rarely addressed tolerance to true self-constituents, which were long impossible to manipulate experimentally. The Aire gene may provide such an experimental handle. Aire is a transcription factor expressed primarily in medullary epithelial cells (MECs) of the thymus, where it promotes the ectopic expression and pre- sentation of peripheral tissue antigens (12). This presentation imparts deletional tolerance, and Aire deficiency results in multi- organ autoimmune disease in both human patients and KO mice. Importantly in the present context, the autoimmune dis- ease that develops in Aire-deficient mice (hereafter Aire disease) is solely controlled by immunological tolerance and does not re- quire microbial triggers (13). Thus, Aire-controlled presentation of self might provide a manipulable system that is more appro- priate for investigating the temporal aspects of T cell tolerance. Figure 1. Conditional Aire transgenic system. (A) Schematic of the Dox-controlled TA and TOA dual transgenes (together referred to as iA). (B) Real- time PCR analysis of Aire transcripts in whole thymus of Aire WT (n = 8), Aire KO (n = 2), and iAON (n = 8) mice. Values are expressed relative to WT thy- mus as a standard. (C) Aire staining (green) thymic epithelial cells (counterstained with anti-keratin5 [blue] and -keratin8 [red]) of WT and iAON mice (100× objective). No Aire staining was detectable in KO mouse samples or WT samples stained with secondary antibody only. (D) Real-time PCR analysis of iA reporter expression in thymic MEC (CD45Ly51/loEpCam+), CEC (CD45Ly51+EpCam+), DC (CD3CD19CD45+CD11c+), and macrophages (CD3CD19CD 45+CD11cCD11b+) of iAON, reporter-only TOA+, and WT mice (n = 2). Expression level is normalized to RNA from whole iAON thymus. (E) Flow cytometry analysis of MHC class II and intracellular Aire in EpCAM+Ly51/lo-gated MECs of one representative WT, KO, iAON, and iA(OFF1wk) sample. The mean percent- age ± SD of the MHCIIhiAire+ population for groups of 5–11 mice is shown. (F) Real-time PCR analysis of whole thymus Aire transcript (top) and percent- age of Aire+ cells within thymic MECs (bottom) at different times after iA turnoff by Dox treatment in iA mice (means of n = 2–3 per time point). (G) Real-time PCR of Aire transcripts in thymus of iA mice that were untreated, treated with 10 µg/ml Dox for 1 wk, or weaned off Dox for 2 wk after a 1-wk Dox treatment (n = 2). Values are relative to iAON thymus. (H) Real-time PCR analysis of iA transcripts in organs of iAON mice (top), Aire transcripts of sorted LN CD45gp38+UEA-1+(bottom left), or spleen CD45 (bottom right) stromal cells (n = 2). Error bars show SD. Results shown are representative of two (D, G, and H) or three or more (B, C, E, and F) experiments. 1246 PERINATAL AIRE IS ENOUGH | Guerau-de-Arellano et al.