Compelling evidence suggests that Foxp3‐expressing CD25⁺CD4⁺ regulatory T cells (Treg) are generated within the thymus as a separate lineage. However, Foxp3⁺CD4⁺ Treg can also be generated de novo in a TGF‐β‐dependent process from naive T cells by TCR triggering. Recently, we have shown that naturally occurring, but not in vitro TGF‐β‐induced Foxp3⁺ Treg display stable Foxp3 expression that was associated with selective demethylation of an evolutionarily conserved element within the Foxp3 locus named TSDR (Treg‐specific demethylated region). Here, we report that inhibition of DNA methylation by azacytidine, even in absence of exogenous TGF‐β, not only promoted de novo induction of Foxp3 expression during priming, but also conferred stability of Foxp3 expression upon restimulation. Most notably, such stable Foxp3 expression was found only for cells displaying enhanced TSDR demethylation. In contrast, in vitro TSDR methylation diminished its transcriptional activity. Foxp3⁺ Treg generated in vivo by DEC‐205‐mediated targeting of agonist ligands to dendritic cells showed long‐term survival in the absence of the inducing antigen and exhibited efficient TSDR demethylation. Together, our data suggest that TSDR is an important methylation‐sensitive element regulating Foxp3 expression and demonstrate that epigenetic imprinting in this region is critical for establishment of a stable Treg lineage.

Supporting Information for this article is available at www.wiley‐vch.de/contents/jc_2040/2008/38105_s.pdf