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Autonomous membrane IgE signaling prevents IgE-memory formation

Nature Immunology volume 17, pages 1109–1117 (2016)Cite this article

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Abstract

Aberrant production of IgE antibodies can lead to allergic diseases. Normally, IgE+ B cells rarely differentiate into memory B cells (Bmem) or long-lived plasma cells (LLPCs), as they only transiently participate in the germinal center (GC), but the mechanism behind this remains elusive. We found that membrane IgE (mIgE) autonomously triggered rapid plasma-cell differentiation and apoptosis independently of antigen or cellular context, predominantly through the mutually independent CD19-PI3K-Akt-IRF4 and BLNK-Jnk/p38 pathways, respectively, and we identified the ectodomains of mIgE as being responsible. Accordingly, deregulated GC IgE+ B cell proliferation and prolonged IgE production with exaggerated anaphylaxis were observed in CD19- and BLNK-deficient mice. Our findings reveal an autonomous mIgE signaling mechanism that normally prevents IgE+ Bmem and LLPC formation, providing insights into the molecular pathogenesis of allergic diseases.

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Figure 1: Ag-independent mIgE signaling induces PC differentiation and apoptosis.
Figure 2: The CD19-PI3K-Akt-IRF4 axis mediates spontaneous PC differentiation of IgE+ B cells.
Figure 3: IgE+ B cell response in immunized Cd19+/− mice.
Figure 4: BLNK facilitates both PC differentiation and apoptosis of IgE+ B cells.
Figure 5: IgE+ B cell response in immunized BLNK-deficient mice.
Figure 6: Enhanced active anaphylaxis in BLNK-deficient mice.
Figure 7: mIgE domains responsible for CD19- and BLNK-mediated PC differentiation and apoptosis.

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Acknowledgements

We thank K. Rajewsky and colleagues (Max Delbrück Center for Molecular Medicine) for B1-8f, Cγ1-Cre and Cd19−/− mice, F. Alt (Harvard Medical School) and T. Tsubata (Tokyo Medical and Dental University) for Igκ−/− mice, T. Azuma (Research Institute for Biomedical Sciences) for N1G9 cDNA, M. Ohkura and J. Nakai (Saitama University) for G-CaMP6, T. Kitamura (University of Tokyo) for pMXs-IRES-GFP and Plat-E cells, J. Yagi (Tokyo Women's Medical University School of Medicine) for CA-Akt, RIKEN BRC for Balb/c 3T3 cells, J. Nakayama, M. Yamamoto and S. Horiuchi for plasmid constructs, T. Nojima, R. Goitsuka, M. Kubo and other members of the Research Institute for Biomedical Sciences for technical advice and comments, and P. Burrows for critical reading. This work was supported by the Japan Society for the Promotion of Science (JSPS) KAKENHI (25293116 to D.K., 15K19138 and 12J08178 to K.H.) and Takeda Science Foundation (D.K.). K.H. was a JSPS Fellow when he started this study.

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Authors and Affiliations

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

    Kei Haniuda, Saori Fukao, Tadahiro Kodama, Hitoshi Hasegawa & Daisuke Kitamura

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  1. Kei Haniuda
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  2. Saori Fukao
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Contributions

K.H. conceived the project, designed and performed all of the experiments, analyzed data, and wrote the manuscript. S.F. performed experiments, analyzed data, gave critical advice and edited the manuscript. T.K. and H.H. provided technical support. D.K. supervised the study, designed experiments and wrote the manuscript.

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Correspondence to Daisuke Kitamura.

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Integrated supplementary information

Supplementary Figure 1 Verification of the class-swapping system and confirmation of autonomous mIgE signaling.

