FIGURE 4.

The middle‐aged thymic environment does not support efficient induction of Tregs in response to TRAs. (a) Representative flow cytometry plots of Treg precursors (CD25⁺ Treg‐P and FOXP3^lo Treg‐P) and Tregs (CD25⁺FOXP3⁺) recovered from OVA(‐), WT slices with addition of 1 μM of OVAp_323‐339, RIP‐mOVA, and RIP‐OVA^hi 1MO versus 12MO thymic slices after 72 hr. Normalized cell numbers (top) and percentages (bottom) of (b) CD25⁺ Treg‐P, (c) FOXP3^lo Treg‐P and (d) Treg CD4SP OT‐II subsets were quantified. Data in (b‐d) show mean ± SEM compiled from 3‐4 independent experiments, with three thymic slices per experiment. Each data point represents one thymic slice. Values were normalized to the mean of triplicate OVA‐ slices. Analyzed by two‐way ANOVA with Šídák's correction for multiple comparisons, p‐values: * <0.05, ** <0.01, *** <0.001, **** <0.0001, ns: not significant