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Originally published online as doi:10.1189/jlb.0906561 on July 12, 2007

Published online before print July 12, 2007
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(Journal of Leukocyte Biology. 2007;82:849-860.)
© 2007 by Society for Leukocyte Biology

High transfection efficiency, gene expression, and viability of monocyte-derived human dendritic cells after nonviral gene transfer

Abdolamir Landi, Lorne A. Babiuk and Sylvia van Drunen Littel-van den Hurk1

Vaccine and Infectious Disease Organization, University of Saskatchewan, Saskatoon, Saskatchewan, Canada

1 Correspondence: Vaccine and Infectious Disease Organization, University of Saskatchewan, 120 Veterinary Rd., Saskatoon, SK, S7N 5E3, Canada. E-mail: sylvia.vandenhurk{at}usask.ca


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ABSTRACT
 
Dendritic cells (DCs) are bone marrow-originated, professional antigen-capturing cells and APCs, which can function as vaccine carriers. Although efficient transfection of human DCs has been achieved with viral vectors, viral gene products may influence cellular functions. In contrast, nonviral methods have generally resulted in inefficient gene transfer, low levels of gene expression, and/or low cell viability. Monocyte-derived DCs are the most common source of DCs for in vitro studies and for in vivo applications. We hypothesized that reduction of the time to generate immature DCs (iDCs) might result in higher viability after transfection. Therefore, we established a protocol to generate human iDCs from CD14+ monocytes within 3 days. These "fast" iDCs were phenotypically and functionally indistinguishable from conventional iDCs, showing high endocytic ability and low antigen-presenting capacity. Furthermore, the fast iDCs matured normally and had similar antigen-presenting capacity to conventional mature DCs. To optimize transfection of iDCs, we compared nonviral transfection of plasmid DNA and in vitro-transcribed (IVT) RNA with transfection reagents, electroporation, and nucleofection. Nucleofection of IVT RNA with the X1 program of an Amaxa Co. Nucleofector resulted in the most efficient transfection, with an average of 93% transfected iDCs, excellent long-term viability, and strong protein expression. Furthermore, the IVT RNA-transfected iDCs retained all phenotypic and functional characteristics of iDCs. This method is applicable to most purposes, including in vitro functional assays, in vivo DC immunotherapy, and DC-based vaccines.

Key Words: plasmid DNA • in vitro-transcribed RNA • nonviral transfection


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INTRODUCTION
 
Dendritic cells (DCs) are bone marrow-originated, professional antigen-capturing cells and APCs, which initiate primary immune responses in vivo. DCs have unique functions, as they are capable of priming naïve T cells and cross-presenting antigens. One of the characteristics of the DC family is maturation, a process during which they change morphologically and functionally. Immature DCs (iDCs) are phagocytic cells capable of sampling antigens at the site of injury or infection. Phagocytosis by DCs is a process, which is much more complicated than that of other phagocytic cells and allows them to present the correct antigenic peptides on MHC I and II molecules. After processing the antigen, they mature, migrate toward local lymph nodes, and present the antigen to naïve T cells. This central role in cell-mediated immunity has made them attractive targets for cancer immunotherapy [1 2 3 4 5 6 ]. DCs loaded with tumor-associated antigens (TAAs) and injected into patients induce anti-tumor responses, which do not occur under natural conditions because of low tumor antigen expression or inaccessibility of the antigen to DCs. DCs are also potential vectors for the treatment of chronic infectious diseases, evading the immune system. Moreover, recent advances in generating DCs have provided strategies for the design of DC-based vaccines [7 ].

Because of their unique properties, DCs are of interest for studies about the mechanisms of antigen processing and presentation. As DCs are professional cells and strictly control the trafficking of molecules across their cell membranes, it is difficult to transfer genes into the nucleus. Many studies have been performed on loading DCs with different antigens for in vitro assays or cancer immunotherapy, and efficient transfection has been achieved with viral vectors [8 9 10 11 12 ]. However, the viral vectors may influence DC functions and interact with the host immune system [13 14 15 16 17 18 ], which may affect the interpretation of in vitro studies. Furthermore, there is a need for DC-based immunotherapy without risk of exposing individuals to other viral genes or integration of the viral genome, which supports the importance of establishing an effective and safe, nonviral method for DC transfection. However, inefficient gene transfer to DCs, low levels of gene expression, or low cell viability by nonviral transfection methods have limited studies about DCs [8 , 19 20 21 22 23 24 25 26 ]. To date, the highest nonviral transfection efficiency of plasmid DNA has been achieved by nucleofection. This method resulted in up to 56% efficiency but only 37% viability of the transfected DCs after 24 h [27 ]. In contrast, several studies have demonstrated efficient transfection of DCs with mRNA [22 , 28 29 30 31 ]. However, protein expression levels were generally low or not discussed, and little information was provided with respect to short- and long-term cell viability. As was concluded recently in an extensive recent review about this subject, strategies for efficient transfection still need to be established for human DCs [7 ] and in particular, monocyte-derived DCs (Mo-DCs).

