression by IL-10 or IL-10 B cells from wild type and nude mice. Data are representative of 2 independent experiments with three mice in each group. Horizontal and vertical gates delineate background staining using unreactive isotype-matched control mAbs. (D) The presence of T cells during in vitro cultures does not influence B cell IL-10 production. Wild type splenocytes or purified B220 B cells were cultured with L+PIM for 5 h, then stained with CD19 mAb, permeabilized, and stained using IL-10 mAb with flow cytometry analysis. Representative results demonstrate the frequency of IL-10-producing cells within the indicated gates among total CD19 B cells. Bar graphs indicate mean (±SEM) percentages and numbers of B cells that produced IL-10 in one of two independent experiments with three mice in each group. (E) CD1d and CD5 expression by spleen CD19 B cells from specific pathogen free (SPF) and gnotobiotic mice. Bar graphs indicate mean (±SEM) percentages and numbers of CD1dCD5 B cells in three mice. (F) IL-10 production by B cells from specific-pathogen-free (SPF) and gnotobiotic mice cultured as in (B). Bar graphs indicate mean (±SEM) percentages and numbers of B cells that produced IL-10 in three mice. (A, B, D–F) Significant differences between sample means are indicated: *p<0.05, **, p<0.01.

To determine whether environmental factors influence B10 cell development, germ-free mice were assessed. CD1dCD5 B cell frequencies and numbers were similar, if not identical, in age-matched mice reared in gnotobiotic and specific pathogen-free colonies (Fig. 3E). Cytoplasmic IL-10 B cell frequencies and numbers were also similar (Fig. 3F) and the majority of IL-10 B cells had a CD1dCD5 phenotype (data not shown). Thus, environmental flora and gut-associated bacteria are not required for spleen B10 cell development.

Autoimmunity promotes B10 cell development

The influence of autoimmunity on B10 cell development was assessed in the NOD, NZB/W F1, MRL/lpr, DBA/1, and SJL mouse strains. NOD mice are a spontaneous model of type 1 diabetes (36). DBA/1 mice develop CIA after collagen immunization (37). SJL mice are susceptible to EAE after myelin proteolipid protein immunization (38). MRL/lpr and NZB/W mice spontaneously develop lupus-like disease (39). Most B cells in NOD (85±2%, n>3), MRL/lpr (80±12%, n=3), and SJL (94±1% n=3) mice expressed cell surface CD5 at levels that were significantly higher than background control mAb staining in comparison with B cells from B6 (25±2%, n>3), NZB/W (28±1%, n=3), and DBA/1 (14±1%, n=3) mice in side-by-side comparisons (Fig. 4A). Nonetheless, the frequency of CD1dCD5 B cells was limited, but 3- to 9-fold higher in NZB/W, MRL/lpr, NOD, and SJL mice than in 2-mo-old B6 mice. CD1dCD5 B cell numbers were also 3.8- to 5.9-fold increased in NZB/W, MRL/lpr, and NOD mice. Thus, the CD1dCD5 B cell subset increased in frequency in mice predisposed to autoimmunity.

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Autoimmunity promotes B10 cell expansion. (A) CD1d and CD5 expression by spleen B cells from 2 mo-old wild type B6, NZB/W F1, MRL/lpr, NOD, DBA/1, and SJL/J mice. Representative results demonstrate the frequency of CD1dCD5 B cells within the indicated gates among total CD19 B cells. Horizontal and vertical gates are set to delineate the CD1dCD5 B cell subset. Bar graphs indicate mean (±SEM) percentages and numbers of CD1dCD5 B cells in one of two independent experiments with 3 mice in each group. (B) IL-10 production by B cells. Splenocytes were cultured with L+PIM for 5 h, then stained with CD19 mAb, permeabilized, and stained using IL-10 mAb with flow cytometry analysis. Representative results demonstrate the frequency of IL-10-producing cells within the indicated gates among total B cells. Bar graphs indicate mean (±SEM) percentages and numbers of B cells that produced IL-10 in one of two independent experiments with 3 mice in each group. (A, B) Significant differences between sample means are indicated: *p<0.05, **, p<0.01. (C) CD1d and CD5 expression by IL-10 or IL-10 B cells. Horizontal and vertical gates are set to delineate the CD1dCD5 B cell subset as in (A). Data are representative of 2 independent experiments with 3 mice in each group.

The numbers of cytoplasmic IL-10 B cells were 2- to 4-fold higher in NZB/W, MRL/lpr, and NOD mice than in B6 wild type mice after L+PIM-stimulation (Fig. 4B). By contrast, IL-10-producing B cell numbers were 49% and 55% lower in DBA/1 and SJL mice, respectively, relative to wild type mice (p<0.01). In all cases, the majority of cytoplasmic IL-10 B cells also retained a CD1dCD5 phenotype (Fig. 4C). Thus, B10 cell numbers were significantly higher in diabetes- and lupus-prone mice, but significantly below wild type levels in DBA/1 and SJL mice that are susceptible to exogenous autoantigen-induced autoimmune disease.