(a) Flow cytometry analyzing the surface expression of λ LC and CD19 on iGB cells from Igκ−/− B1-8f or Igκ−/− B1-8f Cγ1-Cre mice at the indicated time points of culture with IL-4. (b) GFP and surface BCR expression (NP-binding) in Igκ−/− B1-8f Cγ1-Cre B cells transduced with the indicated membrane immunoglobulin HC (mIgH) isotype as in Fig. 1a. (c) Frequencies of Pax5loCD138+ PCs among GFP+ cells in Igκ+/+ B1-8f Cγ1-Cre iGB-4 cells transduced as in b and cultured plain with anti-κ LC Ab for 1 d. (d-g) Flow cytometric analyses of Blnk+/+ or Blnk−/− B1-8f Cγ1-Cre B cells transduced simultaneously with mIgH (pMXs-IRES-mCherry) and G-CaMP6 (pMXs-IRES-hNGFR) on day 2 and further cultured for 1 d. (d) A hNGFR vs. mCherry expression profile of mIgG1-transduced Blnk+/+ cells showing a gating strategy (left) and G-CaMP fluorescence (middle) and hNGFR expression (right) among the mCherry+hNGFR+ cells (a gate indicated by a rectangle of black line (left)) in each sample. A histogram shaded in gray: mCherry−hNGFR−cells in mIgG1-transduced Blnk+/+ cells (a gate indicated by a rectangle of gray line (left). (e) Cell surface expression levels of IgG1 (left) or IgE (right) on mCherry+ Blnk+/+ and Blnk−/− cells in comparison with the cells transduced with an empty Rv (–). (f,g) Sequential change of the ratio of G-CaMP to hNGFR fluorescence (normalized G-CaMP fluorescence) of mCherry+hNGFR+ cells gated as in d. After baseline recording for 150 seconds, cells were stimulated with anti-κ LC Ab at 10 μg/ml. Mean of the normalized G-CaMP fluorescence is collectively shown as a kinetic diagram (g). (h) Immunoblot analysis of Flag-tagged CD19 (Flag) and λLC in whole cell lysate (WCL) or proteins purified by NP-beads (IP:NP-beads) from the lysates of Igα/β-expressing BALB/c 3T3 cells transduced with mIgH and λ LC with or without Flag-tagged CD19. Data are representative of three (a-c) or two (d-h) independent experiments (mean and s.d. of triplicate transductions in c).

Supplementary Figure 2 CD19 and IRF4 are required for mIgE-induced PC differentiation.

(a) Fold-increase of cell numbers (expansion factor) in 4 d of C57BL/6 (B6, control), Blnk−/− or Cd19−/− iGB cells. (b) Flow cytometry analyzing the expression of IgG1 and IgE on day 4 of the same cells as in a. Numbers in outlined areas indicate percent IgG1+ or IgE+ cells. (c,d) Flow cytometric analyses of Cd19+/+ or Cd19−/− iGB cells after the plain culture for 1 d in the presence or absence of anti-κ LC Ab (c) or medium only (d). (c) Frequency of Pax5loCD138+ PCs among IgG1+ or IgE+ cells. (d) Annexin V (AnnV) staining profiles of IgE+CD138− cells, with numbers above bracketed lines indicating percent AnnV+ cells (left), and frequency of AnnV+ cells among IgG1+ or IgE+, CD138− cells (right). (e) Expression of IRF4 in CD138−, IgG1+ or IgE+ gated B6 iGB cells on day 4. (f) Flow cytometry analyzing the expression of CD138 and Pax5 in IgG1+ or IgE+, GFP+ gated cells of B6 iGB cells transduced with Rv (pMXs-IRES-GFP) encoding IRF4 or empty vector (Ev) and cultured plain. Numbers above outlined areas indicate percent PCs among IgG1+ or IgE+, GFP+ cells. (g) RT-qPCR analysis of Irf4 mRNA expression in hNGFR+IgE+CD138− B6 iGB cells transduced with Rv (pSIREN-hNGFR) expressing shRNA for Luciferase (shControl) or two independent shRNAs for Irf4 (shIRF4-1 and -2) and sorted on day 5. AU, arbitrary units. (h) Frequency of CD138+ PCs in hNGFR+IgE+ cells transduced as in g and cultured plain. NS, not significant (P > 0.05); * P < 0.05, ** P < 0.01 (Student’s t-test). Data are pooled from ten (control in a) or six (Blnk−/− and Cd19−/− in a) independent experiments (a, mean and s.d.), or are representative of three (b,d,e) or two (c,f) independent experiments (mean and s.d. of triplicate culture in c,d,g).

