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I M M U N O L O G Y

Interleukin-10–producing CD5+ B cells inhibitmast cells during immunoglobulin E–mediatedallergic responsesHyuk Soon Kim,1* A-Ram Kim,1* Do Kyun Kim,1 Hyun Woo Kim,1 Young Hwan Park,1

Geun Hyo Jang,1 Bokyung Kim,1 Yeong Min Park,1 Jueng Soo You,1 Hyung Sik Kim,2

Michael A. Beaven,3 Young Mi Kim,4 Wahn Soo Choi1†

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Subsets of B cells inhibit various immune responses through their production of the cytokine interleukin-10 (IL-10). We found that IL-10–producing CD5+ B cells suppressed the immunoglobulin E (IgE)– andantigen-mediated activation of mast cells in vitro as well as allergic responses in mice in an IL-10–dependent manner. Furthermore, the suppressive effect of these B cells on mast cells in vitro and in vivodepended on direct cell-to-cell contact through the costimulatory receptor CD40 on CD5+ B cells and theCD40 ligand on mast cells. This contact enhanced the production of IL-10 by the CD5+ B cells. Throughactivation of the Janus-activated kinase–signal transducer and activator of transcription 3 pathway, IL-10decreased the abundance of the kinases Fyn and Fgr and inhibited the activation of the downstream ki-nase Syk in mast cells. Together, these findings suggest that an important function of IL-10–producingCD5+ B cells is inhibiting mast cells and IgE-mediated allergic responses.

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INTRODUCTION

Allergic disorders are widespread, particularly in developed countries (1).Allergic responses are associated with increases in the number of T helper2 (TH2) cells and immunoglobulin E (IgE) antibody production. Presen-tation of allergen to antigen-presenting cells (APCs) causes TH2 cells toproduce TH2-type cytokines. In particular, the cytokine interleukin-4 (IL-4)is essential for the isotype switching of B cells to produce allergen-specificIgE antibodies (2), which then bind specifically to the multimeric high-affinity IgE receptor FceRI (3) on mast cells and basophils.

B cells are generally known for their capacity to regulate effector T cellresponses and to produce antibodies (4); however, studies in murine dis-ease models revealed some distinct B cell subsets that exhibit immunosup-pressive functions (5–8) and thus are named regulatory B (Breg) cells (9).Other subsets of Breg cells have also been identified to suppress variousimmune responses in an IL-10–dependent manner (10), whereas helminthinfection–induced IL-10–producing B cells inhibit allergic reactions inanimal models, specifically ovalbumin-mediated anaphylaxis (11) and al-lergic asthma (12). IL-10 also suppresses mast cell activation in vitro andin vivo and may thus counteract their excessive activation and the devel-opment of chronic inflammation (13, 14). Despite these reports, andgiven the role of mast cells in these and other allergic diseases (15, 16),surprisingly little is known about the mechanism of interaction betweenBreg cells and mast cells or about the consequences of these interactionsfor IgE-mediated allergic responses.

Mast cells are the key effector cells in IgE-mediated allergic reactions.These cells are widely distributed in vascularized tissues, especially nearsurfaces exposed to the environment, such as the skin, airways, and thegastrointestinal tract (15). Mast cells are commonly activated by the mul-

1School of Medicine, Konkuk University, Chungju 380-701, Korea. 2College ofPharmacy, Sungkyunkwan University, Suwon 440-746, Korea. 3Laboratory ofMolecular Immunology, National Heart, Lung, and Blood Institute, NationalInstitutes of Health, Bethesda, MD 20892, USA. 4College of Pharmacy, DuksungWomen’s University, Seoul 132-714, Korea.*These authors contributed equally to this work.†Corresponding author. E-mail: wahnchoi@kku.ac.kr

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tivalent binding of antigen to FceRI-bound IgE, with the subsequent re-lease of various allergic mediators, including histamine, eicosanoids, andinflammatory cytokines. Release of these mediators leads to immediate,and sometimes delayed, symptoms of allergic diseases, such as allergic rhini-tis, anaphylaxis, and atopic dermatitis (17, 18). We found that CD5+ B cellsinhibited IgE-mediated mast cell activation and anaphylaxis in mice in an IL-10–dependent manner. Furthermore, we found that IL-10–producing CD5+ Bcells inhibited the activation of the tyrosine kinase Syk (spleen tyrosine ki-nase) in mast cells. Together, our findings suggest that IL-10–producing CD5+

B cells inhibit IgE-mediated allergic responses in physiological settings.

RESULTS

CD5+ B cells suppress IgE-mediated anaphylaxisin vivo and mast cell activation in vitroIgE-dependent mast cell activation is regarded as one of the cardinal me-chanisms in the development of anaphylaxis. Here, we administrated IgEantibody and antigen intravenously to mice to induce passive systemicanaphylaxis (PSA), and these responses are essentially associated withextensive mast cell activation in vivo. We found that the numbers of IL-10–producing CD5+ B cells were increased in the spleen, peritoneal cavity,lymph node, and blood of IgE- and antigen-induced PSA mice (fig. S1A),which suggests that these cells are associated with the progression of symp-toms. We next examined CD19-decifient mice in which IL-10–producingB cells are substantially depleted (fig. S1B) (7). We found that IgE-mediatedanaphylaxis responses (Fig. 1A) and increases in the concentration of his-tamine in the blood (Fig. 1B) were substantially enhanced in the CD19-deficient mice compared to wild-type mice.

To assess the effects of CD5+ B cells in IgE-mediated allergic re-sponses in vivo, we first induced PSA in CD19-deficient mice. Three daysafter they received purified CD5+ or CD5− B cells from wild-type miceby adoptive transfer (fig. S1C), IgE-sensitized CD19-deficient mice wereintravenously challenged with antigen. The presence of CD5+ B cells,but not CD5− B cells, markedly alleviated the decline in temperature in

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response to antigen (Fig. 1C). Serum histamine concentrations were in-creased in antigen-challenged mice compared to those in unchallengedmice, but this increase was substantially reduced after the adoptive transferof CD5+ cells (Fig. 1D).

