Secreted IgD Amplifies Humoral T Helper 2 Cell Responses by Binding Basophils via Galectin-9 and CD44

Abstract

B cells thwart antigenic aggressions by releasing immunoglobulin M (IgM), IgG, IgA, and IgE, which deploy well-understood effector functions. In contrast, the role of secreted IgD remains mysterious. We found that some B cells generated IgD-secreting plasma cells following early exposure to external soluble antigens such as food proteins. Secreted IgD targeted basophils by interacting with the CD44-binding protein galectin-9. When engaged by antigen, basophil-bound IgD increased basophil secretion of interleukin-4 (IL-4), IL-5, and IL-13, which facilitated the generation of T follicular helper type 2 cells expressing IL-4. These germinal center T cells enhanced IgG1 and IgE but not IgG2a and IgG2b responses to the antigen initially recognized by basophil-bound IgD. In addition, IgD ligation by antigen attenuated allergic basophil degranulation induced by IgE co-ligation. Thus, IgD may link B cells with basophils to optimize humoral T helper type 2-mediated immunity against common environmental soluble antigens.

Keywords: B cell; CD44; IgD; Th2; allergy; antibody; basophil; galectin; humoral immunity; mucosal immunity.

Figure 1.. IgD Ligation by Antigen Elicits Basophil Activation

(A) ELISA of serum IgD from WT Balb/c (n=29), WT C57BL/6 (n=15), Ighd^−/− Balb/c (n=20) or Ighd^−/− C57BL/6 (n=10) mice. (B) Left: FCM of CD49b and IgE on basophils (black gate) from circulating CD45⁺NTNB cells of a WT Balb/c mouse. NTNB, non-T non-B. Right: FCM of IgD on circulating basophils (blue open profile) from a WT Balb/c mouse. Ctrl, isotype-matched control (gray solid profile). (C) FCM of IgD⁺ basophils from peripheral blood, spleen, bone marrow (BM), lung or mesenteric lymph nodes (MLNs) of WT Balb/c mice (n=5). (D) Imaging FCM of IgD, FcεRI and CD49b from a representative viable Ghost Dye Violet 510 (GV510)⁻ splenic basophil of a WT Balb/c mouse. Scale bar, 5 μm. (E) FCM quantification of FcεRI⁺CD49b⁺ basophils from BM, blood, spleen or lung CD45⁺NTNB cells of WT Balb/c (n=10) or Ighd^−/− (n=10) mice. (F) FCM of Il4-driven GFP expression in splenic or lung FcεRI⁺CD49b⁺ basophils from WT Balb/c Il4^GFP (n=5) or Il4^GFPIghd^−/− (n=5) mice. MFI, mean fluorescence intensity. (G) ELISA of IL-4 and IL-13 from serum of WT Balb/c or Ighd^−/− mice (n=10). (H) Schematics of i.v. reconstitution μMT or Rag2^−/− mice with NP-reactive IgD (NP-IgD) followed by i.p. injection of anti-IgD or i.n. inoculation of NP-OVA. (I) FCM of IgD on splenic FcεRI⁺CD49b⁺ basophils from a μMT mouse before (ctrl) or after reconstitution as in (H). PBS, control phosphate buffer solution (PBS). (J, K) FCM quantification of total, CD200R3⁺ or IL-4⁺ basophils from the spleen of μMT C57BL/6 mice (n=10) (J) or the lungs of IgD-deficient Rag2^−/− (n=10) C57BL/6 mice (K) treated as in (H). (L) FCM quantification of total, IgD⁺, IgE⁺ or IL-4⁺ basophils from the lungs of WT Balb/c (n=5) or Ighd^−/− (n=5) mice 5 d following i.n. exposure to PBS or recombinant TSLP. (M) ELISA of serum IgD from Balb/c mice following i.v. injection of control empty or TSLP-encoding plasmids (n=10). Data summarize two-three experiments with 5–10 mice per each experimental group (A, C, E-G, J-M) or show one experiment of at least three with similar results (B, D, I). Results are presented as mean ± SEM; *p < 0.05, **p < 0.01 (two-tailed unpaired Student’s t test). See also Figure S1.

