Extended Data Fig. 7: Analysis of CD4 T-cell subsets in the liver and spleen of control and Eomes-KO mice.

a. The percentages of CD4-EOMES (Left) and CD4⁺GzmB⁺ cells (Right) in spleens of Control (n = 6) and Eomes-KO (n = 7) mice after the TMX regimen was administered. The regimen included 3 days of IP TMX injection (100 µl) followed by an alternating dietary regimen of two weeks on TMX chow and two weeks on regular chow, lasting for 6 weeks. b-e. The percentages of CD45⁺ (b; Left), CD45⁺CD3⁺ (b; Right), CD3⁺CD8⁺ (c; Left), CD3⁺CD4⁺ (c; Right), naive (d; Left), effector (d; Right), Treg (e; Left), and CD44⁺PD1⁺ (e; Right) cells in the livers of Control (n = 6) and Eomes-KO mice (n = 7). f-h. Graphs showing the percentages of CD3⁺ (f; Left), CD3⁺CD4⁺ (f; Right), Treg (g; Left), naive (g; Right), effector (h; Left), and CD44⁺PD1⁺ (h; Right) cells in the spleens of Control (n = 6) or Eomes-KO mice (n = 7). Subsets were defined as indicated in Supplementary Figure 1. Data were analyzed using a two-tailed, unpaired Student’s t-test. The exact P-values are presented in the graphs. i. A representative flow cytometry histogram plot illustrating the expression of β-Galactosidase (β-Gal) staining in liver cells (light purple), unstained liver cells (grey) and Fluorescence Minus One Controls (FMO) liver cells (light blue). j. A representative flow cytometry histogram plot illustrating the expression of p16^ink4a+ (Upper) and p21 (Lower) out of non-immune CD45⁻ liver cells. k-l. The percentages of CD45⁺p16⁺ (k; Left), fold change of p16 MFI (k; Right), percentages of CD45⁺p21⁺ (l; Left), fold change of p21MFI (l; Right) in the livers of Control (n = 6) or Eomes-KO mice (n = 7). m. Tracking of mice weight (Y-axis) over 40 weeks (X-axis) in the Control and Eomes-KO groups (n ≥ 4 mice per time point). * = P < 0.05. n-r. Survival experiment: 18 month-old control and Eomes-KO mice were subjected to a TMX regimen that included 3 days of IP TMX injection (100 µl) followed by an alternating dietary regimen of two weeks on TMX chow and two weeks on regular chow, lasting for 30 weeks. n. Representative spleen size of Control and Eomes-KO mice. o. Comparison of spleen weight (mg) in Control (n = 4) and Eomes-KO (n = 6) mice. p-q. The percentages of CD3 cells (Left; p), CD4 cells (Right; p) and CD4-EOMES cells (q) in spleens of Control (n = 4) and Eomes-KO mice (n = 6). r. Representative flow cytometry plots showing CD4⁺EOMES⁺CCL5⁺ cells in the spleen of Control (Upper) and Eomes-KO mice (Lower) after TMX treatment. Bars indicate mean ± SEM from one (a-h, k-l) or two (m-r) independent experiments. Data were analyzed using two-tailed Student’s t-tests. The exact P-values are presented in the figures.