(a) Proportion of IgD⁺IgM⁻ plasmablasts in various tissues calculated by immunofluorescence as described in Methods. PLN, peripheral lymph nodes; BM, bone marrow. (b) Tonsil tissue stained for IgD (green) and IgM (red). DAPI (blue) counterstains nuclei. Arrowheads point to IgD⁺IgM⁻ plasmablasts. Original magnification ×10. (c) Representative IgD⁺IgM⁻ B cells from tonsils stained for IgD (green), Pax-5, Blimp-1, BCMA, CD138, κ, λ, AID or CD19 (red), and IgM or DAPI (blue). (d) Diagram of CSR from S_μ to σ_δ. Germline I_μ-C_μ and I_μ-C_δ transcripts, σ_δ-S_μ switch circles, and post-switched I_μ-C_δ transcripts are shown. Arrows indicate primers. (e) Southern blots of σ_δ-S_μ switch circles PCR amplified from mononuclear cells of various tissues and hybridized with σ_δ or S_μ probes. Rightmost lane shows a typical multi-banded σ_δ-S_μ smear in enriched tonsillar IgD⁺ B cells. Kb, kilobases. (f) Germline I_μ-C_μ transcripts and germline or post-switch I_μ-C_δ transcripts in Reh IgD⁻IgM⁺ pre-B-like cells, 2E2 pre-germinal center IgD⁺IgM⁺ B cells, Ramos germinal center-like IgD⁻IgM⁺ B cells, and MM-M1 IgD⁺IgM⁻ plasma cells. Genomic β-actin is a loading control. (g) Representative σ_δ-S_μ and I_μ-C_δ DNA sequences from tonsillar IgD⁺IgM⁻ and IgD⁺IgM⁺ B cells, respectively. Panel a summarizes 1 of 3 experiments (bars indicate s.e.m.), whereas panels b, c, d, f, g and h show 1 of 5 experiments yielding similar results.

(a) Flow cytometric analysis of IgD on circulating or tonsillar CD19⁺CD3⁻ B cells, CD19⁻CD3⁺ T cells, CD16^highCD56^low or CD16^lowCD56^high NK cells, CD19⁻CD14⁺ monocytes, CD11c⁺CD83⁻ or CD11c⁺CD83⁺ myeloid dendritic cells (mDCs), CD15⁺CD123⁻ neutrophils, CD15⁺CD123^low eosinophils, CD123⁺HLA-DR⁺ or CD123⁺FcεRI^low plasmacytoid dendritic cells (pDCs), CD123⁺HLA-DR⁻ or CD123⁺FcεRI⁺ basophils, and CD117⁺FcεRI⁺ mast cells. Gray and black histograms depict control unstained cells and IgD, respectively. A F(ab’)₂ pAb was used to detect IgD. Dead cells were excluded using 7-AAD staining. (b) Immunofluoresence analysis of circulating and tonsillar basophils stained for IgD (green) and FcεRI, CD123 or tryptase (red). DAPI (blue) counterstains lobated nuclei of basophils. A F(ab’)₂ pAb was used to detect IgD. (c) Flow cytometry of IgD levels on circulating basophils before (black histogram) and after exposure to exogenous monoclonal IgD (10 μg/ml, brown histogram), treatment with acidic buffer (blue histogram), and treatment with acidic buffer followed by addition of exogenous IgD (red histogram). A F(ab’)₂ pAb was used to detect IgD. Unstained basophils were used as control (gray histogram) and dead cells were excluded using 7-AAD staining. (d) Binding of labeled monoclonal IgD to circulating basophils stripped of endogenous IgD and incubated with 0 μg/ml (green histogram), 100 μg/ml (orange histogram) or 500 μg/ml (purple histogram) of unlabelled monoclonal IgD. A F(ab’)₂ pAb was used to detect IgD. Unstained basophils were used as control (gray histogram) and dead cells were excluded using 7-AAD staining. Panels a-d show 1 of 5 experiments yielding similar results.

(a) Flow cytometry of CD63 on basophils exposed to microbeads alone (control), microbead-bound monoclonal anti-IgD, or microbead-bound monoclonal anti-IgE for 30 min or 5 h in the presence or absence of IL-3. (b) ELISA of histamine from basophils exposed to microbeads alone (open bar), microbead-bound monoclonal anti-IgD (gray bar), microbead-bound monoclonal anti-IgE (black bar), or microbead-bound isotype-matched control monoclonal antibody (striped bar) for 30 min in the presence or absence of IL-3. (c) ELISA of IL-4, IL-13 or BAFF from basophils exposed to microbeads alone (open bar), microbead-bound monoclonal anti-IgD (gray bar), or microbead-bound monoclonal anti-IgE (black bar) for 16 h in the presence or absence of IL-3. IL-8 and CXCL10 were measured after 48 h. (d) Intracellular Ca²⁺ levels of basophils treated as in c. The arrow indicates addition of the cross-linking reagent and 0 sec indicates the start of the kinetic measurement. (e) IgM, IgA and IgG production by peripheral blood IgD⁺IgM⁺ B cells exposed for 7 d to basophils treated as in c. Panels a and d show 1 of 3 experiments yielding similar results, whereas panels b, c and e summarize 3 experiments (bars indicate s.e.m.; *, p < 0.01; **, p < 0.001).

