Panel A: Three box plots compare the mean somatic hypermutation (S H M) across I g D, I g M, I g G, and I g A memory effector (M E) B cells. The horizontal axis labels the B cell subsets, and the vertical axis represents the mean S H M count. Panel B: A vertical bar graph quantifies in vivo divisions by naive or M E B cell subsets using the K R E C assay. The horizontal axis labels the B cell subsets, and the vertical axis shows the number of in vivo divisions. Error bars indicate standard deviation. Panel C: A heatmap visualizes the differential expression of genes involved in somatic hypermutation (S H M) from single-cell R N A sequencing (s c R N A-s e q) analysis of tonsillar I g D germinal center (G C) versus I g G G C B cells. The heatmap rows represent genes, and columns represent B cell subsets. Panel D: A box plot compares the mean expression ratio between mismatch repair (M M R) and base excision repair (B E R) pathways in I g D-G C and I g G-G C B cells. The horizontal axis labels the B cell subsets, and the vertical axis shows the expression ratio. Panel E: A heatmap shows the I G H V gene usage pattern across samples. Rows represent I G H V genes, and columns represent samples. Bold genes are discussed in the text. Panel F: Five box plots compare the usage of specific I G H V genes by M E B cell subsets. The horizontal axis labels the I G H V genes, and the vertical axis shows the percentage of gene usage. Panel G: Three box plots compare the I G H J 6 gene usage by naive, G C B cell, M E B cell, and plasma cell (P C) subsets. The horizontal axis labels the B cell subsets, and the vertical axis shows the percentage of gene usage. Panel H: Three violin plots compare the clonal complementarity-determining region 3 heavy chain (C D R 3 H) length among antibody isotypes expressed by B cell and P C subsets. The horizontal axis labels the antibody isotypes, and the vertical axis shows the C D R 3 H length in amino acids. Panel I: A heatmap shows the mean pairwise clonal overlap percentage across tonsillar B cell and P C subsets. Each cell within the heatmap shows the mean clonal overlap value and standard deviation.
Tonsillar IgD-ME B cells show an increased ratio of transcripts linked to MMR and BER pathways of SHM and share a unique IGHV gene repertoire with IgD-GC B cells and IgD-PCs. (A) Mean SHM across IgD/IgM/IgG/IgA-ME B cells (N = 4). Numbers below plots represent the median value per group. (B) KREC assay quantifying in vivo divisions by naive or ME B cell subsets. Error bars, SD. (C) Heatmap visualizing the DE of a custom panel of genes involved in SHM from scRNA-seq analysis of tonsillar IgD-GC vs. IgG-GC B cells. DEGs with |log2FC| >1 and adj. P <0.05 are marked with an asterisk. (D) Mean expression ratio between the genes included in the MMR GO term and the genes from the BER GO term in the IgD-GC and IgG-GC B cells from scRNA-seq dataset. (E) VH gene usage pattern across samples shown as a heatmap where rows and columns represent IGHV genes and samples, respectively. Bold genes, discussed in the text. (F) IGHV3-13, IGHV3-48, IGHV3-7, IGHV4-30-2, and IGHV4-31 gene usage by ME B cell subsets (N = 4). Numbers below boxes represent median values of average gene usage. (G) IGHJ6 gene usage by naive, GC B cell, ME B cell, or PC subsets (N = 4). Numbers below boxes represent median values of average gene usage. (H) Comparison of clonal H-CDR3 length (aa, amino acids) among antibody isotypes expressed by B cell and PC subsets. Numbers in violin plots represent median CDR3 length. (I) Values of mean pairwise clonal overlap (%) across tonsillar B cell and PC subsets. Each cell within the heatmap shows the mean clonal overlap value ± SD (N = 4). Data show one experiment with multiple biological replicates (A, C–I) or multiple experiments summarized as mean ± SD (B). Significance was determined by the Kruskal–Wallis test followed by the post hoc pairwise Mann–Whitney test with P value adjustment following the Benjamini–Hochberg method (A, F–H), Kruskal–Wallis test followed by Dunn’s correction (B), or Wilcoxon signed-rank test (D). *P < 0.05 and ****P < 0.0001. KREC, κ-deleting recombination excision circle.