Figure 2.. IgD Ligation by Antigen Induces Basophil Expression of IL-4

(A) Schematics of s.c. immunization with NP-OVA combined with control PBS, papain or NP-IgD. (B) Confocal imaging of CD169 (subcapsular sinus macrophage molecule, red), B220 (B cell molecule, blue) and Mcpt8 (YFP, yellow) from draining lymph node (DLN) of a C57BL/6 Mcpt8^YFP mouse immunized for 6 h as in (A). FO, follicle; dashed line, follicular border. Original magnification, ×5; rightbottom panel, ×40. Scale bars, 50 μm (top and bottom-left panels) or 5 μm (bottom-right panel). (C-E) FCM quantification of total YFP⁺ or GFP⁺ basophils and qRT-PCR quantification of Il4 transcripts encoding IL-4 from the DLN of C57BL/6 Mcpt8^YFP (n=5) mice (C, D) or Balb/c Il4^GFP (n=10) mice (E) 6 h following s.c. immunization as in (A). qRT-PCR results (D, bottom graph) are presented as relative expression (RE) compared to mRNA for glyceraldeheyde phosphate dehydrogenase (GAPDH). (F) Schematics of s.c. immunization with NP-OVA combined with papain, alum or CFA. (G) ELISA of serum NP-specific IgD from WT Balb/c mice (n=17) following s.c. immunization with NP-OVA and papain. PI, pre-immune (day −1 relatively to the onset of immunization (day 0). (H) ELISA of serum NP-specific IgG1 and IgE from WT Balb/c (n=12) or Ighd^−/− (n=16) mice following s.c. immunization with NP-OVA and papain. (I) ELISA of serum NP-specific IgG1 and IgE from WT Balb/c (n=15) or Ighd^−/− (n=15) mice following s.c. immunization with NP-OVA and alum. (J) ELISA of serum NP-specific IgG2a and IgG2b from Balb/c (n=10) or Ighd^−/− (n=10) mice following s.c. immunization with NP-OVA and CFA. Data show one experiment of three with similar results (B, C) or summarize results from two experiments with 5–10 (D, E) or 10–17 (G-J) mice per experimental group. Results are presented as mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001 (two-tailed unpaired Student’s t test).

Figure 3.. Ligation of Basophil-Bound IgD by Antigen Enhances IgG1 and IgE Responses

(A) Schematics of i.p. immunization with NP-OVA and papain. (B) ELISA of serum OVA-specific IgG1 and IgE from WT Balb/c (n=10) or Ighd^−/− (n=10) mice immunized as in (A). Dashed line, maximum antibody concentration in pre-immune (PI) mice. PI, day −1 relatively to the onset of immunization (day 0). (C-D) FCM quantification of total and IL-4-expressing splenic PD-1^highCXCR5^high Tfh cells from WT Balb/c (n=5) or Ighd^−/− (n=5) mice immunized as in (A). (E, F) ELISA of serum NP-specific IgG1 from WT Balb/c (n=10) or Ighd^−/− (n=10) mice immunized as in (A). BSA haptenated with 4 or 16 NPs was used to measure high-affinity (HA) and both HA and lowaffinity (LA) IgG1, respectively. (G) IFA of splenic tissue from immunized Balb/c mice stained for IgD (green), IgM (red) and nuclei (blue) following i.p. immunization as in (B). Inset: IgD⁺IgM⁻ plasmablast next to IgD⁻IgM⁺ plasmablast. FO, follicle. Original magnification, ×10 with ×2 enlargement. Scale bar, 50 μm. (H) ELISPOT of spleen ASCs expressing NP-specific IgD from WT Balb/c (n=5) or Ighd^−/− (n=5) mice 3 d following i.p. immunization with PBS or NP-OVA and papain. (I) Schematics of i.v. reconstitution with NP-reactive IgD (NP-IgD) followed by i.p. immunization with NP-OVA and papain. (J) ELISA of serum OVA-specific IgG1 and IgE from WT Balb/c controls (n=10), Ighd^−/− mice (n=10) or NP-IgD-reconstituted Ighd^−/− mice (n=10) following i.p. immunization with NP-OVA and papain as in (I). (K) FCM of circulating FcεRI⁺IgE⁺ basophils from WT Balb/c mice after i.v. injection of a control (ctrl) IgG2b antibody or a basophil-depleting anti-CD200R3 antibody. (L) ELISA of serum total IgD as well as serum NP-specific IgG1 and IgE in control (n=10) or basophil-depleted (n=10) WT Balb/c mice after i.p. immunization with NP-OVA and papain. Data summarize results from two experiments with 5–10 mice per experimental group (B-F, H, J, L) or show one experiment of three-five with similar results (C, G, K). Results are presented as mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001 (two-tailed unpaired Student’s t test). See also Figure S2.

