Transcriptomic analysis of IgM^hi and IgM^lo TS B cells demonstrates different upstream regulators of phenotype. (A) UMAP plot of IgM^hi and IgM^lo TS B cells from five HCDs (see Fig. S2, A–D) clustered according to differentially expressed genes identified using the Seurat SCTransform workflow (Hafmeister and Satija, 2019Preprint). (B) Heatmap of selected genes from the top 60 differentially expressed genes in IgM^hi and IgM^lo TS B cells. (C) Scatter plot demonstrating genes differentially expressed in IgM^hi and IgM^lo TS B cells. (D) A PCA-based approach based on differentially expressed genes identified six clusters among the IgM^hi and IgM^lo TS B cells that were demonstrated by UMAP. (E) Quantification of the frequency of IgM^hi or IgM^lo TS B cells within the clusters demonstrated by UMAP in D reveals that IgM^hi TS B cells dominate in clusters 0 and 2 and IgM^lo TS B cells in clusters 1 and 3. (F) Dot plot demonstrating expression of selected genes within clusters 0–3. (G) Flow cytometry dot plots demonstrating CD27⁻IgD⁺CD10⁺ cells as gated in Fig. S1 D. T2 cells were gated as CD24⁺⁺CD38⁺⁺ and IgM^hi and IgM^lo subsets as 30% of cells with the highest and lowest expression of IgM respectively. (H) Scatter plots demonstrating CD1c mean fluorescence intensity (MFI) in T1, IgM^hi, and IgM^lo T2 B cells gated in G (mean ± SD, paired t test). *, P < 0.05; ***; P < 0.001; ****, P < 0.0001. (I) Histograms demonstrating CD1c MFI in B cell subsets as gated in Fig. S1 D. (J) Dot plot of flow cytometry data demonstrating CD27⁻IgD⁺CD10⁻ cells as gated in Fig. S1 D. Differential expression of CD45RB and R123 allowed the identification of CD45RB^hi T3 (R123⁺) and MZP (R123⁻) cells. (K) A histogram showing CD1c MFI on subsets as gated in J. (L) Dot plots demonstrating CD1c MFI on subsets gated in J (mean ± SD, paired t test). In J–L, RBHI refers to CD45RB^hi and RBLO refers to CD45RB^lo. ***, P < 0.001; ****, P < 0.0001. FSC-A, forward scatter area.