Functional comparison of FOXP3⁺CD4 T cells in tumors versus peripheral blood. Tumor digests from patients and peripheral blood samples from healthy donors were thawed and immediately stimulated with PMA/I for 6 to 8 hours in the presence of monensin. Cells were subsequently stained with anti-CD3, anti-CD8, and anti-FOXP3 mAb along with either (A) anti–IL-2 or (B) anti–IFN-γ mAbs. The dotplots were gated on CD3⁺ T cells. The numbers represent the percentages of T cells in each quadrant. These results are representative of multiple independent experiments using tumor digest samples from different patients (n = 26) and PBL samples from healthy donors (n = 3) and the same patients (n = 5; Figure 5).