Levels of intracellular EBI3, IL-12p35, Foxp3, IL-10, and TGF-β in the unactivated and activated CD4+CD25+ Tregs. CD4+CD25+ Tregs were activated with anti-CD3, anti-CD28, and rhIL-2 for 0, 3, 5, and 7 days. mRNA levels of IL-35 two subsets, EBI3 and IL-12p35 in CD4+CD25+ Tregs, at different activation times were detected by reverse transcription polymerase chain reaction. Levels of EBI3 and IL-12p35 increased gradually in accordance with the activation time, which suggested that both activated and unactivated CD4+CD25+ Tregs can express IL-35. IL-10 and TGF-β mRNA, which are known to be secreted by CD4+CD25+ Tregs, were also assayed, and as anticipated, the levels were higher after activation. The levels of Foxp3, the marker of Treg cells, became higher as the activation time increased (Table 5). CD4+CD25+ Treg and CD4+CD25− T-cells were acquired through MACS from fresh PBMCs.