Aire impact on thymocyte and mTEC maturation. (A) The percentages of four main thymocyte populations in the thymi of Aire-deficient (KO) and control (HE) rats as determined by flow cytometry. Representative flow cytometry plots (above) and percentages of DN, DP, and SP cell populations in Aire-deficient and control rats (below, n = 3 for both KO and HE animals, combined from two independent experiments). (B) Representative flow cytometry plots of thymic Tregs (above), the percentages of Treg population in the thymi determined by flow cytometry (left below, n = 3 for both KO and HE rats, combined from two independent experiments) and relative Foxp3 mRNA expression in the thymus determined by qPCR (right below, n = 5 for both KO and HE animals, combined from two independent experiments). (C) Representative flow cytometry plots (above) and the percentages of three TEC subpopulations in the thymi as determined by flow cytometry (n = 8 for both KO and HE rats, combined from two independent experiments). (D) Thymic sections were stained for MHC II, UEA1, and Involucrin; dashed line represents the medulla–cortex border; red circle represents Involucrin⁺ Hassall’s corpuscles. Shown are representative samples from three independent experiments, scale bar = 150 μm. (E) Relative Involucrin (Inv) and Cytokeratin 10 (CK10) expression in the thymus as determined by qPCR (n = 5 for both KO and HE rats, combined from two independent experiments). (F) Average number of Involucrin⁺ Hassal’s corpuscles per 1 mm² medulla (n = 5 for KO and n = 4 for HE animals, combined from three independent experiments). Throughout the figure, symbols indicate individual rats, horizontal lines with whiskers indicate mean values with SEM. All reported P values are based on t tests. P values are adjusted for multiple comparisons using the Holm–Bonferroni method.