Increase of FOXP3 and TGFB1 expression after hT-cell culture can be prevented by initial depletion of CD25⁺ hT cells. Relative expression of FOXP3 (A) and TGFB1 (B) genes in hT cells (▪) and CD25-depleted hT cells (□) were analyzed by real-time quantitative RT-PCR before culture and at different time points of the culture in the presence of CD3 + IL2. Results from a representative experiment performed in duplicate are expressed in arbitrary units (AU). FOXP3 and TGFB1 gene expressions in calibrator cells (dashed line) were arbitrarily set at 1. (C) Foxp3 protein expression in CD3⁺ T cells and CD3⁺ CD25-depleted T cells cultured in the presence of CD3 + IL2. Intracellular staining of Foxp3 was analyzed by flow cytometry in CD3⁺ gated cells from total cultured T cells (▪) and from CD25-depleted cultured T cells (□) at different time points. One representative curve of 2 separate experiments is shown. Right panels illustrate a typical Foxp3 analysis of T cells and CD25-depleted T cells cultured for 6 days and a negative isotypic control (gray histogram).