Ex vivo depletion of CD25⁺ cells from HSC grafts had the same impact on survival as in vivo CD4 cell depletion, but CD25 cell depletion did not compromise memory to tumor antigens. (A) Experimental design. (B) Survival curves are shown for mice given donor T cells (normalized to 1 × 10⁶ CD8 T cells per recipient for all experimental groups) presensitized to tumor antigens that were either unmanipulated or depleted of CD25⁺ or CD4⁺ cells ex vivo using immunomagnetic sorting. Some mice given unmanipulated T cells were treated with in vivo–depleting anti-CD4 mAb (250 μg intraperitoneally) on days −1, 2, 5, and 8. A group of mice given bone marrow only (no T cells or vaccines) was included as a control. (C) Tumor-free survivors from the experiment in panel B were rechallenged with 10⁵ live AGN2a tumor cells at day 60 after HSC transplantation and followed for another 60 days. The data were combined from 2 independent experiments, and the survival curves were compared using the log-rank test.