FIG. 1.

Lentiviral vectors, transgene expression in vitro. (A) Lentiviral vector (pHRSINCSGW) expressing GFP under the SFFV promoter was used as a control. Experimental vectors were made by replacing SFFV with the dectin-2 gene promoter and GFP with NY-ESO-1 antigen. cPPT, central polypurine tract; WPRE, woodchuck hepatitis virus posttranscriptional regulatory element; LTR, long terminal repeat; RRE, Rev response element. (B) Human 293T cells and murine NIH 3T3 cells were transduced (MOI of 1) with SFFV-GFP or DEC-GFP lentivectors, and GFP expression was analyzed by FACS 72 h postinfection (solid profiles); nontransduced cells are shown as empty profiles. Numbers indicate percentage of transduced cells, MFI values, and the number of copies of transgene per cell (as determined by TaqMan PCR). (C) Bone marrow-derived DC (BM-DC) were transduced with SFFV-GFP or DEC-GFP lentivectors (MOI of 10), stained with anti-CD11c-PE antibody on day 6 postinfection, and then analyzed for GFP expression by FACS. Cell surface staining shows that 95% of the cell population was CD11c⁺. Numbers in the upper right quadrants indicate the percentage of GFP-expressing cells among the CD11c⁺ cells, with MFI values for GFP as 4,161 and 2,256 for the SFFV and dectin-2 promoters, respectively. This is a representative result out of four experiments performed. (D) Lentiviral transduction of primary human LC and dDC. Both cell populations were characterized by surface staining with cell-specific makers. The LC were CD45⁺ DR⁺ CD1a⁺ Langerin⁺ CD14⁻ and the dDCs were CD45⁺ DR⁺ CD1a^low CD14⁻ (not shown). Only DR staining is shown for the sake of simplicity. Cells were transduced with SFFV-GFP or DEC-GFP lentivectors (MOI of 60), and GFP expression was detected by FACS after gating on forward-scatter high HLA-DR⁺ cells in the preparation. Comparison of uninfected controls and cells exposed to heat-inactivated virus (HI) and live virus are shown. Numbers indicate percentages of transduced cells and MFI values. This experiment was performed 3 times with similar results. FSC, forward scatter; SSC, side scatter.