From cancerous and non-cancerous patients, we derived stable clones of CD4(+) Treg, defined as clones that expressed high CD25 at rest, were anergic in vitro, and suppressed the proliferation of co-cultured CD4(+) cells. A conserved region of FOXP3 intron 1 was demethylated in all Treg clones, whereas it was methylated in non-regulatory Th and CTL clones. In our panel of human clones, this stable epigenetic mark correlated better with suppressive activity than did FOXP3 mRNA or protein expression. We used expression microarrays to compare Treg and Th clones after activation, which is required for suppressive function. The transcriptional profile that is specific of activated Treg clones includes a TGF-beta signature. Both activated Treg and Th clones produced the latent form of TGF-beta. However, SMAD2 phosphorylation was observed after activation in the Treg but not in the Th clones, indicating that only activated Treg clones produced the bioactive form of TGF-beta. A TGF-beta signature was also displayed by a Th clone "suppressed" by a Treg clone. In conclusion, the hallmark of our panel of activated human Treg clones is to produce bioactive TGF-beta which has autocrine actions on Tregs and can have paracrine actions on other T cells.