In this study, we first established a protocol to generate Mo-iDCs in 3 days and then carried out an extensive comparison of nonviral transfection methods, including transfection of plasmid DNA and in vitro-transcribed (IVT) RNA with transfection reagents, conventional electroporation, and nucleofection. Finally, we optimized some protocols as the methods of choice for transfecting human DCs for in vitro studies and in vivo trials.


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MATERIALS AND METHODS
 
In vitro generation of Mo-DCs
Human venous blood was obtained following informed consent through the protocol approved by the Biomedical Research Ethics Board at the University of Saskatchewan (Saskatoon, Canada). PBMCs were isolated by density gradient separation using lymphocyte separation medium (LSM; MP Biomedicals, LLC, Irvine, CA, USA). Briefly, 4 ml blood diluted 1:2 in endotoxin-free PBS (Invitrogen Canada Inc., Burlington, ON, Canada) was layered on 3 ml LSM and centrifuged at 400 g for 25 min. PBMCs were collected, washed, and incubated with human CD14-specific antibody conjugated to paramagnetic MicroBeads (Miltenyi Biotech, Auburn, CA, USA). The CD14+ monocytes were isolated on LS columns (Miltenyi Biotech). Conventional iDCs were generated by resuspending monocytes at 1 x 106 cells/ml in complete RPMI [CRPMI; RPMI 1640 (Invitrogen Canada Inc.), supplemented with 10% heat-inactivated FBS, 2 mM L-glutamine, 100 µM nonessential amino acids, 1 mM sodium pyruvate, 50 µM 2-ME, 10 mM HEPES, and 50 µg/ml gentamycin]. Recombinant human (rh)GM-CSF (100 ng/ml, PeproTech Inc., Rocky Hill, NJ, USA) and rhIL-4 (≤100 ng/ml, PeproTech Inc.) were added, and then, the CD14+ monocytes were dispensed into the wells of 24- or 12-well plates (Corning Inc., Corning, NY, USA). To generate "fast" iDCs, the monocytes were resuspended at 1 x 106 cells/ml in CRPMI made with phenol-red-free RPMI 1640 (Invitrogen Canada Inc.), a lower concentration of heat-inactivated FBS (5%), and a higher amount of L-glutamine (4 mM). In addition, the concentration of IL-4 was increased to 200 ng/ml. In conventionally generated iDC cultures, the medium was replaced with fresh medium containing cytokines every second day. Cultured CD14+ cells were checked daily for marker expression by flow cytometry. The phagocytic and antigen-presentation capacities of the monocytes and DCs were examined at different time-points to confirm the exact times of differentiation and maturation. Conventional iDCs collected on Day 7 and fast iDCs collected on Day 3 were evaluated in functional assays, and the fast iDCs were used for transfections. To mature the iDCs, a cocktail of 10 ng/ml rhIL-1ß (PeproTech Inc.), 10 ng/ml rhIL-6 (PeproTech Inc.), 20 ng/ml rhTNF-{alpha} (PeproTech Inc.), and 500 ng/ml PGE2 (Sigma-Aldrich Canada Ltd., Oakville, ON, Canada) was used for 16–48 h.

Flow cytometry
PBMCs, monocytes, and DCs were washed twice in PBS and resuspended in FACS buffer (PBS, pH 7.2, with 0.2% gelatin and 0.03% sodium azide) at 105 cells/100 µl in a round-bottom, 96-well plate (Corning Inc.). Subsequently, 2 µl FITC- or PE-conjugated mAb (BD Biosciences, Oakville, ON, Canada) were added to each well, and the plate was incubated on ice for 15–30 min. After incubation, cells were washed three times with FACS buffer, fixed with 2% formaldehyde in PBS, and analyzed by FACScan (BD Biosciences).

To check the transfection efficiency and intensity, iDCs transfected with plasmid encoding GFP (pmaxGFP, Amaxa Co., Köln, Germany) were washed once in PBS and analyzed by flow cytometry. The cells were gated electronically according to the control, nontransfected cells for forward-scatter (FSC) and side-scatter (SSC) properties to include the main population of the cells and exclude dead cells. To check the cell viability, the cells were stained with propidium iodide (PI; Sigma-Aldrich Canada Ltd.), and more than 10,000 events were analyzed by FACScan without gating. Cell Quest software (BD Biosciences) was used for analysis of flow cytometry data.

Phagocytosis assay
A phagocytosis assay was performed as described elsewhere [32 ] with minor modifications. iDCs and mature DCs (mDCs) were collected, washed once in PBS, and resuspended in CRPMI at 1 x 106 cells/ml. The cell suspension (200 µl) was cultured in a 24-well plate, and 200 µl FITC-conjugated dextran (FITC-DX; Sigma-Aldrich Canada Ltd.) in PBS (1 mg/ml) was added to the DCs. The treatment plate was incubated at 37°C, and the control plate was incubated on ice for 1–2 h in the dark. After incubation, the cells were collected, washed three times with cold PBS, and checked by flow cytometry for FITC-DX fluorescence as an indicator of phagocytosis.

