HUMAN GENE THERAPY 10:775± 786 (March 20, 1999) Mary Ann Liebert, Inc.
Efficient Gene Delivery into Human Dendritic Cells by Adenovirus Polyethylenimine and Mannose Polyethylenimine Transfection SANDRA S. DIEBOLD, 1 HEIKE LEHRMANN,2 MARGARETHA KURSA,3 ERNST WAGNER,3 MATT COTTEN,2 and MARTIN ZENKE1
ABSTRACT Gene-modified human dendritic cells (DCs) were generated by transfection with adenovirus polyethylenimine DNA (Ad/PEI/DNA) and mannose polyethylenimine DNA (ManPEI/DNA) complexes. Ad/PEI/DNA complexes have plasmid DNA bound to adenovirus particles by PEI and deliver DNA into cells via the adenovirus infection route. Such transfection complexes yield high transduction levels and sustained expression of luciferase and green fluorescent protein reporter genes and were almost as effective as recombinant adenovirus vectors. ManPEI/DNA complexes rely on uptake by receptor-mediated endocytosis via mannose receptor, which is highly expressed on DCs. While gene delivery by ManPEI/DNA complexes was less efficient than by Ad/PEI transfection, incorporation of adenovirus particles in ManPEI/DNA transfection complexes further enhanced transduction efficiencies and transgene expression. We also demonstrate that Ad/PEI-transfected DCs are competent in stimulating T cell proliferation in allogeneic and autologous mixed lymphocyte reactions, and in activating T cells from T cell receptor (TCR)-transgenic mice in an antigen-specific manner. Thus, the present study establishes the following relative order of transduction efficiencies of viral and nonviral gene delivery systems for primary human DCs: recombinant adenovirus . Ad/PEI 5 Ad/ManPEI . ManPEI . PEI. Ad/PEI and ManPEI transfection modes represent particularly versatile transduction systems for DCs, with ManPEI being built up exclusively of synthetic compounds.
INTRODUCTION
OVERVIEW SUMMARY Dendritic cells (DCs) are professional antigen-presenting cells that represent a particularly attractive cell type for use in immunotherapy of diseases, such as cancer. In peripheral organs such as skin, DCs are exposed to a variety of pathogens such as viruses and bacteria, which they capture through specific cell surface receptors. The present study capitalizes on using such surface receptors for gene delivery into DCs by receptor-mediated endocytosis. We demonstrate that polyethylenimine/DNA (PEI/DNA) complexes can be transduced effectively into primary human DCs in the form of adenovirus or mannose PEI/DNA transfection complexes.
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(DCs) are professional antigen-presenting cells (APCs) that occur in peripheral organs, where they are exposed to and capture antigens. They then migrate to the lymphoid tissue and present processed antigens in the context of major histocompatibility complex (MHC) class I and II molecules to elicit an antigen-specific T cell response (Peters et al., 1996; Cella et al., 1997; Austyn, 1998; Banchereau and Steinman, 1998). Other cell types, such as B cells and macrophages, are also competent in capturing and presenting antigens; however, DCs are much more effective and they are unique in their ability to prime naive T cells. Accordingly, DCs express high levels of MHC class I and II, and of the costimulatory mole-
1 Max-Delbrück-Center
for Molecular Medicine, D-13125 Berlin, Germany. for Molecular Pathology, A-1030 Vienna, Austria. & Co. GmbH, A-1121 Vienna, Austria.
2 Institute 3 Bender
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cules B7.1 and B7.2 (CD80 and CD86, respectively) that are required for antigen presentation. Given their unique antigen-presenting properties, DCs represent a particularly attractive cell type for immunotherapy of diseases such as cancer (Girolomoni and Ricciardi-Castagnoli, 1997; Schuler and Steinman, 1997; Tuting et al., 1997; Austyn, 1998; Banchereau and Steinman, 1998). Therefore peptide/protein-pulsed or gene-modified DCs have been used in experimental model systems of cancer and shown to induce strong anti-tumor immune responses (Boczkowski et al., 1996; Celluzzi et al., 1996; Condon et al., 1996; Paglia et al., 1996; Zitvogel et al., 1996; Ribas et al., 1997; Song et al., 1997a; Specht et al., 1997; and references therein). The application of genemodified DCs is especially appealing since an ever-increasing number of tumor cell-specific and/or associated antigens have been identified and molecularly cloned (Boon et al., 1997). In addition, in contrast to peptide/protein pulsing, transduction of DCs has the potential advantage that multiple and/or undefined peptide epitopes are expressed and presented, possibly in the context of both MHC class I and class II, and with any MHC allele. Furthermore, gene modification of DCs offers the opportunity to express, in addition to tumor-specific antigens, chemokines and cytokines that attract T cells and/or modulate T cell responses, or affect DC viability and function. However, so far efficient gene transfer into primary immunocompetent DCs has remained difficult. Standard gene delivery techniques including calcium phosphate coprecipitation, DEAE-dextran transfection, or electroporation were found to be rather inefficient and the application of cationic lipids for gene delivery was associated with high unspecific cytotoxicity resulting in low transgene expression (Arthur et al., 1997; Diebold et al., 1997). Recently recombinant retrovirus and adenovirus vectors have been successfully used for transduction of DCs (Aicher et al., 1997; Bello-Fernandez et al., 1997; Gong et al., 1997; Ribas et al., 1997; Song et al., 1997a; Specht et al., 1997; Dietz and Vuk-Pavlovic, 1998; Westermann et al., 1998; and references therein). While residing in peripheral organs, DCs are exposed to a variety of viral and bacterial pathogens that they capture through specific cell surface receptors. We argued that such surface receptors, including adenovirus or mannose receptors (Jiang et al., 1995; Sallusto et al., 1995; Steinman and Swanson, 1995; Avrameas et al., 1996; Cella et al., 1997), might represent ideal targets for delivery of DNA into DCs by receptormediated endocytosis. In this article we tested this idea and demonstrate that polyethylenimine/DNA (PEI/DNA) complexes can be effectively transduced into primary human DCs as adenovirus PEI/DNA transfection complexes yielding high transduction levels and sustained transgene expression. Mannose PEI conjugates that are exclusively built of synthetic compounds were also synthesized and shown to be effective in gene delivery into DCs.
