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

Nature Immunology volume 19, pages 1025–1034 (2018)Cite this article

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Abstract

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.

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Fig. 1: Expression of IL-9R on B cells.
Fig. 2: Function of IL-9R on B cells in vitro.
Fig. 3: Attenuated recall antibody responses to TD antigen in Il9r–/– mice.
Fig. 4: The formation and maintenance of Bmem cells in the primary TD response.
Fig. 5: Attenuated population expansion and differentiation of Il9r–/– Bmem cells during a recall response.
Fig. 6: Augmented formation of GC B cells by Il9r–/– Bmem cells during a recall response.
Fig. 7: Bmem cells produce IL-9 during a recall response, but Tmem cells do not.

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Acknowledgements

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.).

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  1. Shogo Takatsuka

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

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

Authors and Affiliations

  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

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Contributions

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.

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Correspondence to Daisuke Kitamura.

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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

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)

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)

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)

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)

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)

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

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Takatsuka, S., Yamada, H., Haniuda, K. et al. IL-9 receptor signaling in memory B cells regulates humoral recall responses. Nat Immunol 19, 1025–1034 (2018). https://doi.org/10.1038/s41590-018-0177-0

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