Panel A: Volcano plots showing differentially expressed genes between I g D-M E and I g G or I g A-M E B cells. Panel B: Heatmap showing immune response gene expression differences between I g D-M E and combined I g G-I g A-M E B cells. Panel C: Heatmap showing overlap of differentially expressed genes with A B C gene signatures in I g D-M E B cells. Panel D: Gene set enrichment plots showing A B C gene set enrichment in I g D-M E compared to other B cell subsets. Panel E: Heatmap showing gene expression differences between I g D-M E, I g G-I g A-M E, and A B C populations. Panel F: t S N E plots showing expression of markers C D 11 c, C D 95, C X C R 3, C D 27, C D 69, C D 21, and C D 43. Panel G: Confocal images showing I g D, I g M, C D 11 c, and D N A staining highlighting I g D-M E B cells in tonsil tissue.
Tonsillar IgD-ME B cells are heterogeneous and exhibit ABC-like properties. (A) Volcano plots showing up (red)- and downregulated (blue) DEGs (|log2FC| >1 and adj. P <0.05) in IgD-ME B cells when compared to IgG- or IgA-ME B cells. (B) Heatmap showing manually curated immune response genes differentially expressed (adj. P <0.05 and |log2FC| >1) by IgD-ME vs. combined IgG-ME and IgA-ME B cells. Bold genes, discussed in the text. (C) Heatmap visualizing DEGs with |log2FC| >1 and adj. P <0.05 from IgD-ME (N = 3) vs. IgG-IgA-ME B cells (N = 6 each) overlapping with the published ABC dataset (Holla et al., 2021). Bold genes, discussed in the text. (D) GSEA of the published ABC dataset (Holla et al., 2021) in IgD-ME B cells vs. IgG/IgA-ME B cells. ES, enrichment score. (E) Heatmap visualizing DEGs with |average log2FC| >0.585 and adj. P <0.05 in IgD-ME or IgG-IgA-ME B cells vs. ABCs. (F) Flow cytometry–determined CD11c, CD95, CXCR3, CD27, CD69, CD21, and CD43 relative expression intensity projected on t-SNE plots of ME B cell subsets. (G) Confocal imaging of tonsil stained for IgD (green), IgM (red), CD11c (white), and nuclear DNA (blue). Insets from top panel, IgD-ME and naive B cells digitally magnified in middle (50×) and bottom (4×) panels. Scale bars, 50 µm (top), 10 µm (middle), and 2 µm (bottom). Data represent one experiment with multiple biological replicates (A–E) or show results from one representative of at least three experiments (F and G).