Allostimulatory MLR
iDCs and mDCs were collected, washed in PBS once, and resuspended at 1 x 106 cells/ml in CRPMI. The cell suspension (100 µl) was dispensed in the wells of a flat-bottom, 96-well ELISA plate (Corning Inc.). Subsequently, 3 x 105 CD14-negative cells (70–80% CD3+ cells) in 100 µl CRPMI from a different, HLA-mismatched, healthy donor were added to each well followed by addition of 50 µl CRPMI. The plate was incubated at 37°C and 5% CO2. After 48–72 h, 20–40 µl MTT (Sigma-Aldrich Canada Ltd.), at a concentration of 5 mg/ml in PBS, was added to each well, and the cells were incubated for an additional 30–60 min until the uptake of dye by the cells was visible under the light microscope. Subsequently, the plates were centrifuged at 400 g for 10 min, and 150 µl supernatant was replaced with 100 µl acidified isopropanol (0.375% HCl in isopropanol). The plates were mixed for 4 min and read by an ELISA reader (Molecular Devices Corp., Sunnyvale, CA, USA) at a wavelength of 595 nm.

Transfection with plasmid DNA
ExGen 500 (Fermentas, Burlington, ON, Canada), FuGene 6 (Roche Diagnostics, Laval, QC, Canada), GeneJuice (EMD Biosciences, Novagen Brand, Madison, WI, USA), SuperFect (Qiagen Inc., Mississauga, ON, Canada), and TransFast transfection reagent (Promega Corp., Madison, WI, USA) were used to transfect DCs with pmaxGFP. As no protocols were available for human DCs, optimized protocols were developed. Electroporation of DNA was performed according to a protocol described previously [33 ].

Transfection with IVT RNA
The pGEM4Z-5'UT-eGFP-3UT-64A vector was kindly provided by Dr. Eli Gilboa (Miller School of Medicine, University of Miami, Miami, FL, USA). This plasmid contains the GFP gene flanked by the 5' and 3' untranslated regions and a poly A tail. There is a SpeI site after the poly A stretch to allow linearization of plasmid for in vitro transcription. In vitro transcription was performed by using the mMESSAGE mMACHINE kit (Ambion, Austin, TX, USA). IVT RNA was stored at –70°C until use.

For electroporation, iDCs were collected on Day 3 and washed with PBS, followed by serum-free Opti-MEM. Subsequently, 3 x 106 cells were resuspended in 200 µl serum-free Opti-MEM. IVT RNA was added to the cell suspension at different concentrations, and the mixtures were transferred into a 4-mm cuvette and electroporated at different conditions (250–400 V voltage, 125–960 µF capacitance, and 100–1000 {Omega} resistance) in an electroporation apparatus (GenePulserTM Model 1652076, Bio-Rad Laboratories, Mississauga, ON, Canada). Immediately after transfection, the DCs were resuspended at 5 x 105/ml in 37°C CRPMI, supplemented with 50 ng/ml rhGM-CSF and 100 ng/ml rhIL-4, and incubated at 37°C until FACS analysis.

Transfection with TransMessenger transfection reagent (TTR; Qiagen) was performed according to the manufacturer's instructions with some modifications. iDCs were collected on Day 3 and washed with PBS. The Enhancer R solution and different amounts of IVT RNA were diluted in an appropriate amount of Buffer EC-R and incubated for 5 min at room temperature (RT). TTR was added to the mixtures, and the complexes were incubated for 10 min at RT. RPMI 1640 was added to the IVT RNA-TTR complexes, which were then added immediately to the iDCs. Finally, the iDCs were transferred to a 24-well plate and incubated at 37°C. After 1 h, the iDCs were washed with PBS and fresh CRPMI supplemented with 50 ng/ml rhGM-CSF, 100 ng/ml rhIL-4 was added. Subsequently, the iDCs were incubated again until analysis by flow cytometry.

Nucleofection of DNA and IVT RNA
Nucleofection of iDCs was performed according to the manufacturer's instructions with some modifications, by using the Nucleofector machine (Amaxa Co.). The iDCs were collected on Day 3 and washed twice in PBS. Subsequently, 2 x 106 cells were resuspended in 100 µl human DC nucleofection solution (Amaxa Co.). pmaxGFP (5 µg) were added per 2 x 106 cells, and the samples were transferred into certified cuvettes (Amaxa Co.) and transfected by using programs K2, M2, Q2, T2, U1, U2, U8, X1, or X10. The same programs were used for transfection of IVT RNA at a concentration of 5–10 µg per 1 x 106 cells. RPMI 1640, supplemented with 10% heat-inactivated FBS, 2 mM L-glutamine, 1 mM sodium pyruvate, 100 µg/ml streptomycin, and 100 µg/ml penicillin (SRPMI), was warmed to 37°C, and 500 µl was added immediately after transfection to each cuvette. The iDCs were collected, dispensed in the wells of a 24-well plate containing 2.2 ml prewarmed SRPMI, supplemented with 50 ng/ml rhGM-CSF and 100 ng/ml rhIL-4, and divided into four wells to be analyzed at 8, 24, 48, and 72 h. The plate was incubated at 37°C until analysis by flow cytometry. All samples were checked for transfection efficiency and intensity as well as cell viability with an Axiovert 200M fluorescent microscope (Carl Zeiss Canada Ltd., Toronto, ON, Canada) and a FACScan.

