ATRA enhances FoxP3 expression independent of IL-2. (A) Peripheral CD4 cells from Rag2^−/− transgenic (OTII) mice were polyclonally stimulated for 3 days under Th17 conditions or TGF-β1 alone, in the presence of either IL-2 or anti–IL-2. All stimulations were performed with (lower panels) or without (upper panels) 1μM ATRA. IL-17 and FoxP3 expression were measured on fixed cells by intracellular staining. Data are representative of 2 independent experiments. Numbers on plots are percentages of total cells. (B) CD4⁺CD25⁻ MACS-purified FoxP3-GFP cells were polyclonally stimulated for 3 days in the presence of TGF-β1, ATRA, and anti–IL-2. FoxP3-positive cells were isolated by flow cytometry and used to inhibit CD4⁺CD25⁻ cells sorted by flow cytometry and stimulated with soluble anti-CD3 in the presence of irradiated Thy1.1⁻ cells. Proliferation was measured by ³H-thymidine uptake and was compared with CD4⁺CD25⁺ (natural Treg) cells purified by flow cytometry. Error bars denote the standard error of the mean (n = 3), and significance was determined by an unpaired t test, and data are representative of 2 independent experiments.