TCR stimulation of human CD4+CD25- T cells induces Foxp3. Highly purified CD4+CD25- T cells (98–99%) were stimulated with the indicated regimen in X-Vivo 20 serum-free medium, and Foxp3 mRNA and protein were examined. A. Cells were cultured for 48–72 hours. RNA was isolated and cDNA synthesized for assessing the expression of Foxp3 by real-time PCR. Freshly isolated CD4+CD25+ T cells were used as a positive control for Foxp3 expression. Values are expressed as the normalized ratio of Foxp3 to GAPDH. B. Analysis of Foxp3 protein with Western blot. The experiments were repeated three times with similar results. C-E. Analysis of intracellular Foxp3 protein at the single-cell level by FACS. Freshly isolated CD4+CD25- cells (Fresh) or cultured cells at the indicated time were stained with FITC-anti-CD25 (surface) and PE-anti-Foxp3 (intracellular) and analyzed on FACScalibur. A representative FACS profile is shown as dot plots of CD25 versus Foxp3 (C). The quadrant gates were set according to the negative isotype control antibodies in the respective cells. The kinetics of the percentage (D) and total number (E) of CD25+Foxp3+cells are shown as Mean ± SD of each group at each time point (n = 3 to 6). Med: Medium; αCD3: anti-CD3 mAb; αCD28: anti-CD28 mAb. * indicates a different donor.