Article | Published:

IL-9 receptor signaling in memory B cells regulates humoral recall responses

Nature Immunologyvolume 19pages10251034 (2018) | Download Citation


Memory B cells (Bmem cells) are the basis of long-lasting humoral immunity. They respond to re-encountered antigens by rapidly producing specific antibodies and forming germinal centers (GCs), a recall response that has been known for decades but remains poorly understood. We found that the receptor for the cytokine IL-9 (IL-9R) was induced selectively on Bmem cells after primary immunization and that IL-9R-deficient mice exhibited a normal primary antibody response but impaired recall antibody responses, with attenuated population expansion and plasma-cell differentiation of Bmem cells. In contrast, there was augmented GC formation, possibly due to defective downregulation of the ligand for the co-stimulatory receptor ICOS on Bmem cells. A fraction of Bmem cells produced IL-9. These findings indicate that IL-9R signaling in Bmem cells regulates humoral recall responses.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


  1. 1.

    McHeyzer-Williams, M., Okitsu, S., Wang, N. & McHeyzer-Williams, L. Molecular programming of B cell memory. Nat. Rev. Immunol. 12, 24–34 (2011).

  2. 2.

    Shlomchik, M. J. & Weisel, F. Germinal center selection and the development of memory B and plasma cells. Immunol. Rev. 247, 52–63 (2012).

  3. 3.

    Chan, T. D. & Brink, R. Affinity-based selection and the germinal center response. Immunol. Rev. 247, 11–23 (2012).

  4. 4.

    Suan, D., Sundling, C. & Brink, R. Plasma cell and memory B cell differentiation from the germinal center. Curr. Opin. Immunol. 45, 97–102 (2017).

  5. 5.

    Ise, W. et al. Memory B cells contribute to rapid Bcl6 expression by memory follicular helper Tcells. Proc. Natl. Acad. Sci. USA 111, 11792–11797 (2014).

  6. 6.

    Crotty, S. The 1-1-1 fallacy. Immunol. Rev. 247, 133–142 (2012).

  7. 7.

    Schaerli, P. et al. CXC chemokine receptor 5 expression defines follicular homing T cells with B cell helper function. J. Exp. Med. 192, 1553–1562 (2000).

  8. 8.

    Kim, C. H. et al. Subspecialization of CXCR5+ T cells: B helper activity is focused in a germinal center-localized subset of CXCR5+ T cells. J. Exp. Med. 193, 1373–1381 (2001).

  9. 9.

    Linterman, M. A. et al. IL-21 acts directly on B cells to regulate Bcl-6 expression and germinal center responses. J. Exp. Med. 207, 353–363 (2010).

  10. 10.

    Ozaki, K. et al. A critical role for IL-21 in regulating immunoglobulin production. Science 298, 1630–1634 (2002).

  11. 11.

    Bessa, J., Kopf, M. & Bachmann, M. F. Cutting edge: IL-21 and TLR signaling regulate germinal center responses in a B cell-intrinsic manner. J. Immunol. 184, 4615–4619 (2010).

  12. 12.

    Rasheed, M. A. et al. Interleukin-21 is a critical cytokine for the generation of virus-specific long-lived plasma cells. J. Virol. 87, 7737–7746 (2013).

  13. 13.

    Zotos, D. et al. IL-21 regulates germinal center B cell differentiation and proliferation through a B cell-intrinsic mechanism. J. Exp. Med. 207, 365–378 (2010).

  14. 14.

    Rankin, A. L. et al. IL-21 receptor is critical for the development of memory B cell responses. J. Immunol. 186, 667–674 (2011).

  15. 15.

    McGuire, H. M. et al. IL-21 and IL-4 collaborate to shape T-dependent antibody responses. J. Immunol. 195, 5123–5135 (2015).

  16. 16.

    Harada, Y. et al. The 3′ enhancer CNS2 is a critical regulator of interleukin-4-mediated humoral immunity in follicular helper T cells. Immunity 36, 188–200 (2012).

