A critical role for type I IFN in CpG-dependent reversal of tumor-specific T tolerance after transplantation. (A-C) Syngeneic transplantations were set up as described in Figure 1A. Fourteen days after transplantation, mice were vaccinated with either TNF-α–matured DC-HA or DC-Con with (CpG in vivo) or without (no CpG) coadministration of CpG in vivo. Some mice received DCs matured with CpG ex vivo followed by HA pulsing (CpG ex vivo). Seven days after vaccination, the mean absolute number (± SD; n = 4) of CD4⁺CD25⁺Foxp3⁺ T cells per spleen in each group is indicated (A). CD4⁺CD25⁺ T_Reg cells (Suppressor) were isolated by FACS sorting and assayed for their suppressive capacity on naive CD4⁺CD25⁻ T cells (Responder) at the indicated ratios of suppressor to responder (S:R) using an in vitro suppression assay. Cultures were labeled with [³H]thymidine and harvested for scintillation counting. Results are expressed as mean CPM ± SD (B). Sera were harvested 12 hours after administration of CpG and measured for IFN-α by ELISA (C). (D) Fourteen days after transplantation, mice were vaccinated with DC-HA or DC-Con coadministered with CpG in vivo. Six hours before and 24 hours after vaccination, mice were treated with neutralizing antibodies to mouse IFN-α and IFN-β (IFN-αβ Ab) or a control Ab. Seven days after vaccination, splenocytes were stained with anti-CD8, anti-Thy1.1, and anti–IFN-γ antibodies, and the mean percentage (± SD; n = 4) of IFN-γ–secreting HA-specific T cells among total CD8⁺ T cells is indicated (D). Representative results of 2 independent experiments are shown.