Cytokine-induced IFN-γ⁺ Th cells are controlled by CD25⁺⁺ Tregs. (A) CD25⁺⁺ Tregs (II) and CD45RA⁻IL-18Rα⁺ memory-effector (III) or CD45RA⁺IL-18Rα⁻ naive (IV) responder Th cells were sorted from PBMCs (I). (B) CFDA-labeled CD45RA⁺IL-18Rα⁻ naive responder Th cells were cultured with T-cell stimulation beads in the presence of equal numbers of CFDA⁻CD45RA⁺IL-18Rα⁻ control cells or CD25⁺⁺ Tregs. Proliferation was assessed after 84 hours according to loss of CFDA by FACS. (C) CFDA-labeled CD45RA⁻IL-18Rα⁺ responder Th cells were stimulated for 36 hours with IL-12 + IL-18 + IL-15 or with the cytokine cocktail or for 18 hours with T-cell stimulation beads in the presence of equal numbers of CFDA⁻CD45RA⁻IL-18Rα⁺ control cells or CD25⁺⁺ Tregs. Frequencies of CFDA⁺IFN-γ⁺ responder cells were assessed intracellularly by FACS; cocultures with control cells were set to 100%. One representative experiment of 3 (B) or 4 (C), respectively, is shown.