Examination of the role and function of CD4⁺CD25⁺Foxp3⁺ Tregs in PGK.142T-mediated gene transfer. Mice were injected with the PC61 mAb (anti–mouse CD25, 1 mg/mouse) 5 days before treatment with PGK.142T. (A) At the time of vector injection, the number of Tregs was determined by FACS analysis of splenocytes immunostained for CD4, CD25 (clone 7D4), and Foxp3. (B) At 6 weeks after vector injection, mice were killed and GFP expression in the liver was monitored by confocal microscopy. GFP was visualized by anti-GFP staining (green), and nuclei by Topro-3 staining (blue). Also shown is the mean ± SD C/G from the livers of treated mice (n = 9). (C-D) The frequency of IFN-γ–producing, GFP-specific CD8⁺ T cells in the spleen (C) and liver (D) of treated mice was determined by ELISPOT assay. Data are expressed as the mean ± SD number of GFP-specific CD8⁺ T cells per 10⁶ total CD8⁺ T cells. For the spleen, analysis was performed on isolated CD8⁺ T cells, whereas for the liver, analysis was performed on total intrahepatic leukocytes, and the amount of CD8⁺ T cells was determined by FACS analysis (data not shown). The frequency of GFP-specific CD8⁺ T cells in the spleen (C) and liver (D) was determined by FACS analysis of splenocytes and intrahepatic leukocytes, respectively, stained with a H-2K^d pentamer loaded with the immunodominant epitope of GFP, HYLSTQSAL (GFP_200-208). A representative dot plot is shown from 1 of 3 separate experiments (n = 3/group per experiment; n = 9/vector). (E) Transaminase levels (ALT U/L) were determined in the sera of treated mice at the time of sacrifice; data are expressed as mean ± SD (n = 6/group). Normal ALT levels, detected in the sera of vehicle-injected mice, are 60 ± 40 U/L (n = 3, gray area).