Receptors that regulate B10 cell development in vivo

B cell development is regulated through the BCR and other molecules that inform B cells of their extracellular microenvironment, including CD19, CD21, CD22, and CD40 (40). Whether cell surface signals influence B10 cell development was determined by assessing CD1dCD5 and IL-10 producing B cell development in IL-10, MD4, CD19, CD21, CD40, MHC-I/II, hCD19Tg, CD22, CD40L/BTg, and CD40L/BTg/CD22 mice. MD4 transgenic mice have a fixed BCR specific for hen egg lysozyme (28). MHC-I/II mice are deficient in cell surface MHC class II, and most MHC class I and CD1 molecules due to combined disruption of the H2-Ab1 and β2-microglobulin genes (29, 41). B cells from CD40L/BTg mice express ectopic cell surface CD40L constitutively, with some mice developing lupus-like disease (35).

CD1dCD5 B cells were present at similar frequencies and numbers in IL-10, wild type, and MD4 mice (Fig. 5A). However, both the frequencies (65% decrease, p<0.01) and numbers (90% decrease, p<0.01) of L+PIM-induced cytoplasmic IL-10 B cells were reduced in MD4 mice when compared with wild type mice (Fig. 5B). In CD19 mice, the frequency and number of CD1dCD5 B cells was 87–92% lower than in wild type littermates, while L+PIM-induced IL-10 B cell frequencies and numbers were 73% and 89% lower, respectively (p<0.01). By contrast, CD21- or CD40-deficiencies did not affect the frequencies or numbers of CD1dCD5 or IL-10 producing B cells. CD1dCD5 B cell frequencies could not be assessed in MHCI/II mice that do not express CD1d, but IL-10 producing spleen B cell frequencies and numbers were normal. Spleen CD1dCD5 B cell frequencies and numbers were normal in MyD88 mice (Fig. 5A), while L+PIM-induced cytoplasmic IL-10 B cell frequencies and numbers were reduced by 40% and 46%, respectively, in 5 h assays (Fig. 5B). Thus, BCR diversity, and CD19- and MyD88-generated signals were critical for normal IL-10 producing CD1dCD5 B10 cell development and/or peripheral expansion in vivo, or visualization in vitro.

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Cell surface molecules that regulate B10 cell development in vivo. (A) CD1d and CD5 expression by spleen B cells from wild type, IL-10, MD4, CD19, CD21, CD40, MHC-I/II, MyD88, hCD19Tg, CD22, CD40L/BTg, and CD40L/BTg/CD22 mice. Splenocytes were stained with CD1d, CD5, and CD19 or CD20 mAbs (for CD19 mice). Representative results demonstrate the frequency of CD1dCD5 B cells within the indicated gates among total CD19 or CD20 B cells. Bar graphs indicate mean (±SEM) percentages and numbers of CD1dCD5 B cells in one of two independent experiments with 3 mice in each group. The horizontal dashed line is provided for reference to wild type mice. (B) IL-10 production by B cells. Splenocytes were cultured with L+PIM for 5 h, stained with CD19 or CD20 mAb, permeabilized, and stained using IL-10 mAb with flow cytometry analysis. Representative frequencies of IL-10-producing cells within the indicated gates among total CD19 or CD20 B cells. Bar graphs indicate mean (±SEM) percentages and numbers of B cells that produced IL-10 in one of two independent experiments with 3 mice in each group. The horizontal dashed line is for reference.

The frequencies and numbers of CD1dCD5 B cells were 5.8- and 1.5-fold higher in hCD19Tg mice than in wild type littermates, respectively (Fig. 5A). IL-10-producing B cell frequencies and numbers were 7.9- and 2.1-fold higher in hCD19Tg mice, respectively (Fig. 5B). Similarly, the frequency and number of CD1dCD5 B cells was 2.7- and 1.9-fold higher in CD22 mice than in wild type mice, while the frequency and number of IL-10-producing B cells was 4.1- and 2.8-fold higher, respectively. The frequency and number of CD1dCD5 B cells was 1.4- and 3.9-fold higher in CD40L/BTg mice than in wild type mice, while the frequency and number of IL-10-producing B cells was 1.4- and 3.7-fold higher, respectively. Thus, CD19 overexpression, CD22-deficiency, and ectopic CD40L expression on B cells significantly enhanced B10 cell numbers in vivo.

