Functional comparison of FOXP3⁺CD4 T cells in tumor versus peripheral blood in the same patient. Tumor digests and peripheral blood samples from patients with metastatic melanoma 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 anti–IL-2, anti–IFN-γ, anti–TNF-α, or anti–IL-10 mAbs. The dotplots were gated on CD3CD4 (CD3⁺CD8⁻) T cells. The numbers represent the percentages of CD4 T cells in each quadrant. These results are representative of multiple independent experiments using tumor digest samples from different patients (n = 8) and PBL samples from the same patients (n = 5).