Increased mTORC1 signaling due to abundance of transitional and ABC-like aN B-cell clusters in patients with CTLA4 deficiency. (A) Volcano plot of shared differential gene expression analysis between bulk RNA-seq samples of flow-sorted resting FO and TrB populations (T1/2, T3; n = 3 healthy controls; significant differentially expressed genes defined by adjusted P value ≤ .01). (B) Adjusted P value of ingenuity pathway analysis pathway overlaps with significantly differentially expressed genes in TrB vs resting FO B cells. (C) Uniform manifold approximation and projection (UMAP) projection of total naïve (IgD⁺CD27^–) B cells profiled by scRNA-seq from patients with CTLA4 deficiency (n = 4) and healthy controls (n = 3), annotated by naïve B-cell subtype. (D) Dot plot showing the marker gene expression of FO, TrB, and aN B-cell types by cell type annotation; color represents average normalized gene expression and size represents the percent cells with non-zero gene expression. (E) Heat map of scaled gene expression of top 30 differentially expressed genes between annotated cell types. (F) UMAP projection of total naïve (IgD⁺CD27^–) B cells profiled by scRNA-seq, annotated by control or patients with CTLA4 deficiency. (G) Fraction representation of each annotated cell type within each donor scRNA-seq sample. (H) Average log₂-fold change of significant differentially expressed genes (adjusted P value ≤ .05) between distinguishing CD38⁺ TrBs vs CCR7⁺ FO B cells plotted on the x-axis and average log₂-fold change of significant differentially expressed genes between ITGAX⁺ ABC-like aN B cells vs CCR7⁺ FO B cells plotted on the y-axis, showing correlation in differential gene expression distinguishing both TrBs and ABC-like aN B cells from FO B cells. Differentially expressed genes that overlap with significant differentially expressed genes from the bulk RNA-seq analysis in panel A are outlined. (I) Single-cell module scores defined by TrB and ABC-like aN cells shared differentially expressed genes in both TrB and ABC-like aN cells plotted on the x-axis (TrB coexpression score), vs module scores defined by shared FO differentially expressed genes in FO cells on the y-axis (FO B score). Single cells from healthy controls are plotted separately from single cells from patients with CTLA4 deficiency to demonstrate consistent trends in cell type population scores. Pearson correlation rho values (−0.313 for healthy controls, −0.316 for patients with CTLA4 deficiency) were both significant (P < 2E−16). (J) Violin plots of module scores over msigDB hallmark oxidative phosphorylation and mTORC1 signaling pathways (see “Methods”) in healthy controls and patients with CTLA4 deficiency. Violin plots of single-cell module scores shown and evaluated for significance separately for healthy controls and patients with CTLA4 deficiency; ∗∗P < .001; ∗∗∗P < .0001. DEG, differentialy expressed gene; ERK, extracellular signal-regulated kinase; IL4, interleukin 4; msigDB, molecular signatures database; NAD, nicotinamide adenine dinucleotide.