Determination of the contribution of LSECs and hepatocytes to the stability of gene transfer. Mice were injected with either the hepatocyte-specific vector, ET.142T, or a LV-containing target sequence for miR-122 and miR-142, PGK.142T.122T. (A) Confocal fluorescent microscopy was performed on liver section to analyze the pattern of GFP expression (green). Sections were stained with anti-CD31 (red) to detect liver sinusoidal endothelial cells. Note that the majority of GFP-positive cells are also positive for CD31 (yellow), indicating that PGK.142T.122T expression is confined to endothelial cells. (B) Confocal fluorescent microscopy of the liver at 1 and 6 weeks after vector injection. GFP was visualized by anti-GFP staining, and nuclei by Topro-3 staining (blue). Scale bar = 100 μm. A representative image of 2 experiments (n = 3/group per experiment) is shown. (C-D) Leukocytes infiltrating the liver were isolated (C) 1 and (D) 6 weeks after vector administration, and the frequency of GFP-specific CD8⁺ T cells was evaluated by H-2K^d-GFP_200-208 pentamer staining. Data are expressed as the mean ± SD percentage of GFP_200-208-H2-K^d+CD8⁺ T cells. To the right, a representative dot plot, gated on CD8⁺ cells, is shown from 1 of 2 experiments (n = 3/group per experiment). (E) The frequency of CD4⁺CD25⁺Foxp3⁺ Tregs was quantified by FACS analysis. Data are expressed as mean ± SD percentage of positive cells. To the right, a representative dot plot, gated on CD4⁺ T cells, is shown from 1 of 2 experiments (n = 3/group per experiment).