Anti-CD25 Ab treatment alters cytokine production toward a Th1 phenotype. (a) Intracellular staining. LNC from PLP 139-151-immunized B10.S mice that were pretreated with anti-CD25 or rat IgG Ab were restimulated twice with PLP 139-151. Viable lymphoblasts were restimulated with anti-TCR and anti-CD28 Ab for 4-6 h. After staining with anti-CD4 Ab and 7-AAD, the frequency of IL-2-, IL-4-, IL-5-, IL-10-, and IFN-γ-secreting cells was determined in the live (7-AAD-) CD4+ subset by using flow cytometry. A representative sample from six individual experiments is shown. *, Isotype control for IL-2 and IL-10; **, Isotype control for IL-4 and IL-5 and IFN-γ. (b) Cytokine ELISA. Supernatants from the above cultures were analyzed by ELISA for production of IL-2, IL-4, IL-10, and IFN-γ. Each bar represents mean ± SEM values for a group of experiments (anti-CD25 Ab, n = 5; rat IgG, n = 6).