mRNA-electroporated mature dendritic cells retain transgene expression, phenotypical properties and stimulatory capacity after cryopreservation

Abstract

Genetically modified dendritic cells (DC) are increasingly used in vitro to activate cytotoxic T lymphocyte (CTL) immune responses. Because T cell activation protocols consist of multiple restimulation cycles of peripheral blood lymphocytes with antigen-loaded mature DC, continuous generation of DC is needed throughout the experiment. Therefore, cryopreservation of DC loaded with antigen is a valuable alternative for weekly generation and modification of DC. Recently, we described an antigen loading method for DC based on electroporation of defined tumor antigen mRNA. In this study, we demonstrate that mRNA-electroporated DC can efficiently be prepared for cryopreservation. Using an optimized maturation and freezing protocol after mRNA electroporation, we obtained high transgene-expressing viable mature DC. In addition, we showed that these modified cryopreserved DC retain stimulatory capacity in an influenza model system. Therefore, cryopreservation of mature mRNA-electroporated DC is a useful method for continuous availability of antigen-loaded DC throughout T cell activation experiments.

Introduction

Dendritic cells (DC) are the most potent professional antigen-presenting cells of the immune system and are capable of initiating immune responses in vitro and in vivo.1 One of the great challenges in immunotherapy protocols is to introduce relevant antigens into DC for stimulation of major histocompatibility complex (MHC) class I-restricted antitumoral immunity. Recently, we2 and others3,4 described ex vivo loading methods for DC based on the use of defined mRNA as a source of antigen. These transfection protocols provide several safety advantages for clinical perspectives. Transfection of mRNA avoids introduction of immunostimulatory DNA5 and can be achieved without the use of viral vectors. Also, as compared to peptide-pulsed DC, the use of defined mRNA overcomes the need for characterization of immunogenic epitopes and of the HLA haplotype of the patient. Alternatively, total mRNA derived from tumor samples can be used to load DC with the full spectrum of antigens in order to activate an immune response against patient-specific tumour antigens.6 As protocols are developed for inducing in vitro immune responses, it seems that several stimulation cycles of peripheral blood lymphocytes are needed for in vitro expansion of sufficient numbers of antigen-specific T cells. This implies that DC need to be generated and loaded with an antigen on a weekly basis, which could be a time-consuming procedure. Therefore, protocols have been developed for the cryopreservation of DC.7,8 Recently, it was shown that mature DC, pulsed with immunodominant MHC class I-restricted peptides, could be frozen and efficiently used after thawing for inducing in vitro immune responses.9 In this study, we investigated whether mRNA-electroporated DC could be efficiently prepared for cryopreservation. First, we investigated the effect of a freezing cycle on intracellular mRNA by monitoring the expression level of an enhanced green fluorescent protein (EGFP) transgene introduced by mRNA electroporation (EP) in leukemic K562 cells. Next, we monitored transgene expression of the reporter gene in mRNA-electroporated monocyte-derived DC (Mo-DC) after cryopreservation. To our knowledge, this is the first report describing the influence of freezing Mo-DC on the expression level of a transgene introduced by mRNA electroporation. In addition, we compared phenotypical properties and survival rate of frozen mRNA-transfected immature and mature Mo-DC. We also show that influenza matrix protein M1 mRNA-electroporated cryopreserved DC retain their stimulatory capacity for inducing MHC class I-restricted influenza-specific primary T cells.

Materials and methods

Cell lines

K562 cells were obtained from the American Type Culture Collection (ATCC No. CCL-243, Rockville, MD, USA). T2 cells (TAP-deficient, HLA-A2+, TxB hybrid) were kindly provided by Dr Pierre Van der Bruggen (Ludwig institute for Cancer Research, Brussels, Belgium). Cells were cultured in complete medium consisting of Iscove's modified Dulbecco's medium (IMDM) supplemented with L-glutamine (2 mM), penicillin (100 U/ml), streptomycin (100 μg/ml), amphotericin B (1.25 μg/ml Fungizone) and 10% fetal calf serum (FCS; Sera Lab, Sussex, UK). Cells were maintained in logarithmic phase growth at 37°C in a humidified atmosphere supplemented with 5% CO2. All cell culture reagents were purchased from Gibco BRL (Paisley, UK).

