Correlative analysis of Treg functional activity and frequency of IL-2–expressing sorted CD4⁺ T cells. Sorted CD4⁺CD25⁻ T cells from the patient cohort were stimulated with phorbol myristate acetate and ionomycin for 5 hours in the presence of protein transport inhibitor, and intracellular expression of IL-2 was measured by flow cytometry. The frequency of IL-2-expressing CD4⁺ cells negatively correlated with the patients' Treg-suppressive activity at 1:1 ratio of CD4⁺CD25⁻ and CD4⁺CD25^hi (r = −0.47, P = .04).