Efficient tumor infiltration and augmentation of the intratumor effector T cell/T reg cell ratio depends on timing of anti-CD25 administration. Mice challenged with B16/BL6 tumor cells were either left untreated or received anti-CD25 mAb 4 d before or after tumor challenge, plus Gvax/αCTLA-4 on days 8 and 11 and then killed 14 d after challenge. (A) Analysis of frozen tumor sections by confocal microscopy. Anti–CD8-Alexa-488 (green), anti-CD31 PE (red), and anti-CD11c APC (blue) are shown in the top row. Anti–CD4-Alexa-488 (green), anti-CD31 PE (red), and anti-Foxp3 (blue) are shown in the bottom row. Bar, 40 μm. (B, C, and D) In parallel experiments, tumors were harvested, processed for enrichment of TILs, and analyzed by flow cytometry for the expression of CD8, Foxp3, and CD45.1 (pmel transgenic cells). Tumors were pooled for each group, and data show the ratio of CD8⁺ cells to Foxp3⁺ cells infiltrating the tumors (B), the number of total CD8⁺ cells per gram of tumor (C), and the number of CD8⁺ pmel transgenic cells per gram of tumor (D). In E, mice were treated as described in A and analyzed for the expression of ICAM (green), CD31 (red), and VCAM (blue) within the tumors 14 d after challenge. Bar, 40 μm. All images were acquired with a 20× water immersion objective. Data are representative of 3 independent experiments (n = 3 mice per group).