A Regulatory B Cell Subset with a Unique CD1dhiCD5+ Phenotype Controls T Cell-Dependent Inflammatory Responses

B cells mediate multiple functions that influence immune and inflammatory responses. In this study, T cell-mediated inflammation was exaggerated in CD19-deficient (Cd19−/−) mice and wild-type mice depleted of CD20+ B cells, whereas inflammation was substantially reduced in mice with hyperactive B cells as a result of CD19 overexpression (hCD19Tg). These inflammatory responses were negatively regulated by a unique CD1dhiCD5+ B cell subset that was absent in Cd19−/− mice, represented only 1%–2% of spleen B220+ cells in wild-type mice, but was expanded to ∼10% of spleen B220+ cells in hCD19Tg mice. Adoptive transfer of these CD1dhiCD5+ B cells normalized inflammation in wild-type mice depleted of CD20+ B cells and in Cd19−/− mice. Remarkably, IL-10 production was restricted to this CD1dhiCD5+ B cell subset, with IL-10 production diminished in Cd19−/− mice, yet increased in hCD19Tg mice. Thereby, CD1dhiCD5+ B cells represent a unique subset of potent regulatory B cells. B cells mediate multiple functions that influence immune and inflammatory responses. In this study, T cell-mediated inflammation was exaggerated in CD19-deficient (Cd19−/−) mice and wild-type mice depleted of CD20+ B cells, whereas inflammation was substantially reduced in mice with hyperactive B cells as a result of CD19 overexpression (hCD19Tg). These inflammatory responses were negatively regulated by a unique CD1dhiCD5+ B cell subset that was absent in Cd19−/− mice, represented only 1%–2% of spleen B220+ cells in wild-type mice, but was expanded to ∼10% of spleen B220+ cells in hCD19Tg mice. Adoptive transfer of these CD1dhiCD5+ B cells normalized inflammation in wild-type mice depleted of CD20+ B cells and in Cd19−/− mice. Remarkably, IL-10 production was restricted to this CD1dhiCD5+ B cell subset, with IL-10 production diminished in Cd19−/− mice, yet increased in hCD19Tg mice. Thereby, CD1dhiCD5+ B cells represent a unique subset of potent regulatory B cells. B cells play a central role in humoral immunity, but they also regulate CD4+ T cell responses to foreign and self-antigens (Bouaziz et al., 2007Bouaziz J.-D. Yanaba K. Venturi G.M. Wang Y. Tisch R.M. Poe J.C. Tedder T.F. Therapeutic B cell depletion impairs adaptive and autoreactive CD4+ T cell activation in mice.Proc. Natl. Acad. Sci. USA. 2007; 104: 20882-20887Crossref Scopus (249) Google Scholar, Xiu et al., 2008Xiu Y. Wong C.P. Hamaguchi Y. Wang Y. Pop S. Tisch R.M. Tedder T.F. B lymphocytes depletion by CD20 monoclonal antibody prevents diabetes in NOD mice despite isotype-specific differences in FcγR effector functions.J. Immunol. 2008; 180: 2863-2875PubMed Google Scholar), function as antigen-presenting cells (Constant et al., 1995Constant S. Schweitzer N. West J. Ranney P. Bottomly K. B lymphocytes can be competent antigen-presenting cells for priming CD4+ T cells to protein antigens in vivo.J. Immunol. 1995; 155: 3734-3741PubMed Google Scholar), produce cytokines (Harris et al., 2000Harris D.P. Haynes L. Sayles P.C. Duso D.K. Eaton S.M. Lepak N.M. Johnson L.L. Swain S.L. Lund F.E. Reciprocal regulation of polarized cytokine production by effector B and T cells.Nat. Immunol. 2000; 1: 475-482Crossref PubMed Scopus (629) Google Scholar), provide costimulatory signals (Linton et al., 2003Linton P.J. Bautista B. Biederman E. Bradley E.S. Harbertson J. Kondrack R.M. Padrick R.C. Bradley L.M. Costimulation via OX40L expressed by B cells is sufficient to determine the extent of primary CD4 cell expansion and Th2 cytokine secretion in vivo.J. Exp. Med. 2003; 197: 875-883Crossref PubMed Scopus (193) Google Scholar), and promote naive CD4+ T cell differentiation into T helper 1 (Th1) or Th2 subsets (Harris et al., 2000Harris D.P. Haynes L. Sayles P.C. Duso D.K. Eaton S.M. Lepak N.M. Johnson L.L. Swain S.L. Lund F.E. Reciprocal regulation of polarized cytokine production by effector B and T cells.Nat. Immunol. 