  17. 17.

    Reinhardt, R. L., Liang, H. E. & Locksley, R. M. Cytokine-secreting follicular T cells shape the antibody repertoire. Nat. Immunol. 10, 385–393 (2009).

  18. 18.

    Vijayanand, P. et al. Interleukin-4 production by follicular helper T cells requires the conserved Il4 enhancer hypersensitivity site V. Immunity 36, 175–187 (2012).

  19. 19.

    Yu, A. et al. Efficient induction of primary and secondary T cell-dependent immune responses in vivo in the absence of functional IL-2 and IL-15 receptors. J. Immunol. 170, 236–242 (2003).

  20. 20.

    Wilhelm, C. et al. An IL-9 fate reporter demonstrates the induction of an innate IL-9 response in lung inflammation. Nat. Immunol. 12, 1071–1077 (2011).

  21. 21.

    Noelle, R. J. & Nowak, E. C. Cellular sources and immune functions of interleukin-9. Nat. Rev. Immunol. 10, 683–687 (2010).

  22. 22.

    Stassen, M. et al. Murine bone marrow-derived mast cells as potent producers of IL-9: costimulatory function of IL-10 and kit ligand in the presence of IL-1. J. Immunol. 164, 5549–5555 (2000).

  23. 23.

    Licona-Limón, P. et al. Th9 Cells drive host immunity against gastrointestinal worm infection. Immunity 39, 744–757 (2013).

  24. 24.

    Gounni, A. S. et al. IL-9 expression by human eosinophils: regulation by IL-1β and TNF-α. J. Allergy Clin. Immunol. 106, 460–466 (2000).

  25. 25.

    Renauld, J. C. et al. Expression cloning of the murine and human interleukin 9 receptor cDNAs. Proc. Natl. Acad. Sci. USA 89, 5690–5694 (1992).

  26. 26.

    Bauer, J. H., Liu, K. D., You, Y., Lai, S. Y. & Goldsmith, M. A. Heteromerization of the γc chain with the interleukin-9 receptor α subunit leads to STAT activation and prevention of apoptosis. J. Biol. Chem. 273, 9255–9260 (1998).

  27. 27.

    Kaplan, M. H., Hufford, M. M. & Olson, M. R. The development and in vivo function of T helper 9 cells. Nat. Rev. Immunol. 15, 295–307 (2015).

  28. 28.

    Petit-Frere, C., Dugas, B., Braquet, P. & Mencia-Huerta, J. M. Interleukin-9 potentiates the interleukin-4-induced IgE and IgG1 release from murine B lymphocytes. Immunology 79, 146–151 (1993).

  29. 29.

    Dugas, B. et al. Interleukin-9 potentiates the interleukin-4-induced immunoglobulin (IgG, IgM and IgE) production by normal human B lymphocytes. Eur. J. Immunol. 23, 1687–1692 (1993).

  30. 30.

    Vink, A., Warnier, G., Brombacher, F. & Renauld, J. C. Interleukin 9-induced in vivo expansion of the B-1 lymphocyte population. J. Exp. Med. 189, 1413–1423 (1999).

  31. 31.

    Nojima, T. et al. In-vitro derived germinal centre B cells differentially generate memory B or plasma cells in vivo. Nat. Commun. 2, 465 (2011).

  32. 32.

    Steenwinckel, V. et al. IL-13 mediates in vivo IL-9 activities on lung epithelial cells but not on hematopoietic cells. J. Immunol. 178, 3244–3251 (2007).

  33. 33.

    Wang, Y. et al. Germinal-center development of memory B cells driven by IL-9 from follicular helper T cells. Nat. Immunol. 18, 921–930 (2017).

  34. 34.

    Fawaz, L. M. et al. Expression of IL-9 receptor α chain on human germinal center B cells modulates IgE secretion. J. Allergy Clin. Immunol. 120, 1208–1215 (2007).

