In vitro depletion of CD25⁺ T regulatory cells from adoptively transferred T cells enhances antitumor immunity. Tumor-bearing recipients (as in Figure 1) were imaged on day −1 to assess AGN2a-Rluc bioluminescence intensity. This allowed us to ensure that all mice in the experiments had comparable tumor burden levels before HSC transplantation and immunotherapy. (A) Survival curves for tumor-bearing recipients given HSC grafts consisting of 5 × 10⁶ bone marrow cells plus the indicated number (2 × 10⁶, 6 × 10⁶ or 12 × 10⁶) of nondepleted or CD25-depleted T cells from tumor-bearing nonvaccinated donors. The recipients were vaccinated as in Figure 1. A group of tumor-bearing recipients given bone marrow cells only (no T cells or vaccines) was included as controls. (B) Same experiment as panel A except the T cells coadministered with the graft were from tumor-bearing vaccinated (presensitized) donors. (C) Survival curves for tumor-bearing recipients given HSC grafts consisting of bone marrow cells plus 2 × 10⁶ unmanipulated T cells, CD25-depleted T cells, or T cells plus added CD25⁺ cells (at a 1:2 CD25⁺cell-to-T cell ratio; CD25-supplemented) from tumor-bearing nonvaccinated donors; or (C right panel) 0.67 × 10⁶ unmanipulated T cells, CD25-depleted T cells, or T cells plus added CD25⁺ cells (at a 1:2 CD25⁺cell-to-T cell ratio; CD25-supplemented) from tumor-bearing vaccinated (presensitized) donors. The recipients were vaccinated as in Figure 1. Survival curve comparisons were done using the log-rank test. The data represents the combined results of 3 independent experiments with a combined 10-15 mice per group.