CCR4⁺ Tregs have hallmarks of activated memory cells. (A) Splenocytes from B6 mice were stained with CD4, CD25, CCR4, CD44, and ICOS mAbs. Expression of CD44 and ICOS was compared among CD4⁺CD25⁺CCR4⁺ and CD4⁺CD25⁺CCR4⁻ cells. Isotype controls were included in each experiment. (B) CD4⁺CD25⁺CCR4⁻ cells were sorted and activated by anti-CD3 + anti-CD28 mAbs and IL-2 for 36 hours. Expression of CCR4 was then detected on activated cells by flow cytometry. These experiments were reproduced twice.