MATERIALS AND METHODS Preparation and cell cultures of DCs Buffy coats from healthy donors were obtained from the local blood bank and used for preparation of human DCs ac-
cording to published procedures (Romani et al., 1994, 1996; Sallusto and Lanzavecchia, 1994; Bender et al., 1996). Briefly, peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Hypaque centrifugation (density 1.077 g/cm3; Eurobio, Paris, France) followed by T cell depletion with aminoethylthiouronium bromide (AET; Sigma, St. Louis, MO)-treated sheep red blood cells. The T cell-depleted cell fraction was then depleted of B cells and residual T cells using anti-CD19 and anti-CD2 ferromagnetic beads (Dynabeads M-450 Pan-T, CD2 and Pan-B, CD19; Dynal, Great Neck, NY). Cells were then cultured in RPMI 1640 medium (GIBCO-BRL, Gaithersburg, MD) containing 10% inactivated fetal calf serum (FCS; GIBCO-BRL) or 2.5% inactivated autologous serum, 2 mM glutamine, and penicillin and streptomycin (100 U/ml) in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF, 500 U/ml; kindly provided by Novartis, Vienna, Austria) and interleukin 4 (IL-4, 500 U/ml; kindly provided by Schering-Plough, Kenilworth, NJ) at 37°C in a 5% CO2 atmosphere for 6±10 days. This cell population routinely contained 96±98% DCs as determined by flow cytometry and was used for transfection on days 7±9 of culture. For generation of mouse DCs, bone marrow suspensions were prepared from 8- to 12-week-old C57BL/6 mice as described (Inaba et al., 1992). Cells were cultured in RPMI 1640 medium (GIBCO-BRL) containing 10% inactivated FCS (GIBCO-BRL), 50 m M 2-mercaptoethanol, and penicillin and streptomycin (100 U/ml) in the presence of recombinant mouse GM-CSF (300 U/ml; kindly provided by Novartis). The nonadherent cell fraction consisting of DCs was harvested on day 7 of culture and was used for transfection.
Flow cytometry DCs were analyzed for expression of specific surface markers by flow cytometry. Briefly, DCs were incubated in staining buffer (phosphate-buffered saline [PBS] plus 1% bovine serum albumin [BSA], fraction V; Sigma) containing 1% human IgG (Beriglobin [Behringwerke, Marburg, Germany]; 30 min at 4°C) for human DCs to block unspecific binding; unconjugated or fluorescein isothiocyanate (FITC)-labeled rat and mouse monoclonal antibodies were added and cells were incubated for 1 hr at 4°C. Samples containing unlabeled antibodies were then stained with FITC- or phycoerythrin (PE)-conjugated goat antimouse or anti-rat antibody (Sigma) for 45 min at 4°C. Cells were washed three times, resuspended in staining buffer and propidium iodide (PI, 2 m g/ml; Sigma) for gating on viable cells, and analyzed by flow cytometry using a FACScalibur devise with CELLQuest software (Becton Dickinson, Mountain View, CA). The following antibodies were employed: MHC class I (HLA-A, -B, -C, clone G46-2.6; PharMingen, San Diego, CA), MHC class II (HLA-DQ, clone SPVL3 [Immunotech S.A., Marseille, France] and HLA-DR, clone CR3/43 [Dako, Carpinteria, CA]), CD3 (anti-LEU-4, clone SK7; Becton Dickinson), CD14 (IOM2, clone RM052; Immunotech), CD19 (HD37; Dako), CD71 (Ber-T9; Dako), CD80 (B7/BB1, clone MAB104; Immunotech), CD83 (clone HB15A; Immunotech), and CD86 (B70/B7-2, clone 2331; PharMingen) for staining of human cells and I-Ab,d,q/I-Ed,k (ATCC No. TIB-102), H-2Db (clone KH95; PharMingen), CD44 (Pgp-1, clone IM7; PharMingen), CD54 (ICAM-1, clone 3E2; PharMingen), CD80 (B7-1, clone
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Ad/PEI AND ManPEI TRANSFECTION OF DCs 1G10, PharMingen), and DEC-205 (NLDC-145, ATCC No. HB-290) for analysis of murine cells. Mannose receptor expression was determined by reacting cells with FITC-conjugated mannosylated BSA (Sigma) as described (Avrameas et al., 1996). For determination of cell viability after transfection, cells were washed and resuspended in PBS plus 1% BSA and analyzed by flow cytometry after addition of PI.
Plasmid DNA and recombinant adenovirus vectors The following reporter constructs were used: pCluc plasmid (Plank et al., 1992) encoding the Photinus pyralis luciferase gene, pEGFP-C1 (Clontech, Palo Alto, CA) bearing a mutated variant of green fluorescent protein (GFP), and pHook-1 (Invitrogen, San Diego, CA) encoding a single-chain variable fragment (sFv) antibody to a specific hapten that allows selection of pHook-1-expressing cells by magnetic bead affinity purification. The construct pcDNA3-OVA encoding the chicken ovalbumin gene was prepared by cloning the EcoRI±XbaI fragment of pGEM-OVA (Boczkowski et al., 1996) into the pcDNA-3 vector (Invitrogen). Expression of ovalbumin was confirmed by transient transfection of K562 cells followed by immunoprecipitation and immunoblotting with polyclonal antiOVA and monoclonal anti-OVA antibody (Sigma), respectively. Plasmid DNA was prepared by alkaline lysis followed by Triton X-114 purification to remove lipopolysaccharide (LPS; Cotten et al., 1994a). The recombinant E1-defective Ad5 vector AdLuc1 contains the P. pyralis luciferase gene in the BamHI site of the pDE1sp1B vector (Bett et al., 1994). AdLuc1 was grown on 293 cells and high-titer virus was purified by banding twice in CsCl as described (Cotten et al., 1993a). AdGFP is a recombinant E1-defective Ad5 virus containing the GFP cDNA of pEGFP-C1 (Michou et al., 1998); preparation of AdGFP virus was the same as for AdLuc1. Viral titers were determined by UV absorbance measurement of extracted viral DNA (1 absorbance unit at 260 nm equals 1012 virus particles per milliliter; Chardonnet and Dales, 1970).