Supplementary Figure 3 Phenotypic analyses of Cd19+/– iGB cells and Cd19+/– mice.

(a,b) Flow cytometric analyses of Cd19+/+ or Cd19+/– iGB cells after the plain culture. (a) Expression profiles of IgG1 vs. IgE (left) and CD138 vs. Pax5 in IgE+ gated cells (middle), with numbers adjacent outline areas indicating percentages, and frequency of Pax5loCD138+ PCs among IgG1+ or IgE+ gated cells (right). (b) Annexin V staining profiles of IgE+CD138− cells, with numbers above bracketed lines indicating percent AnnV+ cells (left), and frequency of AnnV+ cells among IgG1+ or IgE+, CD138− cells (right). (c) Gating strategy for flow cytometric analysis of fixed inguinal LN cells from mice unimmunized or immunized s.c. with NP-CGG in alum 1 week previously. Fixable viability dye (FVD) negative, singlet cells in a lymphocyte gate defined by forward and side scatters were subdivided into class-switched GC B cells (B220+IgD−CD138−GL7+CD38lo) and PCs (IgG1hi or IgEhi, CD138+). IgG1 and IgE were stained intracellularly. Numbers adjacent outlined areas indicate percentage of the cells depicted in each panel. (d) Anaphylaxis reactions of Cd19+/+ and Cd19+/– mice primed i.p. with NP-CGG in alum 74 days earlier. Changes in rectal temperature (Δ °C) of the mice injected i.v. with 20 μg NP-CGG are shown. *P < 0.05, **P < 0.01 (Student’s t-test). Data are representative of three independent experiments (a,b,c; mean and s.d. of triplicate culture in a,b) or are from one experiment (d, mean ± s.d. (n = 6)).

Supplementary Figure 4 Syk and BLNK are required for mIgE-induced PC differentiation and apoptosis.

(a,b) Flow cytometric analyses of frequency of Pax5loCD138+ PCs in IgE+ cells (a) and AnnV+ cells in IgE+CD138− cells (b) of B6 iGB cells after the plain culture with Bay 61-3606 (Bay) or vehicle only (Veh, –) for 1 d. Four-fold serial dilutions of the each inhibitor starting from 10 μM (indicated by wedges) were added, and representative data of the cells treated with 2.5 μM are shown (each left). (c-g) Flow cytometric analyses of Blnk+/+ or Blnk−/− B1-8f Cγ1-Cre B cells transduced with mIgG1, mIgE or empty (–) Rv as in Fig. 1a and cultured plain. GFP expression (c), frequency of CD138+ cells in GFP+ gated cells (d,e) and frequency of AnnV+ cells in GFP+CD138− gated cells (f,g). Numbers above outlined areas and bracketed lines indicate percentage of the cells depicted in each panel. * P < 0.05, ** P < 0.01 (Student’s t-test). Data are representative of two independent experiments (mean and s.d. of triplicate culture in a,b,g; mean and s.d. of two independent experiments in e).

Supplementary Figure 5 BLNK suppresses formation of IgE+ LLPCs in vivo by a B cell–intrinsic mechanism.

(a) A representative ELISPOT data of Fig. 5h. Spots of anti-NP IgE AFCs from mice immunized with NP-CGG in alum 100 days previously are shown (input: 5 x 106 cells per well). (b) Strategy for single cell sorting of NP-specific plasma cells from mice immunized with NP-CGG in alum. From pre-enriched CD138+ cells, IgG1+ or IgE+, NP-binding CD138+ single cells were sorted. VH186.2 gene was amplified from each single cell and sequenced. (c) Strategy to generate bone marrow (BM) chimeric mice with BM cells from Blnk+/+ or Blnk−/− and μMT mice, mixed at 20:80, or from μMT mice alone. (d,e) ELISPOT assay of NP-specific IgG1 (d) or IgE (e) AFCs in the spleens of the BM chimeric mice immunized i.p. with NP-CGG in alum ten weeks previously. (f-h) ELISPOT assay of splenocytes from mice transferred with follicular (Fo) B cells of Blnk+/+ or Blnk−/− B1-8f mice and immunized i.p. with NP-CGG in alum 4 weeks previously (f). The number of NP-specific IgG1 (g) or IgE (h) AFCs. * P < 0.05, ** P < 0.01 (Student’s t-test). Data are representative of two (a) or three (b) independent experiments, or data are from one experiment (d,e,g,h, each dot indicates one mouse and bar indicates the mean of each group; n = 4 (d,e) or n = 5 (g,h) mice for each group).