Combined CD22-deficiency and CD40L expression dramatically expanded the B10 cell subset in CD40L/BTg/CD22 mice (Fig. 5). The frequency and number of CD1dCD5 B cells was 7.0- and 16-fold higher in CD40L/BTg/CD22 mice, while the frequency and number of IL-10-producing B cells was 11- and 26-fold higher in CD40L/BTg/CD22 mice than in wild type mice, respectively (p<0.01). Thus, the absence of CD22 regulation combined with CD40L expression by B cells dramatically increased B10 cell numbers in vivo. In all mouse lines except MHC-I/II mice, L+PIM-induced IL-10 B cells maintained a CD1dCD5 phenotype when present (data not shown). Thus, spleen B10 cell development or expansion in vivo is not intrinsic, but depends in part on transmembrane signals.

LPS and CD40 stimulation induce B cell cytoplasmic IL-10 production in vitro

Signals that regulate B cell IL-10 production were assessed by culturing wild type spleen B cells with LPS, agonistic CD40 mAb, or mitogenic anti-IgM Ab at predetermined optimal concentrations (data not shown). PMA, ionomycin, and monensin (PIM)-stimulation for 5 h induced cytoplasmic IL-10 expression by 0.5–2% of B cells, which was 8 to 13-fold higher than for media alone and >5-fold higher than for LPS alone (Fig. 6A). The addition of CD40 mAb or anti-IgM Ab to PIM-stimulated cultures did not significantly increase IL-10 B cell frequencies. However, L+PIM-stimulation for 5 h induced >2-fold higher frequencies of IL-10 B cells than PIM, or CD40 mAb plus PIM, or anti-IgM Ab plus PIM (p<0.01). Thus, L+PIM stimulation induced optimal B cell cytoplasmic IL-10 expression in 5 h assays.

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In vitro B cell stimulation induces IL-10 production and secretion. CD19 splenocytes were purified from (A, B) wild type mice, or (C, D) wild type (filled bars) and MyD88 (open bars) littermates. Purified B cells were cultured with media alone, LPS, L+PIM, agonistic CD40 mAb, mitogenic anti-IgM Ab, or various combinations of these stimuli for the times indicated. For cytoplasmic IL-10 staining, PIM was added as indicated during the last 5 h of all cultures before the cells were isolated, stained with CD19 mAb, permeabilized, and stained with IL-10 mAb for flow cytometry analysis. (A) Values within representative histograms indicate the percentage of IL-10-producing cells within the gates shown among total B cells. Monensin was added for 5 h to media-only and LPS-only cultures. (B, D) For measuring secreted IL-10, culture supernatant fluid was harvested from cultured cells at the times indicated, with IL-10 concentrations determined by ELISA. Bar graphs indicate mean (±SEM) percentages or mean IL-10 (±SEM) concentrations from (A, B) one of 3 independent experiments with 3 mice in each group, or (C, D) one experiment with 3 mice in each group. (A–D) Significant differences between sample means are indicated: *p<0.05, **, p<0.01.

Culturing B cells with LPS or CD40 mAb for 48 h with PIM added during the last 5 h of culture induced significantly higher frequencies of cytoplasmic IL-10 B cells than anti-IgM Ab with PIM added during the last 5 h of culture (Fig. 6A). LPS stimulation was also significantly more robust than CD40 mAb stimulation. Unexpectedly however, the combination of LPS plus CD40 mAb for 48 h, or anti-IgM Ab plus either LPS or CD40 mAb, or all three together with PIM stimulation during the last 5 h did not increase IL-10 B cell frequencies significantly beyond what was normally observed with 5 h PIM stimulation alone. Thus, culturing B cells with LPS or CD40 mAb for 48 h before PIM stimulation induced the highest numbers of B cells with cytoplasmic IL-10 expression.

Spleen B cells stimulated with CD40 mAb for 48 h plus L+PIM for 5 h did not induce significantly higher numbers of cytoplasmic IL-10 B cells than LPS for 48 h plus PIM for 5 h (Fig. 6A). However, this sequential combination of stimuli induced the most robust levels of cytoplasmic IL-10 expression when compared with independent LPS or CD40 mAb stimulation. By contrast, adding L+PIM during the last 5 h of anti-IgM Ab, or CD40 mAb plus anti-IgM Ab cultures only induced ~2-fold higher numbers of IL-10 B cells than anti-IgM Ab or CD40 mAb alone. Thus, CD40 ligation with subsequent 5 h L+PIM stimulation was the most potent strategy for inducing the highest numbers of cytoplasmic IL-10 B cells with the highest levels of cytoplasmic IL-10.

LPS but not BCR or CD40 ligation induces B cell IL-10 secretion in vitro

Signals that regulate B cell IL-10 secretion were assessed by culturing spleen B cells with LPS, agonistic CD40 mAb, or mitogenic anti-IgM Ab, with culture supernatant fluid IL-10 levels determined by ELISA. LPS stimulation of spleen B cells for 24 h induced 3.5- to 3.8-fold more IL-10 than unstimulated cells, or cells cultured with CD40 mAb or anti-IgM Ab (p<0.01; Fig. 6B). LPS stimulation alone for 72 h induced significant B cell IL-10 secretion in contrast to CD40 mAb, anti-IgM Ab, or CD40 mAb plus anti-IgM Ab (p<0.01). In fact, simultaneous CD40 mAb or anti-IgM Ab treatment reduced LPS-induced IL-10 secretion by >68%. Furthermore, B cells cultured with CD40 mAb, anti-IgM Ab, and CD40 mAb plus anti-IgM Ab did not secrete significantly more IL-10 when LPS was added during the last 24 h of culture. Thus, LPS was the most potent stimulus for inducing both IL-10 production and secretion, while CD40-generated signals promoted cytoplasmic IL-10 generation but inhibited its secretion.

