1 Introduction Kyewski and Klein, 2006 Derbinski et al., 2001 Derbinski et al., 2001; Gotter and Kyewski, 2004 Kyewski and Klein, 2006 Nagamine et al., 1997 Kumar et al., 2001 Pitkanen et al., 2000 Bjorses et al., 1999; Heino et al., 1999 Everett and Chelbi-Alix, 2007 Blechschmidt et al., 1999; Heino et al., 1999 Perheentupa, 2006; Peterson and Peltonen, 2005 Anderson et al., 2002; Kuroda et al., 2005; Ramsey et al., 2002 Liston et al., 2003 Anderson et al., 2002; Derbinski et al., 2005; Jiang et al., 2005 This study aims to further clarify whether Aire can directly regulate the TRA expression by analyzing the expression of four antigens in several experimental settings where Aire's expression has been modulated. The study aims to establish whether there is a dose-dependent correlation between the number of Aire allele copies and TRA expression level in thymic epithelial cells, and whether TRAs are co-expressed with Aire during thymic development and involution. We also studied whether the over-expression of Aire as a sole factor is sufficient to induce TRA expression and whether thymic microenvironment plays a role in the expression of Aire and TRAs. 2 Material and methods 2.1 Mice and cell cultures Aire Fig. 1 Mizuochi et al., 1992 2.2 EGFP and Aire adenovirus construction and infection Aire Heino et al., 2000 in-trans He et al., 1998 12 8 ® 2.3 Thymic stromal cell isolation 8 2.4 Cell sorting − + − b 2.5 Immunofluorescence and microscopy 2 2.6 Thymic reaggregate organ culture Jenkinson et al., 1992 2.7 Real-time PCR 2 C t C t 2 − Δ Δ C t C t 3 Results 3.1 Decrease in Aire expression down-regulates the TRA expression in a dose-dependent manner Derbinski et al., 2005 Aire Aire Tff3 Ins2 Mup1 Spt1 Derbinski et al., 2005 Anderson et al., 2002; Derbinski et al., 2005 Fig. 1 Aire Fig. 1 Fig. 2 Fig. 2 Fig. 3 Fig. 3 Tff3 Ins2 Mup1 Spt1 3.2 TRAs follow the expression of Aire during normal development and involution Fig. 4 3.3 Over-expression of Aire results in an increase in TRA expression Fig. 5 Fig. 5 Fig. 5 3.4 Thymic microenvironment is needed for the expression of Aire and TRAs Anderson et al., 2006 ex vivo Fig. 6 4 Discussion Anderson et al., 2002; Derbinski et al., 2005 Hoffmann and Jagla, 2002; Karam et al., 2004 Dickinson et al., 1989 Shaw et al., 1983 Chentoufi and Polychronakos, 2002; Derbinski et al., 2005 Chentoufi and Polychronakos, 2002 Cook et al., 1998; Kaern et al., 2005 Liston et al., 2004 Jay et al., 2005 Lee et al., 2007 Sousa Cardoso et al., 2006 Gray et al., 2006 Aire dependent TRA expression is further illustrated by adenoviral experiments enforcing Aire expression in thymic epithelial cells. We show that over-expression of Aire as a single factor is sufficient to induce the expression of all four TRAs studied, providing evidence that modulation of Aire can directly lead to alterations in TRA levels and may thus also affect the maintenance of central tolerance. The finding that Aire, in addition to primary thymic stromal cells, can also modulate TRA levels in the thymic medullary epithelial cell line, suggests that there is no need for other cell types for the Aire-induced up-regulation of TRAs to occur. Van Ewijk et al., 1994 Jenkinson et al., 2005 Jenkinson et al., 2003 Boehm et al., 2003; Chin et al., 2003 Akiyama et al., 2005; Burkly et al., 1995; Kinoshita et al., 2006; Zhang et al., 2006 Gillard et al., 2007 Jenkinson et al., 2005; Zuklys et al., 2000 In conclusion, we show that Aire has a dose-dependent effect on TRA expression in thymus but not in the lymph nodes. Both, Aire as well as TRAs localize in the thymic medulla and are co-expressed during normal development and involution. We also show that Aire can directly induce TRA expression in medullary epithelial cells although the thymic microenvironment plays a crucial role for the maximal expression to occur. Our data suggest a clear correlation between the expression of Aire and TRAs and indicate that approaches to stimulate Aire expression in thymic epithelium could be considered to modulate tolerance induction to peripheral antigens.