(a, b) Flow cytometric analysis of circulating IgD⁺IgM⁻ B cells and CD123⁺HLA-DR⁻ basophils in a healthy subject and hyper-IgD patients with MVK (HIDS), unknown (PFAPA), NALP3 (MWS) and TNFRSF1A (TRAPS) gene defects. Numbers indicate percentage of IgD⁺IgM⁻ B cells in CD19⁺ B cells and of CD123⁺HLA-DR⁻ basophils in mononuclear cells. (c) Immunofluorescence analysis of a tonsil tissue specimen from healthy individual or a PFAPA syndrome patient stained for IgD (green), tryptase (red) and DAPI (blue). Dashed lines demarcate lymphoid follicles. (d) BAFF (green) expression by basophils co-stained for IgD (green), CD123 (red) and FcεRI (blue). Arrowheads show both IgD⁺CD123⁻ plasmablasts (B) and IgD⁺CD123⁺ basophils (Basø). (e) ELISA of TNF and IL-1β from basophils cultured with IL-3 and microbeads alone (control, open bar), microbead-bound monoclonal anti-IgD (gray bar) or microbead-bound monoclonal anti-IgE (black bar) for 48 h in the presence or absence of monocytes. Panels a-d show 1 of 3 experiments yielding similar results, whereas panel e summarizes 3 experiments (bars indicate s.e.m.).

(a) σ_δ-S_μ switch circles from circulating IgD⁺IgM⁺ B cells stimulated with or without CD40L, BAFF or APRIL plus IL-15 and IL-21 for 4 d. Genomic β-actin is a loading control. Kb, kilobases. (b, c) Flow cytometric analysis of surface IgD and IgM and ELISA of secreted IgD from circulating IgD⁺IgM⁺ B cells stimulated as in a for 7 d. Control indicates medium alone. (d) Percentage of circulating IgD⁺IgM⁻ B cells in a healthy donor and patients with TNFSF5 (HIGM1), AICDA (HIGM2), TNFRSF5 (HIGM3), TNFRSF13b (CVID) or MVK (HIDS) gene defects. (e) Induction of σ_δ-S_μ switch circles in circulating mononuclear cells from a HIGM2 patient and a control healthy donor incubated with medium alone (control) or BAFF, IL-15 and IL-21 (stimulated) for 4 d. An anti-IgM antibody was added to optimize the activation and expansion of IgD⁺IgM⁺ B cells. (f) Binding of secreted IgD to MID, CPS, LPS, M. catarrhalis, and H. influenzae type a or type b as determined by ELISA. IgD secretion was obtained by incubating circulating IgD⁺IgM⁺ B cells with or without BAFF, IL-15 and IL-21 for 7 d. Panels a, b, d and e show 1 of 5 experiments yielding similar results, whereas panels c and f summarize 3 experiments (bars indicate s.e.m.; *, p < 0.05).

(a) Binding of monoclonal IgD (50 μg/ml) to various lymphoid and myeloid cell lines. A F(ab’)₂ pAb was used to detect IgD. Mo, MC and Basø indicate monocytic, mast cell and basophilic cell lines, respectively. (b) Binding of increasing amounts (0.5, 5 and 50 μg/ml) of monoclonal IgD to the mast cell line HMC-1 and binding of monoclonal IgD (50 μg/ml) to the basophilic cell line KU812 before and after treatment with IL-4 for 1 d and with IL-3 for 4 d. Control indicates medium alone. A F(ab’)₂ pAb was used to detect IgD. Gray histogram depicts background fluorescence of cells that were not incubated with monoclonal IgD. (c) Binding of fluorochrome-conjugated monoclonal IgD to HMC-1 or KU812 cells in the presence of increasing amounts (0, 5, 50, 250 or 1000 μg/ml) of unlabeled monoclonal IgD. (d) Binding of untreated or denatured monoclonal IgD (50 μg/ml) to HMC-1 cells pre-incubated or not with IgG, IgA, IgE, mannose, mannan, trypsin, pepsin, papain, or papain plus leupeptin. A F(ab’)₂ pAb was used to detect IgD. In all the experiments dead cells were excluded using 7-AAD staining. Panel a summarizes 3 experiments (bars indicate s.e.m.), whereas panels b-d show 1 of 3 experiments yielding similar results.

(a) QRT-PCR of DEFB103A, CAMP, SPAG11A/F, and SPAG11D/G transcripts from basophils exposed to microbeads alone (control, open bar), microbead-bound monoclonal anti-IgD (gray bar), or microbead-bound monoclonal anti-IgE (black bar) for 6 h. PTX3 and CRP transcripts were measured after 16 h. mRNAs were normalized to ACTB mRNA. (b) ELISA of LL-37 from basophils stimulated as in a in the presence or absence of IL-3 for 8 h. (c) Growth of Moraxella catarrhalis and Haemophilus influenzae type-a and type-b upon 2-h exposure to culture supernatants from basophils stimulated as in a for 8 h or 16 h. CFU, colony forming unit. Panels a-c summarize 3 experiments (bars indicate s.e.m.; *, p < 0.03; **, p < 0.02; ***, p < 0.01).