Source of primary cells

Peripheral blood mononuclear cells (PBMC) were obtained from healthy volunteers or hemochromatosis patients. Mononuclear cells were isolated by Ficoll–Hypaque gradient separation (LSM, ICN Biomedicals, Costa Mesa, CA, USA).

In vitro culture of DC

Immature monocyte-derived DC (iMo-DC) were generated from PBMC as described by Romani et al.10 Briefly, PBMC were allowed to adhere in AIM-V medium (Gibco BRL) for 2 h at 37°C. The non-adherent fraction was removed, and adherent cells were further cultured for 6 days in IMDM supplemented with 10% FCS. One hundred ng/ml granulocyte–macrophage colony-stimulating factor (GM-CSF; Leucomax, Novartis Pharma, Basel, Switzerland) and 1000 U/ml interleukin (IL)-4 (R&D Systems, Minneapolis, MN, USA) were added to the cultures every 2–3 days starting from day 0. Maturation of iMo-DC was induced after day 6 by adding a maturation cocktail consisting of 2.5 ng/ml tumour necrosis factor (TNF)-α (Roche Molecular Biochemicals, Mannheim, Germany), 10−7 M prostaglandine E2 (PGE2) (Sigma, St Louis, MO, USA), 500 U/ml IL-6 (Biosource Europe, Nivelles, Belgium), 100 U/ml IL-1 (Biosource, Camarillo, CA, USA).

Immunophenotyping of DC

Immunophenotyping was performed as described previously.11 The following monoclonal antibodies were used: CD1a-phycoerythrin (PE) (Caltag Laboratories, San Francisco, CA, USA), CD14-PE, HLA-DR-PE, CD80-PE (Becton Dickinson, Erembodegem, Belgium), CD86-PE (PharMingen, San Diego, CA, USA), Nonreactive isotype-matched antibodies (Becton Dickinson) were used as controls.

Production of in vitro transcribed mRNA

The pGEM4Z/EGFP/A64 (kindly provided by Prof Dr E Gilboa, Duke University Medical Center, Durham, NC, USA) and pGEM4Z/M1/A64 (kindly provided by Prof Dr A Steinkasserer, University of Erlangen, Erlangen, Germany) plasmids were propagated in E. coli supercompetent cells (Stratagene, La Jolla, CA, USA) and purified on endotoxin-free QIAGEN-tip 500 columns (Qiagen, Chatsworth, CA, USA). The plasmids were linearized with SpeI (MBI Fermentas, St Leon-Rot, Germany), purified using a PCR purification kit (Qiagen) and used as DNA templates for in vitro transcription reaction. Transcription was carried out in a final 20–100 μl reaction mix at 37°C using the T7 MessageMachine Kit (Ambion, Austin, TX, USA) to generate 5′ capped in vitro transcribed (IVT) 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. RNA concentration was assayed by spectrophotometrical analysis at OD260. RNA was stored at −80°C in small aliquots.

Cell transfections

Electroporation of mRNA was done as described previously2 with minor modifications. Briefly, prior to electroporation, K562 cells or iMo-DC were washed twice with Optimix Washing Solution (EquiBio, Ashford, UK) and resuspended to a final concentration of 50 × 106 cells/ml in Optimix electroporation buffer (EquiBio). Subsequently, 0.2 ml of the cell suspension was mixed with 20 μg of IVT mRNA and electroporated in a 0.4 cm cuvette at 300 V and 150 μF using an Easyject Plus device (EquiBio). After electroporation, fresh complete medium (including cytokines for DC) was added to the cell suspension and cells were further incubated at 37°C in a humidified atmosphere supplemented with 5% CO2.