Figure 4.. Basophil-Bound IgD Interacts with Galectin-9 and Its Ligation Induces Th2 Cell-Associated Cytokine Expression But Inhibits Cytoskeleton Remodeling

(A) Schematics of i.p. anti-IgD treated mice i.v. reconstituted or not with secreted IgD. (B-C) ELISA of serum IgG1, IgE (B), IL-4 and IL-13 (C) from WT Balb/c (n=10) or Ighd^−/− (n=10) mice in the presence or absence of i.v. reconstitution with secreted IgD (NP-IgD), followed by i.p. injection of anti-IgD. Dashed line, maximum antibody concentration in pre-immune (PI) mice. PI, day −1 relatively to onset of anti-IgD treatment (day 0). (D) IFA of human tonsillar tissue stained for IgD (green), IgM (red), cytokeratin (purple) and nuclei (blue). EP, epithelium; FO, follicle; PC, plasma cell. Original magnification, ×20 (top) or ×63 (bottom). Scale bars, 50 μm (top) and 5 μm (bottom). (E) ELISA of IgD specific to α-s-casein, β-lactoglobulin (β-LGB) or α-lactalbumin (α-LAL) from plasma of FPIES children with (n=5) or without (n=5) dietary milk restrictions. (F) FCM of IgD (red open profile) bound to human tonsillar or circulating CD123⁺FcεRI⁺ basophils (red gate). Gray open profiles, control isotype-matched antibody with irrelevant binding activity. (G) Microarray analysis of genes expressed by human basophils upon IgD cross-linking. The Volcano plot represents genes differentially expressed by basophils treated with anti-IgD or a F(ab’)2 control antibody (ctrl). Red and blue dots, up-regulated and down-regulated genes, respectively; FC, fold change. (H) Heat map of coordinated gene sets identified by gene set enrichment analysis in human basophils treated as in (G). NES (normalized enrichment score) indicates correlation between individual gene sets. Positive correlation, NES > 0 (yellow gradient); negative correlation, NES < 0 (blue gradient). (I) Heat maps depicting changes in FC expression of coordinated “Basophil-Th2 pathway” (left) or “Actin filament-based processes” (right) gene sets from human basophils treated as in (G). The corresponding gene set enrichment analysis is further detailed in Fig. S4D and S4E. Bottom numbers, biological replicates. Right numbers, functions. Red, high expression; blue, low expression. (J) Heat map of mRNAs for IgD-bound proteins identified by mass spectrometry after immunoprecipitation of human basophil lysates with ctrl or anti-IgD. Letters indicate functional groups. Data summarize results from two-three experiments with 10 mice per experimental group (B, C), show one experiment of at least three with similar results (D, F), or depict results from one experiment with at least three biological replicates (G, H, I, J). Results are presented as mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001 (two-tailed unpaired Student’s t test). See also Figures S3 and S4.

Figure 5.. IgD Binds to Basophils through Galectin-9 and CD44

(A) IB of galectin-9 following IP of human IgD and galectin-9 protein mix with control (ctrl) or anti-IgD antibodies. Prior to IP, the protein mix was supplemented with control PBS, glucose or lactose. (B) FCM of human IgD on human KU812 cells cultured for 30 min with control medium alone (ctrl), IgD or an IgD-galectin-9 complex formed by pre-incubating IgD with galectin-9 for 10 min. (C) FCM of human IgD or galectin-9 on KU812 cells cultured with IgD-galectin-9 in the presence of medium alone (ctrl), glucose or lactose for 30 min. (D) FCM of human IgD or CD44 on KU812 cells treated with scrambled (ctrl) or CD44-targeting small interfering RNA (siRNA) and later incubated with or without (ctrl) IgD-galectin-9. MFI, mean fluorescence intensity. (E) Confocal imaging of human basophils stained for IgD (blue), galectin-9 (green) and CD44 (red). Scale bar, 0.5 μm. (F) FCM of IgD on human basophils incubated with or without (ctrl) IgD-galectin-9 for 30 min. (G) ELISA of IL-4 from human basophils incubated with medium alone (ctrl) and with or without the IgD-galectin-9 complex in the presence or absence of IgD cross-linking by anti-IgD for 18 h. (H, I) FCM of IgD⁺ basophils from the spleen or lung of WT C57BL/6, Lgals9^−/− (H) or Cd44^−/− (I) mice. (J, K) ELISA of serum IgG1 and IgE to NP from WT Balb/c (n=10), Lgals9^−/− (n=8) (J) or Cd44^−/− (n=10) (K) mice following s.c. immunization with NP-OVA and papain. Dashed line, maximum antibody concentration in pre-immune (PI) mice. PI, day −1 relatively to the onset of anti-IgD treatment (day 0). (L) ELISA of serum total IgG1 and IgE from WT C57BL/6 controls (n=10) and Lgals9^−/− mice (n=8) following i.p. injection of anti-IgD. Data show one experiment of at least three with similar results (A, B, E, F) or summarize three experiments (C, D), one experiment with three biological replicates per experimental group (G) or twothree experiments with 8–10 mice per experimental group (H-L). Results are presented as mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001 (two-tailed unpaired Student’s t test). See also Figures S5.

