Characterization of the anti-GFP immune response in C57BL/6 mice injected with LV-CMV-GFP. C57BL/6 mice were injected with LV-CMV-GFP and were killed 1, 2, 4, and 6 weeks after injection. Liver sections from C57BL/6 mice were analyzed by confocal microscopy, after immunostaining for GFP (green) and Topro-3 (blue), 2 weeks (A, left panel) and 4 weeks (A, right panel) after LV administration. Original magnification, × 400. (B) CD8⁺ T cells infiltrating the liver of LV-CMV-GFP–injected mice were detected by FACS after mechanical destruction of the tissue. Data are expressed as average of percentage of CD8⁺ T cells gated on lymphocytes (± SD). One representative experiment of 6 is presented (3 animals per group per experiment). Dotted line represents the average of CD8⁺ T cells detected in livers of vehicle-injected mice (± SD, gray area) (n = 8). (C) CD8⁺ T cells infiltrating the liver of C57BL/6 mice killed at 4 weeks after LV-CMV-GFP injection were detected by confocal immunofluorescence analysis of liver sections immunostained with anti-GFP (green) and anti-CD8 (red) mAbs. The image is representative of 15 images analyzed from tissues of 2 mice. Original magnification, × 1000. (D) CD8⁺ T cells in the spleen of LV-CMV-GFP–injected mice were detected by FACS. Data are expressed as average of percentage of CD8⁺ T cells gated on lymphocytes (± SD). One representative experiment of 6 is presented (3 animals per group per experiment). Dotted line represents average of CD8⁺ T cells detected in spleens of vehicle-injected mice (n = 8) (± SD, gray area). (E) GFP-specific IFN-γ–producing CD8⁺ T cells present in the spleen of LV-CMV-GFP–injected mice were counted by ELISPOT. Data are expressed as average of CD8⁺ GFP-specific T cells of 1 × 10⁶ total CD8⁺ T cells (± SD). One representative experiment of 6 is presented (3 animals per group per experiment). (F) GFP-specific antibodies present in the sera of LV-CMV-GFP–injected mice were measured by ELISA. Absorbance of 1:10 000 dilution are shown as average (± SD). One representative experiment of 6 is presented (3 animals per group per experiment). Dotted line represents the cut off, which was calculated as average OD of vehicle-injected mice (± 3 SD). Based on the experimental data, (G) depicts the kinetics of GFP expression in immunocompetent mice after LV-CMV-GFP injection (green solid line). As soon as 1 week after LV injection, the anti-GFP cellular immune response takes place (gray dotted line) followed by the humoral immune response (red solid line), which mediates clearance of GFP⁺ cells 6 weeks after vector administration.