Effect OX40 stimulation on survival and Foxp3 expression of CD4⁺GFP(Foxp3)⁺ Tregs. (A) Sorted CD4⁺GFP(Foxp3)⁺ Tregs were stimulated with anti-CD3 plus wt and OX40Ltg APCs. Cells were stained with PE-annexin V 24 hours later and analyzed by FACS. Freshly prepared Foxp3⁺ Tregs stained with PE-annexin V were included as a control. The annexin V profile in the GFP⁺ fraction was shown. (B) Sorted CD4⁺GFP(Foxp3)⁺ Tregs were stimulated with anti-CD3 and wt or OX40Ltg APCs. Foxp3 gene transcripts were analyzed 4 days later by real-time PCR. Data shown are mean A.U. of 4 experiments in each group. Error bars represent the SD of triplicate assays. (C) Sorted CD4⁺GFP(Foxp3)⁺ Tregs were stimulated with anti-CD3 and wt or OX40Ltg APCs, levels of GFP(Foxp3) expression in the CD4⁺ fraction were determined by FACS 4 days later and shown. The plot shown is one of 3 experiments. (A,C) Numbers are the relative percentage of cells in each region given by the flow cytometer.