Next, we found that culturing BMMCs with CD5+ B cells, but notCD5− B cells, substantially inhibited their degranulation in response toantigen in a time- and cell concentration–dependent manner (Fig. 1, Eand F), as well as inhibited their release of other allergic mediators, suchas histamine, TNF-a, and IL-4 (Fig. 1, G to I). This inhibitory action ofCD5+ B cells was not dependent on mouse strain or tissue source becauseCD5+ B cells isolated from the peritoneal cavity of C57BL/6 mice and

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from the spleen of BALB/c mice also inhibited the antigen-stimulated de-granulation of BMMCs (fig. S1, D and E).

IL-10 from CD5+ B cells is critical for mast cell inhibitionThe immunoregulatory role of Breg cells, also called B10 cells, is gen-erally dependent on IL-10 (10). We found that the passive cutaneous an-aphylaxis (PCA) reaction was increased in IL-10−/− mice compared to thatin wild-type mice (fig. S2, A and B, top). The numbers of degranulatedmast cells in the ear tissues of IL-10−/− mice were increased, albeit notstatistically significantly, compared to those in the ear tissues of wild-type mice (fig. S2B, bottom). These results led us to investigate whether

Fig. 1. CD5+ B cells suppress IgE-mediated activation of mouse mast cells the absence or presence of CD5+ or CD5− B cells at a BMMC/B cell ratio of+ −

in vivo and in culture. (A and B) PSA was induced by injecting wild-type

(WT) mice or CD19−/− mice with dinitrophenyl (DNP)–IgE (3 mg) 24 hoursbefore injecting them with DNP–bovine serum albumin (DNP-BSA; 250 mg;Ag) as indicated. Mice were then subjected to analysis of rectal tempera-tures at the indicated times (A) and serum concentrations of histamine 30 minafter stimulation (B) (n ≥ 5 mice per experiment). (C andD) PSA was inducedin CD19−/− mice 3 days after they received CD5+ B cells or CD5− B cells byadoptive transfer. Mice were then subjected to analysis of rectal tempera-tures (C) and serum concentrations of histamine (D) (n≥ 5mice per experiment).(E and F) Bone marrow–derived mast cells (BMMCs) were preincubated in

1:5 for the indicated times (E) or CD5 or CD5 B cells at the indicatedBMMC/B cell ratios for 24 hours (F). Cells were then treated with or with-out IgE and antigen (Ag) as indicated, and the extent of release of b-hexosaminidase from the BMMCs was determined. (G to I) BMMCs werecultured alone or together with CD5+ or CD5− B cells and then were treatedwith the indicated combinations of IgE and antigen. The amounts of hista-mine (G), tumor necrosis factor–a (TNF-a) (H), and IL-4 (I) released into theculture medium were determined by enzyme-linked immunosorbent assayELISA). Data are means ± SEM of three independent experiments. *P < 0.05;**P < 0.01; n.s., not significant.

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IL-10 generated by CD5+ B cells inhibited mast cell activation and IgE-mediated anaphylaxis. First, we found that monoclonal antibodiesagainst IL-10 or the IL-10 receptor (IL-10R) blocked the suppressiveeffect of CD5+ B cells on the antigen-induced degranulation of BMMCs(Fig. 2A). Second, CD5+ B cells from IL-10−/− mice failed to inhibit theIgE- and antigen-stimulated degranulation of BMMCs in vitro (Fig. 2B).

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CD5+ B cells from either wild-type or IL-10−/− mice showed no statisti-cally significant differences in the amounts of their cell surface markers,including IgD, IgM, CD19, CD21, CD1d, CD11b, CD40, and B220 (fig.S2C). Third, CD5+ B cells also inhibited the antigen-stimulated degra-nulation of IL-10−/− BMMCs (fig. S2D). Notably, flow cytometric anal-ysis revealed that IL-10–producing B cells were found mostly within a

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Fig. 2. IL-10 production by CD5+ B cells is critical for the suppression of mast+

PSA was induced in IL-10−/−mice 3 days after they received CD5+ or CD5−−/−

cell activation. (A and B) BMMCs were preincubated for 24 hours with CD5

B cells in the presence or absence of the indicated combinations of anti-IL-10 (a-IL-10), anti-IL-10R (a-IL-10R), or isotype control monoclonal anti-body (mAb) (A) or with the indicated combinations of CD5+ or CD5− B cellsfrom WT or IL-10−/− mice (B). Cells were then treated with the indicatedcombinations of IgE and antigen, and the amount of b-hexosaminidase re-leased into the culture medium was determined. Data are means ± SEM ofthree independent experiments. (C and D) PSA was induced in IL-19−/−

mice 3 days after they had received CD5+ or CD5− B cells from WT orIL-10−/− mice, as indicated, by adoptive transfer. Mice were then analyzedto determine rectal temperatures at the indicated times (C) and serum his-tamine concentrations at 30 min (D) (n ≥ 5 mice per experiment). (E and F)

B cells from WT or IL-10 mice by adoptive transfer, as indicated. Micewere then analyzed to determine rectal temperatures at the indicated times(E) and serum histamine concentrations at 30 min (F) (n ≥ 5 mice per ex-periment). (G to I) Equal numbers of CD5+ or CD5− B cells were incubatedalone or in the presence of unstimulated or IgE- and antigen-stimulatedBMMCs from WT (G) or IL-10−/− mice (H and I), as indicated. Cells werethen analyzed by flow cytometry to determine the percentages of IL-10+ Bcells (CD19+) (G), the percentages of CD5+ or CD5− B cells that containedIL-10 (were IL-10+) (H), and the amount of IL-10 in the culture medium (I).Data in (G) are representative of three independent experiments. Data in(H) and (I) are means ± SEM of three independent experiments. *P < 0.05;**P < 0.01.

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CD5+CD19high B cell subset (fig. S2E). We also found that the amounts ofIgM, CD1d, and CD21 were greater on the surface of IL-10+ CD19+ Bcells compared with the amounts of those markers on IL-10− CD19+ Bcells (fig. S2F). Finally, the adoptive transfer of wild-type CD5+ B cells,but not IL-10−/− CD5+ B cells, suppressed PSA reactions and blocked in-creases in serum histamine concentrations in CD19-deficient mice (Fig. 2,C and D) and IL-10−/− mice (Fig. 2, E and F), which supports the ideathat IL-10 produced by CD5+ B cells inhibits mast cell activation andIgE-mediated anaphylaxis.

