Fig. 4. Dynamics of RECs across age and immune sites.

(A to C) Prevalence of RECs and naïve cells measured via flow cytometry (representative panels, left) in mice at the age of 2, 6, 12, 16, and 24 months (right). Color-shaded area of each graph represents ± SEM. Spearman correlation coefficients (r) are shown. (A) Exhausted cells (pink), defined as PD1⁺CD62L⁻ cells, and naïve cells (blue), defined as CD62L⁺PD1⁻ out of CD4⁺FOXP3⁻EOMES⁻CCL5⁻cells. (B) aT_regs cells, defined as CD81⁺ cells out of CD4⁺FOXP3⁺ cells. (C) Cytotoxic cells, defined as EOMES⁺CCL5⁺ out of CD4⁺ cells. Gating was based on unstained samples and fluorescence minus one. Data include two (A) or three (B and C) different experiments, n = 5 to 15 per time point. (D and E) Absolute numbers of total CD4 (D) and RECs (E) cells out of total live cells in each spleen, calculated as specific cell count per gram of spleen in young (2 months, n = 7) and old (24 months, n = 9) mice. (F to H) Percentages of RECs in different immune sites: (F) exhausted cells, (G) aT_regs cells, and (H) cytotoxic cells. LNs represent the mean cell frequencies of four LN sites. Data include three different experiments, n = 2 to 3 per experiment. P values computed via one-way analysis of variance (ANOVA) test with Tukey correction for multiple comparisons (*P < 0.05, **P < 0.01, and ****P < 10⁻⁴).