Normal B10 cell development in MyD88 mice

L+PIM-induced cytoplasmic IL-10 B cell frequencies and numbers were reduced in MyD88 mice (Fig. 5B). Whether this represented a developmental defect in vivo or reflected the absence of LPS-induced IL-10 production was therefore assessed in vitro. The frequency of cytoplasmic IL-10 MyD88 spleen B cells was also significantly reduced after 48 h of LPS stimulation relative to wild type B cells (Fig. 6C). By contrast, the frequency of CD40 mAb-induced cytoplasmic IL-10 B cells was equivalent in MyD88 and wild type littermates. Adding LPS to MyD88 B cell cultures during the last 5 h did not increase the frequency of CD40 mAb-induced cytoplasmic IL-10 B cells. IL-10 secretion was also significantly reduced in LPS-stimulated cultures of MyD88 B cells (Fig. 6D). Therefore, MyD88 expression was not required for normal B10 cell development and/or expansion in vivo, but MyD88 was required for optimal IL-10 production and secretion following LPS stimulation.

LPS and CD40 stimulation promotes B cell competence for cytoplasmic IL-10 production

Although CD5 B cells predominate in the spleens of neonatal wild type mice (Fig. 2), IL-10 production was not constitutive since culturing neonatal spleen B cells with monensin alone did not result in detectable cytoplasmic IL-10 staining (data not shown). Nonetheless, relatively high frequencies of IL-10-producing B cells were generated after 5 h of L+PIM stimulation (Fig. 2 and and7A).7A). Whether additional neonatal B cells could be induced to produce IL-10 was therefore assessed by culturing spleen B cells with LPS or agonistic CD40 mAb for 48 h. IL-10 B cells were 40% more frequent after prolonged LPS stimulation (p<0.05) despite lower level cytoplasmic IL-10 staining (Fig. 7A). Culturing neonatal splenocytes with CD40 mAb induced significantly fewer IL-10 B cells (p<0.05). The combination of CD40 mAb for 48 h with L+PIM stimulation during the last 5 h of culture generated similar numbers of IL-10 B cells as in the 48 h LPS cultures, but the overall intensity of cytoplasmic IL-10 staining was highest. Therefore, the majority of CD5 neonatal B cells were already competent for L+PIM-induced IL-10 production, with additional in vitro stimulation increasing B10 cell numbers significantly.

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LPS and CD40 signals induce the maturation of B10 progenitor cells. LPS and CD40 mAb induce IL-10 production by (A) neonatal spleen or (B) adult blood B cells from wild type mice. (A–B) Cells were cultured with LPS, agonistic CD40 mAb, or both for the times indicated, with PIM added during the last 5 h of each culture. The cultured cells were isolated, stained with CD19 mAb, permeabilized, and stained using IL-10 mAb with flow cytometry analysis. Values within representative histograms indicate the percentage of IL-10-producing cells among CD19 B cells within the gates shown. Bar graphs indicate mean (±SEM) percentages of IL-10 producing B cells in one of two independent experiments with 3 mice in each group. Significant differences between sample means are indicated: *p<0.05, **, p<0.01. (C) CD40 stimulation induces B cell CD5 expression. Cell surface CD1d and CD5 expression by wild type CD19 cells was determined by immunofluorescence staining. Neonatal splenocytes, or adult blood and spleen B cells were freshly isolated, or cultured for 48 h with LPS or agonistic CD40 mAb (plus or minus LPS for the last 5 h of culture). Values indicate the percentage of CD1dCD5 B cells among total B cells within the indicated gates. Single color histograms are representative of two independent experiments with 3 mice in each group.

CD1dCD5 or IL-10-competent B cells are not commonly observed in the blood or peripheral lymph nodes of naïve wild type mice, even after 5 h of L+PIM stimulation in vitro (Fig. 7B, ref. 18). Whether prolonged LPS or CD40 stimulation could induce B cell competence for IL-10 production was therefore examined. LPS or agonistic CD40 mAb stimulation induced 6–9-fold higher frequencies of cytoplasmic IL-10 B cells in 48 h cultures than in 5 h L+PIM cultures (p<0.01; Fig. 7B). The combination of CD40 mAb for 48 h with L+PIM stimulation during the last 5 h of culture also generated high numbers of IL-10 B cells with the highest intensity of cytoplasmic IL-10 staining. Similar results were obtained using peripheral lymph node B cells (data not shown). These results suggest that prolonged LPS or CD40 stimulation can promote the maturation of CD5 progenitor B10 cells into an IL-10 competent state.

