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The Journal of Immunology, 2002, 169: 1669-1675.
Copyright © 2002 by The American Association of Immunologists

Messenger RNA Electroporation of Human Monocytes, Followed by Rapid In Vitro Differentiation, Leads to Highly Stimulatory Antigen-Loaded Mature Dendritic Cells1

Peter Ponsaerts*, Glenn Van den Bosch*, Nathalie Cools*, Ann Van Driessche*, Griet Nijs*, Marc Lenjou*, Filip Lardon{dagger}, Christine Van Broeckhoven, Dirk R. Van Bockstaele*, Zwi N. Berneman* and Viggo F. I. Van Tendeloo2,*

* Laboratory of Experimental Hematology, University of Antwerp, Antwerp University Hospital, Edegem, Belgium; {dagger} Laboratories of Cancer Research and Clinical Oncology and Molecular Genetics, University of Antwerp, Antwerpen, Belgium


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Dendritic cells (DC) are professional Ag-capturing and -presenting cells of the immune system. Because of their exceptional capability of activating tumor-specific T cells, cancer vaccination research is now shifting toward the formulation of a clinical human DC vaccine. We developed a short term and serum-free culture protocol for rapid generation of fully mature, viable, and highly stimulatory CD83+ DC. Human monocytes were cultured for 24 h in serum-free AIM-V medium, followed by 24-h maturation by polyriboinosinic polyribocytidylic acid (polyI:C). Short term cultured, polyI:C-maturated DC, far more than immature DC, showed typical mature DC markers and high allogeneic stimulatory capacity and had high autologous stimulatory capacity in an influenza model system using peptide-pulsed DC. Electroporation of mRNA as an Ag-loading strategy in these cells was optimized using mRNA encoding the enhanced green fluorescent protein (EGFP). Monocytes electroporated with EGFP mRNA, followed by short term, serum-free differentiation to mature DC, had a phenotype of DC, and all showed positive EGFP fluorescence. Influenza matrix protein mRNA-electroporated monocytes cultured serum-free and maturated with polyI:C showed high stimulatory capacity in autologous T cell activation experiments. In conclusion, the present short term and serum-free ex vivo DC culture protocol in combination with mRNA electroporation at the monocyte stage imply an important reduction in time and consumables for preparation of Ag-loaded mature DC compared with classical DC culture protocols and might find application in clinical immunotherapy settings.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Dendritic cells (DC),3 also called nature’s adjuvant, are professional Ag-capturing and -presenting cells of the immune system (1, 2). Mouse studies have demonstrated their exceptional in vitro and in vivo capability of activating tumor-specific T cells (3, 4). Strong activation signals for Ag-specific CD4+ helper T cells and CD8+ cytotoxic T cells, as provided by highly stimulatory mature DC, might break possible T cell tolerance against tumor Ags (5, 6). Bypassing this T cell tolerance will be one of the major concerns in the development of a DC-based cancer vaccine. One of the priorities of current research is the formulation of a human DC vaccine. To reach this goal, the production of a DC-based cancer vaccine has to fulfill three major prerequisites: 1) in vitro culture of human DC, 2) Ag loading into human DC, and 3) maturation of human DC.

Human DC can be cultured starting from hemopoietic progenitor cells using different long and short term culture systems (7, 8). However, for a clinical DC vaccine, preference is likely to be given to the differentiation of DC starting from monocytes (9), because monocytes represent a readily accessible source of DC precursors and can easily be obtained in relatively large numbers from patients. The classical culture system of monocyte-derived DC (Mo-DC) is based on a 6- to 7-day culture of peripheral blood monocytes in medium containing GM-CSF and IL-4. Although this culture system has been used for most in vitro studies and for the first human clinical trials, novel culture systems have been developed for Mo-DC (10, 11, 12). These methods capitalize on the fact that monocytes can rapidly differentiate into immature dendritic-like cells in serum-free medium under the influence of GM-CSF and can further be matured with calcium ionophore, bacterial products, or cytokines including TNF-{alpha} and IFN-{gamma}. Under such conditions, mature DC can be differentiated during a time span of 2–3 days. Because of their significant reduction in preparation time, short term DC culture protocols are likely to become important for large scale clinical DC preparation.