Supplementary Figure 6 The function of the cytoplasmic tail and the EMPD of mIgE.

(a) The expression of BCR (NP-binding, red curves) on day 5 in GFP+ cells (red rectangles) of Igκ−/– B1-8f Cγ1-Cre B cells transduced with the chimeric HCs (used in Fig. 7 and Supplementary Fig. 6b-d) as in Fig. 1a. (b) Schematic of mIgG1, mIgE and mIgE ΔTail chimera. The latter consists of ectodomains and a transmembrane region of mIgE and a cytoplasmic tail of mIgM (three amino acids). (c,d) Flow cytometry analyzing the frequencies of Pax5loCD138+ PCs among GFP+ cells (c) and AnnV+ cells among GFP+CD138− cells (d) of cells transduced with the HCs shown in b as in a and cultured plain. (e) Schematic of mIgG1, mIgE and EMPD-E. The latter is a chimeric mIgG1 in which the EMPD is replaced with that of mIgE. (f-j) Flow cytometric analysis of Igκ−/– B1-8f Cγ1-Cre B cells transduced with the HCs shown in e as in a. The expression of BCR (NP-binding) in GFP+ cells at day 5 (f), and the frequencies of Pax5loCD138+ PCs among GFP+ cells (g,i) and AnnV+ cells among GFP+CD138− cells (h,j) of cells cultured plain. Gray shaded curves, mock-transduced cells (the histogram on top in a,f). Numbers above outlined areas and bracketed lines indicate percentage of the cells depicted in each panel. Data are representative of three (a-d,g-j) or two (f) independent experiments (mean and s.d. of triplicate transductions in i,j).

Supplementary Figure 7 The EMPD of mIgE is sufficient for the interaction with CD19.

(a-d) Immunoblot analyses of the indicated molecules in Igκ−/– B1-8f Cγ1-Cre B cells transduced with the HCs (used in Supplementary Fig. 6e) as in Fig. 1a and lysed on day 5. Proteins purified by NP-beads incubated with the cell lysates (a), proteins immunoprecipitated (IP) from the lysates with anti-CD19 Ab (b), whole cell lysates (WCL) (c) and proteins immunoprecipitated from the lysates with anti-BLNK Ab (d). (e,f) Immunoblot analyses of the indicated molecules in Blnk+/+ or Blnk−/−, B1-8f Cγ1-Cre B cells transduced with mIgG1 or mIgE as in Fig. 1a and lysed on day 5. Whole cell lysates (e) and proteins immunoprecipitated from the lysates with anti-CD19 or with isotype-matched control Ab (f). (g) Flow cytometric analysis of the expression of IgG1 and IgE (left), and immunoblot analysis of proteins immunoprecipitated with anti-BLNK or isotype-matched control Abs from the lysates (right), of Cd19+/+ or Cd19−/− iGB cells cultured for 5 d. (h) A model of autonomous mIgE signaling. In B cells having switched to IgE, mIgE is auto-activated and triggers the Syk-BLNK-JNK/p38 signaling axis that induces apoptosis. Simultaneously, mIgE constitutively recruits CD19 through its EMPD and activates the CD19-PI3K-Akt axis, which elevates IRF4 expression and induces PC differentiation in conjunction with the former signaling axis. By these mechanisms, mIgE expressing GC B cells quickly differentiate into short-lived PCs and are scarcely maintained as GC B cells, which prevents generation of IgE+ Bm cells or LLPCs. Data are representative of three (a,b,d) or two (c) independent experiments, or data are from one experiment (e-g).

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Haniuda, K., Fukao, S., Kodama, T. et al. Autonomous membrane IgE signaling prevents IgE-memory formation. Nat Immunol 17, 1109–1117 (2016). https://doi.org/10.1038/ni.3508

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