GFP expression in differentiated macrophages and DCs from human blood monocytes after LV transduction.

(A) Adherent monocytes enriched from PBL were induced in culture to differentiate into macrophage by M-CSF. Adherent cells were transduced with DR.GFP or EF.GFP twice in 2 days. For mock transduction, the medium used to collect LVs was added. Transduced cells were continuously cultured with M-CSF, and adherent cells were harvested 7 days after transduction to analyze GFP and MHC II (HLA-DR) expression (A). (B) For DC differentiation and maturation, adherent monocytes were first cultured with GM-CSF and IL-4. Cultured monocytes were transduced twice as before. Four days after transduction, motile cells were collected and cultured for 2 more days with TNF-α in addition to GM-CSF and IL-4. At day 8 of culture, transduced cells were harvested and stained by PE-conjugated antibodies to characterize GFP-transduced cells (B). In addition to MHC II staining (top panels), the DC phenotypes were further confirmed by the expression of a DC-specific marker CD83 (bottom right panels) and lacking of a monocyte/macrophage marker CD14 (middle panels). Quadrant setting was based on background staining by isotype-matched control antibodies. Percentages of various cell subsets in each quadrant are indicated. (C) Photomicrographs of DR.GFP-transduced DCs. After MHC II staining (B), DCs were spun onto glass slides and examined by fluorescence microscopy (left, × 1000 magnification) of DC morphology. Cells with GFP transgene expression (green to yellow, open arrows) within transduced cells and MHC II on cell surface (red, closed arrows) are illustrated. Background green fluorescence was seen in nuclei of all the cells. The remaining cytospin slides were stained with the Wright-Giemsa solution (right, × 400 magnification). Note that the staining diminished GFP fluorescence signals but it better revealed DC morphology.