BMMCs stimulate the production of IL-10 fromCD5+ B cellsWe next found that the percentage of IL-10–producing B cells among thewhole population of B cells was increased by the coculture with BMMCsand was even further increased if the cocultured BMMCswere stimulated withIgE and antigen (fig. S3A). However, the production of IL-10 by the BMMCsthemselves was minimal when they were cocultured with B cells (fig. S3, Band C), indicating that most of the IL-10 in cocultures was produced by the Bcells. We further found that BMMCs enhanced the production of IL-10 fromCD5+ B cells, but not from CD5− B cells (Fig. 2, G and H), and that thisincreased production was further enhanced by stimulating wild-type orIL-10−/− BMMCs with antigen (Fig. 2, H and I). The stimulatory effect ofBMMCs on IL-10 production was also apparent with CD5+ B cells fromthe spleen, inguinal lymph node, and blood, but not from the peritonealcavity (fig. S3, D and E). These results suggest that BMMCs stimulate IL-10production by CD5+ B cells from various lymphoid organs.

Direct cell-cell contact is essential to inhibit mast cellactivation and enhance IL-10 production by CD5+ B cellsAlthough degranulation was inhibited when mast cells were cultured withCD5+ B cells, this was not the case when the cell types were physicallyseparated in transwell culture flasks (Fig. 3A). Note that conjugation ofCD5+ B cells and mast cells with or without IgE (~20% of cells conjugated)was observed in cocultures, and the extent of conjugation was increased (to

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~25%) by the addition of antigen (Fig. 3, B and C). We also observedconjugation between CD5− B cells and mast cells (fig. S3F), which wassimilar to that between CD5+ B cells and mast cells. Furthermore, immu-nohistochemical analysis of mouse spleens after PSAwas induced revealedthat some CD5+CD19+ B cells were in close proximity to mast cells, whichraises the possibility of crosstalk between these two cell types in vivo (Fig.3D). Similarly, the increase in IL-10 production by CD5+ B cells was ob-served only in cocultures and not when both cell types were separated intranswell flasks (Fig. 3, E and F).

CD40 on CD5+ B cells and CD40 ligand (CD154) onmast cells are required for the production of IL-10 by CD5+

B cells and the suppression of mast cell activationCD40-generated signals in IL-10–producing B cells participate in the reg-ulation of various inflammatory diseases and possibly in the production

Fig. 3. Suppression of mast cell activation by IL-10–producing CD5+ Bcells requires cell-to-cell contact. (A) BMMCs were cultured for 24 hours

alone or together with CD5+ or CD5− B cells either in direct contact (filledbars) or separated in transwell plates (empty bars). Cells were then treatedwith the indicated combinations of IgE and antigen before b-hexosaminidaserelease was determined. (B and C) CD5+ B cells were incubated in vitroalone or with BMMCs that were untreated, treated with IgE, or treated withIgE and antigen, as indicated. (B) After 45 min, the cocultured cells wereanalyzed by flow cytometry. (C) The percentages of BMMC-CD5+ B cellconjugates that formed were calculated. (D) Spleens of PSA-induced WTmice were analyzed by immunohistochemistry to detect CD5 [black, nickel-diaminobenzidine (N-DAB)], CD19 (red, Novared), and mast cells (pur-ple, toluidine blue). CD5+ B cells are indicated by green arrows; mastcells are indicated by red arrows. Images are shown at ×400 magnifica-tion; however, the area in the yellow box is shown in the bottom right panelat a magnification of ×1000. Scale bar, 100 mm. (E and F) CD5+ or CD5− Bcells were cultured for 24 hours alone or with unstimulated or IgE- andantigen-stimulated BMMCs under conditions of cell-to-cell contact or intranswells. (E) B cells were analyzed by flow cytometry to detect intracel-lular IL-10. (F) Top: The percentages of IL-10+ B cells were determined byflow cytometry. Bottom: The amounts of IL-10 in the culture medium of theindicated cells were determined by ELISA. Data in (B), (D), and (E) are rep-resentative of three independent experiments. Data in (A), (C), and (F) aremeans ± SEM of three independent experiments. *P < 0.05; **P < 0.01.

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of IL-10 (10). Because the abundance of CD40 ligand (CD40L) on thesurface of mast cells is increased by stimulation with antigen (19), wedetermined whether the enhanced IL-10 production by B cells wasdependent on the CD40-CD40L interaction. The abundances of CD40on B cells and IL-10 in B cells, as well as the amount of IL-10 secretedfrom B cells, were substantially increased by recombinant CD40L (Fig. 4,A to D). Whereas CD40 abundance and IL-10 production in whole B cellswere increased when they were cocultured with BMMCs (fig. S4, A andB), IL-10 production by B cells was largely blocked in the presence of ananti-CD40L monoclonal antibody (fig. S4, B and C). In addition, in co-cultures of CD5+ B cells and BMMCs, the increased abundances of cellsurface CD40 (Fig. 4E) and IL-10 (Fig. 4, F and G) in CD5+ B cells weremarkedly inhibited by the anti-CD40L antibody, and the suppressiveaction of CD5+ B cells on BMMC degranulation was also blocked by thisantibody (Fig. 4H).

The interaction between CD5+ B cells and mast cellsthrough CD40-CD40L is critical for the suppression ofIgE-mediated mast cell activation and anaphylaxisWe next observed whether CD5+ B cells and mast cells interacted di-rectly under cocultured conditions. The extent of conjugation of CD5+

B cells and BMMCs in coculture that we noted earlier (Fig. 3B) wassubstantially reduced when CD40−/− CD5+ B cells, CD40L−/− BMMCs,

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or both were substituted for the corresponding wild-type cells (Fig. 5,A and B), suggesting that CD40-CD40L contact was necessary forsuch conjugation to occur. A similar pattern of effects on the produc-tion of IL-10 by CD5+ B cells was observed when cells deficient inCD40 or CD40L were substituted for the corresponding wild-type cells(Fig. 5, C and D), which was consistent with the data obtained from exper-iments with recombinant CD40L and the anti-CD40L antibody (Fig. 4), aswell as with the requirement of CD40-CD40L interaction for IL-10 pro-duction by CD5+ B cells. Moreover, this interaction appeared to be nec-essary irrespective of whether the BMMCs were stimulated with antigen(Fig. 5, C and D). Loss of either CD40 or CD40L did not alter the pat-terns of cell surface markers of CD5+ B cells or BMMCs, respectively(fig. S4D).

