IL-2 increased the frequency of CD4⁺CD25⁺Foxp3⁺ T cells in peripheral blood. (A) RNA was extracted from highly purified CD4 T cells (> 97%-99%) isolated from PBMCs and collected through leukapheresis of 8 patients pretreatment (PRE), during rebound phase (REB), and 2 to 3 weeks after treatment (POST). Foxp3 levels were quantified using real-time RT-PCR and normalized for endogenous β-actin. The P value was calculated using paired t test and adjusted for multiple comparisons. (B) To determine Foxp3 expression per cell, cryopreserved PBMCs from patients or healthy donors were initially stained with allophycocyanin-conjugated anti-CD25, FITC-conjugated anti-CD4 or anti-CD8, and PerCP-conjugated anti-CD3 antibodies, followed by intracellular staining for Foxp3 protein. The dot plots were gated on CD3⁺CD4⁺ T cells. The quadrants for Foxp3 were based on the isotype control antibody. This result is representative of 6 patients and 3 healthy donors.