EGFP analysis

EGFP-transfected cells were checked for EGFP expression at different time points before freezing and after thawing of the cells. Briefly, cells (1–5 × 106) were washed once in phosphate-buffered saline (PBS) supplemented with 1% FCS and resuspended in 0.5 ml of PBS supplemented with 1% BSA and 0.1% sodium azide. Ethidium bromide was added at a final concentration of 10 μg/ml directly prior to FCM analysis on a FACScan analytical flow cytometer (Becton Dickinson) to assess cell viability. For EGFP analysis in DC cultures, gating was performed on cells exhibiting a large forward scatter (FSC) and large side scatter (SSC) profile, in order to allow exclusion of contaminating autologous lymphocytes. Gated DC were then evaluated for EGFP expression. For phenotypical data, viable EGFP+ DC were gated based on a positive EGFP fluorescence.

Freezing procedure

K562 cells or DC were resuspended in cryotubes (Nunc CryoTube Vials; Nalgene Nunc International, Roskilde, Denmark) at a concentration of 10 × 106 per ml in pure FCS. Next, the suspension was mixed on ice with an equal volume of FCS supplemented with 20% DMSO (Sigma). Cell suspensions were slowly frozen (−1°C/min) to −80°C by using a cryo freezing container (Nalgene Nunc International). Cells cryopreserved for EGFP analysis or phenotyping were used after a frozen period of 24–36 h. Cells used for T cell activation were used after a frozen period of 7 days.

Thawing procedure

Frozen cells were thawed quickly in a 37°C water bath, followed by the addition of 100 μg/ml DNaseI (Boehringer-Mannheim) and 50 μl/ml 3.79% MgSO4 for 10 min. Next, cells were centrifuged and resuspended at 0.5 × 106 per ml in IMDM for 15 min to remove residual DMSO. Finally, cells were washed once and resuspended in full medium containing FCS and cytokines.

Synthetic peptides

An influenza virus-specific HLA-A*0201-restricted matrix protein M1 peptide was used for detection of matrix protein-specific T cells (M1; amino acids (aa) 58–66, GILGFVFTL). A human papillomavirus (HPV) HLA-A2-restricted E7 protein-specific peptide was used in control experiments (E7; aa 11–20, YMLDLQPETT). The peptides (>95% pure) were purchased from Sigma-Genosys (Cambridge, UK). 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 T2 cells

T2 cells were washed twice with IMDM and subsequently incubated with 20 μM peptide in serum-free medium supplemented with 2.5 μg/ml β2 microglobulin (Sigma) for 2 h at room temperature in 15 ml conical tubes. Afterwards, cells were washed twice and used as stimulators in cytokine release assays.

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

Immature Mo-DC were electroporated with influenza matrix protein mRNA on day 6 of culture, followed by a 24-h maturation step. Next, these mature DC were used for stimulation of peripheral blood mononuclear cells (PBMC). Briefly, 2 × 106 DC were cocultured with 20 × 106 PBMC in IMDM supplemented with 10% FCS. No additional cytokines were added. After 6–7 days of culture, cells were analyzed in a cytokine release assay. Influenza matrix protein mRNA-electroporated mature DC, that had been frozen on day 7 of culture and had been cryopreserved for 7 days, were thawed and directly used for stimulation of PBMC as described above.

Interferon (IFN)-γ ELISA

T2 cells, pulsed with an MHC class I M1 matrix protein-derived peptide, were used as stimulators of cultured influenza matrix protein-specific T cells. Stimulators were washed twice and resuspended in IMDM containing 10% FCS. Responder primed PBMC, that were cultured for 6–7 days with M1 matrix protein mRNA-electroporated DC, were washed twice and resuspended in IMDM containing 10% FCS. Then, responder primed PBMC (1 × 105 cells) were coincubated with stimulator cells (1 × 104 cells) in 96-well round-bottom plates for 6 h at 37°C in a total volume of 100 μl. Experiments were done in triplicate. Supernatant samples from these cocultures were tested for specific IFN-γ secretion by IFN-γ ELISA (Biosource). As controls, unpulsed or irrelevant E7 peptide-pulsed T2 cells were used as stimulators and fresh PBMC as responders. In some experiments, before analysis of antigen specificity, CD8+ cells were isolated from the cultured PBMC using CD8 Microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany).