Figure 6.. IgD Attenuates Acute Lung Inflammation Following Secondary Antigen Exposure

(A) Schematics of i.p. OVA sensitization followed by a challenge consisting of four consecutive i.t. inoculations of OVA or control PBS. (B) ELISA of OVA-specific IgD and IgE from serum of WT Balb/c (n=10) or Ighd^−/− (n=10) mice treated as in (A). Dashed line, maximum antibody concentration 4 hs after the last i.t. inoculation of PBS. (C) FCM quantitation of IgE on l basophils from lungs of WT Balb/c (n=5) or Ighd^−/− (n=5) mice treated as in (A). (D) Microscopic quantitation of total cells from the bronchoalveolar lavage (BAL) of WT Balb/c (n=5) or Ighd^−/− (n=5) mice treated as in (A). (E, F) FCM quantitation of CD49b⁺IgE⁺ basophils from lungs of WT Balb/c (n=10) or Ighd^−/− (n=10) mice treated as in (A). NTNB, non-T non-B. (G) ELISA of Mcpt8 from serum of WT Balb/c (n=5) or Ighd^−/− (n=5) mice treated as in (A). (H, I) FCM quantitation of CD45⁺Siglec-F⁺ eosinophils from lungs of WT Balb/c (n=5) or Ighd^−/− (n=5) mice treated as in (A). Data summarize two experiments with at least five mice per experimental group. Results are presented as mean ± SEM; *p < 0.05, **p < 0.01 (two-tailed unpaired Student’s t test).

Figure 7.. IgD Ligation by Antigen Attenuates IgE-induced Basophil Degranulation

(A) ELISA of IgD, IgG4 and IgE to PLA2 or tetanus toxin (TT) from plasma of healthy non-allergic controls (n=4) or beekeepers (n=4). (B) ELISA of IgD to α-casein or β-lactoglobulin from plasma of allergic children at baseline (0 months) and following 22 months of treatment with placebo (n=5) or OIT (n=5). (C) ELISA of total IgD or IgD to OVA from plasma of allergic children at baseline (0 months) and following 22 months of treatment with placebo (n=15) or OIT (n=40). Left and right panels: total (top) or OVA-specific (bottom) IgD concentrations by treatment group and treatment outcome, respectively. (D) Microarray analysis of genes from human basophils exposed for 3 h to anti-IgE and control (ctrl) IgG or anti-IgD. Red and blue dots in volcano plot show up-regulated and down-regulated genes, respectively; FC, fold change. (E) Heat map showing enhancement or depletion of coordinated gene sets identified by gene set enrichment analysis of human basophils treated as in (D). NES (normalized enrichment score) > 0 (yellow gradient) or < 0 (blue gradient) indicates positive or negative correlation between individual gene sets. (F, G) FCM of surface CD63 (F) and ELISA of histamine (G) from IL-3-stimulated human basophils incubated with control (ctrl) medium alone, ctrl irrelevant IgG, anti-IgD and/or anti-IgE for 6 h. (H) ELISA of β-hexosaminidase (β-HEX) and galectin-9 from human KU812 cells exposed to NP-OVA following incubation with irrelevant IgG (ctrl) or NP-IgE combined or not with NP-IgD complexed to galectin-9. Data show one experiment with 4–15 donors per treatment group (A-C), depict one experiment with three biological replicates per condition (D-G), or summarize two experiments with three biological replicates per experimental group (H). Results are presented as mean ± SEM; *p < 0.05, **p < 0.01, *** p < 0.001 (Wilcoxon matched pairs signed-rank test or two-tailed Student’s t test). See also Figures S6 and S7.