Induction of CD25 expression by T cells following IL-2 administration. Cryopreserved PBMCs were triple-stained with allophycocyanin-conjugated anti-CD4, PE-conjugated anti-CD25, and FITC-conjugated anti-CD3 antibodies. (A) The dot plots were gated on PI^-CD3⁺ T cells. The gate for CD4⁺CD25^hi T cells was drawn based on the top 4% of CD4⁺ T cells expressing high levels of CD25 in the peripheral blood of each patient prior to IL-2 therapy (PRE). The same gate was used for rebound and post-treatment samples for each patient. The number represents the percentage of CD4⁺CD25^hi T cells per total CD4 T cells. The gate for CD8⁺CD25⁺ (CD4^-CD3⁺) was based on the isotype control antibody, and the number represents the percentage of CD8⁺CD25⁺ T cells per total CD8 T cells. The percent induction of CD25 expression of CD4 (B) and CD8 (C) T cells in 8 patients were quantified by using the gate for CD4⁺CD25^hi and for CD8⁺CD25⁺ T cells, respectively. To enhance for consistency and accuracy, PBMCs from PRE, REB, and POST for each patient were stained and analyzed at the same time. Leukaphereses samples for POST were available only for 5 patients. Each symbol represents one patient. The P values were calculated using paired t test and adjusted for multiple comparisons.