Figure 3.

CD4⁺CD25⁺ regulatory T cells can be induced by activation of human CD4⁺CD25– PBMCs. (a) Schematic for generation of regulatory cells from CD4⁺CD25– cells, including a typical FACS plot with percentages for activated CD4⁺CD25– cells before sorting. Cells were incubated on plate-bound Ab (Y) for 24 hours and then transferred to a new well without Ab for 9 days. (b) Dot plots showing CD4 versus CD25 staining on CD4⁺ PBL before and after sorting with CD25 MACS microbeads. (c) Percent of CD4⁺CD25⁺ cells over time when CD4⁺CD25– cells were stimulated with plate-bound anti-CD3/soluble anti-CD28 or media. (d) Proliferation and suppression of freshly isolated CD4⁺CD25– T cells by CD4⁺CD25⁺ cells, which were generated by activating CD4⁺CD25– PBMCs with plate-bound anti-CD3/soluble anti-CD28 for 3 or 10 days. (e) Western blot analysis of FoxP3 expression on day 3 or day 10 after activation of CD4⁺CD25– T cells. Control cells were 293T cells transfected with a human FoxP3 cDNA clone. These data are from one experiment and are representative of six separate experiments with a suppression range of 60–95%.