2000; 1: 475-482Crossref PubMed Scopus (629) Google Scholar). Abnormal B cell function can also drive the development of autoimmunity (Sato et al., 1996Sato S. Ono N. Steeber D.A. Pisetsky D.S. Tedder T.F. CD19 regulates B lymphocyte signaling thresholds critical for the development of B-1 lineage cells and autoimmunity.J. Immunol. 1996; 157: 4371-4378PubMed Google Scholar). B cells play protective roles as well because both B cell-deficient and CD19-deficient (Cd19−/−) mice develop a severe nonremitting form of experimental autoimmune encephalomyelitis (EAE), a model of human multiple sclerosis (Fillatreau et al., 2002Fillatreau S. Sweenie C.H. McGeachy M.J. Gray D. Anderton S.M. B cells regulate autoimmunity by provision of IL-10.Nat. Immunol. 2002; 3: 944-950Crossref PubMed Scopus (1198) Google Scholar, Matsushita et al., 2006Matsushita T. Fujimoto M. Hasegawa M. Komura K. Takehara K. Tedder T.F. Sato S. Inhibitory role of CD19 in the progression of experimental autoimmune encephalomyelitis by regulating cytokine response.Am. J. Pathol. 2006; 168: 812-821Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). In addition, B cells play a protective role during T cell-mediated inflammation because Cd19−/− mice have augmented contact hypersensitivity (CHS) responses (Watanabe et al., 2007Watanabe R. Fujimoto M. Ishiura N. Kuwano Y. Nakashima H. Yazawa N. Okochi H. Sato S. Tedder T.F. Tamaki K. CD19 expression in B cells is important for suppression of contact hypersensitivity.Am. J. Pathol. 2007; 171: 560-570Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). CHS is an inflammatory immune reaction that is mediated by T cells in sensitized individuals after subsequent contact with the sensitizing antigen. During CHS sensitization, skin antigen-presenting Langerhans cells migrate to draining lymph nodes and prime antigen-specific T cells (Kripke et al., 1990Kripke M.L. Munn C.G. Jeevan A. Tang J.M. Bucana C. Evidence that cutaneous antigen-presenting cells migrate to regional lymph nodes during contact sensitization.J. Immunol. 1990; 145: 2833-2838PubMed Google Scholar). Subsequent antigen challenge at a separate skin site results in cutaneous infiltration of antigen-primed T cells and in edema or spongiosis that peaks 24–48 hr after challenge and then decreases (Wang et al., 2000Wang B. Fujisawa H. Zhuang L. Freed I. Howell B.G. Shahid S. Shivji G.M. Mak T.W. Sauder D.N. CD4+ Th1 and CD8+ type 1 cytotoxic T cells both play a crucial role in the full development of contact hypersensitivity.J. Immunol. 2000; 165: 6783-6790PubMed Google Scholar). Although antigen-specific IgM production by peritoneal B-1 cells is important during CHS initiation (Itakura et al., 2005Itakura A. Szczepanik M. Campos R.A. Paliwal V. Majewska M. Matsuda H. Takatsu K. Askenase P.W. An hour after immunization peritoneal B-1 cells are activated to migrate to lymphoid organs where within 1 day they produce IgM antibodies that initiate elicitation of contact sensitivity.J. Immunol. 2005; 175: 7170-7178PubMed Google Scholar), this does not explain enhanced inflammation in Cd19−/− mice because they are B-1a cell-deficient and generate modest responses to most transmembrane signals and antigens (Sato et al., 1995Sato S. Steeber D.A. Tedder T.F. The CD19 signal transduction molecule is a response regulator of B-lymphocyte differentiation.Proc. Natl. Acad. Sci. USA. 1995; 92: 11558-11562Crossref PubMed Scopus (146) Google Scholar). B cell deficiency delays the emergence of regulatory T cells and interleukin-10 (IL-10) production in the central nervous system (CNS) during EAE (Mann et al., 2007Mann M.K. Maresz K. Shriver L.P. Tan Y. Dittel B.N. B cell regulation of CD4+CD25+ T regulatory cells and IL-10 via B7 is essential for recovery from experimental autoimmune encephalomyelitis.J. Immunol. 