Different strategies have been developed for loading DC with tumor or viral Ags (13). The use of antigenic peptides derived from target proteins, which can be pulsed directly on the DC MHC class I proteins, is a very efficient loading strategy (14), but is only applicable in case the patient’s HLA type corresponds to the known MHC class I-restricted peptide. Alternatively, tumor lysate-pulsed DC (15) or DC-tumor hybrids (16) can be used to obtain full-spectrum tumor Ag-loaded DC. However, the latter methods are highly dependent on the amount of tumor tissue available. To overcome this restriction, many non-viral and viral gene transfer technologies have been developed for DC. However, plasmid DNA transfer into DC has not been very efficient (17), and the use of viral vectors for transduction of DC implies a more complex and laborious manipulation associated with safety issues. Moreover, DC transduced by viral vectors have been reported to have impaired function in terms of stimulatory capacity (18, 19). Recently, we (20) and others (21, 22, 23) developed novel non-viral Ag loading technologies based on the passive pulsing, lipofection, and electroporation of mRNA. These technologies represent safe and clinically applicable Ag-loading strategies for DC.

The final step in the development of a DC vaccine is to obtain mature DC. Under in vitro culture conditions, it is well accepted that mature DC are potent stimulators of T cell activation (24). For in vivo vaccination, the potency of Ag-loaded immature vs mature DC still has to be established in carefully designed clinical trials. Interestingly, recent vaccination trials by Bhardwaj and colleagues (25) have showed that vaccination with immature Ag-loaded DC results in Ag-specific tolerance, while infusion of mature DC leads to an effective CTL-mediated immune response (26). For DC, various maturation agents have been described (27), but it is believed that the optimal in vitro mixture has yet to be formulated. Of recent interest is the focus on the use of synthetic dsRNA (polyriboinosinic polyribocytidylic acid (polyI:C)) as a single and very potent maturation stimulus for DC (28).

In this study we show that the combination of a serum-free culture protocol and a polyI:C maturation stimulus for peripheral blood monocytes results in the rapid generation of fully mature and viable CD83+ DC. Furthermore, we describe an efficient and clinical applicable Ag-loading strategy for these short term cultured DC, based on mRNA electroporation of monocytes. The T cell activation capacity of these short term and serum-free cultured Mo-DC was found to be highly stimulatory in an influenza Ag model system using influenza matrix protein M1 peptide-pulsed and matrix protein mRNA-electroporated DC.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell lines

T2 cells (TAP-deficient, HLA-A2+, TxB hybrid) were provided by Dr. P. Van der Bruggen (Ludwig Institute for Cancer Research, Brussels, Belgium). Cells were cultured in complete medium consisting of IMDM supplemented with L-glutamine (2 mM), penicillin (100 U/ml), streptomycin (100 µg/ml), amphotericin B (1.25 µg/ml; Fungizone), and 10% FCS (Sera Lab, Sussex, U.K.). Cells were maintained in logarithmic phase growth at 37°C in a humidified atmosphere supplemented with 5% CO2. All cell culture reagents, except FCS, were purchased from Life Technologies (Paisley, U.K.).

Source of primary cells

PBMC were obtained from hemochromatosis patients. The six PBMC donors used in this study are designated by letters A–F. Mononuclear cells were isolated by Ficoll-Hypaque gradient separation (LSM, ICN Biomedicals, Costa Mesa, CA). Monocytes were directly isolated and used for DC culture, as described below. PBMC for DC/T cell cocultures were cryopreserved in a solution consisting of 90% FCS and 10% DMSO and were stored at -80°C until use.

DC culture

Monocytes were allowed to adhere in AIM-V medium (Life Technologies) for 2 h at 37°C in six-well culture plates (20 x 106 PBMC/well). After careful removal of the nonadherent fraction, cells were cultured in serum-free AIM-V medium supplemented with 100 ng/ml GM-CSF (Leucomax; Novartis Pharma, Basel, Switzerland) for 2 days. To obtain mature DC, polyI:C (Sigma, Cambridge, U.K.) was added 24 h after starting the culture at a concentration of 25 µg/ml. The typical yield and purity of the DC culture were 1–2 x 106 cells/well containing 60–70% DC. For electroporation experiments monocytes were isolated from PBMC by magnetic isolation using CD14 microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s instructions. Routinely, 4–8 x 106 monocytes were obtained starting from 100 x 106 PBMC with purity levels >=85%.