Stimulation of DCs and cytokine secretion
Four hours after nucleofection with GFP IVT mRNA, iDCs were stimulated for 20 h in the presence of LPS (Sigma-Aldrich Canada Ltd.) at a final concentration of 10 µg/ml, and supernatants were checked for IL-12 (p70), IFN-{gamma}, and IL-10 production by using an OptEIA human ELISA set (BD Biosciences), according to the instructions provided by the manufacturer. Non-nucleofected iDCs as well as iDCs nucleoporated without genetic material were used as controls.

Statistical analysis
Data were analyzed using statistical software (GraphPad Prism Version 4.00). As outcome variables were not distributed normally, differences among all groups were examined using the Kruskal-Wallis test. If a significant difference was found among the groups, the medians between pairs of groups were compared using the Mann-Whitney U-test. Differences were considered significant if the two-tailed P value was lower than 0.05 with the confidence intervals of 95%. All transfection results are shown as mean ± SEM of at least four independent experiments performed with different donors.


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RESULTS
 
In vitro generation of iDCs and mDCs
Transfection of Mo-iDCs, generated over a period of 7–14 days as described previously [34 35 36 37 38 ], resulted in progressively reduced viability over time (data not shown). We hypothesized that generation of DCs in a shorter period of time would result in higher viability, and thus, we established a protocol to differentiate monocytes into iDCs more rapidly. CD14+ monocytes were isolated from PBMCs by MACS, which routinely resulted in at least 95% pure populations (Fig. 1a ). Subsequently, the CD14+ monocytes were cultured with different concentrations of rhGM-CSF and rhIL-4 and checked daily for changes in CD1a, CD14, CD209 [DC-specific ICAM-grabbing integrin (SIGN)], CD83, and CD86 expression to identify the exact time of differentiation and maturation. When the CD14+ cells were cultured in medium supplemented with 100 ng/ml rhGM-CSF and 200 ng/ml rhIL-4 on Day 0, they differentiated into iDCs in 3 days (Fig. 1b) without further addition of cytokines. The expression of CD14, which is a specific monocyte marker, decreased to 5%, CD209 or DC-SIGN as a DC marker increased from 0% to 92%, CD1a as a phagocytic marker increased to 71%, CD86 as a costimulatory receptor decreased from 95% to 57%, and CD83 was expressed at less than 4%, confirming the immature status of the iDCs. When the iDCs were cultured with a cocktail of rhIL-1ß, rhIL-6, rhTNF-{alpha}, and PGE2, CD83, the well-defined marker for maturation [39 40 41 ], increased to 89%, so they became fully mature on Day 5 (Fig. 1c) . In addition, the morphology of these iDCs and mDCs was as expected (Fig. 2c and 2d ). Similar but slower marker changes were observed with the conventional method (Fig. 1b and 1c) . As shown in Figure 1 , the expression of CD1a, CD209, CD83, and CD86 was similar on fast iDCs and conventionally generated iDCs, as well as on the mDCs after maturation. To characterize the iDCs and mDCs further, their phagocytic ability and antigen-presenting capacity were evaluated and compared with conventionally generated DCs. Fast iDCs showed strong phagocytic ability, which decreased upon maturation to mDCs, similar to conventionally generated iDCs (Fig. 3a ). The mDCs, regardless of whether they were generated from conventionally or fast-generated iDCs, had increased antigen-presenting capacity (Fig. 3b) . Overall, this approach generated iDCs more rapidly than methods reported previously using only one treatment with rhGM-CSF and rhIL-4. These data demonstrate that the conventional and fast DCs were phenotypically and functionally indistinguishable, before and after maturation.


Figure 1
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  		Figure 1. Phenotypic characterization of fast and conventionally generated Mo-DCs. (a) Expression of cell surface markers on monocytes isolated by anti-CD14 antibody conjugated to paramagnetic MicroBeads. (b) Differentiation of monocytes to iDCs. (c) Maturation of iDCs. Monocytes were differentiated into iDCs with 100 ng/ml rhGM-CSF and ≤100 ng/ml (conventional) or 200 ng/ml (fast) rhIL-4 and matured to mDCs by adding a maturation cocktail (IL-1ß, IL-6, TNF-{alpha}, and PGE2) on Day 3 (fast) or 7 (conventional). The phenotypes of monocytes, iDCs, and mDCs were monitored by flow cytometry. Numbers in the upper-right quadrants represent the percentage of positive cells for each marker and are representative of eight experiments.


Figure 2
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  		Figure 2. Morphology of monocytes, iDCs, and mDCs after fast differentiation and maturation. (a) Lymphocytes (80% of CD14-negative cells flowing through the MACS column); this population was used for MLR assays as responder cells (x100). (b) Monocytes (CD14+ cells) isolated from PBMCs by MACS (x100). (c) iDCs 3 days after culturing monocytes with rhGM-CSF and rhIL-4. (d) mDCs 1 day after addition of maturation cocktail (MC; IL-1ß, IL-6, TNF-{alpha}, and PGE2) to the iDC cultures. Left iDC and mDC panels show the cells in the culture (x40), and right iDC and mDC panels show the cells stained by modified Wright's stain (x100, HemaTek Stain, Bayer Corp., Tarrytown, NY, USA).