  35. 35.

    Kocks, C. & Rajewsky, K. Stepwise intraclonal maturation of antibody affinity through somatic hypermutation. Proc. Natl. Acad. Sci. USA 85, 8206–8210 (1988).

  36. 36.

    Taylor, J. J., Pape, K. A. & Jenkins, M. K. A germinal center-independent pathway generates unswitched memory B cells early in the primary response. J. Exp. Med. 209, 597–606 (2012).

  37. 37.

    Zuccarino-Catania, G. V. et al. CD80 and PD-L2 define functionally distinct memory B cell subsets that are independent of antibody isotype. Nat. Immunol. 15, 631–637 (2014).

  38. 38.

    Tomayko, M. M., Steinel, N. C., Anderson, S. M. & Shlomchik, M. J. Cutting edge: hierarchy of maturity of murine memory B cell subsets. J. Immunol. 185, 7146–7150 (2010).

  39. 39.

    He, J. S. et al. IgG1 memory B cells keep the memory of IgE responses. Nat. Commun. 8, 641 (2017).

  40. 40.

    Dong, C., Temann, U. A. & Flavell, R. A. Cutting edge: critical role of inducible costimulator in germinal center reactions. J. Immunol. 166, 3659–3662 (2001).

  41. 41.

    Wong, S. C., Oh, E., Ng, C. H. & Lam, K. P. Impaired germinal center formation and recall T-cell-dependent immune responses in mice lacking the costimulatory ligand B7-H2. Blood 102, 1381–1388 (2003).

  42. 42.

    Yoshinaga, S. K. et al. T-cell co-stimulation through B7RP-1 and ICOS. Nature 402, 827–832 (1999).

  43. 43.

    Swallow, M. M., Wallin, J. J. & Sha, W. C. B7h, a novel costimulatory homolog of B7.1 and B7.2, is induced by TNFα. Immunity 11, 423–432 (1999).

  44. 44.

    Weisel, F. J., Zuccarino-Catania, G. V., Chikina, M. & Shlomchik, M. J. A temporal switch in the germinal center determines differential output of memory B and plasma Cells. Immunity 44, 116–130 (2016).

  45. 45.

    Kitamura, D., Roes, J., Kuhn, R. & Rajewsky, K. A B cell-deficient mouse by targeted disruption of the membrane exon of the immunoglobulin μ chain gene. Nature 350, 423–426 (1991).

  46. 46.

    Lam, K. P., Kühn, R. & Rajewsky, K. In vivo ablation of surface immunoglobulin on mature B cells by inducible gene targeting results in rapid cell death. Cell 90, 1073–1083 (1997).

  47. 47.

    Barnden, M. J., Allison, J., Heath, W. R. & Carbone, F. R. Defective TCR expression in transgenic mice constructed using cDNA-based alpha- and beta-chain genes under the control of heterologous regulatory elements. Immunol. Cell Biol. 76, 34–40 (1998).

  48. 48.

    Tamura, S. et al. Cross-protection against influenza virus infection afforded by trivalent inactivated vaccines inoculated intranasally with cholera toxin B subunit. J. Immunol. 149, 981–988 (1992).

  49. 49.

    Takatsuka, S., Sekiguchi, A., Tokunaga, M., Fujimoto, A. & Chiba, J. Generation of a panel of monoclonal antibodies against atypical chemokine receptor CCX-CKR by DNA immunization. J. Pharmacol. Toxicol. Methods 63, 250–257 (2011).

  50. 50.

    Haniuda, K., Fukao, S., Kodama, T., Hasegawa, H. & Kitamura, D. Autonomous membrane IgE signaling prevents IgE-memory formation. Nat. Immunol. 17, 1109–1117 (2016).

  51. 51.