Transfection of adenovirus/PEI/DNA complexes Wild-type adneovirus type 5 (Ad5) and the E4-defective Ad5 strain dl1014 (E42 Ad) were used (Baker and Cotten, 1997; Baker et al., 1997). Biotinylation and psoralen-inactivation of dl1014 adenovirus were done as previously described (Cotten et al., 1994b). For formation of Ad/PEI/DNA complexes low molecular weight polyethylenimine (PEI, MW 2000; Aldrich, Milwaukee, WI) was used. Briefly, 25 m l of 10 mM PEI stock solution prepared as described (Boussif et al., 1995) was diluted in 250 m l of 20 mM HEPES, pH 7.4. This solution was mixed with 6 m g of plasmid DNA in 250 m l of 20 mM HEPES, pH 7.4, and incubated for 20 min at room temperature. After addition of active or psoralen-inactivated adenovirus (0.2±5 m l, 2.5 3 109 adenovirus particles per microliter in 150 mM NaCl, 20 mM HEPES [pH 7.4], 40% [v/v] glycerol; Baker and Cotten, 1997; Baker et al., 1997) samples were incubated for an additional 20 min at room temperature; 250-m l aliquots of Ad/PEI/DNA solution were added to 0.5 3 106 DCs in 500 m l of serum-free medium in 24-well plates. After 4 hr transfection medium was replaced by complete culture medium and cells
were incubated under standard culture conditions. All transfections were done in duplicate and reporter gene expression was measured after various periods of time starting on day 1 after transfection.
Transfection of mannose PEI/DNA complexes Mannose PEI (ManPEI) conjugates were synthesized similarly as described for mannose polylysine conjugates (Erbacher et al., 1996). The ManPEI conjugates tested had mannose linked to PEI via a phenylisothiocyanate bridge using mannopyranosylphenylisothiocyanate (manITC; Sigma) as coupling reagent. ManPEI consisted of 25-kDa (low molecular weight) PEI or 800-kDa (high molecular weight) PEI (Aldrich) containing PEI/manITC at a 1.4:1 or 1.3:1 weight ratio, respectively. This represents an average modification of every tenth (25 kDa) or ninth (800 kDa) PEI nitrogen with mannose. For generation of transfection complexes, 4 m g of plasmid DNA in 300 m l of HEPES-buffered saline (HBS: 150 mM NaCl, 20 mM HEPES [pH 7.4]) was mixed with various amounts of ManPEI conjugate in 300 m l of HBS and incubated at room temperature (20 min). For formation of Ad/ManPEI/DNA complexes active and psoralen-inactivated adenovirus particles (see above) were added and samples were incubated for an additional 20 min at room temperature. ManPEI/DNA or Ad/ManPEI/DNA complex solution (300 m l) was added to 0.5 3 106 cells in 500 m l of serum-free culture medium in 24-well plates. After 4 hr transfection medium was replaced by complete culture medium. Culture and further processing of cells were the same as for Ad/PEI transfection.
Reporter gene assays Luciferase assays were performed as previously described (Disela et al., 1991). Briefly, cells were washed once with PBS and then lysed in 100 m l of 0.25 M Tris buffer, pH 7.5, by three cycles of freeze and thaw. Thirty microliters of lysate was recovered and measured for luciferase activity (Lumat LB9501; Berthold, Wildbad, Germany). Luciferase activity was normalized for protein content. All values represent means of two transfection samples with the standard deviations indicated. For detection of GFP expression by flow cytometry, cells were washed, resuspended in PBS plus 1% BSA, and analyzed using a FACScalibur device with CELLQuest software (Becton Dickinson). PI staining was employed for gating on viable cells (see above). For selection of transgene-expressing cells the Capture-Tec system (Invitrogen) was used. Human DCs were transfected with E42 Ad/PEI and pHook-1 plasmid DNA, and on day 1 after transfection pHook-1-expressingcells were selecetd by magnetic bead affinity purification according to the manufacturer specifications. pHook-positive DCs were allowed to recover by culturing cells for 1 day in standard growth medium and were then used as stimulator cells in mixed lymphocyte reaction (MLR) assays.
Mixed lymphocyte reaction Human DCs cultured in RPMI 1640 medium and 2.5% inactivated autologous serum plus GM-CSF and IL-4 were recovered on day 6±10 of culture and used as stimulator cells.
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Transfected DCs were used in MLRs either without selection on day 1 after transfection or after selection for pHook-1-expressing cells by magnetic bead affinity purification and shortterm culture (see above) on day 2 after transfection. Responder cells were autologous or allogeneic T cells obtained after T cell rosetting with AET-treated sheep red blood cells and depletion of MHC class II-expressing cells by immunomagnetic bead purification (Dynabeads M-450 Pan human HLA class II; Dynal). DCs were irradiated (5000 rad) and seeded with increasing cell numbers in 96-well microtiter plates containing 105 responder cells/well. On day 5 of coculture 1 m Ci of [3 H]thymidine (Amersham, Arlington Heights, IL) was added per well, cells were harvested 6 hr later, and [3H]thymidine incorporation was measured in a Microbeta counter (Wallac, Turku, Finland). All values represent means of triplicates.
Detection of ovalbumin-specific T cell stimulation CD81 T cells from OT-I mice express a transgenic T cell receptor (TCR) that recognizes OVA257±264 peptide on H-2Kb (Hogquist et al., 1994; Kurst et al., 1996). Splenocytes of OTI mice were prepared and CD81 T cells were obtained by immunomagnetic bead purification using MACS anti-CD8 microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) according to manufacturer instructions. CD81 T cells were then cocultured with irradiated mouse DCs (5000 rad) in 96-well microtiter plates. Transfected DCs were used on day 1 after transfection; untreated DCs and DCs pulsed with 0.5 m M OVA257±264 peptide (SIINFEKL) were employed as controls. As a further control T cells were stimulated with phorbol myristate acetate (PMA, 25 ng/ml) and ionomycin (1 m g/ml). After 2 days of culture supernatant was harvested and tested by enzyme-linked immunosorbent assay (ELISA) for IL-2 production (R&D Systems, Minneapolis, MN). On day 5 of coculture 1 m Ci of [3H] thymidine (Amersham) was added per well, cells were harvested 6 hr later, and [3H]thymidine incorporation was measured in a Microbeta counter (Wallac). All values of [3H]thymidine incorporation represent means of triplicates.
