RIP-mOVA mTEC show Aire-dependent regulation of mOVA and directly cause deletion of OT-II cells. (A) Aire regulates mOVA mRNA expression in RIP-mOVA transgenic mice. Relative expression of Aire, Ins2, Spna2, and OVA was determined by quantitative-real time PCR on cDNA prepared from CD45⁻-enriched thymic 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 6-10 individual thymi were pooled per experiment. Significance relative to WT: ***P ≤ .001; ns indicates not significant. (B) mTEC are able to present OVA on MHC II. Lethally irradiated 8-week-old B6 or RIP-mOVA mice were grafted with wt or IA^b-deficient OT-II BM. Six 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 (left) show a representative experiment for recipients of IA^b−/− OT-II BM. Histograms (right) show the mean ± SEM for each group. n = number of mice pooled from several experiments. Figure 3B, Figure 5, and supplemental Figure 3 share the same groups lacking expression of the RIP transgene. Significance relative to WT: *P ≤ .05; ***P ≤ .001. In separate experiments, DCs from chimeric mice receiving IA^b-deficient were shown to be virtually all derived from donor BM as evidenced by the absence of cells expressing high levels of MHC II and CD11c (data not shown).