    Végran, F. et al. The transcription factor IRF1 dictates the IL-21-dependent anticancer functions of TH9 cells. Nat. Immunol. 15, 758–766 (2014).

  52. 52.

    Haniuda, K., Nojima, T., Ohyama, K. & Kitamura, D. Tolerance induction of IgG+ memory B cells by T cell-independent type II antigens. J. Immunol. 186, 5620–5628 (2011).

  53. 53.

    Ainai, A. et al. Zymosan enhances the mucosal adjuvant activity of poly(I:C) in a nasal influenza vaccine. J. Med. Virol. 82, 476–484 (2010).

Download references


We thank A. Ainai and H. Hasegawa (National Institute of Infectious Diseases) for the vaccine containing whole inactivated influenza virus; S. Horiuchi, T. Koike, T. Katayama, I. Shiratori and Y. Takai for reagents and technical assistance; other members of the Research Institute for Biomedical Sciences for technical advice and comments; Y. Takatsuka for figure preparation; and P. Burrows for critical reading. This work was supported by the Japan Society for the Promotion of Science KAKENHI, Grant-in-Aid for Scientific Research (B) (16H05206) and Grant-in-Aid for Scientific Research on Innovative Areas (25111512) (all to D.K.).

Author information

Author notes

    • Shogo Takatsuka

    Present address: Department of Chemotherapy and Mycoses, National Institute of Infectious Diseases, Tokyo, Japan

  1. These authors contributed equally: Hiroyuki Yamada, Kei Haniuda.


  1. Division of Molecular Biology, Research Institute for Biomedical Sciences (RIBS), Tokyo University of Science, Noda, Japan

    • Shogo Takatsuka
    • , Hiroyuki Yamada
    • , Kei Haniuda
    • , Hiroshi Saruwatari
    • , Marina Ichihashi
    •  & Daisuke Kitamura
  2. Ludwig Institute for Cancer Research and Experimental Medicine Unit, Universite catholique de Louvain, Brussels, Belgium

    • Jean-Christophe Renauld


  1. Search for Shogo Takatsuka in:

  2. Search for Hiroyuki Yamada in:

  3. Search for Kei Haniuda in:

  4. Search for Hiroshi Saruwatari in:

  5. Search for Marina Ichihashi in:

  6. Search for Jean-Christophe Renauld in:

  7. Search for Daisuke Kitamura in:


S.T. designed and performed experiments, analyzed data and wrote the manuscript; H.Y., K.H., H.S. and M.I. performed experiments and analyzed data; J.-C.R. made the IL-9R-deficient mice; and D.K. supervised the study, designed experiments and wrote the manuscript.

Competing interests

The authors declare no competing interests.

Corresponding author

Correspondence to Daisuke Kitamura.

