Panel A: Scatter plot showing frequency of I g D-coated bacteria from serum polyclonal antibodies (p A b s) in healthy controls. Panel B: Confocal images showing I g D, I g M, I g A, and D N A staining in sinonasal mucosa highlighting plasma cells. Panel C: Flow cytometry plots showing gating and proportions of I g D- and I g A-bound sinonasal microbiota with pie chart summary. Panel D: Vertical scatter plot showing ELISA-measured levels of free I g D and I g A in sinonasal samples. Panel E: Confocal images showing I g D, I g M, I g A, and D N A staining in bronchial mucosa highlighting plasma cells. Panel F: Flow cytometry plots showing gating and proportions of I g D- and I g A-bound bronchoalveolar microbiota with pie chart summary. Panel G: Scatter plots showing frequency of I g D-coated bacteria in sinonasal and bronchoalveolar microbiota samples. Panel H: Scatter plots showing frequency of I g A-coated bacteria in sinonasal and bronchoalveolar microbiota samples. Panel I: Line graphs showing ELISA-based reactivity of I g D polyclonal antibodies (p A b s) to D e r p 1 and alpha-s-casein. Panel J: Schematic diagram summarizing I g D-M E B cell differentiation into plasma cells and antigen targeting across mucosal sites.
IgD responses target commensal and environmental antigens from the sinonasal and bronchial mucosae. (A) Summary of flow cytometry–determined frequency of IgD-coated bacterial isolates upon exposure to serum IgD pAbs from HCs (N = 5). (B) Confocal imaging of nasal mucosa stained for IgD (green), IgM (red), IgA (white), and nuclear DNA (blue). Insets, IgD-PC (bottom right), IgA-PC (middle right), and IgM-PC (top right) upon digital magnification (2×). Scale bars, 20 µm (left) and 5 µm (right). (C) Gating strategy and representative flow cytometry plots of total, IgD-, IgA-, or IgD-IgA–bound sinonasal bacteria. Pie chart shows mean proportions of IgD- and/or IgA-bound bacterial fractions within the total IgD-IgA–bound microbiota (N = 41). Gray profiles, control isotype antibodies. (D) ELISA of sinonasal free IgD and IgA (N = 32). (E) Confocal imaging of bronchial mucosa stained as in B. Insets, IgD-PC (top right) and IgA-PC (bottom right) upon digital magnification (2×). Scale bars, 50 µm (left) and 5 µm (right). (F) Gating strategy and representative flow cytometry of total, IgD-, IgA-, or IgD-IgA–bound bronchoalveolar bacteria. Pie chart shows mean proportions of IgD- and/or IgA-bound bacterial fractions within the total IgD-IgA–bound microbiota (N = 20). Gray profiles, control isotype antibodies. (G and H) Summaries of frequencies of IgD-coated (G) or IgA-coated (H) sinonasal (N = 41) and bronchoalveolar bacteria (N = 20). (I) ELISA-determined reactivity of free IgD pAbs immunoprecipitated from sinonasal samples to Der p 1 and α-s-casein (N = 21). (J) Graphical summary of findings from tonsillar IgD-ME B cells and their IgD-PC progeny. Data summarize one to three experiments with multiple biological replicates (A, pie chart in C, D, pie chart in F, G, H), show results from one representative of at least three experiments (B, plots in C, E, plots in F), or show one experiment with multiple biological variables (I). Significance was determined by a nonpaired, nonparametric Friedman test (G and H). Panel J was created with BioRender.com.