With respect to mast cell function, the IgE- and antigen-induced de-granulation of BMMCs was no longer suppressed when CD40−/− CD5+ Bcells or CD40L−/− BMMCs were substituted for the corresponding wild-type cells (Fig. 5E). Furthermore, the adoptive transfer of wild-type CD5+

B cells, but not CD40−/− CD5+ B cells, into CD19-deficient mice suppressedthe decline in rectal temperatures when PSA was induced (Fig. 5F). Tofurther determine whether the CD40-CD40L interaction between CD5+

B cells and mast cells occurs in vivo, we transferred wild-type orCD40L-deficient BMMCs into KitW-sh/W-sh mice, which do not contain mastcells and, thus, do not exhibit PSA symptoms in response to stimulation

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Fig. 4. IL-10 production by mouse CD5+ B cells is dependent on CD40-CD40L interactions. (A to D) Total B cells from WT mice were left unstim-

(E) and the percentages of IL-10+ B cells (F and G, top) were determinedby flow cytometric analysis. (G, bottom) The amounts of IL-10 in the cul-

ulated or were stimulated with recombinant CD40L (rCD40L;1 mg/ml)for the indicated times. The mean fluorescence intensity (MFI) of CD40staining on B cells (A) and the percentages of IL-10+ B cells (B and C) weredetermined by flow cytometric analysis. (D) The amounts of IL-10 in theculture medium were determined by ELISA. (E to G) CD5+ B cells werecultured alone or with either unstimulated or IgE- and antigen-stimulatedBMMCs in the presence or absence of anti-CD40L antibody or an iso-type control antibody for 24 hours. The MFI of CD40 staining on B cells

ture medium were determined by ELISA. (H) BMMCs preincubated inthe absence or presence of the indicated combinations of CD5+ B cells,anti-CD40L antibody, and isotype control antibody were stimulatedwith IgE and antigen, as indicated. Twenty-four hours later, the extent ofb-hexosaminidase release into the culture medium was determined. Plotsin (B) and (F) are representative of three independent experiments. Datain (A), (C) to (E), (G), and (H) are means ± SEM of three independentexperiments. *P < 0.05; **P < 0.01; n.s., not significant.

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with IgE and antigen (20). Although the antigen-stimulated degranulationof CD40L-deficient BMMCs was comparable to that of wild-typeBMMCs (Fig. 5E), IgE-mediated anaphylaxis responses became more se-vere when the KitW-sh/W-sh mice received CD40L−/− BMMCs instead ofwild-type BMMCs (Fig. 5G). Together, these results suggest that theCD40-CD40L interaction between CD5+ B cells and mast cells is criticalfor the suppression of mast cell activation and IgE-mediated PSA re-sponses in vivo.

IL-10–producing CD5+ B cells inhibit FceRI-mediatedsignaling in mast cellsWe did not observe any decrease in the cell surface abundances of the a,b, or g subunits of FceRI on BMMCs that were cocultured with CD5+ Bcells for 24 hours (Fig. 6, A and B). However, we observed alterations inthe abundances of some tyrosine kinases that transduce initial signals afterantigen-dependent cross-linking of IgE bound to FceRI. Coculture withwild-type CD5+ B cells, but not IL-10−/− CD5+ B cells, substantially reducedthe abundances of the kinases Fyn and Fgr in BMMCs, whereas Lyn andSyk were unaffected (Fig. 6, C and D). The lack of change in the abundanceof Syk was further confirmed by flow cytometric analysis (Fig. 6E). Fur-thermore, wild-type CD5+ B cells, but not IL-10−/− CD5+ B cells, markedly

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inhibited the antigen-induced tyrosine phosphorylation of Syk in BMMCs(Fig. 6, F and G), which suggests that wild-type CD5+ B cells can inhibitthe activation of mast cells by reducing the abundances of Fyn and Fgr.These observations were further confirmed by incubating BMMCs with re-combinant IL-10 for 24 or 72 hours (fig S5, A to D). The essential require-ment for an interaction between CD40 (on CD5+ B cells) and CD40L (onmast cells) to suppress the phosphorylation of the tyrosine kinases afterIgE- and antigen-mediated stimulation was verified in experiments withan anti-CD40L antibody (fig. S5, E and F).

The inhibition of mast cells by IL-10 is dependent on signal transducerand activator of transcription 3 (STAT3) signaling, which leads to the re-duced activation of tyrosine kinases involved in early signaling events(14). We observed that IL-10 stimulated the phosphorylation of STAT3,which was inhibited by the Janus-activated kinase (JAK) inhibitor AG490(Fig. 6H). In addition, AG490 restored the abundance of Fyn and Fgr (Fig.6I) as well as the extent of tyrosine phosphorylation of Syk in IL-10–treatedBMMCs (Fig. 6J). Further experiments showed that STAT3-specific smallinterfering RNA (siRNA) restored the amounts of Fyn and Fgr, as well asthe phosphorylation of Syk, in IL-10–treated mast cells (Fig. 6, K and L),which suggests that the suppression of mast cells by CD5+ B cell–derivedIL-10 is mediated through the JAK-STAT3 pathway.

