CD4⁺CD25⁺ T_Reg cells are critical for posttransplantation tumor-specific T-cell tolerance. (A) Schematic view for a syngeneic transplantation model that used clone 4 HA-specific transgenic T cells. Donor mice were transferred with A20-HA on day −25 and naive clone 4 T cells on day −15. On day −10, recipients were injected with A20-HA. On day 0, splenocytes and bone stem cells were harvested from tumor-bearing donor mice. Recipient mice were irradiated and then injected with a graft composed of Lin⁻c-Kit⁺ stem cells and A20 lymphoma–purged splenocytes. (B) Purging of A20 lymphoma cells. The percentage of B220⁺ B cells before and after purging is indicated. (C) Syngeneic transplantations were set up using A20-HA, A20-WT, or without tumors (No tumor). Fourteen days after transplantation, mice were vaccinated with mature DCs pulsed with either HA peptide (DC-HA) or a control peptide (DC-Con). Seven days after vaccination, splenocytes were analyzed for activation of HA-specific CD8 T cells. The mean percentage (± SD; n = 4) of IFN-γ–secreting HA-specific T cells among total CD8 T cells is shown. (D-F) Syngeneic transplantations were set up with the use of A20-HA, A20-WT, or without tumors (No tumor). Seven days after transplantation, mice were treated with either 0.3 mg PC61 or rat Ig daily for 3 days. Fourteen days after transplantation, mice were vaccinated with DC-HA or DC-Con. Seven days after vaccination, splenocytes were analyzed for the percentage of CD4⁺CD25⁺Foxp3⁺T_Reg (D); the percentage of IFN-γ–secreting HA-specific T cells (Thy1.1⁺IFN-γ⁺) among total CD8 T cells in A20-HA bearing mice is shown (E); and the mean percentage (± SD; n = 4) of IFN-γ–secreting HA-specific T cells among total CD8 T cells is shown (F). Representative results of 3 independent experiments are shown.