Immunophenotype and enhanced green fluorescent protein (EGFP) analysis of DC

For immunophenotyping of DC, the following FITC-labeled mAbs were used: CD1a-FITC, HLA-DR-FITC, CD80-FITC (BD PharMingen, San Diego, CA), CD14-FITC (BD Biosciences, Erembodegem, Belgium), and CD86-FITC (Serotec, Oxford, U.K.). Also, the nonconjugated CD83 mAb (HB-15 clone; Immunotech, Marseilles, France) was used, followed by staining with a secondary rabbit anti-mouse FITC Ab (DAKO, Glostrup, Denmark). Nonreactive isotype-matched Abs (BD Biosciences) were used as controls. Ethidium bromide was added before FCM analysis on a FACScan analytical flow cytometer (BD Biosciences) to assess cell viability and to exclude dead cells from the analysis. Gating was also performed to exclude remaining lymphocytes in the DC cultures. For immunophenotyping of EGFP mRNA-electroporated DC, the following PE-labeled mAbs were used: CD1a-PE (Caltag Laboratories, San Francisco, CA), HLA-DR-PE, CD80-PE, CD14-PE (BD Biosciences), CD86-PE (BD PharMingen), and the secondary rabbit anti-mouse PE Ab (DAKO) for CD83 staining.

Allogeneic MLR

Immature and mature DC were used for stimulation of allogeneic PBMC. Briefly, immature or mature DC were cocultured with 20 x 106 allogeneic PBMC (ratio, 1:10) in 10 ml IMDM supplemented with 5% human (h) AB serum (Sigma) in T25 culture flasks. On day 4 of culture, 5 ml fresh medium (IMDM and 5% hAB serum) was added to the cultures. On day 7 of culture, cells were analyzed for reactivity. For this, stimulated PBMC (1 x 105 cells) were restimulated with PBMC from the DC donor (1 x 104 cells) in 96-well round-bottom plates for 6 h at 37°C in a total volume of 100 µl. Supernatant samples from these cocultures were tested for IFN-{gamma} secretion by IFN-{gamma} ELISA (BioSource, Nivelle, Belgium).

Synthetic peptides

An influenza virus-specific HLA-A*0201-restricted matrix protein M1 peptide (M1; aa 58–66, GILGFVFTL) was used for activation or for detection of matrix protein M1 peptide specific T cells when pulsed on DC and T2 cells, respectively. A human papillomavirus (HPV) HLA-A2-restricted E7 protein-specific peptide (E7; aa 11–20, YMLDLQPETT) was used in control experiments when pulsed on T2 cells. The peptides (>95% pure) were purchased from Sigma-Genosys (Cambridge, U.K.). The peptides were dissolved in 100% DMSO to 10 mg/ml, further diluted to 1 mg/ml in serum-free IMDM, and stored in aliquots at -80°C. The peptides were used at a final concentration of 20 µM.

Peptide pulsing of DC and T2 cells

DC or T2 cells were washed twice with IMDM medium and subsequently incubated (2 x 106 cells/ml) for 1 h at room temperature in 5-ml polystyrene tubes with 20 µg/ml peptide in serum-free IMDM medium supplemented with 2.5 µg/ml {beta}2-microglobulin (Sigma). Afterward, DC or T2 cells were washed and used, respectively, as stimulators for PBMC or as restimulators in cytokine release assays.

Production of in vitro transcribed mRNA

The pGEM4Z/EGFP/A64 (provided by Dr. E. Gilboa, Duke University Medical Center, Durham, NC) and pGEM4Z/M1/A64 (provided by Dr. A. Steinkasserer, University of Erlangen, Erlangen, Germany) plasmids were propagated in Escherichia coli supercompetent cells (Stratagene, La Jolla, CA) and were purified on endotoxin-free Qiagen-tip 500 columns (Qiagen, Chatsworth, CA). The plasmids were linearized with SpeI (MBI Fermentas, St. Leon-Rot, Germany), purified using a PCR purification kit (Qiagen) and were used as DNA templates for the in vitro transcription reaction. Transcription was conducted in a final 20-µl reaction mix at 37°C using the T7 MessageMachine Kit (Ambion, Austin, TX) to generate 5'-capped in vitro transcribed mRNA. Purification of mRNA was performed by DNase I digestion, followed by LiCl precipitation, according to the manufacturer’s instructions. mRNA quality was checked by agarose-formaldehyde gel electrophoresis. The RNA concentration was assayed by spectrophotometric analysis at OD260. RNA was stored at -80°C in small aliquots.

