has particular predilection for the lung, exocrine pancreas, eye, and ovary/prostate. In contrast, NOD.Aire/ mice are protected from the insulitis that normally affects the NOD strain (19, 20). These observations were reproduced in our study (Fig. 2). Aire deficiency was indeed complemented by the transgenes, as iA mice in which the reporter was contin- uously on (iAON) survived to 100 d of age and beyond, de- veloping the typical insulitis of NOD mice, rather than exocrine pancreatitis. Only rare isolated and mild infiltration of the lung or gonads was detected over the course of multi- ple experiments, comparable with that observed in similarly aged WT mice. Aire turnoff by continuous Dox exposure (iAOFF) restored the full array of disease manifestations, con- firming that Aire disease depends on regulated Aire expres- sion. As expected, Dox treatment of either WT or KO mice had no effect. Figure 3. A neonatal time window for Aire-mediated tolerance. Survival curve analysis (left; the numbers are the mean survival times in days) and histopathology (right) for iA mice in which Aire turnoff was initiated at different times or initiated on embryonic day (e) 0 and then allowed to be reex- pressed from birth (day 0), as depicted in the central diagram (black fill, Aire on; white fill, Aire off). Tissues were harvested for histology after a 14–17-wk follow up, or earlier if they had to be sacrificed as a result of wasting disease. WT (A; n = 53), KO (B; n = 49), and Aire/TOA+ reporter-only (B; n = 11) mice were included as controls in individual experiments and are pooled. iAON(e0-e16.5) (C; n = 3), iAON(e0-e19.5) (D; n = 3), iAON(e0-D0) (E; n = 13), iAON(e0-D3) (F; n = 10), iAON(e0-D10) (G; n = 10), iAON(e0-D21) (H; n = 10), and iAOFF(e0-D0) (I; n = 6) are shown. P < 0.002 when comparing KO to iA mice in E–H (Kaplan-Meier test). The histopathology diagram is as described for Fig. 2. All experiments were performed at minimum of three times per condition. 1248 PERINATAL AIRE IS ENOUGH | Guerau-de-Arellano et al.