Fig. 4.

Anti-CD25 treatment enhances tumor-specific T-cell responses induced by B16-gp96 vaccine. Mice were treated as described in Fig. 3, and splenocytes were isolated for detection on day 12. a and b, Splenocytes of different treatment groups were stained with CD3, CD4, CD25 and intracellular Foxp3 (a) or with CD3, CD8 and intracellular IFN-γ (b) and detected by flow cytometric analysis. c Detection of murine tumor-specific T cells by IFN-γ ELISPOT assay. Splenocytes at 1 × 10⁶ cells/well were stimulated with 20 μg of B16 melanoma cell lysates or BSA as negative control for background evaluation. d Cytotoxicity assay. B16 cells were labeled with CFSE as the target cells and seeded into a 96-well U-bottom microtiter plate. Splenocytes were added at different effector/target ratios: 5:1, 10:1 and 20:1. After 4 h, PI staining was then assessed in the CFSE labeled target cell population to quantify apoptosis by flow cytometric analysis. Data show mean ± SD of seven mice. Student’s t-test was used to determine P values. *P < 0.05 and **P < 0.01. Data are representative of two independent experiments