Previous studies demonstrated that polyethylenimine (PEI) effectively condenses DNA and that such PEI/DNA complexes can efficiently transfect many cell types (Boussif et al., 1995; Abdallah et al., 1996). More recently, adenovirus (Ad) particles have been included in the PEI/DNA transfection complexes both to improve DNA uptake and to extend the range of transfected cell types (Baker and Cotten, 1997; Baker et al., 1997; Meunier-Durmort et al., 1997). Accordingly, Ad/PEI/DNA complexes containing a luciferase reporter gene under control of the cytomegalovirus (CMV) promoter/enhancer were generated as described (Baker and Cotten, 1997; Baker et al., 1997). Briefly, PEI and luciferase plasmid DNA were mixed to form PEI/DNA complexes, followed by addition of adenovirus particles to generate the Ad/PEI/DNA transfection complexes (in the following referred to as Ad/PEI/luc). Such Ad/PEI/DNA complexes contain 1±20 PEI/DNA complexes bound per adenovirus particle (Baker et al., 1997). Under standard transfection conditions cells were incubated with 300±3000 Ad/ PEI/DNA transfection complexes per cell (corresponding to 10±100 plaque-forming units per cell if active virus is used). Wild-type Ad5 (wtAd) and the replication-defective E42 Ad5 mutant dl1014 (E42 Ad) were used for production of wtAd/PEI/luc and E42 Ad/PEI/luc complexes, respectively. In some experiments psoralen-inactivated E42 Ad was employed to evaluate the role of gene expression from carrier virus on reporter gene activity. PEI/DNA complexes without adenovirus particles and a luciferase encoding E1-defective Ad5 virus (AdLuc1; Baker and Cotten, 1997; Baker et al., 1997) served as
RESULTS Transfection of Ad/PEI/DNA complexes Human DCs were generated from peripheral blood monocytes in the presence of GM-CSF and IL-4, following standard procedures (Romani et al., 1994; Sallusto and Lanzavecchia, 1994). Starting on day 5 of culture cells exhibited the typical morphology of DCs (Fig. 1A) and expressed high levels of MHC (class I and II) and CD1a, and of the costimulatory molecules B7.1 and B7.2 (CD80 and CD86, respectively; Fig. 1B). A subpopulation of cells also expressed CD83, a marker for mature DCs. As expected the proportion of CD831 cells was further increased after treatment with monocyte-conditioned medium (Bender et al., 1996; Romani et al., 1996) that induces DC maturation (data not shown). No expression of myeloid or B and T cell-specific surface markers (CD14, CD19, and CD3, respectively) was found. In addition, cells were fully competent in stimulating T cell proliferation in allogeneic mixed lymphocyte reactions (MLRs; see below). Cells on day 7±9 of culture were routinely used for transfection.
FIG. 1. Morphology and cell surface marker expression of DCs. (A) Two representative DCs on day 7 of culture are shown. (B) DCs on day 7 of culture were reacted with specific antibodies as indicated and analyzed by flow cytometry. The open areas represent staining with control antibody.
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FIG. 2. Ad/PEI transfection of DCs. (A) DCs were transfected with Ad/PEI/luc complexes using 2-kDa PEI and 300, 1000, and 3000 adenovirus particles per cell of wtAd, E42 Ad, and psoralen-inactivated E42 Ad as indicated (wtAd/PEI/luc, E42 Ad/PEI/luc, and E42 Ad*/PEI/luc, respectively). AdLuc1 (1000 particles/cell) and 2-kDa PEI/luc complexes were used as control. Luciferase activity was determined on day 1 after transfection. (B) DCs were transfected with Ad/PEI/GFP complexes using 2-kDa PEI and 5000 adenovirus particles per cell of wtAd, E42 Ad, and psoralen-inactivated E42 Ad as indicated (wtAd/PEI/GFP, E42 Ad/PEI/GFP, and E42 Ad*/PEI/GFP, respectively). AdGFP (1000 particles/cell) and 2-kDa PEI/GFP complexes were used as control. The percentage of GFP-expressing cells determined by flow cytometry on day 1 after transfection is shown. experimental controls. DCs were treated with Ad/PEI/luc complexes and analyzed for luciferase activity 1 to 8 days later. It was found that Ad/PEI/luc transfection complexes containing either wtAd or E42 Ad were very effective in delivering DNA into DCs, yielding about 0.2±6 3 106 light units/mg protein (Fig. 2A). Psoralen inactivation of the adenovirus component in E42 Ad/PEI/luc reduced luciferase activity by about 30- to 50-fold. In addition, an increasing number of transfection complexes per cell (300, 1000, and 3000 complexes/cell) led to an increase in transgene expression. As observed previously, gene delivery by PEI alone was very low and high transduction efficiencies were seen for the recombinant adenovirus AdLuc1 (Fig. 2A; Diebold et al., 1997). We also note that Ad/PEI transfection of DCs was most effective when cells were transfected after 4±10 days of in vitro culture in GM-CSF plus IL-4. For freshly isolated cells and cells at day 1±3 of culture, transfection efficiencies were low owing to an increased rate of cell death (data not shown). Next we determined the proportion of transgene expressing cells by employing Ad/PEI/DNA transfection complexes containing green fluorescent protein (GFP) expression vector (Ad/PEI/GFP). Complexes were generated and transfected into DCs, using the same transfection conditions as for luciferase vector (see above). DCs were then analyzed for GFP expression by flow cytometry. Ad/PEI/GFP transfection complexes containing wtAd or E42 Ad yielded 9.6 and 9.5% GFP-positive cells, respectively, on day 1 (Fig. 2B). Only a marginal reduction in the number of GFP-expressing cells was observed within the following 4 days, which is in accordance with the sustained luciferase expression shown below (Fig. 3). In addition, an increasing number of transfection complexes per cell (300, 1000, and 3000 complexes/cell) led to an increase in the number of GFP-positive cells (data not shown), which is consistent with the increase in luciferase expression seen above (Fig. 2A). However, high virus load compromised cell viabil-
ity (see below). For Ad/PEI/GFP transfection complexes, containing psoralen-inactivated E42 Ad, the number of GFP-positive cells obtained was rather low (0.8% on day 1) and further decreased in the following 4 days (0.1%). This finding indicates that inactivation of E42 Ad reduces transduction efficiencies, which might be because the reporter plasmid contains a CMV promoter whose stimulation by adenovirus proteins is compromised in psoralen-inactivated viruses. A recombinant Ad5 GFP virus (AdGFP; Michou et al., 1998), used as experimental control, yielded 15.8 and 36% GFP 1 DCs on day 1 (1000 and 3000 particles/cell, respectively; Fig. 2B and data not shown). Increasing the number of AdGFP virus particles per cell (5000 and 10,000 particles/cell) further increased the proportion of GFP-expressing cells to 60±80%, which was, however, associated with augmented cytotoxicity, probably due
FIG. 3. Kinetics of transgene expression following Ad/PEI transfection. DCs were transfected with Ad/PEI/luc complexes using 3000 adenovirus particles per cell of wtAd, E42 Ad, and psoralen-inactivated E42 Ad (E42 Ad*). AdLuc1 (1000 adenovirus particles/cell)-treated cells were used as control. Luciferase activity was determined on days 1, 2, 4, 6, and 8 after transfection.
