Isolated splenocytes and MLN cells from CD25-depleted or control non-depleted RV infected mice (n=3, 21 dpi) were pooled and cultured in anti-CD3 mAb (5.0 µg/ml) precoated culture plates to analyze cell proliferation (4 day assay) and IFN-γ production (16 h assay). For the splenocyte proliferation assay (A), BrdU solution was added to the culture for the last 6 h and single cells were extracellularly stained with anti-mouse CD4 or CD8 PE Ab and then intracellularly stained with anti-mouse BrdU FITC Ab. Total cells (100,000) were gated on lymphocytes and the percentage of BrdU+ cells among the indicated lymphocyte subsets (CD8, CD4) is represented in the upper right quadrant. The dot plots represent one of two independent experiments. For the intracellular IFN-γ staining assay (B), single cells were extracellularly stained with anti-mouse CD4 or CD8 Ab and then intracellularly stained with anti-mouse IFN-γ PE Ab. For analysis, cells (100,000) were gated on CD4+ or CD8+ lymphocytes. Values shown in each plot reflect the percentage of splenocytes or MLNs cells expressing IFN-γ. The dot plots represent one of three independent experiments.