Panel A: Flow cytometry t S N E plots showing I g D M E, G C, Naive, and P C B cell subsets with marker expression. Panel B: Confocal microscopy images showing Ig D, I g M, and DAPI staining highlighting Ig D M E cells in tonsil tissue. Panel C: Vertical bar graph showing proportion of mutated I G H V sequences across B cell and plasma cell subsets. Panel D: Scatter plot showing P C A distribution of transcriptomes from different B cell subsets based on R N A seq data. Panel E: Volcano plot showing differentially expressed genes between I g D M E and naive B cells with up and down regulation. Panel F: Heatmap showing gene expression patterns distinguishing I g D M E from naive B cells across multiple differentially expressed genes. Panel G: Spatial map showing distribution of tonsillar regions including epithelium, germinal center, and interfollicular areas. Panel H: Spatial heatmap showing intensity distribution of I g D M E transcriptional signature across tonsillar tissue regions. Panel I: Vertical bar graph showing quantified spatial signature scores across defined tonsillar regions for I g D M E cells. Panel J: Scatter plot showing frequency of I g D M E B cells across tonsil, blood, and spleen samples.
IgD-ME B cells are hypermutated, transcriptionally unique, and inhabit tonsillar epithelial, subepithelial, and interfollicular areas. (A) Flow cytometry–evaluated t-SNE plot-projected IgD-AN, IgD-GC, IgD-ME, and IgD-PC clusters from total tonsillar IgD+IgM– B cells. Numbers within the first t-SNE plot from the left indicate the % of each IgD+IgM– subset within the total IgD+IgM– population. The following t-SNE plots reflect relative CD38, CD10, and CD27 expression projected on the first t-SNE plot. (B) Confocal microscopy of tonsil stained for IgD (green), IgM (red), and nuclear DNA (blue). Inset, IgD-ME cell digitally magnified (10×) in bottom panels (yellow arrow). Scale bars, 100 µm (top) and 10 µm (bottom). (C) Proportion of mutated IGHV gene sequences across B cell and PC subsets (N = 4 per cell type). (D) PCA of RNA-seq–determined transcriptomes from B cell subsets. (E and F) Volcano plot (E) and heatmap (F) from bulk RNA-seq data showing up (red)- or downregulated (blue) genes in IgD-ME (N = 3) compared with naive (N = 7) B cells. |log2FC| >1 and adj. P <0.05. Bold genes, discussed in the text. (G–I) Spatial gene expression analysis of main tonsillar areas (G), intensity distribution of a custom IgD-ME gene signature (H), and quantification of intensity score per spot (I). EP1, epithelium 1; EP2, epithelium 2; IF, interfollicular area; SUBEP, subepithelium. (J) Frequency of IgD-ME B cells within total CD45+CD19+CD38–CD10–IgM– class-switched ME (ME-CS) B cells from tonsil (N = 22), blood (N = 8), or spleen (N = 5). Data are shown as the mean ± SEM. Data show results from one representative of at least three experiments (A, B, G, and H), one experiment with multiple biological replicates (C–F, I), or multiple experiments (J). Significance was determined by the Wilcoxon signed-rank test (I) or Kruskal–Wallis test followed by the post hoc pairwise Mann–Whitney test (J). **P < 0.01, ***P < 0.001, and ****P < 0.0001.