The anti-GFP immune response in mice coinjected with LV-CMV-GFP and syngeneic wt nTregs. nTregs isolated from spleens of syngeneic wt C57BL/6 mice were sorted by magnetic beads, and (A, left panel) FACS profile of purified CD4⁺CD25⁺ T cells is shown (nTregs purity 82%). (A, right panel) nTregs' suppressive capacity was tested in vitro in coculture assay with CD4⁺CD25⁻ responder T cells stimulated with anti-CD3 mAb. (B) C57BL/6 mice were injected with 2 × 10⁶ purified nTregs the day before LV-CMV-GFP injection. Four weeks after injection, the anti-GFP immune response was analyzed in mice injected with vehicle (□), LV-CMV-GFP (▒), or LV-CMV-GFP + wt nTregs (▨). CD8⁺ T cells infiltrating the liver were detected by FACS after mechanical destruction of the tissue. (C) GFP-specific IFN-γ–producing CD8⁺ T cells present in the spleen were counted by ELISPOT. (D) GFP-specific antibodies present in the sera diluted 10 000-fold were measured by ELISA. Data are expressed as average (± SD). One representative experiment of 3 is presented (3 animals per group per experiment). (E) Liver sections were analyzed by confocal microscopy after immunostaining for GFP (green) and Topro-3 (blue).