RIP-OVA^hi mTEC show Aire-independent regulation of OVA^hi and cause deletion of OT-I cells but not the OT-II cells. (A) Relative expression of Aire, Ins2, Spna2, and OVA^hi was determined by quantitative RT-PCR on cDNA prepared from CD45⁻-enriched cells. Expression values are shown relative to WT after normalization to Hprt. Data shown are the mean ± SEM of 4 independent experiments. A total of 6-8 individual thymi were pooled per experiment. Significance relative to WT: ***P ≤ .001; ns indicates not significant. (B) Thymocytes (3-week-old mice) generated by crossing OT-II.Aire^+/− to RIP-OVA^hi.Aire^+/− 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). (C) Thymocytes (3-week-old mice) generated by crossing OT-I.Aire^+/− to RIP-OVA^hi.Aire^+/− 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. Significance relative to WT: **P ≤ .01; ***P ≤ .001; ns indicates not significant.