Figure 3
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  		Figure 3. Functional evaluation of fast and conventional DCs. (a) Phagocytosis assay showing similar uptake of FITC-DX in fast and conventional iDCs. Numbers in the upper-right quadrants represent the percentage of phagocytic cells and are representative for three experiments. (b) Antigen-presenting abilities in fast and conventional mDCs measured by MTT MLR assay (*, P<0.05). Solid arrows, MC added; open arrows, no MC.

As a result of the fact that GM-CSF alone drives monocytes toward macrophages, we hypothesized that IL-4 might play a key role in promoting differentiation toward DCs and that an increase in IL-4 concentration might be responsible for the expedited differentiation of fast DCs. To investigate this, we checked the effects of GM-CSF or IL-4 on expression of key phenotypic markers. The results indicate that IL-4 is the main cytokine for reduction of CD14 expression, which is a specific marker for monocytes also present on macrophages (Fig. 4a ). Moreover, CD209 (DC-SIGN), an important marker for DCs, increased more dramatically in the presence of IL-4 than with GM-CSF (Fig. 4b) . We also showed that IL-4 was responsible for maintaining stable levels of MHC II expression during differentiation (Fig. 4c) . These observations support the contention that IL-4 plays a major role in the differentiation of monocytes to iDCs and rationalize the use of high IL-4 concentrations during this process.


Figure 4
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  		Figure 4. Effects of GM-CSF and IL-4 on expression of three key markers during differentiation from monocytes into iDCs. (a) CD14. (b) CD209. (c) HLA-DR. Nil, Monocytes cultured without any cytokines. GM-CSF, Monocytes cultured with 100 ng/ml GM-CSF. IL-4, Monocytes cultured with 100 ng/ml IL-4. Numbers in the upper-right quadrants represent the percentage of positive cells for each marker and are representative of at least four experiments.

Transfection with plasmid DNA
To develop an optimal, nonviral transfection method, which would result in high gene expression levels and long-term viability and thus allow functional studies, the iDCs were transfected on Day 3 with pmaxGFP and various transfection reagents or conventional electroporation. The transfection efficiency, GFP intensity, and cell viability were evaluated at different time-points after transfection. Regardless of the reagent selected, the transfection efficiency was low. The highest efficiency was obtained with FuGene 6, and 20% of the iDCs were transfected. However, although to our knowledge, this was the highest efficiency reported for any transfection reagent with plasmid DNA, this was still not sufficient for in vitro or in vivo studies. The other transfection reagents tested, ExGen 500, GeneJuice, SuperFect, and TransFast Transfection reagent, resulted in less than 10% efficiency and low intensity. Electroporation also resulted in less than 10% transfection efficiency, as well as low viability (data not shown). Overall, this demonstrated that regardless of the method or reagent used, transfection with plasmid DNA was inefficient.

Transfection with IVT RNA
As we did not achieve acceptable transfection efficiencies with aforementioned methods, we considered IVT RNA as the next approach to transfect DCs. After generating IVT RNA from the pGEM4Z-5'UT-eGFP-3UT-64A vector, iDCs were transfected with the GFP IVT RNA by conventional electroporation with different settings in terms of voltage, capacitance, and resistance. In comparison with transfection with plasmid DNA, this method was less toxic to the DCs and more effective, resulting in more than 80% transfection efficiency (data not shown), which is comparable with previous reports about RNA electroporation [28 ]. Some electroporation conditions resulted in high transfection efficiency, but the cell viability was not high enough for further in vitro studies or in vivo applications (data not shown). Five electroporation conditions resulted in acceptable efficiency and viability after 72 h (Fig. 5a and 5c ). However, the intensity was low (Fig. 5b) , so although electroporation could be useful for transfection with genes that need to be expressed at low levels, the protein expression was considered insufficient for most applications.


Figure 5
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  		Figure 5. Transfection efficiency, transfection intensity, and cell viability after transfection of GFP IVT RNA. iDCs were transfected by electroporation using five different settings (R-ELA, 300 V, 125 µF, and 100 {Omega}; R-ELB, 300 V, 250 µF, and 100{Omega}; R-ELC, 300 V, 500 µF, and 200 {Omega}; R-ELD, 300 V, 500 µF, and 400 {Omega}; and R-ELE, 350 V, 250 µF, and 200 {Omega}) or with TTR (R-TTR). (a) Transfection efficiency expressed as percentage of iDCs expressing GFP. (b) Intensity expressed as a percentage of the strongest fluorescence achieved with the most effective method, the Nucleofector Program X1 with GFP IVT RNA. The geometric mean intensity was calculated based on FACS results and expressed as a percentage of the highest intensity obtained with Program X1 and IVT RNA, which was calculated as 100%. (c) Cell viability expressed as percentage of live iDCs.

As TTR is a novel transfection reagent recommended for use with IVT RNA, we subsequently tested the efficacy of this reagent with GFP IVT RNA. When the TTR and IVT RNA were incubated for 2 h, up to 61% of the iDCs became transfected, but the viability was very low (data not shown). Therefore, we reduced the incubation time to 1 h, which resulted in 40% efficiency, high levels of GFP expression, and 69% viability after 72 h (Fig. 5) . It is interesting that after transfection of iDCs with TTR, the CD83 expression increased (see Go Go Fig. 8a ), and the cell morphology changed (see Fig. 7c ), indicating that TTR induced the iDCs to become mature. Thus, transfection of DCs with TTR may have effects on the functional properties of the DCs.