780 to high virus levels (data not shown). As expected, PEI/GFP complexes were inactive and no GFP-positive cells were found (Fig. 2B). To monitor sustained transgene expression, luciferase activity was measured after various periods of time after transfection of Ad/PEI/luc complexes. Transfection complexes with active wtAd or E42 Ad adenovirus components showed high luciferase expression that was similar for wtAd and E42 Ad and remained virtually constant over the period of analysis (8 days; Fig. 3). Ad/PEI/luc complexes containing psoralen-inactivated E42 Ad virus exhibited a reduced transgene expression, which declined further in the following days. This finding is in accordance with the decline in the number of GFP-positive cells observed after transfection of the respective E42 Ad/PEI/GFP complexes (see above), indicating that the loss of gene expression from carrier virus might influence reporter gene activity. Current experiments aim at addressing this question in more detail. AdLuc1-treated DCs expressed, as expected, very high luciferase levels that remained constant for more than 15 days (Fig. 3 and data not shown). Thus Ad/PEI/DNA transfection complexes with active wtAd and E42 Ad are efficient in delivering genes into human DCs, yielding high transduction levels and sustained transgene expression, and were only about 10fold less efficient than recombinant adenovirus vectors. In many gene delivery systems effective transgene expression is severely compromised by cytotoxic side effects of the compounds applied. This is particularly evident for the primary human DCs used in this study, and it has so far hampered the application of standard transfection reagents, such as LipofectAMINE and other cationic lipids, calcium phosphate, or DEAE dextran, for gene transfer into DCs (Arthur et al., 1997; Diebold et al., 1997). To determine quantitatively potential cytotoxic effects of Ad/PEI transfection, DC viability was determined on days 1, 2, 4, and 6 posttransfection by staining nonviable cells with propidium iodine (PI) and analyzing by flow cytometry.
FIG. 4. Cell viability following Ad/PEI transfection. DCs were transfected with Ad/PEI/GFP complexes containing wtAd and E42 Ad (3000 adenovirus particles/cell) and with PEI/GFP complexes as control. DCs treated with AdGFP virus (1000 particles/cell) are also shown. Cell viability on days 1, 2, 4, and 6 following transfection was determined by PI staining and flow cytometry. The proportion of viable cells (percent of total cell number) is shown with viability of untreated cells set at 100%. One representative experiment of four is shown. Standard deviation of duplicates was less than 5%.
DIEBOLD ET AL. Following transfection of Ad/PEI/GFP complexes, cell viability was 87 and 84% of untreated DCs for transfection complexes containing wtAd or E42 Ad (3000 complexes/cell), respectively (day 1; Fig. 4). This reduction in cell viability is most probably the combined effect of PEI and adenovirus. Transfection of PEI/DNA complexes, which were essentially inactive in DNA delivery (see above), affected cell viability to some extent and was observed for both luciferase and GFP-bearing transfection complexes. In addition, while Ad/PEI transfection with 3000 transfection complexes per cell only marginally affected cell viability, increasing the number of virus particles in transfection complexes further compromised cell viability (data not shown). As shown in Fig. 4, on days 2, 4, and 6 after transfection viability of E42 Ad/PEI/DNA-transfected cells remained high and unchanged throughout the period of analysis, while on day 6 viability of wtAd/PEI/DNA transfected cells was somewhat reduced (56%). Thus, optimal transfection conditions that include efficient and sustained transgene expression and also high and long-lasting cell viability were achieved with E42 Ad-containing transfection complexes.
