Figure 6. Elevated returning of IFN-γ-producing cells in aged Aire-deficient mice.

(A, B) Flow cytometric analysis of GFP^- returning T cells in the thymus of 6-, 12- and 18-month-old Aire^-/- Rag2p-GFP mice and WT littermates. Representative dot plots (A) and the proportion of GFP^- T cells in CD4⁺ and CD8⁺ T cells in the thymus (B) are shown. (C) The expression levels of CD44 and PD-1 measured by flow cytometry in GFP⁺ CD4 SP thymocytes and GFP^- returning CD4⁺ T cells from18-month-old Aire^-/- mice and WT littermates. Representative histograms are shown. (D, E) Isolated CD4⁺GFP^- returning T cells were stimulated with anti-CD3 and anti-CD28 for 12 hours. IFN-γ-, IL-4- and IL-17A-producing cells in CD4⁺GFP^- returning T cells were analyzed by flow cytometry. Representative density plots (D) and the proportion of IFN-γ-, IL-4- and IL-17A-producing cells in CD4⁺GFP^- returning T cells (E) are shown. The experiments were repeated three times. Data are presented as Mean ± SD (n=8 pairs). Statistical differences between groups were determined by the Student’s t test. **p < 0.01.