Whether LPS or CD40 generated signals induce B cells to express a CD1dCD5 phenotype was therefore assessed. Neonatal spleen, and adult blood and spleen B cells were cultured with LPS or agonistic CD40 mAb for 48 h and examined for CD1d and CD5 expression by immunofluorescence staining. CD40 mAb but not LPS stimulation induced markedly higher CD5 expression on most B cells (Fig. 7C). By contrast, B cell CD1d expression was not induced or changed by LPS or CD40 mAb stimulation or the combination of both treatments for 48 h. Thus, CD5 was an induced marker for CD40-stimulated B10 cells.

IL-10 production by adult spleen B cells is restricted to the CD1dCD5 B cell subset

Splenic B10 cells that express cytoplasmic IL-10 after L+PIM stimulation localize primarily within the CD1dCD5 subset (Fig. 1A). It was therefore determined whether the increased frequency of IL-10 B cells in LPS or CD40 stimulated cultures results from the maturation of B10 cell progenitor cells within the CD1dCD5 subset or other B cell populations. Spleen CD1dCD5 or non-CD1dCD5 B cells from wild type mice were purified and cultured with LPS for 48 h, or with agonistic CD40 mAb for 48 h with LPS added during the last 5 h of culture. The CD1dCD5 B cell subset from B6 mice normally contains ~9–18% IL-10 B cells after 5 h of L+PIM stimulation (Fig. 1A, ref. 18). However, 33–43% of the CD1dCD5 B cells expressed cytoplasmic IL-10 after 48 h LPS or CD40 mAb stimulation, whereas <3% of CD1dCD5 B cells produced IL-10 (Fig. 8A). Thus, splenic B cells capable of producing IL-10 after prolonged LPS or CD40 mAb stimulation predominantly derive from the CD1dCD5 subset.

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Effect of LPS or CD40 ligation on IL-10 production, proliferation, and the phenotype of CD1dCD5 B cells. (A) LPS and CD40 mAb-induced cytoplasmic IL-10 production are restricted to CD1dCD5 B cells. CD1dCD5 or CD1dCD5 B220 B cells were purified from pooled splenocytes of three wild type mice by cell sorting and reassessed for CD1d and CD5 expression (middle panels). The purified B cell subsets were cultured with LPS or CD40 mAb for 48 h, with L+PIM added for the last 5 h of culture before permeabilization, staining for IL-10, and flow cytometry analysis (right panels). The frequencies of IL-10 cells among the sorted CD1dCD5 or CD1dCD5 B cell subsets are shown for one of two independent experiments. (B) Clonal expansion of IL-10-producing B cells after LPS but not CD40 stimulation in vitro for 48 h. Wild type CD19 splenocytes were labeled with CFSE and cultured with LPS or CD40 mAb for 48 h, with L+PIM added for the last 5 h of culture. Histograms (right) represent CFSE expression by the IL-10 or IL-10 B cell subsets. Dashed lines represent CFSE staining of unstimulated B cells. (A–B) Data are representative of 2 independent experiments. (C) Potential B10 developmental pathway leading to the generation of the IL-10 secreting B10 cell subset. Dashed arrows and question marks represent potential maturation steps based on CD5 and CD1d expression patterns.

To determine whether the increased frequency of IL-10 B cells after LPS or CD40 stimulation results from the clonal expansion of existing IL-10-competent B cells or maturation of progenitor B10 cells, IL-10 B cell proliferation was assessed by labeling purified spleen B cells with CFSE before LPS or CD40 mAb stimulation in vitro. LPS stimulation for 48 h induced IL-10 and IL-10 B cell proliferation, although IL-10 B cells proliferated more than IL-10 B cells as measured by reduced CFSE staining (Fig. 8B). By contrast, CD40 mAb stimulation for 48 h plus LPS treatment for the last 5 h of culture only induced modest IL-10 or IL-10 B cell proliferation during these 48 h cultures. CD40 mAb stimulation predominantly induces B cell clonal expansion between 72–96 h as described (31, 42). Thus, LPS stimulation induces and expands the IL-10 B cell subset during 48 h cultures, while CD40 ligation induces B cell competence for cytoplasmic IL-10 production (Fig. 8C).