Fig. 5. Suppression of mast cell activation and PSA by CD5+ B cells dependson CD40-CD40L interactions. (A and B) CD5+ B cells from WT or CD40−/−

from WT or CD40L−/− mice, as indicated. The extent of b-hexosaminidaserelease into the culture medium was determined. (F) CD19−/− mice were

mice were incubated in a 1:1 ratio with BMMCs from WT or CD40L−/− mice,as indicated. (A) After 1 hour, BMMC–B cell conjugates were detected byflow cytometric analysis. (B) The percentages of BMMC–B cell conjugatesthat formed under the four indicated conditions (a to d) were calculated.(C and D) CD5+ B cells from WT or CD40−/− mice were incubated alone orin a 1:1 ratio with unstimulated or IgE- and antigen-stimulated BMMCs fromWT or CD40L−/− mice, as indicated. (C) Cells were subjected to flow cyto-metric analysis to identify IL-10+CD5+ B cells. (D) The percentages ofIL-10+CD5+ B cells under the indicated conditions were calculated. (E)CD5+ B cells from WT or CD40−/− mice were incubated for 24 hours aloneor in a 1:1 ratio with unstimulated or IgE- and antigen-stimulated BMMCs

left untreated or received CD5+ or CD5− B cells from WT or CD40−/− miceby adoptive transfer. Three days later, the mice were treated with IgE andantigen to induce PSA, and rectal temperatures in the indicated mice weremeasured over time (n = 5 mice per experiment). (G) Mast cell–deficientmice (KitW-sh/W-sh mice) were left untreated or received BMMCs from WT orCD40L−/− mice by adoptive transfer. Three days later, the mice weretreated with the indicated combinations of IgE and antigen, and the rec-tal temperatures of the mice were measured over time. Plots in (A) and(C) are representative of three independent experiments. Data in (B) and(D) to (G) are means ± SEM of three independent experiments. *P <0.05; **P < 0.01.

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Fig. 6. CD5+ B cell–derived IL-10 reduces Fyn and Fgr abundance and nant IL-10 (rIL-10; 100 ng/ml) in the absence or presence of 25 mM AG490

inhibits downstream activation of Syk in BMMCs. (A and B) BMMCs fromWT mice were cultured alone or with CD5+ or CD5− B cells, as indicated.(A) Twenty-four hours later, the cells were analyzed by flow cytometry todetect the cell surface expression of the indicated FceRI subunits. (B)The relative MFIs of the indicated FceRI subunits on the BMMCs werecalculated. (C and D) BMMCs were cultured alone or with CD5+ B cellsfrom WT or IL-10−/− mice, as indicated. (C) BMMCs were then analyzedby Western blotting with antibodies against the indicated proteins. (D)Quantification of the relative abundances of the indicated proteins was per-formed by densitometric analysis of Western blots. (E) BMMCs werecultured alone or with CD5+ B cells from WT or IL-10−/− mice at a 1:5 ra-tio. Twenty-four hours later, the cells were treated with the indicated com-binations of IgE and antigen before being analyzed by flow cytometry todetermine the MFI of Syk. (F and G) BMMCs cultured alone or with WTor IL-10−/− CD5+ B cells at a 1:5 ratio for 24 hours were then treated for7 min with the indicated combinations of IgE and antigen. (F) The BMMCswere analyzed by flow cytometry to detect tyrosine-phosphorylated Syk(Tyr352). (G) The MFIs of pSyk in BMMCs under the indicated conditionswere determined. (H) BMMCs were incubated with or without recombi-

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for 24 hours and were then were left unstimulated or were stimulated withantigen for 15 min. Cells were then analyzed by flow cytometry to determinethe MFIs of total STAT3 (top) and pSTAT3 (Tyr705; bottom). (I and J) BMMCswere cultured for 24 hours in medium containing IL-3 in the absence orpresence of rIL-10 (100 ng/ml) or 25 mM AG490. The cells were then stimu-lated with the indicated combinations of IgE and antigen for 7 min beforebeing analyzed by Western blotting to detect total Lyn, Fyn, and Fgr pro-teins (I) and total Syk and phosphorylated Syk proteins (J). Band densitiesare shown as the mean values from three independent experiments in eachlower panel. (K and L) BMMCs were transfected with STAT3-specific siRNAs(siSTAT3) or control siRNAs (siCtrl) 48 hours before the experiment. TheBMMCs were then incubated with the indicated combinations of rIL-10(100 ng/ml) and IgE (500 ng/ml) for 24 hours before being left untreatedor stimulated with antigen for 7 min. (K) BMMCs were analyzed by Westernblotting with antibodies specific for the indicated proteins. Western blots arerepresentative of three independent experiments. (L) Densitometric analysisof the relative abundances of total Fyn and Fgr proteins and of pSyk. Data in(B), (D), (E), (G), (I and J, lower panels), and (L) are means ± SEM of threeindependent experiments. *P < 0.05; **P < 0.01.

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DISCUSSION

B cells have the capacity to produce antibodies, function as APCs, andregulate the activation of CD4+ T cells (21–23). In addition, there is ev-idence that B cells have a regulatory role in various immune responsesthrough their production of cytokines. Katz et al. (24) were the first todemonstrate that the delayed-type hypersensitivity reaction was exacer-bated by the depletion of B cells. Mizoguchi and Bhan (9) introducedthe term “regulatory B cells” to designate negative regulatory subpop-ulations of B cells. Breg cells are now recognized as one of the key regu-latory cell types that suppress inflammatory disorders (10) and variousimmune cells, including dendritic cells (DCs), macrophages, and TH cells(25, 26).

IL-10 was originally identified as the TH2 cell–derived cytokine syn-thesis inhibitory factor (27), and it has broad anti-inflammatory actions.IL-10 suppresses the effector function of T cells and macrophages (28, 29).With respect to Breg cells (B10 cells), IL-10 enables the suppressive func-tions of these cells in various immune disease models (10). Moreover, sub-sets of IL-10–producing Breg cells suppress TH2 cell–mediated allergicresponses, including contact hypersensitivity and allergic airway diseasein mouse models (7, 12). Although mast cells can produce IL-10 throughFceRIII signaling after the receptor is increased in abundance by IL-4 (30),IL-10 is not normally produced by mast cells through IgE- and antigen-mediated stimulation (fig. S3, B and C).