Cell transfections

Electroporation of mRNA was performed as described previously (20) with minor modifications. Briefly, before electroporation, CD14 microbead-isolated monocytes were washed twice with Optimix Washing Solution (EquiBio, Ashford, Middlesex, U.K.) and resuspended to a final concentration of 50 x 106 cells/ml in Optimix electroporation buffer (EquiBio). Subsequently 0.2 ml of the cell suspension was mixed with 20 µg in vitro transcribed mRNA and electroporated in a 0.4-cm cuvette at 300 V and 150 µF using an Easyject Plus device (EquiBio). After electroporation, DC were cultured as described above.

Induction of MHC class I-restricted influenza-specific T cells

M1 peptide-pulsed immature DC, M1 peptide-pulsed mature DC, and matrix protein mRNA-electroporated mature DC were used for Ag-specific stimulation of PBMC. Briefly, 2 x 106 Ag-loaded DC were cocultured with 20 x 106 autologous PBMC (ratio, 1:10) in 10 ml IMDM supplemented with 5% hAB serum in T25 culture flasks. On day 4 of culture, 5 ml fresh medium (IMDM and 5% hAB serum) was added to the cultures. On day 7 of culture, cells were analyzed for Ag specificity.

IFN-{gamma} ELISA

T2 cells pulsed with matrix protein M1-derived peptide were used as restimulators of primed PBMC. Stimulators and responder PBMC were washed and resuspended in IMDM and 5% hAB serum. Then responder PBMC (1 x 105 cells) were coincubated with stimulator cells (1 x 104 cells) in 96-well round-bottom plates for 6 h at 37°C in a total volume of 100 µl. This 6-h incubation was previously determined to be optimal to allow for measurable influenza Ag-specific IFN-{gamma} production and to avoid allo-specific IFN-{gamma} production against the T2 targets (29). Supernatant samples from these cocultures were tested for IFN-{gamma} secretion by IFN-{gamma} ELISA (BioSource, Nivelle, Belgium). Irrelevant HPV E7 peptide-pulsed T2 cells were used as control stimulators. Individual T cell activation experiments were analyzed in duplicate.

IFN-{gamma}-secreting cell assay

PBMC cultured as described above (1 x 106) were restimulated for 3 h in 24-well plates with T2 cells (1 x 105) pulsed with M1 peptide or E7 peptide as a control. Next, IFN-{gamma}-secreting cells were analyzed by a flow cytometric IFN-{gamma} Secretion Assay Detection kit (Miltenyi Biotec) according to manufacturer’s instructions. Cells were also stained with CD8-FITC (BD Biosciences), and 5 x 105 cells were analyzed per sample by flow cytometry. Analysis was performed by gating on the lymphocyte population.

Statistics

Results are expressed as the mean ± SD. Comparisons were validated using Student’s t test. A value of p <= 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Characterization of short term and serum-free in vitro cultured DC with or without polyI:C maturation

After monocyte enrichment from PBMC, cells were cultured for 2 days in AIM-V medium supplemented with GM-CSF only. To obtain mature DC, polyI:C was added after 24 h of culture. Cultured cells were analyzed after a total 48-h culture period by flow cytometry. One observed difference with classical DC cultured for 6–7 days in serum-containing medium supplemented with GM-CSF and IL-4 was a lower forward and side scatter profile of the serum-free cultured cells (Fig. 1Go, upper panels). However, this was not due to serum-free- or polyI:C-induced mortality, since ethidium bromide staining showed cell populations with a mean viability of >80% (Fig. 1Go, lower panels). Immune phenotyping was also performed after 48 h of culture (Fig. 2Go). A majority of the cultured cells showed down-regulation of CD14 expression, demonstrating the loss of a characteristic monocyte marker. This down-regulation is most likely due to serum-free culture of monocytes (11), since in experimental conditions where human AB serum (1%) was added to the DC culture medium, no down-regulation of CD14 was observed (data not shown). Cells cultured without polyI:C showed moderate expression of HLA-DR, and only a small fraction showed expression of CD83 and of the costimulatory molecules CD80 and CD86. This corresponds to a typical immature DC phenotype. In contrast, cells that were exposed to polyI:C, showed a fast up-regulation of HLA-DR, CD83, and the costimulatory molecules CD80 and CD86, corresponding to the typical phenotype of mature DC. CD1a was present on a small proportion of the cells (Fig. 2Go).



