Cell–cell interactions and overexpression of S100a4/Hes1/Cd274(Pd-l1) defines RT gene signature in Eµ-TCL1^Akt-C–enriched B-cell clusters. (A) Flow diagram of scRNA-Seq experimental setup (upper panel). Integrated UMAP from Cd19-Cre (n = 2), Eµ-TCL1 (n = 4), and Eµ-TCL1^Akt-C (n = 4) mice (n = 33 224 cells). Demarked clusters represent B-cell tumor clusters enriched within either Eµ-TCL1 (blue) or Eµ-TCL1^Akt-C (red) and the TME (black). (B) Dot plot representing the number of cells appearing in each cluster per genotype (upper panel). Graphical representation of differential gene expression analysis and heatmap showing the normalized percent cell expression changes of significantly differentially expressed genes (middle panel). Dot plot representing the median disease tendency score of GO term groups with the dot size representing the number of genes associated with all GO terms (lower panel, left graph). Bar chart representing the percentage of GO term groups of the top 25 Eµ-TCL1^Akt-C–associated GO terms (lower panel, right graph). (C) Network visualization of all cell–cell associated GO terms from the analysis conducted in B. GO terms denoted as large gray circles, Eµ-TCL1^Akt-C and Eµ-TCL1 significant genes denoted as small red and blue circles, respectively, and gray lines represent the association between genes and GO terms. (D) Dot plot of selected cell–cell associated GO term genes with biological links to Notch signaling, Pi3k/Akt signaling, or RT. Size of the dots represents the percentage of cells with a normalized expression value >1, with the color of the dots representing the mean expression of each gene across all cells within the cluster. Additional annotation beside the dot plot represents the number of transcription factor–binding sites for all 25 genes for FoxO1, Nfatc1, and Rbpjκ/Notch1, respectively (left panel). Messenger RNA expression validation in Cd19⁺ splenocytes for S100a4 and Hes1 genes in Eµ-TCL1 and Eµ-TCL1^Akt-C mice, as well as S100a4 in Gsk3β^S9A/+ (Nfatc1 nuclear excluded), Cd19-Cre^tg/wt; R26-fl-Notch1-IC (Cd19-Cre^Notch1-IC nuclear Notch1), and Cd19-Cre^tg/wt; R26-fl-FoxO1ADA (Cd19-Cre^FoxO1ADA nuclear FoxO1) via quantitative polymerase chain reaction. Protein expression validation in CD19⁺ splenocytes for Cd274 (Pd-l1) in Cd19-Cre, Eµ-TCL1, and Eµ-TCL1^Akt-C mice (right panel). (E) Percentage of Cd4⁺ T cells per mouse per genotype as a function of all T cells (left panel). Graphical representation of differential gene expression analysis of Cd4⁺ T cells (right panel) and heatmap showing the normalized percent cell expression changes of significantly differentially expressed genes (lower panel panel). (F) Network visualization of all cell death–associated GO terms from the analysis conducted in E. GO terms denoted as large gray circles, Eµ-TCL1^Akt-C and Eµ-TCL1 significant genes denoted as small red and blue circles, and gray lines represent the association between genes and GO terms. (G) Dot plot of selected cell death–associated GO term genes. Size of the dots represents the percentage of cells with a normalized expression value >1, with the color of the dots representing the mean expression of each gene across all cells within the cluster (left panel). Bar chart representing in percent terms how often the genes presented occur in the top 25 Eµ-TCL1^Akt-C–associated GO terms, with red bars for Eµ-TCL1^Akt-C significant genes and blue bars for Eµ-TCL1 significant genes. *P < .05, **P ≤ .01, ***P ≤ .001 (unpaired, 2-sided Student t test). CLP, common lymphoid progenitor; CMP, common myeloid progenitor; MFI, mean fluorescent intensity; NK, natural killer; NKT, natural killer T.