Loss of p53 in TECs reduces specific mTEC-mediated functions. (A) DP (TCRβ⁺CD4⁺CD8⁺CD25⁻) and SP4 (TCRβ⁺CD4⁺CD8⁻CD25⁻) thymocytes isolated from 8-week-old Control (Ctr) and p53cKO mice were analyzed for the expression of Helios and PD1. Numbers indicate the mean percentage of gated cells. Graphs show the total number of Helios⁺PD1⁺ thymocytes within the DP and SP4 stage and represent data from 2 independent experiments (n = 4 to 5 mice per group; mean ± SEM). (B) Flow cytometry analysis of thymic selection in 8-week-old female and male Ctr and p53cKO Marilyn-Rag2^−/− thymus. Numbers indicate the frequencies of SP4 thymocytes (mean ± SEM). Thymic cellularity (top) and the number of SP4 thymocytes (bottom) are depicted on the graphs. Data represent the average of 3 independent experiments (n = 5 to 6 mice per group; mean ± SEM). (C-D) SP4 thymocytes (CD8⁻CD4⁺TCRβ⁺) were analyzed for the expression of CD25 and Foxp3 (C) and CD24 and CD62L (D) at the indicated time points. Numbers indicate the average percentage of gated cells (±SEM). Graphs represent the number of immature (CD25⁺Foxp3⁻) and mature (CD25⁺Foxp3⁺) regulatory T cells (Tregs) (left) (C); the number of immature (CD24^hiCD62L^low) and mature (CD24^lowCD62L^hi) SP4 thymocytes (right) (D). The asterisk compares mature Treg and mature SP4 cells between Ctr and p53cKO mice. In panels C-D, graphs represent data from 3 independent experiments (n = 4 to 13 mice per group). Results are presented as mean ± SEM. Ml, Marilyn-Rag2^−/− thymus. ♀, female; ♂, male; *P < .05; **P < .01; ***P < .001.