Discussion

The majority of adult spleen B cells that were competent for IL-10 production after 5 h L+PIM stimulation were found within the CD1dCD5 subset (Fig. 1A). IL-10 B10 cells preferentially produced IL-10 transcripts relative to other B cells, but did not appear to preferentially produce other known cytokines (Fig. 1E). IL-10-competent B cells were also found within the CD1dCD5 and CD1dCD5 subsets, but at significantly lower (p≤0.05) frequencies and numbers than in the CD1dCD5 subset. Spleen CD1dCD5 B cells also exist that could acquire IL-10 competence in vitro after 48 h stimulation with LPS or agonistic CD40 mAb (Figs. 6A and and8A),8A), potentially reflecting their maturation. By contrast, spleen CD1dCD5 B cells were not rendered IL-10 competent after 48 h stimulation with LPS or agonistic CD40 mAb (Fig. 8A). Progenitor B10 cells may also exist that do not express CD5 or CD1d, yet can be induced to express IL-10 in vitro. Specifically, the vast majority of blood and lymph node B cells in adult mice were CD1dCD5 and did not express IL-10 after 5 h L+PIM stimulation (Fig. 7B). However, a small subset of blood and lymph node B cells acquired IL-10 competence after 48 h CD40 ligation and/or LPS exposure. Neonatal spleen B cells predominantly expressed CD5 and were almost exclusively CD1d, but ~14% were induced to express cytoplasmic IL-10 after 5 h L+PIM exposure (Fig. 2). Consistent with this, neonatal and adult B cells uniformly upregulated CD5 expression after CD40 ligation in vitro (Fig. 7C). Thereby, L+PIM stimulation may induce IL-10 production in small subsets of B cells that have received appropriate competence-inducing signals in vivo or in vitro regardless of their maturation. Alternatively, CD1dCD5 progenitor B10 cells may be induced to mature, express CD5, and acquire competence for activation-induced cytoplasmic IL-10 production as proposed in the maturation scheme outlined in figure 8C. Factors that regulate or induce CD1d expression by some spleen B cells are unknown. Thus, IL-10 competence and the CD1dCD5 phenotype define the spleen B10 cell subset, but may also reflect their maturation, activation status, subset commitment, and/or tissue localization.

Development, maturation, and/or expansion of the spleen B10 cell subset required specific external signals. BCR specificity significantly influenced B10 cell development, with B10 cell numbers reduced by 90% in transgenic mice expressing a fixed Ag-receptor (Fig. 5B). In contrast, B10 cell development did not require the presence of T or NKT cells (Fig. 3). Furthermore, CD1, MHC class I and class II, CD21, or CD40 expression were not required for normal B10 cell development or IL-10 induction (Fig. 5). Nonetheless, CD40 ligation induced cytoplasmic IL-10 production by B cells in vitro (Fig. 6) and ectopic CD154 expression by B cells in CD40L/BTg mice increased B10 cell numbers by 3- to 4-fold (Fig. 5). Thereby, CD40:CD154 interactions may facilitate B10 cell maturation under some conditions, but were not required for B10 cell acquisition of IL-10-competence in vivo. TLR signaling was also critical for B10 cell effector function since LPS induced B10 cells to both produce and secrete IL-10 in vitro, while CD40 ligation only induced cytoplasmic IL-10 production (Fig. 6). B10 cell development was normal in MyD88 mice (Fig. 6C–D), but LPS-induced IL-10 production and secretion were significantly reduced in MyD88 B cells (Fig. 6D). A need for MyD88 in LPS-induced B10 cell function may explain why mice containing only MyD88 B cells develop chronic EAE (43). Thus, intertwined innate and adaptive signals may regulate B10 cell maturation and effector function rather than independently regulating distinct follicular, MZ, and B-1a regulatory B cell subsets.

The B10 cell subset expanded significantly in response to enhanced B cell signaling in vivo, while retaining their CD1dCD5 phenotype. B10 cell numbers were significantly expanded in hCD19Tg mice, but were dramatically reduced in CD19 mice (Fig. 5, ref. 18). B10 cell numbers were also increased 2- to 3-fold in CD22 mice (Fig. 5). CD19 regulates a Lyn kinase amplification loop (44, 45) that enhances transmembrane signals (27, 46, 47), while CD22 dampens B cell and CD19 signal transduction through the recruitment of SHP-1 and SHIP phosphatases (44, 46), resulting in elevated cell surface CD5 expression by B cells in CD22 B6 mice (48). Spleen B10 cells were also significantly expanded in CD40L/BTg mice, with a 26-fold increase in CD22CD40L/BTg mice where up to 20% of spleen B cells were B10 cells (Fig. 5). Since CD22 negatively regulates CD40 signaling (31, 48), enhanced CD40 function may drive B10 cell expansion and/or survival in CD22CD40L/BTg mice (35, 49). Although spleen B1a cells were also expanded in hCD19Tg (3-fold), CD40L/BTg (4.2-fold), and CD22CD40L/BTg (3-fold) mice (data not shown), these frequencies did not parallel B10 cell expansion. Thus, the B10 cell subset responds significantly to transmembrane signals in vivo.

