(A) CV1 cells stably expressing FcγRI and its associated • chain (FcγR1 γ+) were mixed with λ(GFP) particles or λ(GFP) immune complexes at a MOI of 10⁶. 48 hours later, cells were harvested and the number of cells expressing GFP was quantitated by flow cytometry. (B) CV1 FcγR1 γ+ cells were mixed with λ(luc) particles or λ(luc) immune complexes at a MOI of 10⁶, as described above. In parallel, CV1 FcγR1 γ+ cells were subjected to transient transfection with 500 ng of purified λ(luc) DNA using Lipofectamine2000. (A, B) The data represent mean values calculated from triplicate samples; error bars represent the standard deviation. The asterisks (***) denote a statistically significant difference from all other conditions (p value<0.001, one-way ANOVA).

Bacteriophage λ:IgG complex binds and cross-links FcγRI (1), leading to localization of the bound complex to clathrin-coated pits (2). Clathrin-mediated endocytosis of phage-containing immune complexes then occurs, in an actin-dependent fashion (3); drugs that block clathrin-mediated endocytosis (chlorpromazine) or actin polymerization (latrunculin A) therefore inhibit phage-mediated gene transfer. The receptor-associated γ chain (shown in green in (1)) is not required for receptor-mediated internalization of phage-immune complexes. Bacteriophage λ is then trafficked to an early endosome/phagosome (4). Phagosome maturation occurs via the transport of early vesicles along microtubules (5) to late compartments containing degradative enzymes that break down the phage in late endosomes/phagolysosomes (6); drugs that target microtubules (nocodazole, taxol) therefore augment bacteriophage-mediated transduction. Some partially or fully uncoated phage particles/genomes are presumed to escape the endosome via an undefined mechanism (7), leading to nuclear import of phage DNA, and subsequent expression of phage-encoded genes (i.e., luciferase) (8).

Luciferase encoding λ phage particles (λ luc) were pre-incubated with various dilutions of a rabbit antiserum raised against the major λ coat protein, gpD, or with a non-immune pre-bleed from the same animal. Immune complexes containing phage particles were then added to parental (white bars) or FcγRI γ+ expressing (black bars) CV1 cells, at a multiplicity of infection (MOI) of 10⁶. Forty-eight hours later, cells were harvested, and luciferase activity was measured in cell lysates. The results shown represent mean values calculated from triplicate samples; error bars represent the standard deviation. The data are representative of 3 independent experiments that yielded similar results. The asterisks (***) denote a statistically significant difference from control cells/conditions (p value < 0.001, one-way ANOVA).

Luciferase encoding λ phage particles either added directly to target cells (λ luc), or were preincubated with a gpD-specific rabbit antiserum (λ luc + gpD) prior to addition to target cells at a MOI of 10⁶. In some cases, the parental (white bars) or FcγRI γ+ expressing (black bars) CV1 cells were preincubated with 5 µg/ml of a FcγRI ligand-binding blocking antibody, 10.1 (αFcγRI), or IgG1 isotype-matched control antibody (IgG1 isotype) for 30 minutes prior to the addition of λ(luc) or λ(luc) + αgpD. Forty-eight hours later, cells were harvested, and luciferase activity was measured in cell lysates. The results shown represent mean values calculated from triplicate samples; error bars represent the standard deviation. The data are representative of 3 independent experiments that yielded similar results. The asterisks (***) denote a statistically significant difference from control cells/conditions (p value < 0.001, one-way ANOVA).

Luciferase encoding λ phage particles either added directly to target cells (λ luc), or were preincubated with a gpD-specific rabbit antiserum (λ luc + gpD) prior to addition to target cells at a MOI of 10⁶. Phage were then added to COS-7 cells that had been transiently transfected with mammalian expression plasmids encoding the indicated FcγR family members (either alone, or in combination with the common γ chain). Forty-eight hours after the addition of phage, cells were harvested, and luciferase activity was measured in cell lysates. The data represent mean values calculated from triplicate samples; error bars represent the standard deviation. The data are representative of 3 independent experiments that yielded similar results. The asterisks (***) denote a statistically significant difference from control cells/conditions (p value < 0.001, one-way ANOVA).

Luciferase encoding λ phage particles were preincubated with a gpD-specific rabbit antiserum (λ luc + gpD) prior to addition to target cells at a MOI of 10⁶. Phage were then added to parental CV1 cells (open bars) or to CV1 cells stably expressing FcγRI together with the accessory γ-chain subunit (black bars; FcγR1 γ+). Cells were pretreated with medium alone (no drug), bafilomycin A1 (20 nM and 100 nM) or monensin (20 µM), for 30 minutes at 37°C before the addition of bacteriophage. Forty-eight hours after the addition of phage, cells were harvested, and luciferase activity was measured in cell lysates. The data represent mean values calculated from triplicate samples; error bars represent the standard deviation. The asterisks (***) denote a statistically significant difference from control cells (no drug) (p value < 0.001, one-way ANOVA).