Figure 6
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  		Figure 6. Transfection efficiency, transfection intensity, and cell viability after nucleofection of GFP plasmid DNA or GFP IVT RNA. (a–c) Nucleofection with 2.5 µg GFP plasmid DNA per 1 x 106 cells, using programs K2 (D-K2), M2 (D-M2), Q2 (D-Q2), U2 (D-U2), and X1 (D-X1). (d–f) Nucleofection with 10 µg GFP IVT RNA per 1 x 106 cells, using programs K2 (R-K2), M2 (R-M2), Q2 (R-Q2), U2 (R-U2), and X1 (R-X1). (a and d) Transfection efficiency expressed as percentage of iDCs expressing GFP. (b and e) Intensity calculated as for Figure 4 and expressed as a percentage of the strongest fluorescence achieved with the Nucleofector Program X1 with GFP IVT RNA. (c and f) Cell viability expressed as percentage of live iDCs.


Figure 7
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  		Figure 7. Human iDCs transfected on Day 3 and analyzed 8 h after transfection. (a) Nucleofection (program U2) with GFP plasmid DNA (D-U2). (b) Nucleofection (Program X1) with GFP IVT RNA (R-X1). (c) Transfection of GFP IVT RNA with TTR (R-TTR). Left panels, Phase contrast (x40); middle panels, fluorescence (x40); right panels, analysis of GFP expression by flow cytometry on the FL1 channel.


Figure 8
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  		Figure 8. Investigation of the effects of transfection on the maturation and antigen-presentation ability of DCs. (a) Comparison of the expression of CD83 on nontransfected, nucleofected, and TTR-transfected iDCs and mDCs. Numbers in the upper-right quadrants represent the percentage of DCs expressing CD83. (b) Antigen-presentation capacity of nucleofected DCs. Nil, Nontransfected iDCs; X1, nucleofected iDCs without RNA or DNA with program X1; R-X1, nucleofected iDCs with GFP IVT RNA and Program X1; R-TTR, transfected iDCs with GFP IVT RNA and TTR.

Nucleofection of DNA
As there is a report about nucleoporation of human DCs with a Nucleofector [27 ], we evaluated this approach as an alternative for transfection of iDCs with plasmid DNA. To optimize the protocol, we screened nine different programs and further evaluated the five most-promising programs with respect to efficiency, level of protein expression, and viability after nucleofection with pmaxGFP. Reasonably high transfection efficiency and protein expression levels were achieved (Fig. 6a and 6b ), as well as good viability for up to 24 h (Fig. 6c) with some of the programs, in particular, U2 and X1, which led to significantly better transfection efficiencies than K2 and M2 (P<0.05). However, with the U2 and X1 programs, the viability decreased significantly between 24 and 48 h. Program X1 was comparable with Program U2, which is recommended by Amaxa Co. for nucleofecting DCs with DNA (Fig. 6a 6b 6c) .

Nucleofection of IVT RNA
As the transfection efficiency achieved by nucleofection of plasmid DNA was at most 50%, and the viability of the iDCs decreased significantly between 24 and 48 h, we subsequently used the same programs with GFP IVT RNA. Nucleofection with IVT RNA resulted in higher efficiency regardless of the program used (Fig. 6d) , as well as good viability after 72 h (Fig. 6f) . The iDCs expressed higher levels of protein when transfected with Program X1 than with any other transfection method (Fig. 6e) , and the other programs resulted in comparable levels of protein expression with DNA nucleofection (Fig. 6b and 6e) . The transfection efficiency obtained with Program X1 varied between 90% and 98%, with a mean of 93% 8 h after transfection, and after 72 h, the mean efficiency was 78%, and the mean viability was 75%. Furthermore, high protein expression was observed 8 h after transfection, and although there was some decrease, the protein expression level was still reasonably high at 72 h after transfection. In fact, GFP could be detected as early as 1 h and as long as 5 days after transfection (data not shown). In addition, there was less variability in protein expression levels between cells within one experiment (Fig. 7a and 7b ) and less variation between experiments (Fig. 6b and 6e) when iDCs were transfected with IVT RNA instead of DNA. It is interesting that at 8 h after transfection, the GFP intensity in the RNA-transfected iDCs was higher than that obtained after DNA nucleofection, whereas long-term protein expression was equivalent. To check if the nucleofection has any effect on the functional properties of DCs, iDCs were checked for their antigen-presentation ability as well as CD83 expression after nucleofection and addition of maturation cocktail. Normal maturation, similar to that of nontransfected DCs, and comparable antigen-presentation ability were observed after nucleofection with the X1 program (Fig. 8a and 8b ).

