OX40 expression by CD4⁺Foxp3⁺ Tregs and CD4⁺Foxp3^— T effector cells. (A) Spleen cells from naive foxp3gfpKI mice were stained with cychrome-anti-CD4 and PE-anti-OX40. CD4⁺GFP(Foxp3)⁺ cells and CD4⁺GFP(Foxp3)⁻ cells were selectively gated and expression of OX40 on the Foxp3⁺ and Foxp3⁻ subsets was analyzed. A representative plot of 5 experiments is shown. (B) FACS sorted CD4⁺ GFP(Foxp3)⁻ T effector cells were stimulated in vitro with anti-CD3 and anti-CD28, and expression OX40 on the activated T cells was analyzed and shown. Naive CD4⁺ T effector cells were included as a control (the solid histogram). (C) The sorted CD4⁺ GFP(Foxp3)⁻ T effector cells were stimulated with anti-CD3/anti-CD28 plus TGF-β for 4 days, and the induction of GFP(Foxp3)⁺ cells was determined. Expression of OX40 on the GFP(Foxp3)⁻ T effector cells and the converted GFP(Foxp3)⁺ Tregs was shown. (D) CD4⁺GFP(Foxp3)⁻ T effector cells were cocultured with the natural CD4⁺GFP(Foxp3)⁺ Tregs or the converted CD4⁺GFP(Foxp3)⁺ Tregs. The cell mixture was stimulated with anti-CD3 plus APCs, and suppression of T effector cell proliferation was shown as mean (CPM ± SD) of triplicate assays. Representative data of 3 individual experiments are shown.