Panel A shows a UMAP plot and a stacked bar graph illustrating the proportions of CD8 T-cell clusters in wild-type and knockout mice. Panel B shows a heatmap of gene expression across CD8 T-cell clusters. Panel C shows a heatmap comparing CD8 T-cell cluster signatures with previously reported T-cell states. Panel D shows flow cytometry plots and bar plots with x-axis Experimental group and y-axis Percentage of CXCR6-positive cells or TIM3-positive CXCR6-positive cells. Panel E shows a histogram and a bar plot with x-axis Experimental group and y-axis Normalized Programmed Cell Death Protein 1 geometric mean fluorescence intensity. Panel F shows a stacked bar graph showing clonotype distributions across CD8 T-cell clusters in wild-type and knockout mice. Panel G shows heatmaps displaying shared clonotype percentages among CD8 T-cell clusters in wild-type and knockout mice.
scRNA-seq analysis reveals altered differentiation and clonal expansion of islet-infiltrating CD8 T cells in Cre + -Tnfrsf9 fl/fl mice. (A) UMAP plot of islet-infiltrating CD8 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) Gene module scores of the signatures obtained from previously defined CD8 T cell differentiation states in T1D (Collier et al., 2023) and viral infection (Giles et al., 2022). (D) Frequencies of CXCR6+ cells among CD44highCD127low and TIM3+ cells among CD44highCD127lowCXCR6+ CD8 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 < 0.005; ***P < 0.0005, unpaired t test. (E) Relative PD-1 gMFI of CD44highCD127low CD8 T cells from the islets of 8- to 11-wk-old prediabetic Cre+-Tnfrsf9+/+ (WT) and Cre+-Tnfrsf9fl/fl (KO) females. Representative flow cytometry profiles (upper) and summarized results (lower) from four experiments are shown. ***P < 0.0005, unpaired t test. (F) Clonal expansion of Cre+-Tnfrsf9+/+ (WT) and Cre+-Tnfrsf9fl/fl (KO) cells within each CD8 T cell cluster. The percentages of cells in clonotypes with 1, 2–5, 6–10, or >10 cells in the indicated cluster are shown. (G) Tile plots showing the percentage of clonotype overlap across clusters in Cre+-Tnfrsf9+/+ (WT) and Cre+-Tnfrsf9fl/fl (KO) CD8 T cells. The number in each tile indicates the number of unique clonotypes shared by the two clusters. Clonotype overlap across cell clusters was higher in Cre+-Tnfrsf9fl/fl than in Cre+-Tnfrsf9+/+ mice (P = 1.759 × 10−05, Wilcoxon signed rank test). gMFI, geometric mean fluorescence intensity.