Transfected DCs stimulate T cell proliferation To investigate whether transfection of Ad/PEI/DNA complexes affects functional properties of DCs, transfected cells were analyzed (1) for expression of various cell surface molecules that are involved in antigen presentation and (2) for their ability to stimulate T cell proliferation in MLR assays. DCs were treated with Ad/PEI/DNA transfection complexes containing E42 Ad and GFP reporter plasmid (to gate on GFP 1 and GFP 2 cells), and analyzed for expression of MHC class I and II, and of the costimulatory molecules B7.1 and B7.2, by flow cytometry. As expected and shown in Fig. 5, untreated DCs expressed high levels of MHC class I and II, B7.2 (CD86) and of various cell adhesion molecules including LFA-1, VLA4, and ICAM-1 (CD54); expression of B7.1 was low to moderate, presumably because in the experiment shown autologous serum was used. Most importantly, following transfection MHC class I and II, B7.1, and B7.2 expression of GFP 1 cells was the same as for GFP 2 cells, for control (untreated) cells, and for AdGFP-treated DCs. In addition, similarly treated but luc-transfected DCs, using wtAd/PEI and E42 Ad/PEI, were analyzed over a period of 5 days posttransfection. It was found that surface marker expression remained high and unchanged in comparison with untreated and AdLuc1-transduced control cells throughout the period of analysis (data not shown). Transfected cells were then investigated for their capacity to induce T cell activation in MLR assays. DCs were transfected with E42 Ad/PEI/GFP complexes or, as controls, treated with AdGFP virus or left untreated (Fig. 6A and B). On day 1 following transfection GFP expression was verified by flow cytometry and DCs were seeded in MLR assays. Figure 6A shows that transfected DCs are competent in stimulating T cell proliferation in allogeneic MLR assays, although this activity was reduced by 40±50% with respect to control (untreated) DCs. AdGFP-treated DCs also showed a reduced activity in allogeneic MLR assays. We note that in this experiment Ad/PEI/GFP transfection complexes contained a higher number of adenovirus particles than that used for AdGFP (3000 and 1000 particles/cell, respectively), which might well explain the
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FIG. 5. Cell surface marker expression of DCs following Ad/PEI transfection. DCs were transfected with E42 Ad/PEI/GFP complexes (5000 adenovirus particles/cell) or treated with AdGFP virus (1000 particles/cell). Control, untreated DCs. Cells on day 1 after transfection were reacted with specific antibodies as indicated and analyzed by flow cytometry with gating on GFP 1 (gray areas) and GFP 2 cells (open areas), respectively.
FIG. 6. Transfected DCs stimulate T cell proliferation in MLR assays. (A and B) DCs were transfected with active E42 Ad/PEI/GFP complexes (3000 adenovirus particles/cell) or treated with AdGFP virus (1000 particles/cell). Control, untreated DCs. Cocultures with allogeneic (A) and autologous T cells (B) were started on day 1 after transfection, using various responder/stimulator cell ratios (T:APC) as indicated. [3H]Thymidine (TdR) incorporation was measured on day 5 of coculture. Results are means of triplicate values. (C and D) DCs were transfected with E42 Ad/PEI/DNA complexes containing pHook-1 plasmid DNA, and on day 1 after transfection pHook-1-expressing cells were selected by magnetic bead affinity purification. After selection cells were seeded in allogeneic and autologous MLR assays (C and D, respectively) as described above. Controls, untreated DCs and DCs alone as indicated. Note that the GFP and pHook transfections were done with DC preparations from different donors, which may well explain the different proliferation rates observed.
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different effects on MLR activity. Such a conclusion would also be in line with studies of adenovirus-transduced mouse DCs, where an increase in the multiplicities of infection compromises T cell activation in MLR assays (Gong et al., 1997). Interestingly, Ad/PEI/GFP-transfected DCs and AdGFP-transfected control DCs were also active in autologous MLR (Fig. 6B), presumably owing to the presence of viral proteins. To further extend these results, DCs were transfected with E42 Ad/PEI and pHook-1 plasmid DNA encoding a singlechain variable fragment (sFv) antibody specific to a hapten that permits selection of transduced pHook-1 sFv-expressing cells by magnetic bead affinity purification. Following transfection and purification of sFv-positive DCs, cells were allowed to recover for 1 day and then analyzed in MRL assays. Figure 6C and D show that transfected sFv hook-positive DCs were active in both allogeneic and autologous MLR assays. In the allogeneic MLR [3H]thymidine incorporation was lower than observed for untreated cells, probably owing to the extensive manipulations during cell purification and/or the presence of magnetic beads that could not be removed after selection and that might compromise DC activity. In conclusion, our results demonstrate that Ad/PEI transfection does not compromise expression of several cell surface molecules involved in antigen presentation and hence, transfected DCs are competent in stimulating T cell proliferation.
Transfected mouse DCs stimulate TCR-transgenic T cells Mouse DCs were routinely transfected on day 7 of culture, when cells expressed high levels of MHC class I and II, and of the costimulatory molecule B7.1 (data not shown). Transfection of E42 Ad/PEI/luc complexes into mouse DCs yielded high luciferase expression levels, similar to human DCs (Fig. 7A). To investigate whether transfected DCs are fully functional in stimulating T cells in an antigen-specific manner, cells were transfected with a plasmid encoding the model antigen chicken ovalbumin (OVA), using E42 Ad/PEI/DNA complexes. Starting on day 1 after transfection, irradiated DCs were cocultured with freshly isolated CD81 T cells from TCR-transgenic OT-I mice that recognize OVA257±264 peptide on MHC class I H2Kb. IL-2 production was determined after 2 days and T cell proliferation was measured after 5 days of coculture. It was found that Ad/PEI/OVA-transfected DCs effectively stimulated OVA-restricted T cell proliferation whereas untreated or GFPtransfected DCs did not (Fig. 7B). In addition, OVA-transfected DCs were only 1 log less efficient in T cell stimulation than DCs that were pulsed with the respective peptide. In further support of this observation, OVA-transfected DCs induced OTI CD81 T cells to produce high levels of IL-2 (600 pg/ml) while IL-2 levels induced by GFP-transfected control cells were close to background (16 pg/ml).