As DCs may lose their capacity to produce IL-12 p70 after electroporation, iDCs were stimulated with LPS at 10 µg/ml. Supernatants were collected and checked for the presence of IFN-{gamma}, IL-12 (p70), and IL-10. The results demonstrate that DCs, nucleofected with mRNA using the X1 program, were capable of secreting significant amounts of cytokines in comparison with non-nucleofected DCs (Fig. 9a 9b 9c ). Nucleofection had no effect on IFN-{gamma} and IL-10 secretion, and although the ability of the nucleofected iDCs to secrete IL-12 was reduced, ~200 pg/ml was still detected, which is a significant amount.


Figure 9
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  		Figure 9. Evaluation of the capacity of fast-generated iDCs to secrete cytokines before and after nucleofection. (a) IFN-{gamma}. (b) IL-12 (p70). (c) IL-10. Day 3 iDCs were nucleoporated without or with GFP IVT mRNA using program X1 (X1 and R-X1, respectively), and non-nucleofected iDCs (Nil) were used as control. Four hours after nucleofection, 10 µg/ml LPS was added to stimulate cytokine secretion, and cells were incubated for an additional 20 h. Supernatants were checked for cytokine secretion (–LPS, No LPS; +LPS, LPS added).

Comparison of DNA and RNA transfection methods
To compare the different approaches for transfection with plasmid DNA and IVT RNA directly, we selected the best conditions for each method based on statistical analyses in terms of efficiency, intensity, and viability and compared them with IVT RNA nucleofection with the X1 program at 72 h after transfection (Fig. 10 ). The efficiency was higher after nucleofection of IVT RNA with the X1 program than after nucleofection of plasmid DNA (P<0.01), transfection of IVT RNA by conventional electroporation (P<0.05), or transfection of IVT RNA with TTR (P<0.01) but equivalent to nucleofection of IVT RNA with the U2 program (Fig. 10a) . In contrast, the protein expression level was equivalent to that achieved with nucleofection of plasmid DNA or transfection of IVT RNA with TTR but higher than the level achieved by conventional electroporation or nucleofection of IVT RNA with the U2 program (P<0.05; Fig. 10b ). The viability after nucleofection of IVT RNA with the X1 program was higher than after nucleofection of plasmid DNA (P<0.01) but equivalent to the viability achieved with Program U2, conventional electroporation, or transfection with TTR (Fig. 10c) . Overall, nucleofection of IVT RNA with the X1 program resulted in extremely efficient transfection, with good, long-term viability and comparable or higher levels of protein expression with other methods. Although transfection with TTR was less-efficient than nucleofection with IVT RNA with the X1 program, this method resulted in strong protein expression as well as maturation of iDCs.


Figure 10
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  		Figure 10. Comparison of transfection methods for DNA and RNA. The optimal conditions for the different transfection methods were selected and compared with respect to transfection efficiency, transfection intensity, and cell viability after 72 h. D-U2 and D-X1, Nucleofection of GFP plasmid DNA using Programs U2 and X1, respectively. R-U2 and R-X1, Nucleofection of GFP IVT RNA using programs U2 and X1, respectively. R-ELD, Electroporation of IVT RNA at 300 V, 500 µF, and 400 {Omega}. R-TTR, Ttransfection of IVT RNA with TTR. (a) Transfection efficiency expressed as percentage of iDCs expressing GFP. (b) Intensity calculated as in Figure 4 and expressed as a percentage of the strongest fluorescence achieved with the Nucleofector Program X1 with GFP IVT RNA. (c) Cell viability expressed as percentage of live iDCs (*, P<0.05; **, P<0.01).


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DISCUSSION
 
Transfer of genetic material into DCs has become a major focus of research, having applications to the development of DC-based vaccines for treatment of cancer or chronic infectious diseases. It is also an important requirement for in vitro evaluation of the effects of different microbial genes on DCs. Although viral transduction is efficient, increased immunogenicity of transfected DCs, as a result of viral proteins and the risk of oncogenesis from the insertion of the viral genome into host cells in vivo, has encouraged efforts to establish nonviral transfection methods. We hypothesized that if we generate iDCs more rapidly, this may result in higher viability after transfection, so we established a new protocol to generate iDCs. Although there is one report about direct differentiation and maturation of monocytes to mDCs in 48 h [42 ], this protocol is faster than previous reports in terms of generating iDCs. These fast iDCs showed high endocytic ability and low antigen-presenting capacity, comparable with conventional iDCs generated in 7–14 days. Furthermore, after addition of maturation cocktail, the iDCs matured as expected, with an increase in antigen-presenting ability.

As GM-CSF alone drives monocytes toward macrophages [43 , 44 ], we hypothesized that IL-4 plays a key role in differentiation toward DCs. Indeed, when we checked the individual effects of GM-CSF and IL-4, we observed that IL-4 was responsible for the reduction in CD14 expression, enhanced CD209 expression, and maintained MHC II expression on the iDCs, confirming that IL-4 is a critical component for the fast differentiation of monocytes to iDCs. This may be a result of the fact that IL-4 operates through STAT6 (the primary STAT pathway activated in response to IL-4 [45 ]), which is critical in the activation of many IL-4-responsive genes, including those for MHC class II molecules. As IL-4 has been demonstrated to enhance the survival of hematopoietic cells [46 47 48 ] and to play a role in prolonging T and B cell survival in cultures [49 ], IL-4 may also be responsible for better viability of DCs after nucleofection. Activation of PI-3K pathways by IL-4 may enhance cell survival through the production of phosphoinositides and the subsequent activation of kinases critical for cell survival [45 ].