Integrated supplementary information

  1. Supplementary Figure 1 IL-9R expression on activated B cells and various hematopoietic cells.

    Flow cytometric analyses of cell surface IL-9R expression. (a) Evaluation of anti-IL-9R mAbs (RZ-66, -87, -98, -103) by staining B300-19/IL-9R cells (red line) or mock-transfected B300-19 cells (shaded). (b) Change in mean fluorescence intensity (fold MFI) of IL-9R staining on each Il9r+/+ sample as in Fig. 1b, after division by MFI on control Il9r−/−sample (an average when more than one sample were stained in one experiment), is indicated as a circle (the samples in one experiment are indicated as circles with the same color). Mean of collective values at each time point is indicated by a bar graph (white: GC B cells; black: Bmem cells). Note that MFI varies considerably among experiments due to variable settings of flow cytometer. Data are pooled from two (1, 2, 3 weeks), one (4 week) or three (15-32 weeks) independent experiments, with n = 6, n = 5, n = 2, n = 3, n = 9 biological samples (mice) in total for 1, 2, 3, 4, 15-32 weeks, respectively. (c) RZ-66 staining of Bmem, GC-MP and GC B cells in spleens of Il9r+/+ or Il9r−/− mice immunized with NP-CGG in alum 2 weeks earlier (n = 4). Gating strategy for Bmem cells (CD19+ IgG1+ NP+ CD38hi GL-7 FAS), GC-MP (CD19+ IgG1+ NP+ CD38hi GL-7+ FAS+) and GC B cells (CD19+ IgG1+ NP+ CD38lo GL-7+ FAS+) (left panels), representative histograms of the IL-9R staining (center panels; line: Il9r+/+, shaded: Il9r−/−), and averages of the ratios (Il9r+/+ / Il9r−/−) of MFIs of the histograms (right bar graph) are shown. (d) Histograms showing RZ-66 staining of the following gated cells in the spleens of Il9r+/+ (line) or Il9r−/− (shaded) mice immunized with NP-CGG in alum 3 weeks earlier: plasmacytoid dendritic cells (DCs) (pDC: B220+ CD11c+), conventional DCs (cDC: B220- CD11c+), macrophages (Mϕ: F4/80+ CD11b+), basophils (Baso: DX5+ FcεRIα+), T cells (T cell: CD3ε+), mast cells (Mast: c-kit+ FcεRIα+), neutrophils (Neu: Ly6c+ CD11b+ Gr-1+), eosinophils (Eos: Ly6c+ CD11b+ Siglec-F+), natural killer cells (NK: NK1.1+). (e) Naive splenic B cells from Il9r+/+ (line) or Il9r−/− (shaded) mice, stimulated with LPS (left), anti-IgM (center), anti-CD40 (right), for 72 h, were stained with IL-9-Fc (of human IgG1) fusion protein plus human IgG-specific antibody. (f) Naïve splenic B cells of Il9r+/+ (red line) or Il9r−/− (blue line) mice, successively cultured on 40LB feeder cells with IL-4 for 3 d (left), then with IL-21 for 3 d (center), and further 3 d without additives (right), were stained with IL-9-Fc as in e. For the latter two, histograms represent cells gated on IgG1+ CD138 cells. * P < 0.05, ** P < 0.01, *** P < 0.001 (b: two-tailed unpaired Student’s t-test; c: one-way ANOVA test). Data are representative of three (a,f) or two (d,e) independent experiments

  2. Supplementary Figure 2 Antibody class titers in the recall response and SHM profiles in the primary response to TD Ag.

    (a) Titration of NP-specific IgG1, IgG2b, IgG2c, and IgM by ELISA using the same sera of 2 weeks after the secondary immunization that were used in Fig. 3a. IgG3 titer was below the detection limit. Each symbol represents the mean and s.d. of each group, with n = 7 biological samples (mice). (b) SHM in VH186.2 genes in NP+ IgG1+ Bmem cells (CD19+ NP+ IgG1+ CD38hi) from Il9r+/+ and Il9r−/− mice (n = 3, mixed) immunized i.p. with NP-CGG in alum 15 d earlier. Pie charts indicate frequency of VH186.2 sequences with mutation numbers surrounding, and the ratio and frequencies (%) of the sequence with the W33L replacement (center). * P < 0.05, ** P < 0.01, *** P < 0.001 (two-tailed unpaired Student’s t-test)

