Circulating CCR6⁺ T cells stably express CCR6 on in vitro expansion and maintain a demethylated DMR. (A) Carboxyfluorescein succinimidyl ester-labeled CCR6⁺CD4⁺ memory T cells were cultured for 6 days in medium containing recombinant human IL-7 and IL-15 or anti-CD3/anti-CD28 Dynabeads with or without TGF-β and reanalyzed for CCR6 expression. One representative donor of 4 is shown. (B) Ex vivo isolated CCR6⁺CD4⁺ memory T cells were stimulated with anti-CD3/anti-CD28 Dynabeads in the absence or presence of TGF-β. After 6 days, methylation analysis of the DMR was assessed. (C) Ex vivo isolated CCR6⁺CD4⁺ memory T cells were stimulated with anti-CD3/anti-CD28 Dynabeads, sorted on day 6 in CCR6⁻ and CCR6⁺ cells, and cultured for an additional 3 days in medium containing IL-2. Data are representative for 2 donors.