The anti-GFP immune response in mice coinjected with LV-CMV-GFP and GFP-tg nTregs. (A, left panel) nTregs isolated from spleens of GFP-tg mice were sorted by flow cytometry, and FACS profile of sorted CD4⁺CD25⁺GFP⁺ T cells is shown (GFP-tg nTregs purity 99%). (A, right panel) GFP-tg nTregs' suppressive capacity was tested in vitro in coculture assay with CD4⁺CD25⁻ responder T cells isolated from C57BL/6 mice and stimulated with anti-CD3 mAb. (B) C57BL/6 mice were injected with 2 × 10⁶ purified GFP-tg 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 + GFP-tg 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 2 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).