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  		FIGURE 1. Representative flow cytometric analysis of scatter profile and viability of short term, serum-free cultured immature DC and polyI:C-maturated DC. Left, Immature Mo-DC (iMo-DC) cultured for 2 days in AIM-V medium and GM-CSF. Right, PolyI:C-maturated Mo-DC (mMo-DC). The upper dot plots show forward and side scatter profiles of all cells. The R1 gate shows the percentage of DC in the cultures. The lower dot plots show mortality by ethidium bromide staining within the cultured DC (upper numbers, ethidium bromide-positive dead DC; lower numbers, ethidium bromide-negative living DC). The lower dot plots were gated on R1 (upper panel). The data shown are from PBMC donor A. The results are representative for PBMC from donors A, B, and F for immature DC and from donors A–F for mature DC.

 


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  		FIGURE 2. Representative phenotypic analysis of short term, serum-free cultured immature DC and serum-free cultured, polyI:C-maturated DC. Flow cytometric analysis of FITC-labeled mAbs directed against DC and monocyte markers: CD14, HLA-DR, CD86 (left) and CD1a, CD80, CD83 (right). After 2 days of culture in AIM-V medium supplemented with GM-CSF, with or without addition of polyI:C after 1 day of culture, DC were analyzed by outgating remaining lymphocytes. Comparative data are shown in histograms for immature (thin line) and mature (thick line) DC. The data shown are from PBMC donor A. The results are representative for PBMC from donors A, B, and F for immature DC and for donors A–F for mature DC.

 
PolyI:C-maturated serum-free cultured DC are more potent than their immature counterparts in inducing in vitro T cell immune responses

To determine whether the new cultured cell types also had the functional properties of DC, their stimulatory capacity was first evaluated in a modified allogeneic MLR. For this, immature and mature DC were cultured for 7 days with allogeneic PBMC. Next, the stimulated PBMC were restimulated with PBMC from the DC donor, and IFN-{gamma} secretion in the supernatant was analyzed by ELISA (Fig. 3Go). Based on the level of IFN-{gamma} secretion against the PBMC targets, the results show that mature DC were more potent in inducing an allogeneic MLR response than immature DC (3.1 ± 0.1 IU/ml/6 h for immature DC vs 21.3 ± 0.8 IU/ml/6 h for mature DC; p = 0.0004). Autologous Ag-specific stimulatory capacity was evaluated in an influenza model system, DC were pulsed with an HLA-A2-restricted influenza matrix protein M1-specific peptide and cocultured with autologous PBMC. After 7 days of coculture, cultured PBMC were restimulated with M1 peptide- or E7 control peptide-pulsed T2 cells. After a 6-h restimulation, IFN-{gamma} secretion in the supernatant was analyzed by ELISA (Fig. 4Go, A and B). Based on the level of IFN-{gamma} secretion against the influenza M1 target, the results show that mature DC were more potent in inducing an autologous immune response than immature DC (Fig. 4GoA; 2.3 ± 0.3 IU/ml/6 h for immature DC vs 22.9 ± 3.1 IU/ml/6 h for mature DC; p = 0.0006). The specificity of this immune response was shown by significantly lower IFN-{gamma} production against the control HPV E7 target compared with the influenza M1 target (Fig. 4GoA: for mature DC, p = 0.0079; for immature DC, p = 0.0461; Fig. 4GoB: for mature DC, p = 0.0064). To show that IFN-{gamma} was produced by CD8+ T lymphocytes, we used an IFN-{gamma}-secreting assay in which, after restimulation of cultured PBMC with an influenza (T2/M1) or control (T2/E7) target, IFN-{gamma}-secreting cells are directly stained for detection by flow cytometry (Fig. 5Go). Flow cytometric analysis showed detectable M1-specific IFN-{gamma}-secreting T cells within the CD8+ T cell population of PBMC cultures initially stimulated with mature DC pulsed with M1 peptide. This immune response was virtually not seen in cultures initially stimulated with immature DC.



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  		FIGURE 3. Allogeneic stimulatory capacity of short term, serum-free cultured immature DC vs serum-free cultured, polyI:C-maturated DC. Immature and mature short term cultured DC (respectively, iMo-DC and mMo-DC) were used as stimulators for allogeneic PBMC during a 7-day coculture. Afterward, primed PBMC were restimulated with PBMC from the DC donor during a 6-h coculture. Activated T cells in the primed PBMC culture were detected, as shown by IFN-{gamma} production against the target PBMC. Results are shown as the mean ± SD of two individual experiments for cultures initiated with immature DC (iMo-DC) and mature DC (mMo-DC). The significant difference is indicated with an asterisk. Results were obtained with PBMC from donors B and C.