2007; 178: 3447-3456PubMed Google Scholar). Furthermore, IL-10 produced by B cells can downregulate autoimmune disease in EAE (Fillatreau et al., 2002Fillatreau S. Sweenie C.H. McGeachy M.J. Gray D. Anderton S.M. B cells regulate autoimmunity by provision of IL-10.Nat. Immunol. 2002; 3: 944-950Crossref PubMed Scopus (1198) Google Scholar), collagen-induced arthritis (Mauri et al., 2003Mauri C. Gray D. Mushtaq N. Londei M. Prevention of arthritis by interleukin 10-producing B cells.J. Exp. Med. 2003; 197: 489-501Crossref PubMed Scopus (674) Google Scholar), and inflammatory bowel disease (Mizoguchi et al., 2002Mizoguchi A. Mizoguchi E. Takedatsu H. Blumberg R.S. Bhan A.K. Chronic intestinal inflammatory condition generates IL-10-producing regulatory B cell subset characterized by CD1d upregulation.Immunity. 2002; 16: 219-230Abstract Full Text Full Text PDF PubMed Scopus (708) Google Scholar). IL-10-deficient (Il10−/−) mice also have enhanced CHS responses (Berg et al., 1995Berg D.J. Leach M.W. Kuhn R. Rajewsky K. Muller W. Davidson N.J. Rennick D. Interleukin 10 but not interleukin 4 is a natural suppressant of cutaneous inflammatory responses.J. Exp. Med. 1995; 182: 99-108Crossref PubMed Scopus (224) Google Scholar). Neutralizing IL-10 by monoclonal antibody (mAb) treatment also enhances CHS responses, whereas systemic IL-10 administration reduces CHS responses (Ferguson et al., 1994Ferguson T.A. Dube P. Griffith T.S. Regulation of contact hypersensitivity by interleukin 10.J. Exp. Med. 1994; 179: 1597-1604Crossref PubMed Scopus (149) Google Scholar, Schwarz et al., 1994Schwarz A. Grabbe S. Riemann H. Aragane Y. Simon M. Manon S. Andrade S. Luger T.A. Zlotnik A. Schwarz T. In vivo effects of interleukin-10 on contact hypersensitivity and delayed-type hypersensitivity reactions.J. Invest. Dermatol. 1994; 103: 211-216Crossref PubMed Scopus (145) Google Scholar). IL-10 is secreted by multiple cell types, including T cells, monocytes, macrophages, mast cells, eosinophils, and keratinocytes, and can suppress both Th1 and Th2 polarization and inhibit macrophage antigen presentation and proinflammatory cytokine production (Asadullah et al., 2003Asadullah K. Sterry W. Volk H.D. Interleukin-10 therapy-review of a new approach.Pharmacol. Rev. 2003; 55: 241-269Crossref PubMed Scopus (732) Google Scholar). Thus, B cells and IL-10 play important inhibitory roles during T cell-mediated inflammatory responses. The generation of regulatory B cells has been reported in mouse models of chronic inflammation, although their existence in normal mice remains unknown (Mizoguchi and Bhan, 2006Mizoguchi A. Bhan A.K. A case for regulatory B cells.J. Immunol. 2006; 176: 705-710PubMed Google Scholar). Furthermore, it is unknown whether multiple B cell populations or a distinct B cell subset regulates inflammatory responses, whether regulatory B cells produce IL-10 or other cytokines directly, or whether regulatory B cells have potent activities in vivo. Therefore, the importance of B cells during T cell-mediated inflammation was examined with CHS responses as a model for inflammation in Cd19−/− and human CD19 transgenic (hCD19Tg) mice and in wild-type (WT) mice with intact immune systems that were depleted of B cells in vivo (Uchida et al., 2004aUchida J. Hamaguchi Y. Oliver J.A. Ravetch J.V. Poe J.C. Haas K.M. Tedder T.F. The innate mononuclear phagocyte network depletes B lymphocytes through Fc receptor-dependent mechanisms during anti-CD20 antibody immunotherapy.J. Exp. Med. 2004; 199: 1659-1669Crossref PubMed Scopus (487) Google Scholar). CHS was chosen as a model because a balance between B cells and dendritic cells regulates CD4+ T cell expansion in response to antigens in vivo (Bouaziz et al., 2007Bouaziz J.