Panel A shows a UMAP plot and a stacked bar graph illustrating the proportions of CD4 T-cell clusters in wild-type and knockout mice. Panel B shows a heatmap of gene expression across CD4 T-cell clusters. Panel C shows flow cytometry plots and a bar plot with x-axis Experimental group and y-axis Percentage of CXCR6-positive cells. Panel D shows violin plots comparing expression levels of Tnfrsf4, Lag3, Lef1, Klf2, Dnaja1, and Ifngr1 between wild-type and knockout mice. Panel E shows flow cytometry plots and a bar plot with x-axis Experimental group and y-axis Percentage of LAG3-positive TIGIT-positive cells among Foxp3-positive CD4 T cells. Panel F shows violin plots comparing expression levels of Egr1, Egr2, Nr4a2, and Nr4a3 between wild-type and knockout mice. Panel G shows a stacked bar graph showing clonotype distributions across CD4 T-cell clusters in wild-type and knockout mice. Panel H shows heatmaps displaying shared clonotype percentages among CD4 T-cell clusters in wild-type and knockout mice. Overall, dimensionality-reduction plots, heatmaps, flow cytometry plots, bar plots, violin plots, and stacked bar graphs characterize CD4 T-cell subsets, gene expression patterns, and clonotype distributions in wild-type and knockout mice.
scRNA-seq analysis reveals altered differentiation and clonal expansion of islet-infiltrating CD4 T cells in Cre + -Tnfrsf9 fl/fl mice. (A) UMAP plot of islet-infiltrating CD4 T cells isolated from 7- to 10-wk-old prediabetic Cre+-Tnfrsf9+/+ (WT) and Cre+-Tnfrsf9fl/fl (KO) females. Bar plots show the proportions of the clusters. (B) Heatmap of key cluster marker genes. (C) Frequencies of CXCR6+ cells among CD44highCD127low CD4 effector T cells in islets of 8- to 11-wk-old prediabetic Cre+-Tnfrsf9+/+ (WT) and Cre+-Tnfrsf9fl/fl (KO) females. Representative flow cytometry profiles (left) and summarized results (right) from six experiments are shown. ***P < 0.0005, unpaired t test. (D) Differential expression of Tnfrsf4, Lag3, Lef1, Klf2, Dnaja1, Ifngr1 between Cre+-Tnfrsf9+/+ (WT) and Cre+-Tnfrsf9fl/fl (KO) cluster CD4-2 (Treg) cells. Adjusted P value for each comparison is shown in the parentheses. (E) Frequencies of LAG3+TIGIT+ cells within Foxp3+ Tregs in islets of 8- to 11-wk-old prediabetic Cre+-Tnfrsf9+/+ (WT) and Cre+-Tnfrsf9fl/fl (KO) females. Representative flow cytometry profiles (left) and summarized results (right) from five experiments are shown. *P < 0.05 by an unpaired t test. (F) Differential expression of Egr1, Egr2, Nr4a2, and Nr4a3 between Cre+-Tnfrsf9+/+ (WT) and Cre+-Tnfrsf9fl/fl (KO) cluster CD4-1 (naïve) cells. Adjusted P value for each comparison is shown in the parentheses. (G) Clonal expansion of Cre+-Tnfrsf9+/+ (WT) and Cre+-Tnfrsf9fl/fl (KO) cells within each CD4 T cell cluster. The percentages of cells in clonotypes with 1, 2–5, 6–10, or >10 cells in the indicated cluster are shown. (H) Tile plots showing the percentage of clonotype overlap across clusters in Cre+-Tnfrsf9+/+ (WT) and Cre+-Tnfrsf9fl/fl (KO) CD4 T cells. The number in each tile indicates the number of unique clonotypes shared by the two clusters. The overall clonotype overlap across different clusters was increased in Cre+-Tnfrsf9fl/fl mice (P = 0.02 compared with Cre+-Tnfrsf9+/+, Wilcoxon signed rank test).