Aire expression in mTEC and BM-derived cells are required to cause the deletion of OT-II cells. (A) Thymocytes (6 weeks postreconstitution) from chimeric mice generated by reconstituting wt.Aire^+/+, wt.Aire^−/−, RIP-OVA^hi.Aire^+/+, or RIP-OVA^hi.Aire^−/− mice with OT-II BM were analyzed by flow cytometry for expression of CD4, CD8, and Vα2 (representative contour plots, left) and enumerated for SP thymocytes expressing high levels of TCR (CD4⁺CD8⁻Vα2⁺ cells, right). (B) mTECs are not able to present OVA on MHC II. Lethally irradiated 8-week-old B6 or RIP-OVA^hi mice were grafted with IA^b-deficient OT-II BM. At 6 weeks after reconstitution, thymocytes from the indicated mice were analyzed by flow cytometry for expression of CD4, CD8, and Vα2 (representative contour plots, left) and enumerated for SP thymocytes expressing high levels of TCR (CD4⁺CD8⁻Vα2⁺ cells, right). Contour plots show a representative experiment. Histogram shows the mean ± SEM for each group. n = number of mice pooled from several experiments. Figure 5A and supplemental Figure 3 as well as Figures 5B and 3B share, respectively, the same groups lacking expression of the RIP transgene. Significance relative to WT: ***P ≤ .001; ns indicates not significant.