Fig. 5.

Anti-CD25 treatment decreases Treg and increases CD8⁺ T-cell infiltration in tumor. Mice were treated with B16-gp96 as described in Fig. 3 and killed for TIL analyses on day 12. a and b Tumor tissues were digested and the single-cell suspensions were subjected to discontinuous Percoll gradient centrifugation. The separated lymphocytes were analyzed by flow cytometry to identify CD4⁺CD25⁺Foxp3⁺ Treg (a) and CD8⁺CD3⁺ cells (b). c Ratio of CD8⁺ cells to Treg cells was calculated. d Consecutive paraffin sections of tumors were stained with anti-CD8 mAb and counterstained with hematoxylin. i, ii, iii, iv and v represent mice treated with PBS, Anti-CD25, gp96+IgG1, gp96+cy and gp96+Anti-CD25, respectively. Arrow indicates CD8⁺ T cells. Data show mean ± SD of seven mice. *P < 0.05 and **P < 0.01. Data are representative of two independent experiments