a progressively marked increase in the median survival age and in the protection from multiorgan infiltration (Fig. 3, E–H). Mice expressing Aire only until 21 d (iAON(0–21)) were indis- tinguishable from iAON controls, including the appearance of “normal” insulitis (Fig. 3 H). Several iAON(0–21) mice that were observed for >250 d remained similarly free of disease (un- published data). These results demonstrate that Aire expression during the neonatal period is essential for self-tolerance but that it is dispensable beyond weaning age. This conclusion was confirmed by the reverse experiment, in which Aire was turned on only after birth; in spite of continued Aire expres- sion for the remainder of their lives, these animals showed wasting and autoimmune pathology (Fig. 3 I). Thus, expo- sure to self-antigens during the neonatal period is both neces- sary and sufficient to prevent the devastating autoimmunity associated with Aire disease. These data imply that escape of autoreactive lymphocytes into the periphery is more tolerable after weaning age than during the neonatal period, and they raise two connected but different questions: what is special about the neonatal period, and why don’t the intolerant T cells emerging from the adult AireOFF thymus induce pathology? Neonatal T cells are pecu- liar in several respects, any of which could be relevant in the present context: their TCRs are largely devoid of N-region diversity and have a different intrinsic affinity for major histocompatibility complex molecules (21–23); they include relatively few CD4+Foxp3+ regulatory (T reg) cells (24), and they are able to access the parenchymal tissues (25). Fi- nally, neonatal thymocytes emerge into peripheral T cell compartments that are largely empty and, thus, show lym- phopenia-driven proliferation, a state which can provoke ef- fector-like potentialities (26–30). Several experiments were set up to explore some of these explanations. First, iAON(0–21) mice were lethally irradiated and reconstituted with synge- neic bone marrow while Aire was turned (and maintained) off (Fig. 4 A), a setting which should recreate in an adult mouse the lymphopenic conditions of the neonate. In this case, iAON(0–21) mice did develop wasting disease and multiorgan lymphocytic infiltration (Fig. 4 A). Unmanipulated iAON(0–21) mice remained free of disease, and all other controls behaved as expected (Fig. S1). Similarly, partial T cell lymphopenia was induced in iAON(0–21) mice by injection of depleting anti- CD4/CD8 mAbs at 3 wk of age at the time of Aire turnoff. Multiorgan pathology ensued, albeit in the absence of wast- ing disease (Fig. 4 A; and Fig. S2 shows additional controls). Third, Aire/ neonates were infused with boluses of adult spleen and LN cells (10 × 106 T-enriched cells at days 1 and 7), thus reducing their lymphopenic state. Distinct, albeit not complete, protection was achieved with this treatment, with less frequent and delayed wasting and a reduced intensity of histological lesions. Such protection could not be achieved by the transfer of 3 × 105 FoxP3+ T reg cells alone, which is equivalent to the number of T reg cells present within the 10 × 106 T cell–enriched cells’ bolus (Fig. 4 B and Fig. S3). These results are not compatible with a major influence of the neonatal TCR repertoire or with an intrinsic “openness” 1250 of neonatal tissues. Neither are they compatible with an expla- nation based solely on the relative dearth of T reg cells at birth because after irradiation and reconstitution from bone marrow precursors, T cell populations are restored with largely normal proportions of T reg cells (31, 32). An explanation which is purely based on T reg cell deficiency would not account for the autoimmune pathology in reconstituted iAON(0–21) mice. Rather, they indicate that lymphopenia provides a permissive environment for the initiation of autoimmunity, because T cells spontaneously acquire effector functions and/or are able to en- ter tissues, in which escape of autoreactive cells from the thy- mus has deleterious consequences. The autoimmune manifestations of Aire disease have strik- ing similarities to those induced by neonatal thymectomy (d3Tx): widespread but slowly progressing autoimmunity, inter- individual variability, and a clear parallel in the target organs preferentially affected on different genetic backgrounds (19, 33). The autoimmunity induced by d3Tx was initially thought to re- flect defective T reg cells, but it has since been realized that the numbers and activity of T reg cells are normal in d3Tx mice (34, 35). A unifying interpretation is that the T cell compartment re- quires exposure to self-antigens in the thymus during the post- natal period. Eliminating this exposure by either thymectomy or shutdown of Aire expression results in incomplete tolerance. This tolerogenic exposure can be achieved only in the thymus and not in the periphery, a notion which is also supported by the classical studies of embryonic quail/chick embryonic grafts (36); pre/postnatal exposure to peripheral tissues, even as massive as an entire limb, cannot replace the contribution of thymic epi- thelium. In addition, the lymphopenic state inherent to a grow- ing T cell pool exacerbates this dependency, which is consistent with the autoimmune manifestations often observed in lympho- penic situations, such as the “immune reconstitution inflamma- tory syndromes” observed after T cell depletion in HIV or other viral infections (37). In contrast, a fully established adult T cell pool can provide enough control, via regulatory cells or other mechanisms of peripheral tolerance, to buffer incoming self-re- active cells arising from an Aire-deficient thymus. But these buf- fers can be established only if thymic exposure to self has occurred previously in the formative stages. Materials and Methods. Mice. The TOA construct contains Aire cDNA (positions 36–1879 from GenBank/EMBL/DDBJ under accession no. NM_009646.1) and an intron and polyadenylation site from the rabbit -globin gene in the tetO-SB plas- mid. The fragment was prepared for microinjection into NOD embryos by AatII and Eco47III digestion and purification as previously described (38). Progeny were screened by a PCR assay specific for transgenic Aire (primers: 5-GCAACGTGCTGGTTGTTGTG-3 and 5-TGACTCCAAGTTGC- CATCTG-3). TA mice (14) were backcrossed more than seven times onto NOD, fixing NOD alleles at Ag7, Idd3, and Idd5.2. Aire/ mice on the NOD background have been previously described (19). Dox hydrochloride (Sigma-Aldrich) was provided in drinking water at 100 µg/ml, unless other- wise indicated. Equal sweetener was added to the water to mask Dox’s bitter flavor, and water was changed three times per week. For prenatal or neonatal treatment, Dox-supplemented water was provided to pregnant or lactating mothers. To follow wasting disease, mice were weighed three times per week and sacrificed when they lost 15–20% body weight relative to littermates. For PERINATAL AIRE IS ENOUGH | Guerau-de-Arellano et al.