-D. Yanaba K. Venturi G.M. Wang Y. Tisch R.M. Poe J.C. Tedder T.F. Therapeutic B cell depletion impairs adaptive and autoreactive CD4+ T cell activation in mice.Proc. Natl. Acad. Sci. USA. 2007; 104: 20882-20887Crossref Scopus (249) Google Scholar, Xiu et al., 2008Xiu Y. Wong C.P. Hamaguchi Y. Wang Y. Pop S. Tisch R.M. Tedder T.F. B lymphocytes depletion by CD20 monoclonal antibody prevents diabetes in NOD mice despite isotype-specific differences in FcγR effector functions.J. Immunol. 2008; 180: 2863-2875PubMed Google Scholar) but skin Langerhans cells are the exclusive antigen-presenting cells during CHS (Bursch et al., 2007Bursch L.S. Wang L. Igyarto B. Kissenpfennig A. Malissen B. Kaplan D.H. Hogquist K.A. Identification of a novel population of Langerin+ dendritic cells.J. Exp. Med. 2007; 204: 3147-3156Crossref PubMed Scopus (397) Google Scholar). Thereby, using CHS as a model minimized the potential stimulatory roles for B cells during immune responses. This allowed the identification of a potent regulatory B cell subset identified by IL-10 production and a unique CD1dhiCD5+ phenotype. These rare CD1dhiCD5+ B cells negatively regulated antigen-specific T cell-dependent inflammation during CHS responses in vivo. To assess T cell-mediated inflammation responses in mice with altered B cell signaling, Cd19−/−, hCD19Tg, and WT mice were sensitized and challenged with 4-ethyoxymethylene-2-phenyl-oxazoline-5-one (oxazolone). B cells from hCD19Tg mice are hyperresponsive to transmembrane signals, proliferate more to mitogens, generate elevated humoral immune responses to T dependent antigens, and spontaneously produce IgG autoantibodies as they age (Inaoki et al., 1997Inaoki M. Sato S. Weintraub B.C. Goodnow C.C. Tedder T.F. CD19-regulated signaling thresholds control peripheral tolerance and autoantibody production in B lymphocytes.J. Exp. Med. 1997; 186: 1923-1931Crossref PubMed Scopus (156) Google Scholar). Thus, CD19 functions as a general “rheostat” that defines signaling thresholds critical for expansion of the peripheral B cell pool (Tedder, 1998Tedder T.F. Response-regulators of B lymphocyte signaling thresholds provide a context for antigen receptor signal transduction.Semin. Immunol. 1998; 10: 259-265Crossref PubMed Scopus (43) Google Scholar). In WT mice, ear inflammation peaked at 24 hr after challenge and then decreased gradually (Figure 1A). Ear swelling was significantly diminished in hCD19Tg mice compared with WT mice throughout the observation period (39% ± 13%, 48 hr, p < 0.05). By contrast, ear swelling was enhanced and prolonged in Cd19−/− mice (58% ± 8%, 48 hr, p < 0.05) as reported (Watanabe et al., 2007Watanabe R. Fujimoto M. Ishiura N. Kuwano Y. Nakashima H. Yazawa N. Okochi H. Sato S. Tedder T.F. Tamaki K. CD19 expression in B cells is important for suppression of contact hypersensitivity.Am. J. Pathol. 