Spleen B10 cell numbers were increased in mice predisposed to develop autoimmunity. B10 cell numbers expanded significantly in the NZB/W F1 and MRL/lpr mouse models of lupus and the NOD model of diabetes even before obvious autoantibodies and signs of disease were apparent (Fig. 4, data now shown). B10 cell numbers are significantly expanded in CD40L/BTg mice (Fig. 5), although some develop lupus-like disease (35). Spleen B10 cell numbers were also significantly higher in 6 mo-old C57BL/6 mice relative to 2 mo-old mice (Fig. 2), which may combat the development of autoimmunity with age. By contrast, B10 cell numbers were significantly lower in the DBA/1 and SJL mouse models of autoantigen-inducible autoimmunity, where the relative paucity of B10 cells may prevent effective tolerance induction. Thereby, B10 cell expansion may suppress autoimmunity, in contrast to B1a cells that contribute to autoimmune disease (50). As a result, these autoimmune diseases may be worse in the absence of B10 cells as occurs when all B cells are depleted during CHS and EAE (18, 19). Since B10 cell numbers are dynamic, change during development, and increase with age and autoimmunity, alterations in the balance between B10 cell negative regulation and B cell positive contributions to immune responses are likely to vary in different diseases and during the course of disease (4).

Spleen B10 cells and their potential progenitors (Fig. 8C) can account for many of the in vivo activities previously attributed to regulatory B cells (4, 18, 19). Specifically, BCR and CD40 engagement are required for regulatory B cell functions in CIA, CHS, and EAE models (13, 1719), and functional B10 cells required diverse BCRs (Fig. 5) and in vivo Ag sensitization (18, 19) for their generation. Stimulating naïve or autoimmune spleen B cells in vitro with LPS or agonistic CD40 mAb also gives rise to regulatory B cells that inhibit or prevent autoimmunity (15, 24). That CD40 ligation induced IL-10-competance in both CD1dCD5 and some CD1dCD5 B cells (Fig. 6) may also explain how agonistic CD40 mAbs reduce inflammation in the CIA model of rheumatoid arthritis (51). LPS induction of B10 cell competence for IL-10 production and secretion (Fig. 6C) may also explain why LPS pretreatment modulates the course of disease in EAE (52). Similarly, B cells activated with LPS in vitro can protect NOD mice in vivo, although this effect was not attributed to B cell IL-10 production (24). Thus, B10 cells and regulatory B cells identified in previous studies were similar in their responses to polyclonal stimuli such as LPS and CD40.

That BCR diversity was required for B10 cell development in vivo (Fig. 5) supports observations that B10 cell and regulatory B cell function is Ag-specific (13, 18, 19). The activation of arthritogenic splenocytes with collagen alone (17) or collagen plus agonistic CD40 mAb in vitro gives rise to IL-10 producing B cells that prevent arthritis (15). Autoreactive B cell production of IL-10 during EAE also requires simultaneous autoantigen and CD40 stimulation (13). Transfusions of BCR-activated B cells also protects NOD mice from type 1 diabetes in an IL-10-dependent manner (26). However, BCR ligation using mitogenic Ab in vitro negatively regulated cytoplasmic and secreted IL-10 production when combined with LPS or CD40 mAb during in vitro cultures, although BCR ligation alone induced some B cells to express IL-10 at higher than background levels (Fig. 6). These results contrast with the findings of others that BCR ligation using anti-Igκ Ab does not affect simultaneous LPS-induced IL-10 secretion by splenic transitional, follicular, and marginal zone B cells, B1 B cells from the peritoneal cavity, or lymph node B cells (43). However, the strength, nature, or timing of BCR generated signals required for evoking B10 cell development or function may be specifically regulated in vivo. For example, BCR engagement by potent foreign Ags may inhibit B10 cell clonal expansion or divert B10 progenitor cells along a distinct functional pathway, while BCR signals generated by self Ags may promote their expansion. Thereby, LPS or other signals may optimally induce B10 cell effector function (IL-10 secretion) after Ag-selection or CD40-induced maturation in vivo.

It remains difficult to distinguish the relationships between spleen B10, B-1a, and MZ B cells due to their shared phenotypic markers and potentially overlapping developmental pathways. For example, microbial colonization and conventional T cells were not required for spleen B10, B-1a, or CD1d MZ B cell development, and all three subsets require CD19 expression (Figs. 3 and and5,5, and refs. 53, 54, 55). However, spleen CD5 and IL-10-competent B cells were present at high frequencies in newborns, while the splenic CD1d subset was not detectable in newborns (Figs. 12) but develops between 3–7 wks after birth (53). Spleen B10 cell proliferation was also more robust following LPS stimulation than for IL-10 B cells (Fig. 8B). MZ B cells also expand and provide protection early during pathogen challenge (56). Furthermore, some IL-10 producing cells can be induced within the spleen CD1dCD5 and CD1dCD5 subsets (Fig. 1A), but it is hard to discern whether these cells represent contaminating B10 cells or are progenitor B10 cells that have not fully upregulated CD1d or CD5 expression (Fig. 8C). Therefore, it is likely that spleen B-1a and MZ B cells represent subsets of mixed origins, with B10 cells representing either a distinct subset with shared phenotypic markers, or a subset representing different branches of a common lineage.

