B R I E F D E F I N I T I V E R E P O RT The functional importance of Aire in stromal cells of pe- ripheral lymphoid organs remains conjectural (17, 18), al- though the transferability of the autoimmune disease by grafts of Aire-deficient thymi (15) suggests that it is not relevant for Aire disease. Consistent with this notion, there was no corre- lation between peripheral Aire expression, which was largely insensitive to Dox (Fig. 1 H), and protection from disease, which was fully sensitive (Fig. 2). This peripheral expression is thus not relevant to the present study, although it may not be a coincidence that the same peripheral cells that normally express low levels of Aire are also those that show leaky ex- pression of the transgenic reporter. Thus, the inducible Aire system provided us with a means to vary the expression of and tolerance to Aire-controlled self-anti- gens. To dissect the influence of Aire expression on tolerance throughout life, we examined the incidence of wasting disease and autoimmune infiltration in iA mice in which Aire was ex- pressed over different time intervals (Fig. 3). Dox treatment should result in clearance of peripheral tissue antigens within a matter of days, from their own molecular turnover and because Aire-positive cells turn over rapidly, with a half-life of 3–4 d (16). Turning Aire off before birth resulted in full-blown wasting dis- ease and multiorgan autoimmunity (Fig. 3, C and D), but keep- ing it on for an increasing number of days after birth resulted in Figure 4. Aire is required to prevent autoimmunity during lymphopenia. Survival curve analysis (left) and histopathology (right) in supplemented Aire-deficient mice (WT, KO, and additional control mice were included in each experiment and are not shown here for clarity but are explained in detail in Figs. S1–S3). (A) iAON(e0-D21) mice were otherwise untreated (top), lethally irradiated and bone marrow reconstituted (middle), or anti-CD4/CD8 treated at 3 wk of age (bottom) for a 2-wk period. Irradiated/reconstituted iAON(e0-D21) versus KO (P = NS) or WT (P = 0.0024) was calculated by Kaplan-Meier survival analysis. (B) Aire/ mice were untreated (top; n = 6), received 10 × 106 T cell–enriched peripheral LN and spleen cells (middle; n = 5), or received 3 × 105 T reg cells (bottom; n = 5) on days 1 and 7. T cell–supplemented KO mice versus KO (P = 0.005; Kaplan-Meier) or versus WT (NS) and T reg cell–supple- mented KO mice versus KO (NS) or versus WT (P = 0.0001) were compared. Experimental and histopathology diagrams are as in Fig. 3. All results in A and B are representative of two or more independent experiments. JEM VOL. 206, June 8, 2009 1249