Fig. 1.

Anti-CD25 mAb treatment increases T-cell responses in mice immunized with gp96–peptide complexes. a Anti-CD25 mAb treatment reduces the number of CD4⁺CD25⁺Foxp3⁺ Treg cells. BALB/c mice were injected with either 30 μg of anti-CD25 mAb or control IgG1 mAb, and 3 days later, splenocytes were stained with CD3, CD4, CD25 and intracellular Foxp3. CD25 and Foxp3 expression of CD3⁺CD4⁺ cells are shown by flow cytometric analysis. b–f BALB/c mice were immunized three times with gp96–peptide complexes or peptide (PBS) as control, and an additional gp96-immunized group received two intravenous injections of anti-CD25 mAb at 2-week interval, starting 3 days prior to gp96 immunization (five mice per group). Two days after the third immunization, flow cytometric analysis was performed to identify CD4⁺CD25⁺Foxp3⁺ Treg (b), IFN-γ-producing CD8⁺ (c) and CD4⁺ T cells (d) in spleen and lymph node, and CD8⁺CD3⁺ cells in spleen were further analyzed for MHC class-1/K^d Pentamer (e). In the meantime, splenocytes at 1 × 10⁶ cells/well were stimulated with HBcAg_87–95 peptide and analyzed by IFN-γ ELISPOT assay (f). Data show mean ± SD of five mice. Student’s t-test was used to determine P-values. *P < 0.05 and **P < 0.01. Data are representative of three independent experiments