Bacteriophage lambda vectors are capable of transducing mammalian cells in vivo
, and the efficiency of this process is increased when mice are pre-immunized with lambda phage particles (Lankes et al., 2007
). To investigate the underlying basis for this observation, we developed an in vitro
model for the antibody-dependent enhancement of lambda phage-mediated gene expression. Our model comprised (i) a CV1 cell line stably transduced with human FcγRI and its associated γ-chain (FcγRI γ+ cells), plus (ii) phage-containing immune complexes that were incubated with these cells. To facilitate the quantitation of phage-mediated gene transfer, we took advantage of an available phage recombinant containing a luciferase-encoding mammalian expression cassette [λ(luc)]. These phage particles were then mixed with a rabbit antiserum directed against gpD, the major coat protein of bacteriophage λ, in order to generate phage-containing immune complexes ().
GpD-specific antiserum enhances λ-phage mediated luciferase expression in FcγRI positive CV1 cells
The gpD-specific antiserum was found to significantly enhance phage-mediated gene expression in FcγRI γ+ CV1 cells (which have previously been shown to express high levels of FcγRI on their surface; (Rodrigo et al., 2006
)), but not in parental CV1 cells (which do not express FcγRI). This enhancement was greatest at a low dilution of the antiserum (corresponding to an estimated molar ratio close to 1 antibody molecule per gpD monomer on the phage capsid), and declined with serial dilution of the antibody (). No enhancement was detected when phage was mixed with serum from the same rabbit, obtained prior to immunization with lamdba gpD protein (; labeled as “prebleed”). In light of this result, subsequent experiments were performed using phage-containing immune complexes that were prepared using a serum dilution of 1:5.
To confirm that Fc receptor binding was required for the antibody-dependent enhancement of phage-mediated gene transfer in FcγRI γ+ CV1 cells, a follow-up experiment was performed in which the Fc-binding domain of the receptor was saturated with a FcγRI specific monoclonal antibody (10.1) whose binding site overlaps with the Fc-binding site on the receptor. Cultures treated with this blocking antibody showed a reduced level of luciferase reporter gene expression following incubation with phage-containing immune complexes, when compared with non-treated FcγRI γ+ CV1 cells or cells that were treated with an irrelevant, isotype-matched control antibody ().
Antibody-dependent enhancement of λ-phage mediated gene expression in FcγRI-positive CV1 cells requires Fc receptor availability
This result demonstrates that antibody-dependent enhancement of phage-mediated gene transfer required the presence of a Fcγ receptor on the surface of the target cells. Thus, the enhancement of gene transfer by phage-immune complexes cannot be attributed to the formation of a dense DNA-phage precipitate that is more efficiently deposited on cell surfaces by centrifugation, or non-specifically internalized by target cells.
We next wished to know whether Fcγ receptors other than FcγRI might also be capable to permitting antibody-dependent enhancement of phage-mediated gene transfer. In particular, although FcγRI binds IgG isotypes with nanomolar affinity, Fc-binding is not restricted to this receptor, and other receptors such as FcγRIIA have been implicated in antibody-dependent enhancement of susceptible cells by dengue virus (Rodrigo et al., 2006
), and by adenovirus vectors (Leopold et al., 2006
). Experiments were therefore performed to assess the ability of other FcγR family members to support antibody-dependent enhancement of phage-mediated gene transfer. To do this, COS-7 cells were transiently transfected with a panel of mammalian expression plasmids encoding a series of FcγR family members, in the presence or absence of the receptor-associated γ-chain. Surface expression of FcγRs on transiently transfected COS-7 cells was verified by flow cytometric analysis (); γ-chain expression was confirmed by immunoblot analysis of cell lysates using a γ-chain specific antibody (). Stably-transfected CV1 cells expressing FcγRI and the γ-chain subunit were used as a positive control for γ-chain expression (, lane 2). In the case of the γ chain-associated receptors, FcγRI and FcγRIII, cell surface expression in the transiently transfected COS-7 cells was detected even in the absence of a co-expressed γ-chain cDNA (). This is consistent with previous data showing that FcγRI can be expressed on the cell surface without an absolute requirement for the common γ chain (van Vugt et al., 1999
FcγRs and associated γ chains are expressed in transiently transfected COS-7 cells
Having validated that the desired FcγRs were expressed on the surface of the transiently transfected COS-7 cells, and that cytoplasmic receptor-associated γ chain was also expressed, we performed gene transfer experiments using phage-containing immune complexes. As shown in , the only Fc receptor that supported antibody-dependent enhancement of phage-mediated gene transfer was FcγRI. Expression of the receptor-associated γ-chain was not required for this effect. In addition, the other FcγR family members that were studied (FcγRIIA, FcγRIIB and FcγRIII [+/- the γ-chain]) all failed to support antibody-dependent enhancement of phage-mediated gene transfer ().