2007; 171: 560-570Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). Despite this, blood, spleen, and lymph node-regulatory CD25+Foxp3+CD4+ T cell numbers were identical in WT, hCD19Tg, and Cd19−/− mice (not shown). Thus, enhanced or reduced B cell function inversely paralleled T cell-mediated inflammatory responses. Whether B cells were directly responsible for decreased T cell-mediated inflammatory responses in hCD19Tg mice was determined by depleting B cells in hCD19Tg mice with human CD19 mAb as described (Yazawa et al., 2005Yazawa N. Hamaguchi Y. Poe J.C. Tedder T.F. Immunotherapy using unconjugated CD19 monoclonal antibodies in animal models for B lymphocyte malignancies and autoimmune disease.Proc. Natl. Acad. Sci. USA. 2005; 102: 15178-15183Crossref PubMed Scopus (97) Google Scholar). CD19 mAbs depleted the majority of circulating B cells within 1 hr of treatment, with > 95% depletion of spleen and lymph node B cells within 2 days. Mice treated with human CD19 mAb 7 days before primary sensitization showed significantly enhanced CHS responses compared with control mAb-treated mice (p < 0.01, Figure 1B). Mice treated with human CD19 mAb 2 days after primary oxazolone sensitization had comparable CHS responses with control mAb-treated mice at 24 hr after oxazolone elicitation but developed augmented CHS responses by 48 hr. Ear swelling 48 hr after oxazolone challenge in mice treated with human CD19 mAb 7 days before or 2 days after primary oxazolone sensitization was increased by 102% ± 8% or 89% ± 12%, respectively. Thus, B cell depletion in hCD19Tg mice restored CHS responses to levels observed in WT mice. B cells were depleted from mice with intact immune systems by the use of CD20 mAb to examine whether normal B cells regulate T cell-mediated inflammation in WT mice. Mature spleen CD20+ B cells in WT mice are eliminated within 2 days after a single CD20 mAb treatment (Uchida et al., 2004aUchida J. Hamaguchi Y. Oliver J.A. Ravetch J.V. Poe J.C. Haas K.M. Tedder T.F. The innate mononuclear phagocyte network depletes B lymphocytes through Fc receptor-dependent mechanisms during anti-CD20 antibody immunotherapy.J. Exp. Med. 2004; 199: 1659-1669Crossref PubMed Scopus (487) Google Scholar). Mice depleted of B cells 7 days before or 2 days after primary oxazolone sensitization exhibited significantly enhanced CHS responses when compared with control mAb-treated mice: 91% ± 10% and 72% ± 11% increase, respectively, at 48 hr after oxazolone challenge (p < 0.01, Figure 1C). Thus, B cell depletion augmented T cell-mediated inflammatory responses in both WT and hCD19Tg mice. Whether CD19 deficiency completely eliminates B cell negative regulation was determined by depleting B cells from Cd19−/− mice with CD20 mAb. B cell depletion further increased CHS severity in Cd19−/− mice, but the difference was not statistically different from control mAb-treated mice (Figure 1D). This suggests that some regulatory B cells still exist in Cd19−/− mice, but at levels below those found in WT and hCD19Tg mice. Moreover, anti-human CD19 as well as anti-mouse CD20 mAbs do not eliminate all peritoneal B cells (Hamaguchi et al., 2005Hamaguchi Y. Uchida J. Cain D.W. Venturi G.M. Poe J.C. Haas K.M. Tedder T.F. The peritoneal cavity provides a protective niche for B1 and conventional B lymphocytes during anti-CD20 immunotherapy in mice.J. Immunol. 2005; 174: 4389-4399PubMed Google Scholar, Yazawa et al., 2005Yazawa N. Hamaguchi Y. Poe J.C. Tedder T.F. Immunotherapy using unconjugated CD19 monoclonal antibodies in animal models for B lymphocyte malignancies and autoimmune disease.Proc. Natl. Acad. Sci. USA. 2005; 102: 15178-15183Crossref PubMed Scopus (97) Google Scholar). Furthermore, CD20 mAb treatment does not reduce serum or natural antibody levels (DiLillo et al., 2008DiLillo D.J. Hamaguchi Y. Ueda Y. Yang K. Uchida J. Haas K.M. Kelsoe G. Tedder T.F. Maintenance of long-lived plasma cells and serological memory despite mature and memory B cell depletion during CD20 immunotherapy in mice.J. Immunol. 