Panel A: U M A P plots showing expression of P R D M 1, X B P 1, I R F 4, P A X 5, B A C H 2, and F O X O 1 across I g D-M E subclusters. Panel B: Flow cytometry histograms showing B L I M P-1 and B C M A expression in naive and M E B cell subsets. Panel C: Flow cytometry contour plots showing B L I M P-1 and B C M A expression across I g D-M E, I g G-M E, and I g A-M E subsets. Panel D: Lineage tree diagrams showing clonal relationships among I g D class-switched B cells across multiple donors. Panel E: Lineage tree diagrams showing clonal relationships among I g G and I g A class-switched B cells across donors.
Tonsillar IgD-ME B cells encompass heterogeneous cells with increased B cell identity or PC-associated properties and form complex lineage trees that exhibit more clonal relatedness to IgD-PC B cells than to IgD-GC B cells. (A) UMAPs depicting the distribution of PRDM1, XBP1, IRF4, PAX5, BACH2, and FOXO1 within IgD-ME 1 and 2 subclusters. (B and C) Representative flow cytometry histograms (B) and contour plots (C) of BLIMP-1 and BCMA on IgM+IgD+CD10–CD19+CD27–CD38–/low naive B cells and IgM–CD10–CD19+CD27+CD38–/low ME B cell subsets expressing IgDhigh (IgD-ME) or IgA (IgA-ME) or lacking IgD and IgA in addition to IgM (IgG-ME). Vertical lines in B mark the positive gate cutoffs. Black arrow in B indicates a subpopulation of cells enriched in BLIMP-1. (D and E) Examples of lineage trees from the IgD gene repertoire of tonsillar IgD class-switched (D) or IgG/IgA class-switched (E) B cells from study donors. The length of scale bars is equal to 10 mutations. Top numbers next to each donor identify specific clonal trees. Data are from one experiment with multiple biological replicates (A) or depict representative results from an experiment with at least three biological replicates (B–E).