Panel A: A spatial map showing the distribution of I g D-P C s in tonsillar tissue with a color gradient indicating the U Cell score. Panel B: A box plot quantifying the intensity score per spot of an I g D-P C gene signature in different tissue regions, including epithelium 1, epithelium 2, interfollicular area, and sub-epithelium. Panel C: A heatmap visualizing differentially expressed genes with adj.P.Value less than 0.05 and absolute log 2 fold change greater than 1 from M E B cell subsets encoding transcription factors. Panel D: A U M A P plot showing the U Cell score of a gene signature from a tonsillar P C cluster computed on I g D-M E 1 and I g D-M E 2 subclusters, along with a violin plot of the score distribution and expression of key genes. Panel E: A heatmap visualizing differentially expressed genes with adj.P.Value less than 0.05 and absolute log 2 fold change greater than 1 from M E B cell subsets encoding signal transducers. Panel F: Flow cytometry data showing the expression of B L I M P-1, B C M A, I F N A R 2, and T L R 8 in naive and M E B cells. Panel G: A bar plot showing significantly enriched terms in G S E A from bulk R N A-s e q analysis of tonsillar I g D-M E B cells compared to I g G slash I g A-M E B cells. Panel H: Electron microscopy images of representative I g D-M E B cell and I g D-P C, highlighting the perinuclear area and rough endoplasmic reticulum. Panel I: Flow cytometry data measuring P C s from sorted naive or M E B cells stimulated with C D 40 L and I L-21. Panel J: ELISA data measuring I g D secreted by cells from Panel I.
Tonsillar IgD-PCs inhabit epithelial areas co-occupied by an IgD-ME B cell population that encompasses cells showing some properties usually associated with PCs. (A and B) Spatial mapping (A) and quantification of intensity score per spot (B) of an IgD-PC gene signature in tonsillar tissue. EP1, epithelium 1; EP2, epithelium 2; IF, interfollicular area; SUB-EP, subepithelium. (C) Heatmap visualizing bulk RNA-seq–determined DEGs with adj. P <0.05 and |log2FC| >1 from ME B cell subsets encoding transcription factors. Bold genes, discussed in the text. (D) UCell score of the gene signature obtained from a published scRNA-seq–derived total tonsillar PC cluster and computed on IgD-ME 1 and IgD-ME 2 subclusters. Top, middle, and bottom panels show the score on the UMAP, the score distribution in a violin plot, and the expression of several key genes from the PC signature, respectively. (E) Heatmap visualizing bulk RNA-seq DEGs with adj. P <0.05 and |log2FC| >1 from ME B cell subsets encoding signal transducers. Bold genes, discussed in the text. (F) Flow cytometry of BLIMP-1, BCMA, IFNAR2, and TLR8 from naive and ME B cells (N = 4–6). Error bars, SD. (G) Bar plot showing significantly enriched terms (adj. P <0.05) in GSEA from bulk RNA-seq analysis of tonsillar IgD-ME B cells compared with IgG/IgA-ME B. NES, normalized enrichment score. (H) Electron microscopy of representative IgD-ME B cell (top) and IgD-PC (bottom). Circles, perinuclear area; arrowheads, RER. Scale bars, 2 µm (left panels) and 500 nm (right panels). (I) Flow cytometry–measured PCs from sorted naive or ME B cells stimulated for 6 days with CD40L and IL-21 (N = 4). Error bars, SD. (J) ELISA-measured IgD secreted by cells from I (N = 4). Data show results from one representative of at least three experiments (A, H, I [left]), one experiment with multiple biological replicates (B–E, G), or multiple experiments summarized as mean ± SD (F) or mean ± SEM (I [right], J). Significance was determined by the Wilcoxon signed-rank test (B, D [middle and bottom]) or Kruskal–Wallis test followed by the post hoc pairwise Mann–Whitney test (F, I right, J). *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.