 


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  		FIGURE 4. Stimulatory capacity of short term, serum-free cultured immature DC vs serum-free cultured, polyI:C-maturated DC. Influenza matrix protein M1 peptide-pulsed immature and mature DC (respectively, iMo-DC and mMo-DC) were used as stimulators for autologous PBMC during a 7-day coculture. Afterward, primed PBMC were restimulated with T2 cells and pulsed with an MHC class I-restricted influenza matrix protein M1 peptide (T2/M1) during a 6-h coculture. Ag-specific T cells in the primed PBMC culture were detected, as shown by increased IFN-{gamma} production. As a control, irrelevant HPV E7 peptide-pulsed T2 cells (T2/E7) were used as stimulators. Significant differences are indicated with an asterisk. Results were obtained with PBMC from donor B (A; three experiments) and donor F (B; two experiments).

 


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  		FIGURE 5. Stimulatory capacity of serum-free cultured immature DC vs serum-free cultured, polyI:C-maturated DC. Direct staining of IFN-{gamma}-secreting CD8+ T cells after restimulation with an influenza target. Influenza matrix protein M1 peptide-pulsed immature and mature DC (respectively, iMo-DC and mMo-DC) were used as stimulators for PBMC during a 7-day coculture. Primed PBMC were then restimulated for 3 h with T2 cells pulsed with an MHC class I-restricted influenza M1 peptide or an HPV E7 control peptide. Dot plots show IFN-{gamma}-secreting cells within the CD8+ and CD8- lymphocyte populations. The numbers of IFN-{gamma}-secreting cells indicated on the dot plots are percentages of total lymphocytes. Results were obtained with PBMC from donor B.

 
mRNA electroporation of monocytes, followed by differentiation to DC

Using a previously optimized mRNA electroporation protocol, we examined the possibility of genetic modification of the above-described DC. In these experiments the EGFP reporter gene was used to assess mRNA transfection efficiency. After optimization, the following mRNA electroporation and culture protocol resulted in the generation of Ag-loaded mature DC. First, monocytes were isolated from PBMC by CD14 immunobead magnetic separation. After electroporation, cells were resuspended in serum-free AIM-V medium supplemented with GM-CSF. After 24 h of culture, polyI:C was added to the cultures to obtain mature DC. The cultured DC were analyzed 48 h after electroporation of the monocytes. No difference was observed in scatter profile between nonelectroporated and EGFP mRNA-electroporated monocytes that were cultured to DC (Fig. 6GoA). The mean electroporation-related mortality in the DC cultures was 10% (mean of three independent experiments; Fig. 6GoB). This low cell mortality was probably due to the serum-free culture condition, because addition of autologous plasma following electroporation resulted in the lack of electroporation-related mortality (data not shown). Comparing the level of EGFP fluorescence in nonelectroporated and EGFP mRNA-electroporated, short term cultured DC, the data show low, but detectable, EGFP expression in practically all viable mRNA-loaded DC (Fig. 6GoC). The phenotype of the cultured cells was examined by flow cytometry for the characteristic DC markers (Fig. 7Go). We observed no difference in phenotype between nonelectroporated and EGFP mRNA-electroporated short term and serum-free cultured mature DC. Remarkably, compared with the data shown in Fig. 2Go in which DC were cultured from adherent monocytes, less down-regulation of CD14 was observed on DC grown from CD14+ positively isolated monocytes (<10% CD14+ DC generated from adherent PBMC vs ~50% CD14+ DC generated from CD14+ monocytes).



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  		FIGURE 6. Representative flow cytometric analysis of scatter profile, viability, and EGFP expression of EGFP mRNA-electroporated monocytes short term cultured to mature DC. Monocytes, electroporated (EP; lower dot plots) or not (EP; upper dot plots) with EGFP mRNA were cultured for 2 days in AIM-V medium and GM-CSF. Maturation was induced by polyI:C after 24 h of culture. A, Scatter profile of the cultured mature DC. B, Ethidium bromide staining of the cultured mature DC. The dot plots were gated on R1 (scatter profile). The indicated numbers show the percentage of ethidium bromide-negative living DC. C, FL-1 EGFP fluorescence histogram overlay of nonelectroporated mature DC (thin dotted line) and EGFP mRNA-electroporated mature DC (thick line). The data shown are from PBMC donor D. The results are representative for PBMC from donors C–E.