  3. Supplementary Figure 3 Flow cytometric analyses of GC and Bmem cells in the primary TD response.

    (a-b) Representative flow cytometry plots indicating gating strategies for IgG1+ GC B cells (B220+ IgM IgD IgG1+ NP+ CD38lo GL-7+, for Fig. 4a), IgG1+ Bmem cells (B220+ NP+ IgM IgG1+ CD38hi, for Fig. 4b left) and class-switched non-IgG1 Bmem cells (B220+ NP+ IgM IgD IgG1 CD38hi, for Fig. 4b right). (c) Flow cytometric analysis of spleen cells from Il9r+/+ or Il9r−/− bone marrow chimeric mice (n = 8, pooled from two independent experiments) generated as in Fig. 3c, and immunized i.p. with NP-CGG in alum 12 weeks previously. The numbers of Bmem cells (B220+ NP+ IgM IgG1+ CD38hi) per 1 × 107 B cells in the spleens, calculated by the flow cytometric analyses (representative plots shown on the left), are plotted with an average for each group (horizontal bar). (d) Flow cytometric analysis of Bmem cells in the spleens from mice (CD45.2+) having been transferred with 1 × 106 CFSE-labelled CD45.1+ B1-8ki B cells of Il9r+/+ or Il9r−/− mice (n = 6) (with the same but 1 × 107 cells of Il9r+/+ mice for a non-immunized control mouse) and immunized with NP-CGG in alum 24 d earlier. The numbers of IgM+ or IgG1+ Bmem cells (CD19+ CD45.1+ CFSE NP+ CD38hi) per 1 × 107 B cells in the spleens, calculated by flow cytometric analyses (representative plots shown on top), were plotted with an average for each group (horizontal bar). (e) The numbers of CD73+ CD80+ (DP), CD73 CD80+ (SP) and CD73 CD80 (DN) cell subsets in IgG1+ PDL2+ Bmem cells (B220+ NP+ IgM IgG1+ CD38hi) per 1 × 107 B cells in the spleens from Il9r+/+ or Il9r−/− mice immunized with NP-CGG 4 weeks earlier (n = 9). The numbers were calculated by flow cytometric analyses (representative plots shown on top), and plotted with a mean in each group (bar graph). Statistical analysis was performed using the two-tailed unpaired Student t-test (c,d,e). Data are of single experiments (d,e)

  4. Supplementary Figure 4 B cell-intrinsic defect of in vivo recall expansion and normal in vitro survival of Il9r−/− Bmem cells.

    (a) Recall response in bone marrow chimeric mice. Flow cytometric analyses of spleen cells from chimeric mice reconstituted with Il9r+/+ or Il9r−/−bone marrow cells (n = 5), generated as in Fig. 3c, which had been immunized i.p. with NP-CGG in alum 16 weeks previously and were rechallenged i.p. with soluble NP-CGG (boost) or PBS 3.5 d earlier. Right: each symbol represents the ratio of the calculated number of Bmem cells (gating shown in representative plots, left) from each rechallenged mice to an average number of those from PBS-injected mice, and each horizontal bar indicates a mean of the ratios in each group. (b) Cell survival analyses. GC-B and Bmem cells were sorted from C57BL/6 mice immunized i.p. with NP-CGG in alum 8 weeks previously, and naive B cells from unimmunized B6 mice (n = 1), as described in the ONLINE METHODS. Naive B, GC-B and Bmem cells were cultured with ( + ) or without (–) IL-9 (1 ng/mL) in vitro for 12 or 24 h, and then stained with propidium iodide (PI) and analyzed by flow cytometry. Top: representative plots indicating percentages of PI cell fractions. Bottom: percentages of the PI cell fractions (symbol) with a mean in each group (bar graph). Data are representative of two independent experiments (a), or of one experiment, with n = 3 technical replicates per group of one biological sample pooled from n = 32 mice (for GC B and Bmem). * P < 0.05 (two-tailed unpaired Student’s t-test, a)

  5. Supplementary Figure 5 Selective ICOSL down-regulation in vitro by IL-9/IL-9R signaling and ICOSL-dependent augmentation of the recall GC B-cell expansion in vivo by IL-9R-deficiency.

    (a) Flow cytometric analysis of surface expression on iGB cells of various molecules involved in T-B interaction. iGB cells from Il9r+/+ or Il9r−/− mice, retrovirally transduced with IL-9R-encoding (IL-9R) or empty (Ev) vectors, were cultured with IL-9 for 2 d. (b) A representative flow cytometry plots showing gating strategy for GC B cells (B220+ IgM IgD IgG1+ NP+ CD38lo GL-7+) used in Fig. 6e. Data are representative of two independent experiments (a)

  6. Supplementary Figure 6 Detection of IL-9-producing Tfh cells in the primary response, and evaluation of Tmem cells in the adopted experimental system.

