CD4⁺CD25⁺ T_Regs suppress HCV-specific CD8⁺ T cells. (A) Frequency of CD25⁺ T cells in the blood of HCV-naive, HCV-recovered, and persistently HCV-infected chimpanzees. (B) Depletion of CD25⁺ T cells from PBMCs increased HCV-specific IFN-γ responses of HCV-recovered chimpanzees, especially if they had recovered from repeated HCV rechallenges. The data were calculated from duplicate cultures and are representative of 16 of 18 experiments, the remaining 2 of 18 experiments showed no increase in IFN-γ responses. In contrast, the increase of IFN-γ responses was weaker or absent in persistently HCV-infected chimpanzees (P < .01 when the increase in IFN-γ responses was compared between the recovered/rechallenged and the persistently HCV-infected chimpanzees). The data are representative of 12 experiments in persistently HCV-infected chimpanzees. Horizontal bold lines indicate the mean response. (C) Increase of HCV-specific IFN-γ responses after depletion of CD25⁺ T cells from PBMCs. The depleted T-cell populations exerted suppressor function when added back to the culture (Figure S3). (D) CD4⁺CD25⁺ T_Regs of an HCV-recovered chimpanzees inhibited IFN-γ secretion of autologous NS5B peptide-specific CD8⁺ T cells, did not produce IFN-γ and did not require CD4⁺CD25^– cells to exert suppressive function (for additional experiments, see Figure S3). APC indicates antigen-presenting cells (irradiated, CD4- and CD8-depleted PBMCs).