2008; 180: 361-371PubMed Google Scholar). Thereby, induced B cell depletion eliminates most B cell negative regulation but does not eliminate the peritoneal B-1 cell population that also appears important for CHS initiation (Itakura et al., 2005Itakura A. Szczepanik M. Campos R.A. Paliwal V. Majewska M. Matsuda H. Takatsu K. Askenase P.W. An hour after immunization peritoneal B-1 cells are activated to migrate to lymphoid organs where within 1 day they produce IgM antibodies that initiate elicitation of contact sensitivity.J. Immunol. 2005; 175: 7170-7178PubMed Google Scholar). B cells produce multiple cytokines that influence immune responses (Harris et al., 2000Harris D.P. Haynes L. Sayles P.C. Duso D.K. Eaton S.M. Lepak N.M. Johnson L.L. Swain S.L. Lund F.E. Reciprocal regulation of polarized cytokine production by effector B and T cells.Nat. Immunol. 2000; 1: 475-482Crossref PubMed Scopus (629) Google Scholar). Therefore, B cells from WT, hCD19Tg, and Cd19−/− mice were purified (>95% purities, Figure 2A), with cytokine production quantified and compared with T cell-mediated inflammatory responses observed in each mouse line. Whereas B cells cultured without mitogens did not produce cytokines, lipopolysaccharide (LPS)-stimulated B cells from WT, hCD19Tg, and Cd19−/− mice produced tumor necrosis factor-α, IL-1β, IL-10, and IL-6 protein as determined with Luminex assays (Figure 2B, not shown). IgM antibody plus CD40 mAb stimulation also induced the production of these cytokines, but at lower amounts than LPS. Only WT B cells secreted transforming growth factor-β1, but only at very low amounts after IgM antibody plus CD40 mAb stimulation (not shown). Neither LPS nor IgM antibody plus CD40 mAb stimulation induced detectable IL-4, -5, -12, -13, -17, or -23 secretion (not shown). Nonetheless, increased IL-10 production by hCD19Tg but reduced IL-10 production by Cd19−/− B cells was the only cytokine change that was inversely proportional to inflammatory responses in these mice. In Luminex and standard ELISAs, B cells from hCD19Tg mice showed increased IL-10 production compared with WT mice (LPS stimulation, 1.8-fold; p < 0.01), whereas B cells from Cd19−/− mice exhibited reduced IL-10 production (65% of WT, p < 0.05; Figures 2B and 2C). With ELISPOT assays, IL-10-producing B cell frequencies were 2.7-fold higher in hCD19Tg mice than WT mice (p < 0.01) but 74% lower in Cd19−/− mice than in WT mice (p < 0.01; Figure 2D). Thus, frequencies of IL-10-producing B cells were inversely proportional to the inflammatory responses of hCD19Tg and CD19−/− mice. Reciprocal IL-10 production by B cells from hCD19Tg and Cd19−/− mice was verified directly by intracellular cytokine staining. Cytoplasmic IL-10 production was not detected in resting B cells from WT, hCD19Tg, or Cd19−/− mice (Figure 3A). After LPS, phorbol 12-myristate 13-acetate (PMA), and ionomycin stimulation for 5 hr, the frequencies of spleen IL-10-producing B cells was 7.4-fold higher in hCD19Tg mice than in WT mice (p < 0.01), whereas the frequency of IL-10-producing B cells was 85% lower in Cd19−/− mice than in WT mice (p < 0.01; Figure 3B). Interestingly, IL-10 production by non-B cells after LPS, PMA, and ionomycin stimulation was also increased in hCD19Tg mice (Figure S1 online). Peritoneal IL-10-producing B cell frequencies were 3-fold higher in hCD19Tg mice than in WT mice (p < 0.01) but 80% lower in Cd19−/− mice (p < 0.01; Figure 3C). Even though Cd19−/− and hCD19Tg mice have reduced numbers of splenic B cells compared with WT mice (Haas et al., 2005Haas K.M. Poe J.C. Steeber D.A. Tedder T.F. B-1a and B-1b cells exhibit distinct developmental requirements and have unique functional roles in innate and adaptive immunity to S. pneumoniae.Immunity. 