Aire regulates negative selection in RIP-mOVA mice. (A) Thymocytes (3-week-old mice) generated by crossing OT-II.Aire^+/− to RIP-mOVA.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). (B) Thymocytes (6 weeks postreconstitution) from chimeric mice generated by reconstituting wt.Aire^+/+, wt.Aire^−/−, RIP-mOVA.Aire^+/+, or RIP-mOVA.Aire^−/− mice with OT-I 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). Representative contour plots were not all from the same experiment: Aire^+/+ groups are matched, as are Aire^−/− groups. Histograms show the mean ± SEM for each group. n = number of mice pooled from several experiments. Figures 2B and 6 share the same groups lacking expression of the RIP transgene. Significance relative to WT: ***P ≤ .001; ns indicates not significant.