bone marrow chimeras, recipients were irradiated (9 Gy), reconstituted with 10 × 106 bone marrow cells from a syngeneic WT mouse, and kept on with sulfatrim for the remainder of the experiment. For mAb depletion of CD4 and CD8 T cells, mice were treated i.p. with 0.5 mg each of anti-CD4 (clone GK1.5) and anti-CD8 (clone 2.43) every 3 d for a 2-wk period. All mice were maintained under specific pathogen-free conditions at the Harvard Medical School facility (protocol 02954 approved by Harvard Medical School’s Institu- tional Animal Care and Use Committee). For lymphocyte transfer experiments, spleen and LN cells were depleted of non–T cells with the negative selection T cell isolation reagent (Miltenyi Biotec) and 10 × 106 cells were injected i.p. into neonates. For T reg cell transfer experiments, T reg cells from spleen and LN of female Foxp3-GFP (NOD) transgenic mice were isolated by sorting (B220CD4+GFP+) and 3 × 105 T reg cells were injected i.p into neonates. Real-time PCR. RNA was prepared from tissues or sorted cells with Trizol (Invitrogen), treated with DNase (Applied Biosystems) for 30 min at 37°C to remove contaminating DNA, and cDNA synthesized with random or gene- specific priming (GSP). For GSP, the 5-TCATCTCTACCAGGTATAGT- GAC-3 primer was used. cDNA was used as a template for amplification by Taqman RT-PCR (Mx3000P; Agilent Technologies). Both WT and trans- genic Aire transcripts were detected using the 5-GTACAGCCGCCTG- CATAGC-3 and 5-CCCTTTCCGGGACTGGTT-3 primers and the Fam-5-CTGGACGGCTTCCCAAAAGATGT-3-Tamra probe. For de- tection of Aire transcripts only from the TAO transgene, the 5-TGAGA- ACTTCAGGCTCC-3 and 5-GGAAAGGCACTGTCTATGGC-3 primers and the Fam-5-CGTCTGCTGAGGCTGCACCG-3-Tamra probe were used. Whole thymus RNA from WT or iAON (depending on the experi- ment) was used as a reference. Data were visualized and analyzed with MxPro software (Agilent Technologies), using the standard curve method. Stromal cell isolation and intracellular Aire staining. Suspensions of thy- mic stromal cells were prepared and stained for intracellular Aire as previously described (16). For stromal cells from peripheral LNs and spleen, small incisions were made in the tissues, which were agitated in RPMI supplemented with 5% FBS to release free lymphocytes. The remaining tissue was digested in RPMI 5% FBS with 0.5 mg/ml collagenase (Roche), 1 mg/ml dispase (Roche), and 1 mg/ml DNAase (Sigma-Aldrich), and the released cells were stained for FACS analysis. Antibodies and flow cytometry. Anti–mouse gp38–Alexa Fluor 647 and rat anti–mouse Aire (5H12 clone) were gifts from S. Turley (Dana Farber Cancer Institute, Boston, MA) and H. Scott (Walter and Elisa Hall Institute, Melbourne, Australia), respectively. Anti–keratin-5 and –8 antibodies were obtained from Covance. Flow cytometric analysis was performed on an LSRII, sorting on a FACSAria (BD). Data were analyzed with FlowJo software (Tree Star, Inc.). Online supplemental material. Figs. S1–S3 show additional curves for control animals corresponding to Figs. 3 and 4. Online supplemental material is available at http://www.jem.org/cgi/content/full/jem.20090300/DC1. We thank Hamish Scott, Shannon Turley, Nora Mauermann, Shohei Hori, Polly Matzinger, Shannon Turley, and the Aire Group for discussion and reagents, John Stockton for microinjections, Joyce LaVecchio and Giri Buruzala for flow cytometry, Chris Campbell and Angela Wilcox for genotyping, and Vanessa Tran and Kimie Hattori for help with mice. This work was supported by R01 DK60027 and Young Chair funds to D. Mathis and C. Benoist, and by the Joslin’s NIDDK-funded Diabetes and Endocrinology Research Center. The authors have no conflicting financial interests. s ubmitted: 9 February 2009 a ccepted: 29 a pril 2009 REFEREn CES 1. Burnet, F.M., and F. Fenner. 1951. The production of antibodies. J. Immunol. 66:485–486. 2. 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