Transfection of ManPEI/DNA complexes While the studies described above demonstrate the capacity of Ad/PEI/DNA transfection complexes and of recombinant adenoviruses to deliver genes efficiently into DCs, there is clearly a need for nonviral, fully synthetic delivery systems that selectively target DCs (see Discussion). DCs express on their cell surface high levels of mannose receptor (Fig. 8A) that is
FIG. 7. Antigen-specific stimulation of T cells with genemodified DCs. (A) Mouse DCs were transfected with Ad/PEI/luc complexes using 2-kDa PEI and 300, 1000, and 3000 active E42 Ad particles/cell. AdLuc1 (1000 particles/cell) and 2-kDa PEI/luc complexes were used as control. Luciferase activity was determined on day 1 after transfection. (B) DCs were transfected with E42 Ad/PEI/OVA and E42 Ad/PEI/GFP complexes (3000 adenovirus particles/cell). Cocultures with spleenic CD81 T cells from TCR transgenic OT-I mice were started on day 1 after transfection. [3H]Thymidine (TdR) incorporation was measured on day 5 and means of tripilcate values for the responder-to-stimulator ratio of 1:3 are shown. Positive controls, coculture of T cells with peptide-pulsed DCs (0.5 m M OVA257±264 peptide), and PMA plus ionomycin-treated T cells. Negative controls, coculture of T cells with untreated DCs, and T cells only. used for uptake of mannosylated antigens (Jiang et al., 1995; Sallusto et al., 1995; Steinman and Swanson, 1995; Avrameas et al., 1996; Cella et al., 1997). We reasoned that mannose receptor might represent a suitable entry site for delivery of DNA into DCs via receptor-mediated endocytosis by employing appropriate mannose PEI (ManPEI) conjugates and using a strategy that was successfully applied before for gene delivery via transferrin receptor (Cotten et al., 1990, 1993a; Wagner et al., 1990, 1992; Zenke et al., 1990; Kircheis et al., 1997). In related studies mannosylated polylysine conjugates have been
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FIG. 8. Mannose receptor expression and ManPEI transfection of DCs. (A) DCs on day 7 of culture were analyzed for mannose receptor expression by reaction with FITC-conjugated mannosylated BSA at 37°C and analysis by flow cytometry. Control (open area) reaction with FITC-conjugated mannosylated BSA at 4°C. (B) DCs were transfected with ManPEI/luc, PEI/luc, and Ad/ManPEI/luc complexes containing 25-kDa PEI and 3000 adenovirus particles/cell of active or psoralen-inactivated E42 Ad (E42 Ad and E42 Ad*, respectively) or wtAd as indicated. To optimize for efficient transgene expression various conjugate-to-DNA ratios were analyzed as indicated (1:2, 1:1, and 2:1). For PEI/luc the optimized conjugate-to-DNA ratio of 1.5:1 was used (data not shown; and Boussif et al., 1995). In Ad/ManPEI/luc transfection complexes the conjugate-toDNA ratio was 1:1.
used for targeted gene delivery into monocyte-macrophages (Erbacher et al., 1996; Ferkol et al., 1996). In our experiments PEI conjugates were generated, thereby taking advantage of the higher transfection potential of PEI as compared with polylysine (Boussif et al., 1995). Several ManPEI conjugates were synthesized by reductive amination with mannobiose or by coupling with mannosyl-phenylisothiocyanate, and tested for efficient gene transfer into DCs. Finally, a ManPEI conjugate that contained a phenylisothiocyanate bridge for linking the mannose moiety to 25-kDa PEI was selected for further analysis. ManPEI/DNA transfection complexes containing the luciferase reporter gene (in the following referred to as ManPEI/luc) were prepared and applied to DCs, and 1 day later cells were analyzed for luciferase activity. In these experiments an ManPEI conjugate-to-DNA ratio of 1:1 was found to be optimal (Fig. 8B). PEI alone was about 50-fold less efficient than ManPEI. In addition, in control experiments the application of mannosylated bovine serum albumin (ManBSA) specifically reduced luciferase expression by ManPEI/luc conjugates, indicating that ManBSA and ManPEI/luc complexes effectively compete for a limited number of mannose receptor molecules present on the cell surface (data not shown). BSA alone did not compete, thus demonstrating that binding of ManBSA and tar-
geted gene delivery by ManPEI conjugates are receptor specific. However, under optimal conditions transfection efficacies by ManPEI/luc complexes were still about 10- to 100-fold lower than those by Ad/PEI/luc transfection complexes (Figs. 2A and 8B). Essentially the same result was obtained for ManPEI/luc transfection complexes containing 800-kDa PEI (data not shown). To determine whether incorporation of adenovirus particles into ManPEI/luc complexes would augment gene delivery and hence yield elevated luciferase activities, Ad/ManPEI/luc complexes were generated with active and psoralen-inactivated E42 Ad, and wtAd. DCs were transfected by employing otherwise unaltered experimental conditions as described above and 1 day later analyzed for luciferase expression. As shown in Fig. 8B, incorporation of adenovirus (3000 particles/cell) augmented gene transfer efficacy of ManPEI conjugates by 10- to 100-fold and the luciferase activities measured were similar to those observed for Ad/PEI transfection. Thus, while ManPEI conjugates allow targeted gene delivery into DCs via mannose receptor, the efficacy of transfer can be further increased by incorporating adenovirus particles into the complex. Current experiments address the question whether, following endocytosis of ManPEI/ DNA complexes via mannose receptor, adenovirus particles act by facilitating DNA release from the endosomal compartment (as observed, e.g., for transferrin±polylysine complexes; Curiel et al., 1991; Wagner et al., 1992; Cotten et al., 1993a,b) or, alternatively, whether they contribute to transfection complex uptake via the adenovirus internalization route.
