The frequencies of tumor-reactive CD8 and CD4 T cells were increased in the spleens of recipient mice given CD25-depleted T cells presensitized to tumor antigens. The experimental design in Figure 1 was used, except all transplantation recipients were given 3 × 10⁶ presensitized donor T cells (ie, from tumor-vaccinated donors) with the HSC graft. The donor T cells were unmanipulated or depleted of CD25+ cells by immunomagnetic sorting. At days 11 and 18 after transplantation, (A) CD8⁺ or (B) CD4⁺ T cells were isolated from spleens and tested in IFN-γ ELISPOT assays using AGN2a or MHC class II⁺ AGN2a cells as stimulators, respectively. The data are representative of 3 replicate experiments. (C) The percentages of splenic CD25⁺Foxp3⁺ CD4 T cells were assessed on days 5, 8, 11, and 18 after HSC transplantation. Splenocytes from naive nontransplanted mice were analyzed as a control. Each bar is the mean value of 6-8 mice (± SD), and the data are combined from 2-3 experiments. (D) Spleens, lymph nodes, and tumors were harvested 8 days after HSC transplantation, processed into single-cell suspensions, and the percentages of CD8⁺ and CD4⁺Foxp3⁺ cells determined by flow cytometry. The data represent the mean values of 6 spleens and lymph nodes, 4 individual tumors. (E) Numbers of tumor-infiltrating CD8⁺ and Foxp3⁺CD4 T cells. The data represents the mean values of 4 individual tumors. Results were compared using the Student t test: *P < .05, **P < .01, ***P < .001.