Mast cells are the critical effector cells in food allergies, allergic asthma,and allergic rhinitis (16, 18). Although present in multiple tissues, mast cellsare distributed mainly at the major immunologic interfaces, such as the skin,gut, and lungs. Secondary lymphoid organs including spleens, tonsils, andlymph nodes harbor modest numbers of mast cells in physiological settings(19, 31) where they could also regulate inflammation. We previously re-ported that the prevalence of IL-10–producing CD5+ peripheral bloodB cells increased in healthy donors but decreased in patients with milkallergy after challenge with milk antigen (32). Our present finding thatCD5+ B cells inhibited IgE-mediated mast cell activation and anaphylaxisin mice in an IL-10–dependent manner suggests that the interaction be-tween IL-10–producing CD5+ B cells and mast cells provides a mecha-nism for counteracting allergic phenomena. IL-10, in particular, mayprovide another link between IL-10–producing CD5+ B cells and variousimmune cells, such as regulatory T cells, DCs, and eosinophils, in that itinhibits mast cell activation as well as allergic reactions (14, 33, 34). Aswe reported here, production of IL-10 by CD5+ B cells is enhanced uponcoculture with mast cells (Fig. 2, G to I). The possible physical associationbetween mast cells and CD5+ B cells was apparent from immunohisto-chemical analysis of the spleens of mice with PSA (Fig. 3D) and was verifiedby the observation that these cells form conjugates in coculture (Fig. 3,B and C). Therefore, the potential exists for crosstalk between thesetwo cell types in physiological settings. Furthermore, direct cell-to-cellcontact was essential for the production of IL-10 by CD5+ B cells, as wellas for the inhibition of mast cell activation by CD5+ B cells.

The interaction between CD40L and CD40 on T cells and B cells,respectively, is critical for CD4+ T cell activation and the effector func-tions of B cells (35). Such an interaction may also stimulate the proliferationof IL-10–producing Breg cells in mice (36) and in patients with systemiclupus erythematosus (SLE) (37, 38) and thereby suppress the humoral re-sponse. Mast cells were reported to communicate with B cells and astro-cytes through the CD40-CD40L interaction (39, 40). Here, we proposethat mast cells may similarly regulate CD5+ B cell function on the basisthat mast cells express CD40L (fig. S5D) and increase the cell surfaceabundance of CD40 on CD5+ B cells, and that the CD40L-CD40 interac-tion leads to an increase in the number of IL-10–producing CD5+ B cells,

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which in turn suppress mast cell activation and anaphylaxis (Fig. 5). No-tably, the anaphylaxis responses stimulated by IgE and antigen were re-duced in mice that received wild-type CD5+ B cells, but not CD40−/−

CD5+ B cells, by adoptive transfer (Fig. 5F). To further determine whetherCD40L on mast cells was critical for a direct interaction with CD40 onCD5+ B cells, we performed experiments with mast cell–deficient mice(KitW-sh/W-sh mice), which do not show any PSA in response to IgE andantigen (20). Although IgE-mediated anaphylaxis was not observed inKitW-sh/W-sh mice (Fig. 5G) (20), the responses became much more severein mice that received CD40L−/− BMMCs by adoptive transfer compared tothose that received wild-type BMMCs (Fig. 5G). Together, these resultssuggest that CD40L on mast cells is critical to the induction of IL-10–producing CD5+ B cells in physiological settings.

The precise details by which IL-10–producing Breg cells suppress al-lergic and inflammatory responses are unclear. Our results provide someinsight with regard to the interaction of the B cells with mast cells. Earlysignaling events in antigen-stimulated mast cells include the recruitment ofLyn and other Src family kinases, such as Fyn (41) and Fgr (42), to FceRI,which results in the phosphorylation and activation of Syk and the activa-tion of mast cells (43). In our experimental system, coculturing CD5+ Bcells with BMMCs for 24 hours reduced the abundance of Fyn and Fgr inmast cells, and consequently decreased the phosphorylation of Syk (Fig.6, C to G), but had no effect on the abundances of individual FceRIsubunits (Fig. 6B). Similar results were obtained from experiments inwhich BMMCs were treated with recombinant IL-10 under similarconditions. However, more prolonged incubation (72 hours) of mast cellswith IL-10 resulted in a reduction in the amounts of the FceRI subunits, inaddition to a reduction in the abundances of Syk, Fyn, and Fgr in mastcells (fig. S5, B and D), consistent with previous findings (14). We furtherdemonstrated that the effects of CD5+ B cells on the abundances of Fynand Fgr and on the phosphorylation of Syk were blocked by inhibiting theJAK-STAT3 pathway with a typical JAK inhibitor, AG490 (Fig. 6, H to J),and by siRNAs specific for STAT3 in mast cells (Fig. 6, K and L). Theseobservations led us to suggest that CD5+ B cells inhibit mast cell activa-tion through IL-10 by reducing the abundances of Fyn and Fgr throughthe JAK-STAT3 signaling pathway. It would be also of interest to determine

Fig. 7. Proposed scheme for the suppression of mast cell activation by IL-10–+

producing CD5 B cells. Suppression is dependent on direct cell-to-cell con-

tact through the interaction of CD40L on mast cells and CD40 on CD5+

B cells. This interaction results in the production of IL-10 by the CD5+ B cells.IL-10 signaling reduces the abundances of Fyn and Fgr in the mast cells,which thus reduces the extent of activation of Syk, resulting in the inhibitionof mast cell degranulation. Tyk, tyrosine kinase.

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whether the inhibitory effects of IL-10–producing B cells extend to othermast cell stimulants such as IgG1, which may be involved in analphylacticreactions.

In summary, our results demonstrate a mechanism by which IL-10–producing CD5+ B cells inhibit mast cell function as follows: (i) Bothcell types form cell-cell conjugates through an interaction betweenCD40 (on CD5+ B cells) and CD40L (on mast cells). (ii) This interac-tion stimulates IL-10 production by CD5+ B cells. (iii) IL-10 producedby CD5+ B cells inhibits the abundance of Fyn and Fgr in mast cellsthrough activation of the JAK-STAT3 pathway. (iv) As a result, mastcell activation through FceRI is suppressed by the diminished quanti-ties of Fyn and Fgr in mast cells (Fig. 7). These findings suggest thatIL-10–producing CD5+ B cells may provide an additional therapeutictarget to treat IgE-mediated allergic diseases.

