In vitro induced CCR6 expression is unstable and not associated with a demethylated DMR. (A) Naive CD4⁺ T cells were stimulated with anti-CD3/anti-CD28 Dynabeads without (white bar) or with proinflammatory cytokines and TGF-β (black bar) for 4 days, followed by culture in IL-2. After 6 to 7 days, cells were analyzed for CCR6 expression (n = 21, left graph). The methylation profile of CD4⁺ T cells cultured with (+) and without (−) cytokines is shown in the right graph (n = 2). ***P < .001. (B) Cells stimulated with cytokines as in panel A were sorted into CCR6⁺ and CCR6⁻ cells (shown for 1 representative donor) and analyzed for methylation of the DMR. (C) Naive CD4⁺ T cells were stimulated and cultured as in panel A. At day 6, a fraction of the cultured cells were stimulated again under the same conditions. Cells were analyzed for CCR6 expression (one representative donor). (D) Methylation analysis of the CCR6 DMR (one representative donor, left panel) and FACS analysis of CCR6 expression in cells cultured as described in C (n = 11, right panel). **P < .01.