Monitoring the transgene-specific immune response in mice injected with PGK.142T. BALB/c mice were intravenously injected with LV PGK or PGK.142T encoding GFP. (A) Confocal fluorescent microscopy analysis showing GFP (green) expression patterns in the liver at the indicated times. Scale bar = 100 μm. (B) The frequency of IFN-γ–producing, GFP-specific CD8⁺ T cells in the spleen of treated mice was determined by ELISPOT at the indicated times. Data are expressed as the mean ± SD number of GFP-specific CD8⁺ T cells per 10⁶ total CD8⁺ T cells. Measurement of the GFP⁺ cell-specific killing capacity of CTL derived from treated mice was performed by chromium release assay using different ratios of CD8⁺ effector T cells: P815 target cell line (P815 cell line, BALB/c syngeneic mastocytoma-derived cell line, was in vitro transduced to stably express GFP, or untransduced as control). (C) Measurement of the GFP-driven killing capacity of liver-derived CD8⁺ T cell; 10:1 ratio is reported. (D) Measurement of the GFP⁺ cell-specific killing capacity of spleen CD8⁺ T cell. The 100:1 ratio of effector:target cells is shown. Data are expressed as the mean ± SD percentage of target cell lysis. (E-F) The frequency of GFP-specific CD8⁺ T cells in the liver (E) and spleen (F) was determined by FACS analysis of intrahepatic leukocytes and splenocytes, respectively, stained with a pentamer loaded with the H-2K^d immunodominant epitope of GFP, GFP_200-208. Analysis was performed 6 weeks postinjection (n = 3/group). A representative dot plot is shown. Below, the mean ± SD of the experiment is provided. (G) Quantification of the absolute number of CD8⁺ T cells infiltrating the liver 6 weeks after LV injection. Shown as the mean ± SD (n = 3/group).