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MATERIALS AND METHODS

MiceWild-type (~6- to 8-week-old male C57BL/6 mice), CD19−/− [Cd19tm1(cre)Cgn],IL-10−/− (Il10tm1Cgn), CD40−/− (Cd40tm1Kik), CD40L−/− (Cd40lgtm1Imx),and KitW-sh/W-sh mice were purchased from The Jackson Laboratory, housedin a specific pathogen–free animal facility at Konkuk University (Seoul, Korea)and fed with a sterilized diet and autoclaved water before being used forexperiments. All animal experiments were approved by the InstitutionalAnimal Care and Use Committee (IACUC) at Konkuk University.

Preparation and adoptive transfer of B cell subsetsSplenic B cells were presorted with CD19 microbeads (Miltenyi Biotec).Then CD5+ or CD5− B cells were isolated with a FACSAria flow cytom-eter (BD Biosciences). For in vivo adoptive transfer of B cell subsets,the isolated B cells were transferred intravenously [2 × 106 cells/0.2 ml ofphosphate-buffered saline (PBS)] into recipient mice. Three days after theadoptive transfer of cells, PSA was induced in the recipient mice.

Induction of PSA or PCAMice were primed with 3 mg of DNP-specific IgE (SPE-7; Sigma) by in-travenous injection. On the next day, the mice were injected intravenouslywith 250 mg of DNP-BSA (Sigma) in 200 ml of PBS or as indicated inthe figure legends. Rectal temperatures of the mice were measured every10 min for 1 hour and 30 min after they were injected with antigen. Forthe histamine assay, the mice were euthanized with CO2 30 min afterthey were injected with antigen, and serum was obtained by cardiac punc-ture. The concentration of histamine in the serum was measured byELISA according to the manufacturer’s instructions (Beckman Coulter).PCA was induced as described previously (43). All experiments withmice were performed three times, with five mice for each condition usedper experiment.

Flow cytometric analysisSingle-cell suspensions were isolated from the spleen, inguinal lymph node,peritoneal cavity, and blood. To detect intracellular IL-10 in B cells fromeach site, isolated B cells were cultured with medium alone or with mediumcontaining BMMCs, IgE-treated BMMCs, IgE- and antigen-treated BMMCs,or CD40L (1 mg/ml; R&D Systems) for 24 hours or the times indicated inthe figure legends, and phorbol 12-myristate 13-acetate (50 ng/ml; Sigma),ionomycin (500 ng/ml; Sigma), and brefeldin A (3 mg/ml; eBioscience)were added during the last 5 hours of incubation. Before cell surfacemarkers were stained, Fcg receptors were blocked with anti-CD16 andanti-CD32 monoclonal antibodies (2.4G2, BD Biosciences), and

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conjugated and dead cells were excluded from the analysis on the basisof forward and side light scatter parameters and staining with Fixable Vi-ability Dye (eBioscience). Cells were fixed and permeabilized with aCytofix/Cytoperm kit (BD Biosciences) and then were incubated with anti–IL-10 monoclonal antibody (JES5-16E3, eBioscience) at 4°C for 30 min.The antibodies against cell surface proteins were as follows: anti-CD1d(1B1), anti-CD4 (RM4-5), anti-CD5 (53–7.3), anti-CD11b (M1/70), anti-CD19 (eBio1D3), anti-CD21/CD35 (eBioBD9), anti-CD23 (B3B4), anti-CD25 (PC61.5), anti-CD40 (HM40-3), anti-CD86 (GL1), anti-B220(RA3-6B2), anti-IgD (11–26), anti-IgM (eB121-15 F9), anti–c-Kit(2B8), and anti-CD40L (MR1), which were purchased from eBioscience,and anti-FceRI (anti-IgE, R35-72), which was purchased from BD Bio-sciences. To detect FceRI subunits and intracellular Syk, fixed BMMCswere stained with antibodies against FceRIa (G-14), FceRIb (N-18),FceRIg (H-5), and Syk (N-19), which were obtained from Santa CruzBiotechnology, and with anti-STAT3 antibody (M59-50), which was ob-tained from BD Biosciences. To evaluate the extent of phosphorylation ofSyk or STAT3, BMMCs were primed with DNP-specific IgE (500 ng/ml)and cultured with CD5+ B cells for 24 hours before being stimulated withDNP-BSA (100 ng/ml) for 7 min (for pSyk) or 15 min (for pSTAT3).The BMMCs were immediately fixed and permeabilized and then werestained with anti-CD19 (eBio1D3), anti–c-Kit (2B8, eBioscience), and ei-ther anti-ZAP70(Tyr319)/Syk(Tyr352) (17A/P-ZAP70) or anti-STAT3(Tyr705) (4/P-STAT3) antibodies (BD Biosciences). Briefly, after CD19+

B cells were excluded from gated mast cells because of staining with an anti-CD19 monoclonal antibody, c-Kit+ cells that stained with anti-ZAP70(Tyr319)/Syk(Tyr352)+ or anti-STAT3 (Tyr705)+ were analyzed. Cells were analyzedwith a FACSCalibur flow cytometer (Becton Dickinson) and FlowJo version10 software (TreeStar).

BMMC differentiation and transfection withSTAT3-specific siRNABMMCs derived from C57BL/6 or BALB/c mice were cultured in RPMI1640 medium containing 2 mM L-glutamine, 0.1 mM nonessential aminoacids, antibiotics, 10% fetal bovine serum (FBS), and IL-3 (10 ng/ml;PeproTech Inc.). After 4 weeks, >98% of the cells were verified asBMMCs, as previously described (44). BMMCs (5 × 106 cells) were trans-fected with 100 nM STAT3-specific siRNA or scrambled siRNA with anAmaxa Nucleofector (Lonza Cologne AG) with program T-5 in Dulbecco’smodified Eagle’s medium with 20% FBS and 50 mM Hepes (pH 7.5).Cells were used within 48 hours of transfection.

Measurement of degranulation and release of cytokinesBMMCs were primed for 4 hours with DNP-specific IgE (500 ng/ml; Sigma).The IgE-primed BMMCs were then stimulated with antigen [DNP-BSA(100 ng/ml); Sigma] in Tyrode-BSA buffer [20 mM Hepes (pH 7.4), 135mM NaCl, 5 mM potassium chloride, 1.8 mM calcium chloride, 1 mMmagnesium chloride, 5.6 mM glucose, and 0.05% BSA] for 15 min in thepresence or absence of the B cell subsets indicated in the figure legends.Degranulation was determined by measuring the release of the granulemarker b-hexosaminidase as previously described (45). In some cocultureexperiments, BMMCs and B cell subsets were separated by 3.0-mm trans-well membrane plates (Corning Life Sciences). Cells were stimulated withantigen for 24 hours (or the times indicated in the figure legends) incomplete medium to measure the secretion of TNF-a, IL-4, and IL-10 withELISA kits from Invitrogen (BioSource) or R&D Systems Inc.

