Figure 1. Human iTreg generation with TGF-β and ATRA.

Human CD4⁺ T cells were obtained by magnetic bead sorting (CD4⁺ kit, Miltenyi) of PBMC (purity >90%; 5% FoxP3⁺) and stimulated with high-dose IL-2 +/− TGF-β (20 ng/ml) +/− ATRA (10 ng/ml). After 6 days, live CD4⁺ cells were assessed for FoxP3 expression by flow cytometry. Cells cultured in the presence of TGF-β + ATRA showed 50% FoxP3⁺ expression while cells cultured with only IL-2 were only 25% FoxP3⁺; the remaining 75% of the cells showed an intermediate level of FoxP3.

(A) Gating for FoxP3⁺ cells (horizontal bar). Red: isotype; green: no TGF-β, no ATRA; blue: TGF-β only; brown: TGF-β + ATRA.

(B) Quantification of FoxP3⁺ cells: 50% of cells cultured with TGF-β + ATRA acquired FoxP3⁺ phenotype.