Luciferase encoding λ phage particles were preincubated with a gpD-specific rabbit antiserum (λ luc + gpD) prior to addition to target cells at a MOI of 10⁶. Phage were then added to parental CV1 cells (open bars) or to CV1 cells stably expressing FcγRI together with the accessory γ-chain subunit (black bars; FcγR1 γ+). Cells were pretreated with (A) latrunculin A (2.5 µM), or chlorpromazine (5 µg/ml), or (B) nocodazole (5 µM) or taxol (1 µg/ml and 20 µg/ml), for 30 minutes at 37°C before the addition of bacteriophage. As controls, some cells were preincubated in the absence of any exogenous drug, or with DMSO alone. Forty-eight hours after the addition of phage, cells were harvested, and luciferase activity was measured in cell lysates. The data represent mean values calculated from triplicate samples; error bars represent the standard deviation. The data are representative of 3 independent experiments that yielded similar results. The asterisks (**, ***) denote a statistically significant difference from control cells/conditions (p value < 0.05[**] or p value < 0.001[***], one-way ANOVA).

Mammalian expression plasmid vectors encoding cDNAs corresponding to FcγRI, FcγRIIA, FcγRIIB, FcγRIII, or the common γ-chain subunit were transiently transfected into COS-7 cells using Lipofectamine-2000. Cells were collected 48-hours later and subjected to flow cytometric analysis for cell surface FcγR expression (A) or immunoblot analysis for γ chain expression (B). (A) Flow histograms are presented for COS-7 cells that were transiently transfected with expression plasmids encoding the indicated FcγR family members (CD16, CD32A, CD32B and CD64); in some cases, cells were also co-transfected with a plasmid encoding the common γ chain (CD16γ; CD64γ). In each panel, the shaded histogram represents receptor expression on non-transfected COS-7 cells, the dashed line denotes control staining with an irrelevant isotype-matched antibody, and the open histogram bounded by the thick solid line represents surface staining for the respective FcγR family member. (B) An immunoblot analysis of common γ chain expression in the transfected COS-7 cells is shown. Briefly, lysates were prepared from γ chain transfected cells, and equal quantities of cell protein were then electrophoretically separated under reducing conditions, on a 15% SDS polyacrylamide gel. Proteins were then transferred to nitrocellulose, and the membrane was probed with a γ chain-specific antibody. Lane 1 represents molecular weight markers (numbers denote the size of selected marker proteins in kilodaltons), while lanes 2–4 show γ-chain expression in lysates from various transfected cells. Shown are analyses of lysates from: the stably transfected FcγRI γ+ CV1 cell line (lane 2); COS-7 cells transiently transfected with the CD64 expression plasmid plus the γ chain expression plasmid (lane 3); or COS-7 cells transiently transfected with the CD16 expression plasmid plus the γ chain expression plasmid (lane 4). The expected molecular weight of the common γ-chain is ~10kD.

Luciferase encoding λ phage particles were preincubated with a gpD-specific rabbit antiserum (λ luc + gpD) prior to addition to CV1 cells stably expressing FcγRI together with the accessory γ-chain subunit (FcγR1 γ+) at a MOI of 10⁶. Cultures were incubated with phage-immune complexes at 4°C for one hour to allow binding, and then internalization was permitted by a brief (10 min) incubation at 37°C. Cells were then fixed, permeabilized and subjected to immunocytochemical staining. To do this, they were reacted with a polyclonal chicken antiserum directed against early endosome associated (EEA1) protein (Zymed Laboratories) and a polyclonal rabbit antiserum directed against the major phage capsid protein, gpD. Bound antibodies were then detected using an Alexa488 -conjugated goat anti-chicken IgG (Molecular Probes) and a Texas Red-conjugated donkey anti-rabbit IgG (Jackson ImmunoResearch). After washing, bound antibodies were visualized using a Nikon C1 visible light scanning confocal microscope. All images were taken using a 60x/1.4NA objective. A volume-rendered three-dimensional reconstruction of a series of optical sections taken at 0.3 µm steps in the Z dimension was calculated by the Nikon EZ-C1 analysis software. A representative 0.3 µm Z-stack confocal image, taken through the plane of the cell, is shown. The left panel shows immunostaining for EEA1 (green), the center panel shows staining for phage (red) and the right panel shows a composite image of the two. Note the abundant yellow staining, indicating colocalization of phage and EEA1.