 


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  		FIGURE 7. Representative phenotypic analysis of monocytes electroporated with mRNA and short-term, serum-free monocytes cultured to mature DC. Flow cytometric analysis of PE-labeled mAbs directed against DC and monocyte markers: CD14, CD80, CD86, HLA-DR, and CD83. Monocytes, electroporated (EP; lower dot plots) or not (EP; upper dot plots) with EGFP mRNA were cultured for 2 days in AIM-V medium and GM-CSF. Maturation was induced by polyI:C after 24 h of culture. Histograms show the level of marker expression (black overlay) against isotype control staining (dotted line). The data shown are from PBMC donor D. The results are representative for PBMC from donors C–E.

 
Stimulatory capacity of mRNA-loaded short term cultured mature DC

We examined in an influenza model system whether mRNA-electroporated monocytes rapidly differentiated in serum-free medium into mature DC could stimulate Ag-specific T cells upon coculture with PBMC. In these experiments monocytes were electroporated with mRNA encoding influenza matrix protein M1 and further cultured to mature DC as described above. Next, DC were cocultured with autologous PBMC without the addition of exogenous cytokines. After 7 days of culture, primed PBMC were restimulated with T2 cells pulsed with a MHC class I-restricted influenza matrix protein M1 peptide (T2/M1), and IFN-{gamma} secretion was determined after 6 h by ELISA (Fig. 8Go). Upon restimulation with peptide-pulsed T2 cells, the activated T cells in the primed PBMC culture produced IFN-{gamma} against the immunodominant M1 matrix protein peptide. The specificity of this activation was shown by only background IFN-{gamma} production of the primed PBMC culture against HPV E7 peptide-pulsed T2 cells (T2/M1 vs T2/E7; for Fig. 8GoA, p = 0.0002; for Fig. 8GoB, p < 0.0001).



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  		FIGURE 8. Stimulatory capacity of mRNA-electroporated monocytes short term, serum-free cultured to mature DC. Monocytes, electroporated with influenza matrix protein mRNA, were cultured for 2 days in AIM-V medium and GM-CSF. Maturation was induced by polyI:C after 24 h of culture. These mature Ag-loaded DC were used as stimulators for autologous PBMC during a 7-day coculture. Afterward, primed PBMC were restimulated during a 6-h coculture with T2 cells and pulsed with an MHC class I-restricted influenza matrix protein M1 peptide (T2/M1). Ag-specific T cells in the primed PBMC culture were detected, as shown by increased IFN-{gamma} production. As a control, irrelevant HPV E7 peptide-pulsed T2 cells (T2/E7) were used as stimulators. Results are shown as the mean ± SD of three individual experiments for PBMC from donor B (indicated as A) and PBMC from donor C (indicated as B). Significant differences are indicated with an asterisk.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the first part of this study we describe an alternative culture protocol for Mo-DC. The present serum-free culture and polyI:C maturation protocol resulted in the rapid generation of fully mature, viable, and highly stimulatory CD83+ DC. The observed phenotypical properties (Fig. 2Go) are in accordance with previous reported data by Czerniecki et al. (11) using similar DC culture protocols for short term and serum-free cultured DC. The observed difference in CD14 down-regulation between DC cultured from adherent monocytes (Fig. 2Go, CD14+ cells <10%) and DC cultured from magnetic bead-isolated monocytes (Fig. 7Go, ~50% CD14+ cells) could probably be ascribed to the CD14 selection procedure and warrants further investigation, e.g., by comparing positive selections of CD14+ cells and negative selection of CD14+ cells by depletion of B, T, and NK cells. A similar observation was reported by Cavanagh et al. (30) where conventional DC, grown from adherent PBMC, were compared with DC generated from monocytes sorted by CD14 positive selection. The relatively rapid maturation of the presented short term cultured DC, demonstrated by the up-regulation of HLA-DR, CD80, CD86, and CD83, compared with immature DC is, according to previous reports, due to polyI:C signaling via Toll-like receptor 3 (31). This signaling pathway induces IFN-{alpha} production by the DC (32), which can act as a strong maturation stimulus. Furthermore, type I IFNs, like IFN-{alpha}, might induce IL-15 production and in this way strongly promote a Th1 response, which is needed for induction of a strong CD8+ T cell response (33, 34). This might account for the differences we observed when comparing immature and mature short term cultured DC in both allogeneic and autologous T cell stimulatory capacities. These data also confirm a recent report by Bhardwaj and colleagues (24) describing the need for mature DC to activate influenza-specific memory T cells.

