Suppression of IFN-γ production by CD4⁺GFP(Foxp3)⁺ Tregs with or without OX40 stimulation. (A) CD4⁺GFP(Foxp3)⁻ T effector cells sorted from OX40KO foxp3gfpKI mice were stimulated with anti-CD3 plus wt APCs or OX40Ltg APCs, IFN-γ production by the T effector cells in the presence or absence of wt CD4⁺GFP(Foxp3)⁺ Tregs was analyzed by ELISPOT assay. Data shown are mean (± SD) of 3 experiments. (B) OX40 deficient CD4⁺GFP(Foxp3)⁻ T effector cells were stimulated with anti-CD3 plus OX40Ltg APCs. In these cultures, wt or OX40 deficient CD4⁺GFP(Foxp3)⁺ Tregs were added as indicated, and suppression of IFN-γ production was shown. Data shown are mean (± SD) of 3 independent experiments. (C) CD4⁺GFP(Foxp3)⁻ T effector cells from OX40KO foxp3gfpKI mice were transferred into syngeneic Rag^−/− hosts (5 × 10⁵ cells/mouse), groups of host mice were also transferred with equal number of wt CD4⁺GFP(Foxp3)⁺ Tregs. The host mice were then grafted with DBA/2 skin grafts and treated with an agonist anti-OX40 mAb, and skin allograft survival was shown. (*) P < .05