With respect to other culture conditions, including higher glutamine, according to the media expert in Sigma-Aldrich Canada Ltd., glutamine is an important amino acid for highly active cells and can replace serum in culture media. The use of glutamine could promote the differentiation, as the cells need a high source of energy and better culture conditions as a result of being active during the differentiation process. Finally, the use of higher glutamine and IL-4 resulted in healthier cells, which would also reduce the chance of driving monocytes toward macrophages.

To optimize the transfection efficiency of the fast iDCs with plasmid DNA, we evaluated a number of transfection reagents. However, regardless of the reagent or conditions used, the transfection efficiency was low, whereas the reagents were frequently toxic to the DCs. Therefore, we selected electroporation as an alternative method. Conventional electroporation resulted in low transfection efficiency and cell viability, whereas nucleofection with plasmid DNA was an effective method with good viability during the first 24 h [27 ]. Unfortunately, the viability of the iDCs decreased progressively between 24 h and 48 h. We hypothesized that the excess amount of DNA might be an important factor affecting cell viability, so we decreased the amount of DNA and indeed obtained better viability but significantly reduced efficiency. We concluded that using IVT RNA instead of DNA in combination with conventional electroporation or nucleofection might provide a solution. Moreover, as RNA does not need to be transferred through the large pores within the cell membrane or across the nuclear membrane, gentler electroporation conditions can be used, which should result in better viability and fewer functional defects in the DCs. Transfection with IVT RNA would also be of benefit if the DCs were to be used for DC-based vaccination, against infectious agents, or in cancer patients, as there is no danger of possible integration in cellular genetic material. However, although transfection with IVT RNA by electroporation resulted in good efficiency and viability with some of the conditions, protein expression was low, so transfection of DCs with conventional electroporation and IVT RNA would only be useful for proteins, which have to be expressed at low amounts.

Nucleofection of IVT RNA resulted in a mean transfection efficiency of 93%, which to our knowledge, is the highest rate reported for a nonviral transfection method, as well as excellent protein intensity and long-term viability. A concern with IVT RNA would be short stability in the cell cytoplasm; however, GFP was detected for at least 5 days after transfection with IVT RNA, which would be long enough for assays such as MLR. Although this may be a result of high protein stability or high protein expression levels after transfection, there are some explanations supporting enhanced mRNA stability with the nucleofection method. First, there is a possibility that the reagent (patented) used for nucleofection may protect mRNA from degradation in the cytoplasm. Second, as the company claims that the nucleofection method transfers the genetic material directly to the nucleus, there is a possibility that mRNA will be directed to the nucleus, where it could be protected from degradation and released gradually to the cytoplasm. Although nucleofection with IVT RNA generally led to equivalent levels of protein compared with transfection with DNA, with Program X1, higher amounts of protein were produced, which is one of the major advantages of this method, especially where stable and sufficient antigen concentration of TAA is needed [50 51 52 ]. In addition, high protein expression was detected as early as 2 h after nucleofection of IVT RNA, so the iDCs can be collected and used for further assays or immunotherapy soon after transfection. This approach may provide a solution to poor protein production, which may result in a weak, not sustained, immune response, leading to repeated vaccination in cancer immunotherapy trials [53 , 54 ]. Another advantage of transfection with IVT RNA is the uniform and consistent level of protein expression in each cell and for each donor, which implies that all IVT RNA-transfected DCs have similar characteristics.

It is important that the iDCs nucleofected with IVT RNA remained immature, unless maturation cocktail was added. After maturation, the mDCs were capable of antigen presentation, as shown in a MLR assay, which demonstrated that they were functional. In contrast, transfection of iDCs with IVT RNA and TTR induced maturation of the iDCs, so TTR may affect the functional properties of the DCs and would not be suitable for in vitro studies. However, as this method resulted in strong protein expression as well as maturation of iDCs, it is useful when mDCs, which appear to be more effective for cancer immunotherapy [55 ], are required immediately after transfection without further induction of maturation.

In summary, we established a method for the generation of functionally active iDCs from CD14+ monocytes in 3 days, after only one treatment with cytokines. This approach reduces the time required for in vitro functional assays and provides more viable iDCs for transfection. In addition, we developed two methods for transfection of human Mo-iDCs. Nucleofection led to high efficiency, as well as high protein expression and long-term viability and thus, can be applied to in vitro studies, cancer immunotherapy, and DC-based vaccination. Transfection with IVT RNA and TTR was less efficient but resulted in strong protein expression, good long-term viability, and maturation of the iDCs, so this method might be useful in cancer immunotherapy or DC-based vaccines.


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ACKNOWLEDGEMENTS
 
This study was supported by the Canadian Institutes of Health Research and the Saskatchewan Health Research Foundation and was published as Vaccine and Infectious Disease Organization's Journal Series Number 431. We thank Marlene Snider and Laura Latimer for technical assistance and Dr. Hong Yu for advice about IVT RNA transfections, and we thank our blood donors specifically.

Received September 11, 2006; revised May 23, 2007; accepted June 17, 2007.


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