2005; 23: 7-18Abstract Full Text Full Text PDF PubMed Scopus (415) Google Scholar), the numbers of IL-10-producing splenic and peritoneal B cells were 2.1-fold- and 3.1-fold-higher in hCD19Tg mice than in WT mice, respectively (p < 0.01). Splenic and peritoneal IL-10-producing B cell numbers were 80% and 78% lower in Cd19−/− mice than in WT mice, respectively (p < 0.01). By contrast, naive or stimulated B cells from blood, peripheral and mesenteric lymph nodes, and Peyer's patches exhibited little, if any, IL-10 production in WT, hCD19Tg, or Cd19−/− mice (Figures 3D–3F, not shown). Intracellular staining of B cells from Il10−/− mice served as background controls. Thus, IL-10-producing B cells represent a distinct subset that was dramatically reduced in Cd19−/− mice but preferentially expanded in hCD19Tg mouse spleen and peritoneal cavity. Whether IL-10-producing B cells represent a known B cell subset was determined by immunofluorescence staining. Because B cell cytoplasmic IL-10 was only visualized after combined LPS, PMA, ionomycin, and monensin treatment for 5 hr (Figure 3), the effect of this treatment and cell permeabilization on phenotypes was determined. Untreated, treated, or permeabilized B cells from WT and hCD19Tg mice expressed identical IgM, IgD, CD19, CD5, CD1d, CD21, CD24, CD23, CD11b, CD43, and B220 densities (Figure 4A, not shown). These cell-surface molecules were therefore used to categorize IL-10-producing B cells. Spleen IL-10-producing B cells in WT and hCD19Tg mice were part of a CD19hi subset (Figures 3B and 3C). Spleen IL-10-producing B cells exhibited the CD5+CD19hi phenotype characteristic of B-1a cells, but they unexpectedly expressed cell-surface CD1d at high amounts in both WT and hCD19Tg mice (Figure 4B). By contrast, Cd19−/− mice did not have detectable spleen CD1dhiCD5+ or IL-10-producing B cells (Figures 3B and 4C, Table 1). On average, B cells with a CD1dhiCD5+ phenotype represented 2.3% and 12.2% of spleen B220+ cells in WT and hCD19Tg mice, respectively (Figure 4C). CD1dhiCD5+ splenic B cell numbers were 38% higher in hCD19Tg mice than in WT mice (Table 1). Within the CD1dhiCD5+ B cell subset, an average of 18% and 58% expressed IL-10 in WT and hCD19Tg mice, respectively. CD1dloCD5+ B cells did not express IL-10 (Figure 4C). When CD1dhiCD5+ or the remaining spleen B cells were purified and then stimulated, the vast majority of IL-10-producing B cells were found within the CD1dhiCD5+ subset of B cells from WT and hCD19Tg mice (Figure 4D), further excluding the possibility that LPS, PMA, and ionomycin treatment induced their phenotype. Whether the CD1dhiCD5+ B cell phenotype was induced by oxazolone sensitization in vivo was also assessed. B cell CD1d and CD5 expression and CD1dhiCD5+ B cell numbers were similar in oxazolone sensitized and naive WT mice (Figure S2), making it unlikely that the CD1dhiCD5+ B phenotype was induced by oxazolone sensitization.Table 1Spleen B Cell Subsets in Wild-Type, hCD19Tg, Cd19−/−, and Il10−/− MiceMouse GenotypeB Cell Subset Numbers (× 10−5)CD1dhiCD5+B-1aMZFollicularhCD19Tg18 ± 2∗32 ± 416 ± 1∗∗68 ± 4∗Wild-type13 ± 130 ± 342 ± 3362 ± 42Il10−/−12 ± 131 ± 242 ± 1324 ± 17Cd19−/−0.4 ± 0.1∗∗8 ± 1∗∗12 ± 1∗∗148 ± 6∗B cell subsets were as follows: CD1dhiCD5+, B-1a (CD5+B220lo), MZ (CD1dhiCD21hiB220hi), and follicular (CD21intCD23+B220hi). Values (± SEM, n ≥ 4 mice) were significantly different from those of wild type mice; ∗, p < 0.05; ∗∗, p < 0.01. Open table in a new tab B cell subsets were as follows: CD1dhiCD5+, B-1a (CD5+B220lo), MZ (CD1dhiCD21hiB220hi), and follicular (CD21intCD23+B220hi). Values (± SEM, n ≥ 4 mice) were significantly different from those of wild type mice; ∗, p < 0.05; ∗∗, p < 0.01. The phenotype of IL-10-producing spleen B cells was further verified by determining the phenotypes of IL-10+ and IL-10− populations. Most splenic IL-10-producing B cells expressed IgM, CD1d, CD19, and CD24 at