Given that Aire+CD80high and Aire+MHC IIhigh mTECs develop from Aire−CD80low 16, 17 and Aire−MHC IIlow 18 immature mTECs, respectively, and that Aire+ mTECs are postmitotic 18, it is now clear that Aire is expressed in the distal stages of mTEC differentiation. Indeed, Aire+ mTECs are negative for p63 expression, a regulator of the proximal stages of epithelial cell differentiation 19–21; however, it had not been determined in which distal stage (beginning or end) Aire was expressed. In other words, it was not clear whether Aire+CD80high mTECs undergo further differentiation accompanied by phenotypic change(s) before their cell death, or whether they maintain this cellular signature until they die. Therefore, it was necessary to determine more precisely the timing of Aire expression during the course of the mTEC differentiation program. In this respect, a fate-mapping strategy allowing permanent marking of cells expressing a gene of interest, even after extinction of its transcription, was expected to be informative (basis for fate mapping and its application to the study of thymus epithelium is reviewed in 22). In this strategy, bacterial artificial chromosome transgenic mice expressing Cre recombinase under the control of the Aire regulatory element (i.e. Aire/Cre knock-in mice) were crossed with a GFP reporter strain, thereby making it possible to mark the cells that had expressed Aire at some time during their life history with GFP 23. This situation contrasts markedly with Aire/GFP knock-in mice, in which Aire expression can be monitored on a real-time basis 19 (as described in “Two contrasting models” below). Unfortunately, specific marking of Aire-expressing cells in the thymus (Aire+ mTECs) by a fate-mapping strategy proved to be impossible because of Aire expression from epiblast of E6.5 embryos, in which thymic organogenesis has not yet begun 23; however, with the use of a particular transgenic line in which cell marking with GFP subsequent to Aire expression was confined to the mTECs, possibly due to the low Aire/Cre transgenic expression, the existence of additional differentiation stage(s) after Aire+CD80high was revealed. Namely, the Aire+CD80high stage(s) were found to be followed by extinction of Aire expression together with down-regulation of CD80 expression, thereby generating Aire−CD80intermediate mTECs 23. Supporting this finding, a recent study has suggested that Aire expression and terminal differentiation within the mTEC lineage are temporally separable events controlled by distinct mechanisms; initial formation of Aire+ mTECs depends on RANK signaling provided by lymphoid-tissue inducer cells 16, whereas continued mTEC development to the stage of expression of involucrin, a marker of terminally differentiated epithelium 24 (see “Model 2: Promotion of the mTEC differentiation program by Aire”), maps to activation of the lymphotoxin pathway by mature thymocytes 25. Given that mTECs at the Aire+CD80high stage(s) are the most competent for PGE, the functional significance of mTECs at the Aire−CD80intermediate stage(s) needs to be determined. It is possible that Aire-expressing mTEC lineage(s) at this stage(s) contributes to central tolerance by mechanisms beyond PGE, as exemplified by antigen processing/presentation 26, cross-presentation 18, and intrathymic thymocyte migration and maturation 27.
The existence of post-Aire stage(s) during the course of mTEC differentiation is apparently inconsistent with the notion that Aire expression in mTECs results in apoptosis. Aire's proapoptotic activity has been deduced from the increased percentage of the CD80high mTEC population (in which Aire+ mTECs reside) in Aire-deficient mice, despite the lack of Aire's ability to directly cause proliferative arrest of mTECs 18. Furthermore, overexpression of Aire in an mTEC line by gene transfection causes overt apoptosis of the cells, although this effect was delayed by a couple of days after transfection 18. One possible explanation for the discrepancy may be the delayed effect which suggests that the apoptoic events initiated by Aire expression are neither rapid nor direct. Whether or not Aire exerts any proapoptotic activity in mTECs is a critical point when considering the mTEC differentiation program (as discussed in Model 1: Interruption of the mTEC differentiation program by Aire), further study is needed to establish definitively the relationship between Aire and apoptotic events in mTECs.