Antibody-dependent enhancement of λ-phage mediated gene expression requires FcγRI, but not γ chain expression, and is not supported by other FcγR family members
To better understand the mechanisms involved in FcγRI-mediated, antibody-dependent enhancement of phage infectivity, we employed a panel of pharmacologic agents to dissect the intracellular mechanisms required for phage-mediated gene transfer. Drugs known to target clathrin-mediated endocytosis (chlorpromazine), actin polymerization (latrunculin A, cytochalasin D) and microtubules (nocodazole, taxol) were used in these experiments.
Chlorpromazine, an inhibitor of clathrin-coated pit formation at the plasma membrane, significantly decreased phage-mediated gene expression (), as did the actin polymerization inhibitors latrunculin A () and cytochalasin D (data not shown). In contrast, agents that targeted the microtubule network strongly augmented gene expression by phage-immune complexes. Both nocodazole (a microtubule-depolymerizing agent) and taxol (paclitaxel; a microtubule-stabilizing agent) augmented luciferase expression by roughly 10 to 50 fold, in comparison to non-treated cells (). These results suggest that the microtubule network plays an unexpected, inhibitory role in antibody-dependent enhancement of phage-mediated gene transfer.
Antibody-dependent enhancement of λ-phage mediated gene expression in FcγRI-positive cells is inhibited by chlorpromazine and latrunculin A, but augmented by nocodazole and taxol
The inhibitory effect of chlorpromazine on gene expression by phage-immune complexes (), suggested that these complexes may enter cells via an endocytic pathway. We therefore conducted two sets of followup experiments to confirm this.
First, we performed transduction experiments in the presence of well-characterized pharmacologic agents that inhibit endosomal acidification via different mechanisms. To do this, we used bafilomycin A, which is an inhibitor of the vacuolar H+
-ATPase (Bowman, Siebers, and Altendorf, 1988
), and monensin, which is a proton ionophore (Berg et al., 1983
; Harford et al., 1983
; Mollenhauer, Morre, and Rowe, 1990
). Both agents strongly inhibited gene expression by phage immune complexes (), without exerting overt cytotoxic effects ().
Antibody-dependent enhancement of λ-phage mediated gene expression in FcγRI-positive cells is prevented by inhibitors of endosomal acidification
Cell viability is unaffected by pharmacologic agents that modulate antibody-dependent enhancement of λ-phage mediated gene expression
Second, we conducted immunocytochemical staining experiments to determine whether internalized phage particles were colocalized with endosomal marker proteins. To do this, cells were transduced with phage-immune complexes for 10 minutes, and then fixed, permeabilized and stained with antibodies specific for the major phage coat protein (gpD) or for the early endosome associated protein (EEA1) (Mu et al., 1995
; Simonsen et al., 1998
). The results of this analysis, shown in , reveal colocalization of phage and EEA1 (see yellow punctate dots in the "composite" panel). Thus, both sets of followup experiments confirmed that phage-immune complexes enter FcγRI-bearing cells via an endocytic pathway.
Internalized λ-phage immune complexes colocalize with EEA1 in FcγRI-positive cells
Finally, we performed experiments to assess the efficiency of gene expression by phage-immune complexes. To do this, we took advantage of an available λ lysogen that contains a phage genome bearing a mammalian expression cassette encoding the enhanced GFP reporter gene [λ(GFP)] (Eguchi et al., 2001
). λ(GFP) phage were produced and phage-immune complexes were used to transduce FcγRI-bearing CV1 cells. The results, shown in , reveal that about 1.5% of the cell population expressed GFP following exposure to λ(GFP) phage-immune complexes.
Efficiency of gene transfer by λ-phage immune complexes
In order to directly compare the efficiency of gene transfer by phage immune complexes to a standard DNA transfection methodology, we also performed an experiment in which FcγRI-bearing CV1 cells were either exposed to λ(luc) phage-immune complexes or transiently transfected with purified λ(luc) DNA using a cationic lipid (Lipofectamine2000). This experiment was conducted in 96-well plates, using our standard phage MOI (106
) – thus, a total of 1010
phage particles were added into each well. To make the most direct possible comparison with phage-mediated gene transfer, cells were transiently transfected with an amount of purified λ(luc) DNA (500 ng) that corresponds to the DNA content of 1010
phage particles (Lankes et al., 2007
). Measurement of luciferase activity in lysates of transduced or transfected cells revealed that the efficiency of gene transfer by phage-immune complexes was within an order of magnitude of that of lipofectamine-mediated DNA transfection ().