These studies address the ambiguity regarding a major B cell subset that regulates inflammation and autoimmune disease. Evidence for the existence of a distinct natural B10 cell subset that generally suppresses immune responses was not uncovered. Rather, the current data indicate that BCR and other signals are central to B10 cell generation and that polyclonal signals such as CD40 and LPS can induce their maturation and/or regulatory functions. Thereby, immature CD5 progenitor B10 cells may be induced to mature and express CD5 and CD1d through Ag selection, potentially involving CD40 signals that induced CD5 expression (Fig. 7C). BCR ligation is also well characterized to induce CD5 expression (57). That CD40 ligation induces cytoplasmic IL-10 production but not significant cytokine secretion is likely to represent another critical regulatory checkpoint in B10 cell function. Although regulatory B cells and B10 cells have been predominantly described in mouse models where autoantigen plus TLR-agonist-containing adjuvants induce autoimmunity, B10 cells also significantly influence CHS inflammation, where disease is independent of adjuvant challenge (18). Thus, stimuli in addition to LPS are likely to also regulate IL-10 secretion by B10 cells. Although B10 cell development and tolerance regulation are undoubtedly more complex, the current results provide a potential framework (Fig. 8C) for further characterizing B10 cell development.

Acknowledgments

We thank Drs. Susan Harless-Smith, Damian Maseda, David DiLillo, and David Gray for their help and influential comments, and Dr. Scott Plevy for providing gnotobiotic mouse tissues.

Departments of Immunology, Duke University Medical Center, Durham, North Carolina 27710
Department of Immunology, Medical Research Institute, Tokyo Medical and Dental University, Tokyo, Japan
Address correspondence and reprint requests to: Thomas F. Tedder, Box 3010, Dept. Immunology, Duke University Medical Center, Durham, NC 27710. Phone 919-684-3681; FAX 919-684-8982; ude.ekud@reddet.samoht
These authors contributed equally to these studies and share first authorship.

Abstract

Autoimmunity and inflammation are controlled in part by regulatory B cells, including a recently identified IL-10-competent CD1dCD5 B cell subset termed B10 cells that represents 1–3% of adult mouse spleen B cells. In this study, pathways that influence B10 cell generation and IL-10 production were identified and compared with previously described regulatory B cells. IL-10-competent B cells were predominantly CD1dCD5 in adult spleen and were the prevalent source of IL-10 but not other cytokines. B10 cell development and/or maturation in vivo required Ag receptor diversity and intact signaling pathways, but not T cells, gut-associated flora, or environmental pathogens. Spleen B10 cell frequencies were significantly expanded in aged mice and mice predisposed to autoimmunity, but were significantly decreased in mouse strains that are susceptible to exogenous autoantigen-induced autoimmunity. LPS, PMA, plus ionomycin stimulation in vitro for 5 h induced B10 cells to express cytoplasmic IL-10. However, prolonged LPS or CD40 stimulation (48 h) induced additional adult spleen CD1dCD5 B cells to express IL-10 following PMA+ionomycin stimulation. Prolonged LPS or CD40 stimulation of newborn spleen and adult blood or lymph node CD1d and/or CD5 B cells also induced cytoplasmic IL-10 competence in rare B cells, with CD40 ligation uniformly inducing CD5 expression. IL-10 secretion was induced by LPS signaling through MyD88-dependent pathways, but not following CD40 ligation. LPS stimulation also induced rapid B10 cell clonal expansion when compared with other spleen B cells. Thereby, both adaptive and innate signals regulate B10 cell development, maturation, CD5 expression, and competence for IL-10 production.

Keywords: Rodent, B cells, autoimmunity, cell differentiation, tolerance
Abstract

Footnotes

These studies were supported by grants from the National Institutes of Health CA105001, CA96547, AI56363 and AI057157. J.D.B. is supported by grants from Association pour la Recherche contre le Cancer, the Fondation René Touraine and the Philippe Foundation.

Abbreviations used in this paper: Ab, antibody; Ag, antigen; B6, C57BL/6; BCR, B cell Ag receptor; CHS, contact hypersensitivity; CIA, collagen-induced arthritis; EAE, experimental autoimmune encephalomyelitis; hCD19Tg, human CD19 transgenic mice; L+PIM, LPS+PMA+ionomycin+monensin; L+PI, LPS+PMA+ionomycin; MZ, marginal zone; PIM, PMA+ionomycin+monensin; TLR, toll-like receptor.

Disclosure

T.F.T. is a paid consultant for MedImmune Inc. and a paid consultant and shareholder for Angelica Therapeutics, Inc. The other authors have no financial conflict of interest.

Footnotes

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