Panel A: Four line graphs show the reactivity of I g D recombinant monoclonal antibodies (r m A b s) from I g D-M E B cells to food and airborne antigens. The x-axis represents r m A b concentration in micrograms per milliliter, and the y-axis represents optical density at 450 nanometers (O D 450). Each graph shows a different antigen: alpha-s-casein, Bet v 1, whole casein, and alpha-lactalbumin. Dashed lines indicate reactivity thresholds. Panel B: A heatmap summarizes the intensity of binding to various antigens by I g D r m A b s from I g D-M E B cells. The x-axis lists different antigens, and the y-axis lists different r m A b s. The color scale indicates binding intensity. Panel C: Flow cytometry plots show the frequency of I g D-bound bacteria. The first plot shows all events, the second plot shows a control with a 2 R Y A b, and the third plot shows I g D r m A b D 3 with a 2 R Y A b. The x-axis represents side scatter (S S C A), and the y-axis represents P E. Panel D: A heatmap summarizes the frequencies of I g D-bound bacterial isolates upon exposure to I g D r m A b s. The x-axis lists different bacterial isolates, and the y-axis lists different r m A b s. The color scale indicates the percentage of P E-positive bacteria. Panel E: Histograms show the binding of wild type (W T) and germline (G L) r m A b 48 from I g D-G C B cells to nasopharyngeal B. cereus and S. mutans. The x-axis represents P E, and the y-axis represents counts. Panel F: A line graph summarizes the binding of wild type (W T) and germline (G L) r m A b 48 to B. cereus and S. mutans. The x-axis lists the bacterial types, and the y-axis represents the percentage of I g D-positive bacteria. Panel G: A line graph shows the reactivity of wild type (W T) or germline (G L) I g D monoclonal antibodies (m A b s) 47 and 48 from I g D-G C B cells to various antigens. The x-axis lists the antigens, and the y-axis represents optical density at 450 nanometers (O D 450). Panel H: Heatmaps show the binding intensity to various antigens by I g D r m A b s 70, 98, 47, and 48 from I g D-G C B cells. The x-axis lists different antigens, and the y-axis lists different r m A b s. The color scale indicates binding intensity. Panel I: Violin plots show the reactivity to Der p 1 and beta-lactoglobulin of serum I g D polyclonal antibodies (p A b s) from P-H C s, H I D S, and H I E S patients. The x-axis lists the patient groups, and the y-axis represents optical density at 450 nanometers (O D 450).
Secreted IgD recognizes commensal and common environmental antigens in a SHM-dependent manner. (A) ELISA-determined reactivity of IgD rmAbs (N = 7) from IgD-ME B cells to food and airborne antigens. Dashed lines, reactivity thresholds. (B) Heatmap summarizing intensity of binding to food, airborne, fungal, viral, and autologous antigens by IgD rmAbs from IgD-ME B cells at 10 µg/ml, determined as in A. (C) Representative flow cytometry of total and IgD-bound bacteria. Numbers are frequencies of IgD-bound bacteria. (D) Heatmap summarizing frequencies of IgD-bound bacterial isolates upon exposure to IgD rmAbs, determined as in C. (E and F) Representative histograms (E) and summary graph (F) of WT and GL binding to nasopharyngeal B. cereus and S. mutans of rmAb 48 from IgD-GC B cells. (G) ELISA-determined reactivity of WT or GL IgD mAbs 47 (triangles) and 48 (circles) from IgD-GC B cells to antigens from B. (H) Heatmaps showing binding intensity to antigens from B by IgD rmAbs 70, 98, 47, and 48 from IgD-GC B cells at 10 µg/ml, encompassing either Cδ (top) or Cγ1 (bottom) HCs. (I) Violin plots showing reactivity to Der p 1 (left) and β-lactoglobulin (right) of serum IgD pAbs from P-HCs, and HIDS and HIES patients. Data are from one experiment with multiple biological replicates (A, B, D, F, G, H, and I) or show results from one representative of at least three experiments (C and E). Significance was determined by the Kruskal–Wallis test (I). WT, wild type; GL, germline.