RESULTS And dISCUSSIon To this end, we attempted to develop a regulable Aire trans- genic system that could complement an Aire deficiency in a time- and dose-controlled manner. The TA transgene drives expression of the tetTA (tTA) transcriptional activator in thy- mic epithelium (14); its product activates the tetOAire (TOA) reporter transgene, which carries the mouse Aire complemen- Figure 2. The iA transgenic system rescues Aire disease. (A) Survival curve of WT (n = 7), Aire/TOA+ reporter-only (n = 29), iAON (n = 7), and iAOFF (n = 3) mice. Mice were sacrificed upon 15–20% body weight loss. KO versus iAON, P = 0.0002 by Kaplan-Meier survival analysis. (B) Representa- tive histopathology of tissues affected in Aire KO NOD mice performed at 14–17 wk, or earlier for mice that had to be sacrificed as a result of wast- ing disease. EY, eye (retinal degeneration); LU, lung; PR, prostate; EP, exo- crine pancreas; IN, pancreatic islets (insulitis). White arrows point at areas of infiltration/retinal degeneration. (C) Histopathology analysis in WT, KO, iAON, and iAOFF mice at 14 wk of age. Filled squares represent diseased tis- sue, as indicated (i, insulitis typical of Aire-proficient NOD mice). Results in A–C are representative of more than three independent experiments. JEM VOL. 206, June 8, 2009 B R I E F D E F I N I T I V E R E P O RT tary DNA (cDNA) under the dictates of a tTA-responsive pro- moter (Fig. 1 A). When both transgenes are present, Aire is expressed in thymic epithelium, but administration of doxycy- cline (Dox) blocks the interaction of tTA with the reporter gene promoter, turning Aire off. The transgenes were crossed to the Aire KO mutation (15) on the nonobese diabetic (NOD) genetic background to generate Aire/TA+TOA+ mice, which are referred to as inducible-Aire (iA) mice. RT-PCR analysis of Aire transcripts in the thymus showed that the iA system rescued Aire expression to values close to those of WT mice (Fig. 1 B). Similarly, the usual punctate stain- ing of Aire in nuclei from thymic epithelial cells was recovered in iA mice (Fig. 1 C). Transgene-encoded Aire was expressed in medullary and cortical epithelial cells, with little to no expression in DCs and macrophages, as expected from the properties of the driver construct (Fig. 1 D). Expression in these thymic popula- tions was dependent on the presence of both the driver and re- porter transgenes, as the reporter alone yielded no detectable expression (Fig. 1 D). Flow cytometry after intracellular staining confirmed the presence of Aire in MECs from iA mice, in quan- tities slightly higher than those of WT littermates and of an Aire+ MEC population in close to normal proportions (Fig. 1 E). Aire expression in the thymus of iA mice proved sensitive to Dox, with a complete loss of Aire transcripts within the first 24 h of Dox treatment and a disappearance of Aire+ MECs over the subsequent 3 and 5 d (Fig. 1 F), which is consistent with the rapid turnover of Aire+ MHCIIhi MECs (16). Reexpression of Aire after a turnoff period was more sluggish but could be achieved after 1–2 wk of Dox withdrawal, particularly when low doses were initially used (Fig. 1 G and not depicted). Because the turnoff with Dox treatment was sharper than the turnon in this model, as in other Dox-controlled transgenic settings, regulated Aire extinction was the strategy primarily used in the studies described in the subsequent paragraphs. It was recently reported that Aire is expressed in the stro- mal cells of peripheral lymphoid organs (17, 18). Aire-encod- ing transcripts from the iA transgenes were detected almost exclusively in the thymus, with very low expression in the spleen and LNs (<3–5% of thymus values; Fig. 1 H, top). In the CD45gp38+UEA-1+ stromal cell population described by Lee et al. (17), Aire transcripts were 10× more abundant in iAON than in WT mice but were maintained in iAOFF ani- mals, likely because of the leakiness occasionally observed with Dox-controlled systems (Fig. 1 H, bottom left). Similarly, significant transgene-driven Aire transcripts were detected in CD45 spleen cells that correspond to the eTAC cells of Gardner et al. (18), but this expression was also only partially affected by Dox, with residual expression in treated iA mice comparable to that of WT (Fig. 1 H, bottom right). No ex- pression was detected in other tissues, including the lung, kidney, heart, or brain (Fig. 1 H, top, and not depicted). The key test of complementation was whether the iA transgenes could protect, in a Dox-sensitive manner, from Aire disease. Mice with the Aire-null mutation on the NOD back- ground develop wasting disease, with a median survival time of 55 d (19) because of multiorgan lymphocytic infiltration that 1247