The second part of this study focused on the genetic modification of these short term, serum-free cultured DC. Previously, we developed an Ag-loading strategy based on electroporation of mRNA into DC cultured in the presence of serum and GM-CSF and IL-4. This transfection technology resulted in high level transgene expression in Mo-DC using an EGFP reporter gene. More than 70% of the transfected cells showed high level EGFP expression (relative fluorescence between 102 and 103 decade as measured by flow cytometry) and retained their phenotypical properties after transfection (20, 29). However, the use of this technology for transfection of short term, serum-free cultured DC, as presented in this study, resulted in substantial cell mortality among transfected cells (data not shown). Because to be effective a DC vaccine should have a high DC viability, we attempted to transfect fresh monocytes, followed by rapid differentiation to DC. As shown by the data in Fig. 6Go, cell viability was high, and Ag was still detectable in the DC 2 days after the initial electroporation in virtually all cells when using this strategy. Compared with our previous results (20, 29), related to the electroporation of DC cultured in serum in the presence of GM-CSF and IL-4, the level of protein expression, e.g., EGFP, was much lower in monocytes after 24 h (data not shown) and 48 h (Fig. 6GoC). Monocytes that were electroporated with EGFP-mRNA and subsequently differentiated to mature DC showed only a small shift of EGFP fluorescence compared with nonelectroporated control DC. This can be explained by the difficulty of obtaining high protein expression levels in primary uncultured mononuclear cells (data not shown). However, there is no consensus yet that a high level of Ag expression in DC is mandatory for the induction of a stronger immune response. Here we provide functional evidence that despite the lower level of Ag expression in these short term cultured DC, a specific immune response in an influenza model system could be initiated very efficiently. Previous experiments in our laboratory (29) focused on the stimulatory capacity of influenza matrix protein mRNA-electroporated conventional DC, i.e., DC cultured for 6–7 days in serum-containing medium supplemented with GM-CSF and IL-4, and maturated with a mixture consisting of TNF-{alpha}, PGE2, IL-1, and IL-6. Comparing the final outcome in terms of autologous influenza-specific T cell activation, Ag mRNA-loaded conventional DC and short term cultured DC gave similar results, indicating the validity of this protocol.

In conclusion, this combined serum-free culture and polyI:C maturation (and optional mRNA electroporation) of peripheral blood monocytes results in the rapid generation of fully mature, viable, and highly stimulatory CD83+ DC. This ex vivo protocol results in an important reduction in time and consumables for preparation of mature DC compared with classical culture protocols. This might be of importance not only for laboratory experiments, but also for clinical immunotherapy protocols.


   Acknowledgments
 
We thank Kevin Ariën and Dr. Guido Vanham of the Laboratory of Immunology at Institute of Tropical Medicine (Antwerp, Belgium) for helpful technical suggestions.


   Footnotes
 
1 This work was supported by Grant G.0313.01 from the Fund for Scientific Research-Flanders, Belgium (FWO-Vlaanderen), grants from the Scientific Committee of the Fortis Bank Verzekeringen-financed Cancer Research, and grants from the Belgian Federation against Cancer. P.P., N.C., and A.V.D. hold Ph.D. fellowships from the Flemish Institute for Science and Technology. G.V.d.B. holds a Ph.D. fellowship from the FWO-Vlaanderen. V.F.I.V.T. is a postdoctoral fellow of FWO-Vlaanderen. Back

2 Address correspondence and reprint requests to Dr. Viggo F. I. Van Tendeloo, Laboratory of Experimental Hematology, University of Antwerp, Antwerp University Hospital, Wilrijkstraat 10, B-2650 Edegem, Belgium. E-mail address: viggo.van.tendeloo{at}uza.be Back

3 Abbreviations used in this paper: DC, dendritic cell; EGFP, enhanced green fluorescent protein; h, human; HPV, human papillomavirus; Mo-DC, monocyte-derived DC; polyI:C, polyriboinosinic polyribocytidylic acid. Back

Received for publication March 26, 2002. Accepted for publication May 31, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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