ImmunohistochemistryParaffin-embedded spleen sections were subjected to immunohistochem-ical analysis with specific antibodies and isotype controls according to a

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regular protocol. The signal was amplified with horseradish peroxidase–or alkaline phosphatase–conjugated streptavidin with a Vectastain Elite ABCkit (Vector). The sections were visualized with N-DAB or VectorRed, andthen the mast cells were stained with toluidine blue.

Analysis of CD5+ or CD5− B cell–mast cell conjugationIgE-primed or untreated BMMCs were stained with 1 mM CellTrackerGreen probe (BODIPY, Invitrogen), and isolated splenic CD5+ B cells werestained with 1 mM CellTracker Red probe (CMPTX, Invitrogen). Thestained BMMCs (1 × 106 cells in 0.5 ml) were combined with 0.5 ml ofstained CD5+ B cells (1 × 106 cells) at a cell/cell ratio of 1:1. The mixedcells were incubated with or without antigen (100 ng/ml) at 37°C for45 min. Cells were then immediately fixed in 4% paraformaldehyde.Cell conjugates were determined by flow cytometric analysis with aFACSCalibur flow cytometer (Becton Dickinson).

Western blottingAfter BMMCs were cocultured with wild-type or IL-10−/− CD5+ B cellsfor 24 hours, CD19 microbeads (Miltenyi Biotec) were used to purifyBMMCs by negative selection according to the manufacturer’s instruc-tions. The BMMCs were lysed in 100 ml of ice-cold lysis buffer containinga protease inhibitor cocktail tablet. The cell lysates were subjected to West-ern blotting analysis according to a standard protocol. Antibodies againstFceRg and actin were purchased from Upstate Biotechnology; antibodiesagainst FceRa, FceRb, Lyn, Fyn, Fgr, and Syk were obtained from SantaCruz Biotechnology; and antibody against the phosphorylated form ofSyk (pSyk) was purchased from Cell Signaling Technology.

Statistical analysisData were expressed as means ± SEM. Statistical analysis was performedby one-way analysis of variance and Dunnett’s test. Statistical signifi-cance (*P < 0.05 and **P < 0.01) was determined with SigmaStat soft-ware (Systat Software Inc).

SUPPLEMENTARY MATERIALSwww.sciencesignaling.org/cgi/content/full/8/368/ra28/DC1Fig. S1. Analysis of the population of IL-10–producing CD5+ B cells involved in PSA.Fig. S2. Characterization of CD5+ B cells in IL-10−/− mice and of IL-10–producing B cellsstimulated by mast cells.Fig. S3. Analysis of the production of IL-10 by B cells, mast cells, and various tissue-derivedCD5+ B cells.Fig. S4. The CD40-CD40L interaction is critical for mast cell–mediated IL-10 production byB cells.Fig. S5. IL-10 reduces the abundances of several critical signaling molecules in mast cells.

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Funding: This work was supported by the National Research Foundation of Korea (NRF)grant [Ministry of Science, ICT, and Future Planning (MSIP), No. 2012R1A2A1A03670516]and in part by an NRF grant (MSIP, NRF-2013R1A4A1069575) funded by the Korean gov-ernment. M.A.B. was supported by the Intramural Program of the National Heart, Lung, andBlood Institute, NIH. Author contributions: W.S.C. and Y.M.K. designed the experiments,analyzed the data, and wrote the paper; Hyuk S.K. and A.-R.K. performed most of the ex-periments; D.K.K. and G.H.J. collected and analyzed flow cytometry data; H.W.K. and Y.H.P.performed in vivo experiments; J.S.Y. and Hyung S.K. performed all experiments for cellsignaling analysis; B.K. and Y.M.P. contributed reagents and knockout animals and designedthe experiments; and Y.M.K. and M.A.B. provided intellectual input and wrote the paper.Competing interests: The authors declare that they have no competing interests.

Submitted 29 August 2014Accepted 27 February 2015Final Publication 17 March 201510.1126/scisignal.2005861Citation: H.S.Kim, A.-R. Kim,D. K.Kim,H.W. Kim,Y.H.Park,G. H. Jang, B. Kim, Y.M. Park,J. S. You, H. S. Kim, M. A. Beaven, Y. M. Kim, W. S. Choi, Interleukin-10–producing CD5+ Bcells inhibit mast cells during immunoglobulin E–mediated allergic responses. Sci. Signal. 8,ra28 (2015).

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.SCIENCESIGNALING.org 17 March 2015 Vol 8 Issue 368 ra28 11

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allergic responsesmediated− B cells inhibit mast cells during immunoglobulin E+producing CD5−Interleukin-10

Park, Jueng Soo You, Hyung Sik Kim, Michael A. Beaven, Young Mi Kim and Wahn Soo ChoiHyuk Soon Kim, A-Ram Kim, Do Kyun Kim, Hyun Woo Kim, Young Hwan Park, Geun Hyo Jang, Bokyung Kim, Yeong Min

DOI: 10.1126/scisignal.2005861 (368), ra28.8Sci. Signal. 

target to treat allergic diseases.producing B cells might provide a therapeutic−IL-10, which inhibited tyrosine kinase signaling in mast cells. Thus, IL-10mediated inhibition of mast cells depended on−with the B cells, which stimulated the B cells to produce more IL-10. B cell

lacking these special B cells had more severe symptoms of anaphylaxis. Mast cell inhibition required physical contact cells inhibited the activation of mast cells, immune cells that are critical regulators of allergic reactions. Indeed, mice

. found that these Bet alanti-inflammatory cytokine interleukin-10 (IL-10) and have immunosuppressive properties. Kim B cells promote immune responses by producing antibodies; however, subsets of B cells secrete the

Limiting allergic responses with B cells

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REFERENCES

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