DISCUSSION Gene-modified DCs represent particularly promising tools for immunotherapy of various diseases (Girolomoni and Ricciardi-Castagnoli, 1997; Schuler and Steinman, 1997; Tuting et al., 1997; Banchereau and Steinman, 1998). Here we describe an efficient DNA delivery system for human DCs based on Ad/PEI and ManPEI transfection. Ad/PEI/DNA complexes contain plasmid DNA that is condensed and bound to adenovirus carrier via PEI, and thus deliver DNA into DCs by the adenovirus infection route. Such Ad/PEI/DNA transfection complexes were found to exhibit high transduction efficiencies and to yield sustained transgene expression in DCs. In contrast, ManPEI/DNA transfection complexes represent a fully synthetic, nonviral delivery system that capitalizes on gene transfer by receptor-mediated endocytosis via surface-bound mannose receptor that is highly expressed in DCs. Transgene expression by ManPEI/DNA complexes was lower than that obtained by Ad/PEI/DNA complexes, but incorporation of adenovirus particles into transfection complexes further enhanced gene transduction and expression. Thus the present study establishes the following relative order of transduction efficiencies for DCs: recombinant adenovirus . Ad/PEI 5 Ad/ManPEI . ManPEI . PEI. Human DCs can readily be obtained from peripheral blood monocytes by in vitro differentiation in the presence of GMCSF and IL-4 (Romani et al., 1994; Sallusto and Lanzavecchia, 1994; see also review by Peters et al., 1996; Cella et al., 1997; Austyn, 1998; Banchereau and Steinman, 1998). Other DC types isolated from bone marrow, CD341 peripheral blood stem
784 cells, and lymphoid precursors were also described (Peters et al., 1996; Cella et al., 1997; Austyn, 1998; Banchereau and Steinman, 1998). DCs from peripheral blood monocytes are particularly attractive for potential use in medical therapy, since they can be easily obtained in large numbers and are highly competent in antigen presentation. However, gene transfer into DCs has remained difficult. Standard techniques were found to be mostly inefficient and associated with high unspecific cytotoxicity that results in low transgene expression (Arthur et al., 1997; Diebold et al., 1997). In addition, DCs from peripheral blood monocytes are largely postmitotic and difficult to infect with recombinant retroviruses that require replicating cell populations. Thus, several sequential rounds of infection with virus were employed to yield good transduction efficiencies (Aicher et al., 1997; Westermann et al., 1998). Alternatively, bone marrow cells or CD341 stem cells, which exhibit higher proliferative potential, were used for retroviral infection and then differentiated into DCs (Henderson et al., 1996; Reeves et al., 1996; Bello-Fernandez et al., 1997). Again, several sequential rounds of infection were routinely employed for efficient transduction. More recently adenovirus vectors were applied, since they also effectively infect mostly quiescent DCs (Arthur et al., 1997; Diebold et al., 1997; Ribas et al., 1997; Song et al., 1997a; Dietz and Vuk-Pavlovic, 1998) and, furthermore, can act as adjuvants to boost T cell responses against heterologous transgenes (Rodrigues et al., 1997; Song et al., 1997a,b). In this study we took advantage of the high transduction efficiencies into DCs observed for adenovirus and recombinant adenovirus vectors (Arthur et al., 1997; Diebold et al., 1997; Gong et al., 1997; Ribas et al., 1997; Song et al., 1997a). However, rather than driving transgene expression from promoters within the adenoviral genome, plasmid DNA is bound to the outside of adenovirus particles via PEI (Baker and Cotten, 1997; Baker et al., 1997). In such Ad/PEI/DNA transfection complexes PEI serves both as a DNA-condensing agent and as linker for binding the PEI/DNA complex to virus particles through charged interactions with negative domains on the viral hexon. The Ad/PEI transfection method is particularly versatile and offers several advantages over conventional adenovirus vectors: (1) transgene expression does not rely on a transcriptionally active viral genome and, thus, genetically and chemically inactivated adenovirus can be employed (such as the psoralen-inactivated E4-defective dl1014 adenovirus mutant used in this study); (2) the same adenovirus component can be used to transduce different plasmid DNAs, thereby abolishing the need to prepare different adenovirus vectors for each individual cDNA to be transfected; (3) even large DNA molecules (e.g., bacterial artificial chromosomes [BACs]), more than 100 kb in size, can be effectively transfected to ensure long-lasting transgene expression (Baker and Cotten, 1997); (4) different plasmids can be transduced simultaneously. This might be particularly desirable when, e.g., tumor-specific antigens are to be expressed together with chemokines and cytokines that attract T cells and stimulate T responses, and/or prolong DC viability, to enhance antigen presentation (e.g., in cancer patients, who are often immunosuppressed). In preliminary experiments we found that Ad/PEI transfection of IFN-g and IL-2 expression vectors leads to cytokine production by DCs (S. Diebold, E. Wagner, and M. Zenke, unpublished, 1998). The impact of cytokine-transduced human DCs in enhancing T cell activation in vitro has been
DIEBOLD ET AL. shown (Westermann et al., 1998). Finally, we emphasize that Ad/PEI transfection did not compromise expression of MHC class I and II, and of the costimulatory molecules B7.1 and B7.2, and hence transfected DCs were active in allogeneic and autologous MLR, and most importantly in activating T cells from TCR-transgenic mice in an antigen-specific manner. While a single dose of adenovirus- or retrovirus-transduced DCs can be effective and sufficient for immunization (Song et al., 1997a; Specht et al., 1997), repeated vaccination, if required, might be hampered by antiviral immune responses. In this instance, nonviral gene delivery systems, which are most likely not immunogenic or exhibit low immunogenicity, are more suited for transduction of DCs. As a first step in this direction we have explored the possibility of delivering DNA into DCs by ManPEI/DNA complexes, which are built exclusively of synthetic compounds. DCs express high levels of mannose receptor and ManPEI/DNA transfection complexes are readily taken up by receptor-mediated endocytosis. Gene transduction and expression of a luciferase reporter were lower than observed for Ad/PEI/DNA complexes but enhanced by incorporating adenovirus particles into the complex, indicating that the system still needs to be further optimized. An obvious question is whether the transduction efficiencies achieved with ManPEI/DNA complex alone are in fact sufficient for expression of, e.g., tumor-specific or -associated antigens, for instance, simultaneously with immunomodulatory cytokines (see above), to elicit an antigen-specific T cell response. Experiments to address this question are currently in progress. The Ad/PEI and ManPEI transfection systems described in this article represent a particularly versatile gene delivery system for human DCs and should be useful for generation of gene-modified DCs to be used in medical therapy and to study DC biology and function.
ACKNOWLEDGMENTS We thank Novartis (Vienna, Austria) for recombinant human and mouse GM-CSF and Schering-Plough (Kenilworth, NJ) for recombinant human IL-4. We are most grateful to R. Holzhauser for conjugate synthesis; to F.R. Carbone, M. Lutz, and G. Schuler for OT-I mice; to R. Förster for advice on flow cytometry; to T. Blankenstein, C. Esslinger, A. Pezzutto, J. Westermann, and G. Wolff for careful review of the manuscript; and to I. Gallagher for expert secretarial assistance. This work was funded in part by a grant from the Deutsche Forschungsgemeinschaft (DFG, SFB 506) to M.Z. S.D. was funded by a grant from the MDC Gene Therapy Program.
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Address reprint requests to; Dr. Martin Zenke Max-Delbrück-Center for Molecular Medicine MDC Robert-Rössle-Str. 10 D-13125 Berlin, Germany E-mail:
[email protected] Received for publication March 11, 1998; accepted after revision January 11, 1999.
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