    (a) Evaluation of anti-IL-9 antibody for intracellular staining by flow cytometry. IL-9-producing TH9 cells were generated from splenocytes in vitro and stained as described in the ONLINE METHODS. (b) Flow cytometric analysis of TFH (CXCR5+ PD-1+) and non-TFH (CXCR5lo PD-1lo) cells from mice (n = 4) immunized i.p. with NP-CGG in alum 7 d earlier. Cells were stimulated for 4 h in vitro with PMA and ionomycin before staining intracellularly with eFluor660-conjugated IL-9-specific or isotype-matched control (Isotype) antibodies. Frequencies of eFluor660+ cells in the TFH and non-TFH cells (representative plots shown on the left) are plotted with averages (horizontal bars) on the right. (c) Schematic presentation of an experimental system used for Fig. 7a-d. Naïve CD4+ T cells (CD3ε +, CD4+, CD62L+) purified from OT-II CD45.1/CD45.2 F1 mice were transferred i.v. into C57BL/6 mice (4 × 105 per mouse), which were then immunized i.p. with NP-OVA in alum on the next day. (d, e) Flow cytometric analysis of the same spleen cells that is shown in Fig. 7a-d. (d) Expression of CXCR5 and CD62L on Tmem (CD45.1+) cells from mice that had no booster immunization. (e) Each symbol represents of the calculated number of the CD45.1+ cells (gating shown in Fig. 7b), with a mean in each group (horizontal bar). * P < 0.05 (two-tailed unpaired Mann-Whitney test). Data are representative of three independent experiments (a, b), or are pooled from three independent experiments, with n = 5 (PBS) and n = 6 (NP-OVA) biological samples in total (e)

  7. Supplementary Figure 7 A small fraction of IL-9+ cells appear within IgG1+ Bmem cells during primary response.

    (a, b) Flow cytometric analysis for IL-9 expression of spleen cells from C57BL/6 mice immunized i.p. with NP-CGG in alum 1, 2, 3 and 4 weeks earlier. Spleen cells were stained as in Fig. 7c with additional antibody against IgD. (a) Representative plots of a mouse 4 weeks after immunization, showing the gating for Bmem and GC B cells. (b) Frequency of IL-9+ cells among Bmem and GC B cells, analyzed as in a. Each symbol indicates the mean and s.d. of each group (n = 5 mice). (c) ELISA of IL-9 in the supernatant of Bmem cells cultured for 6 d on 40LB feeder cells (5,000 cells per well of 48-well plate). The Bmem cells (B220+ IgG1+ NP+ CD38hi) were isolated from pooled spleens of C57BL/6 mice immunized i.p. with NP-CGG in alum 25 weeks previously. Symbols indicate n = 2 technical replicates (bar graph indicates a mean) per group of one biological sample pooled from n = 10 mice. (d) Flow cytometric analysis for IL-9 expression of spleen cells from C57BL/6 mice (n = 5) immunized i.p. with NP-CGG in alum 12 weeks earlier. Splenocytes were stained intracellularly with IL-9-specific or isotype-matched control antibodies after being stained with mAbs against B220, IgM, IgD and IgG1. Percentages of IL-9+ cells among NP+ IgG1+, NP+ IgM+ IgD+ or NP+ IgM+ IgD- cells (representative plots shown on the left) were plotted on the right panel, with a mean (horizontal bar) for each group. A mean of the percentages of cells stained with the control antibody is indicated by a dashed horizontal line. All data are representative of two independent experiments

Supplementary information

  1. Supplementary Figures

    Supplementary Figures 1-7, Supplementary Table 1

  2. Reporting Summary

About this article

Publication history