high levels (Figures 4B and 4E, not shown). Approximately half of splenic IL-10-producing B cells expressed high-density CD21 (44.3% ± 2.6% and 54.8% ± 1.6% in WT and hCD19Tg mice, respectively). Peritoneal IL-10-producing B cells were CD19hiIgMhiIgDloCD5+CD23−CD11b+CD43+B220lo, a phenotype shared by B-1a cells. Thus, splenic IL-10-producing B cells shared features common to marginal zone (MZ), T2-MZ precursor, and B-1a B cells but were localized within a unique CD1dhiCD5+ subset. Whether B cell IL-10 production might regulate T cell-mediated inflammation was determined by assessment of IL-10 production by B cells during CHS responses in WT, hCD19Tg, and Cd19−/− mice. Spleen and draining axillary and inguinal lymph node B cells were purified 2 days after ear challenge with oxazolone, with IL-10 mRNA expression quantified by real-time polymerase chain reaction (PCR). Relative IL-10 transcripts in B cells from spleen and peripheral lymph nodes of unchallenged hCD19Tg mice were significantly increased relative to B cells from WT mice (spleen 4.5-fold, p < 0.01, lymph node 1.5-fold, p < 0.05; Figure 5A). During CHS responses, spleen B cells from both WT and hCD19Tg mice expressed more IL-10 transcripts than naive B cells (6-fold, p < 0.01 and 2.2-fold, p < 0.01, respectively), and hCD19Tg B cells produced higher IL-10 transcripts than WT B cells (1.8-fold, p < 0.01; Figure 5A, left panel). IL-10 transcripts in spleen B cells from challenged Cd19−/− mice increased significantly during CHS responses, but only up to 16% of the amount seen in WT B cells (p < 0.01). By contrast, B cell IL-10 mRNA expression in draining lymph nodes did not change during CHS response (Figure 5A, right panel). In similar experiments, IL-10 transcripts produced by the spleen CD1dhiCD5+ B cell subset were increased 7.1-fold during CHS responses in comparison with naive CD1dhiCD5+ B cells, whereas IL-10 transcripts were lower in non-CD1dhiCD5+ B cells with or without sensitization and challenge (Figure 5B). Thus, B cell IL-10 production in the spleen but not lymph nodes was increased during CHS responses, with the amount of IL-10 production by B cells being inversely proportional to the severity of inflammatory responses. IL-10 production by blood B cells was assessed to determine whether IL-10-producing B cells enter the circulation during CHS responses. IL-10-producing B cells were not observed in Il10−/− or naive mice before oxazolone sensitization (Figures 3D and 5C). However, circulating IL-10-producing B cells were found in the blood after sensitization, with the percentage of circulating IL-10-producing B cells peaking before challenge and gradually decreasing after challenge (Figure 5C). Thus, IL-10-producing B cells enter the circulation during CHS responses. Blocking IL-10 function in vivo with an IL-10 receptor-specific mAb enhances CHS responses in WT mice (Ferguson et al., 1994Ferguson T.A. Dube P. Griffith T.S. Regulation of contact hypersensitivity by interleukin 10.J. Exp. Med. 1994; 179: 1597-1604Crossref PubMed Scopus (149) Google Scholar). Therefore, whether the enhanced CHS responses observed in hCD19Tg mice were dependent on IL-10 was assessed with a function-blocking mAb against the IL-10 receptor. Blocking IL-10 receptor function 1 hr before oxazolone challenge significantly augmented CHS responses in hCD19Tg mice when compared with control mAb-treated mice (Figure 5D, p < 0.05 at 48 hr). In fact, blocking IL-10 receptor function restored CHS responses to the degree normally observed in WT mice. Thus, the suppression of T cell-mediated inflammation observed in hCD19Tg mice was IL-10 dependent. The relative contribution of WT IL-10-producing B cells to CHS suppression was assessed by compariso