Journal: Nature immunology

Article Title: Immunoglobulin D enhances immune surveillance by activating antimicrobial, pro-inflammatory and B cell-stimulating programs in basophils

doi: 10.1038/ni.1748

Figure Lengend Snippet: ( a ) Flow cytometric analysis of IgD on circulating or tonsillar CD19 + CD3 − B cells, CD19 − CD3 + T cells, CD16 high CD56 low or CD16 low CD56 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.

Article Snippet: Polyclonal or monoclonal IgD antibodies purified from the plasma of healthy individuals or multiple myeloma patients (Athens Research & Technology) were incubated at the indicated concentrations with various cell types (1 × 10 5 ) for 20 min at 4 °C.

Techniques: Control, Staining, Flow Cytometry, Binding Assay, Labeling, Incubation

Journal: Nature immunology

Article Title: Immunoglobulin D enhances immune surveillance by activating antimicrobial, pro-inflammatory and B cell-stimulating programs in basophils

doi: 10.1038/ni.1748

Figure Lengend Snippet: ( 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.

Article Snippet: Polyclonal or monoclonal IgD antibodies purified from the plasma of healthy individuals or multiple myeloma patients (Athens Research & Technology) were incubated at the indicated concentrations with various cell types (1 × 10 5 ) for 20 min at 4 °C.

Techniques: Binding Assay, Control, Fluorescence, Incubation, Staining

Journal: Nature immunology

Article Title: Immunoglobulin D enhances immune surveillance by activating antimicrobial, pro-inflammatory and B cell-stimulating programs in basophils

doi: 10.1038/ni.1748

Figure Lengend Snippet: ( 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 Ca 2+ 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).

Article Snippet: Polyclonal or monoclonal IgD antibodies purified from the plasma of healthy individuals or multiple myeloma patients (Athens Research & Technology) were incubated at the indicated concentrations with various cell types (1 × 10 5 ) for 20 min at 4 °C.

Techniques: Flow Cytometry, Control, Enzyme-linked Immunosorbent Assay

Journal: Nature immunology

Article Title: Immunoglobulin D enhances immune surveillance by activating antimicrobial, pro-inflammatory and B cell-stimulating programs in basophils

doi: 10.1038/ni.1748

Figure Lengend Snippet: ( 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).

Article Snippet: Polyclonal or monoclonal IgD antibodies purified from the plasma of healthy individuals or multiple myeloma patients (Athens Research & Technology) were incubated at the indicated concentrations with various cell types (1 × 10 5 ) for 20 min at 4 °C.

Techniques: Quantitative RT-PCR, Control, Enzyme-linked Immunosorbent Assay

Journal: Nature immunology

Article Title: Immunoglobulin D enhances immune surveillance by activating antimicrobial, pro-inflammatory and B cell-stimulating programs in basophils

doi: 10.1038/ni.1748

Figure Lengend Snippet: ( 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.).

Article Snippet: Polyclonal or monoclonal IgD antibodies purified from the plasma of healthy individuals or multiple myeloma patients (Athens Research & Technology) were incubated at the indicated concentrations with various cell types (1 × 10 5 ) for 20 min at 4 °C.

Techniques: Immunofluorescence, Staining, Expressing, Enzyme-linked Immunosorbent Assay, Cell Culture, Control

Journal: Cell

Article Title: Protection from SARS-CoV-2 Delta one year after mRNA-1273 vaccination in rhesus macaques coincides with anamnestic antibody response in the lung

doi: 10.1016/j.cell.2021.12.002

Figure Lengend Snippet: mRNA-1273 provides durable protection in the lower airway from B.1.617.2 (A and B) Representative images of lung samples 7 days after B.1.617.2 challenge from 4 NHPs that received mRNA-1273 (A) or mRNA control (B). Top row, detection of SARS-CoV-2 antigen by immunohistochemistry with a polyclonal anti-N antibody. Antigen-positive foci are marked by red arrows. Bottom row, hematoxylin and eosin (H&E) staining illustrating the extent of inflammation and cellular infiltrates. Images at 10× magnification with black bars for scale (100 μm). (C) SARS-CoV-2 antigen and inflammation scores in the left cranial (Lc) lobe, right middle (Rmid) lobe, and right caudal (Rc) lobe of the lungs 7 days after B.1.617.2 challenge. Antigen scoring legend: –, no antigen detected; +/−, rare to occasional foci; +, occasional to multiple foci; ++, multiple to numerous foci; +++, numerous foci. Inflammation scoring legend: –, minimal to absent inflammation; +/−, minimal to mild inflammation; +, mild to moderate inflammation; ++, moderate to severe inflammation; +++, severe inflammation. Horizontal rows correspond to individual NHPs depicted above (A and B).

Article Snippet: Goat anti-human IgD-FITC (polyclonal) , Southern Biotech , Cat#2030-02; RRID: AB_2795624.

Techniques: Control, Immunohistochemistry, Staining

Journal: Cell

Article Title: Protection from SARS-CoV-2 Delta one year after mRNA-1273 vaccination in rhesus macaques coincides with anamnestic antibody response in the lung

doi: 10.1016/j.cell.2021.12.002

Figure Lengend Snippet:

Article Snippet: Goat anti-human IgD-FITC (polyclonal) , Southern Biotech , Cat#2030-02; RRID: AB_2795624.

Techniques: Labeling, Virus, Neutralization, Recombinant, Transfection, Staining, Multiplex Assay, Diagnostic Assay, Software