Fig. 1 |. Deep transcriptomes from mouse exT_reg cells and T_reg cells identify differentially expressed candidate genes.

a, Frequency of mouse exT_reg cells (red) and T_reg cells (blue) among all CD4⁺T cells in spleen and LNs of FoxP3^{eGFP-Cre-ERT2}ROSA26^{CAG-fl-stop-fl-tdTomato} Apoe^−/− mice at 4 (T_reg n = 7; exT_reg n = 5), 8 (T_reg n = 4; exT_reg n = 4), 12 (T_reg n = 5; exT_reg n = 5) and 20 (T_reg n = 6; exT_reg n = 5) weeks after tamoxifen injection. All mice were on regular CD. b, Median fluorescence intensity (MFI) of CD25 expression on exT_reg cells (red circles), T_reg cells (blue circles) and conventional CD4⁺T cells (T_con, black circles) in spleen (n = 4) and LNs (n = 6) from 16-week-old FoxP3^{eGFP-Cre-ERT2}ROSA26^{CAG-fl-stop-fl-tdTomato} Apoe^−/− mice on CD. Results (a and b) are represented as the mean ± s.e.m. Spleen (b) *P = 0.0286; LN (b) **P = 0.0022; two-tailed Mann–Whitney U test. c, PCA of bulk RNA-seq data from sorted mouse exT_reg cells and T_reg cells from spleen and LNs of 20-week-old FoxP3^{eGFP-Cre-ERT2}ROSA26^{CAG-fl-stop-fl-tdTomato} Apoe^−/− mice on CD. Results (a–c) are from independent biological replicates. d, Volcano plot of differentially expressed mouse exT_reg and T_reg cell-classifying genes, identified by the SVM trained on mouse transcripts with human orthologs and filtered for those present in the human scRNA-seq targeted gene panel. y and x axes capped at 20 (P = 10⁻²⁰) and ±4 (log₂FC), respectively. Horizontal line at −log₁₀ (P adjusted) = 1.3 (same as P_adj = 0.05). Statistical analyses were performed using a two-tailed Wald test with Benjamini–Hochberg P-value adjustment. e, Mouse aortas with carotid artery branches from FoxP3^{eGFP-Cre-ERT2}ROSA26^{CAG-fl-stop-fl-tdTomato} Apoe^−/− mice were fixed and imaged using a Leica SP8 multiphoton microscope. GFP⁺ T_reg cells (pseudocolored green) and tdTomato⁺ exT_reg cells (pseudocolored pink) in the adventitia (top). Bottom image is a zoomed-in view of the white box. Blue-green indicates a second harmonic generation microscopy analysis of extracellular matrix. Scale bar, 100 μm. Data are representative of four independent experiments.