Depletion of CD4⁺CD25⁺ T_Reg cells from the donor lymphocyte graft fails to overcome posttransplantation tolerance. (A) Syngeneic transplantations were set up as described in Figure 1A. On day 0, splenocytes were purged of A20-HA. Some A20-purged donor lymphocytes were further depleted of CD25⁺ T cells by microbead selection. Percentages of CD4⁺CD25⁺ T cells (left), and CD4⁺CD25⁺Foxp3⁺ and CD4⁺CD25⁻Foxp3⁺ T cells (right) before and after depletion are indicated. (B,C) On day 0, recipients were transferred with either T_Reg cell–depleted (Donor T_Reg depletion) or unmanipulated (no T_Reg depletion) donor lymphocytes. Some recipients that received an unmanipulated lymphocyte graft were depleted of T_Reg cells in vivo after transplantation by PC61 injection (Recipient T_Reg depletion) as described in Figure 1. Fourteen days after transplantation, mice were vaccinated with DC-HA or DC-Control. Seven days after vaccination, splenocytes were stained with anti-CD8, anti-Thy1.1, and anti–IFN-γ antibodies and subjected to FACS analysis. The percentage of IFN-γ–secreting HA-specific T cells (Thy1.1⁺IFN-γ⁺) among total CD8 T cells is indicated (B). The mean percentage (± SD; n = 4) of IFN-γ–secreting HA-specific T cells among total CD8 T cells is indicated (C). Representative results of 3 independent experiments are shown.