Statistical analysis

Results are expressed as mean ± standard deviation or standard error as indicated in the legends. Comparisons were validated using Student's t-test. A P value 0.05 was considered to be statistically significant.

Results and discussion

EGFP expression in cryopreserved mRNA-electroporated K562 cells

In order to optimize cryopreservation and monitor its influence on stability and functionality of electroporated intracellular mRNA, we used leukemic K562 cells, as these cells are highly transfectable with mRNA, as demonstrated previously.2 K562 cells were electroporated with enhanced green fluorescent protein (EGFP) mRNA as a reporter system and transgene expression was followed in non-frozen samples after electroporation and in frozen samples after thawing (Table 1). In these experiments we explored two alternative protocols for cryopreservation, based on the time point of freezing following EGFP mRNA electroporation. In protocol 1, cells were electroporated with mRNA followed by a culture period of 3 h, in order to allow for recovery and for transgene expression to start. After 3 h, transgene expression was determined by flow cytometry, and aliquots of cells were frozen as described in Materials and methods. In protocol 2, cells were incubated for 24 h after electroporation, in order to allow the cells to translate the introduced mRNA. Cells were frozen after 24 h of culture. When cells were thawed after being frozen at different time points, we observed less cell survival (P = 0.0025) in cultures which were frozen 3 h after the electroporation (protocol 1) as compared to 24 hours after electroporation (protocol 2), when analyzed 3 h after thawing. However, cells survived this freezing cycle, since the proportion of dead cells in the culture decreased over time. To demonstrate that intracellular EGFP mRNA was still functional, we compared the level of EGFP fluorescence by looking at the mean fluorescence intensity (MFI), as determined by flow cytometric analysis. In cultures that had been frozen 3 h after electroporation, MFI of expressed EGFP almost doubled between 3 and 24 h after thawing (P = 0.0009), implying that new protein was made and that electroporated mRNA can survive freezing. In cultures frozen 24 h after electroporation (protocol 2), this effect was not seen, presumably because an almost maximal level of translation had occurred and because of the short half-life time of mRNA.

Table 1 Transgene expression in cryopreserved mRNA- electroporated K562 cells

EGFP expression in cryopreserved mRNA-electroporated DC

Based on the observation that immature (i)Mo-DC are more susceptible to mRNA electroporation than mature (m)Mo-DC,2 we focused on transfection of DC in an immature state. As DC are more difficult to handle for cryopreservation and given the fact that freezing of cells directly after electroporation is associated with a higher mortality, we first allowed DC to express the transgene before cryopreservation (similar to protocol 2 for K562 cells). After mRNA electroporation, immature DC cells were cultured in the presence of GM-CSF + IL-4, with or without addition of a maturation cocktail (TNF-α + PGE2 + IL-1 + IL-6), the former in order to obtain mature DC. As seen in a non-frozen control of immature and mature DC, viability is not significantly affected by this electroporation in function of time (Table 2: 24 h vs 48 h after electroporation, P value 0.1849 and 0.1362 for control 1 and control 2, respectively) and cells express high levels of EGFP (Table 2; Figure 1a and b). Immature DC that were frozen 18 h after electroporation seemed to survive the freezing cycle well 6 h after thawing. There was an increase in cell mortality (+14%, P = 0.0008), but the MFI of EGFP-expressing cells was approximately the same as in non-frozen control DC (P = 0.5185). However, 24 h after thawing, there was a high level of cell mortality in the frozen cultures as compared to non-frozen control DC that had been cultured for 48 h after electroporation (64% vs 24%, P = 0.0017) (Table 2; Figure 1a). Still, the remaining living DC expressed similar high levels of EGFP as compared to non-frozen control DC (Table 2, MFI values, P = 0.1917). We also investigated transgene EGFP expression in frozen mature DC. Immature Mo-DC were electroporated, followed by a 2 h incubation in medium supplemented with GM-CSF + IL-4, in order to allow transgene expression to start. Following this, the DC maturation cocktail was added and the level of EGFP expression and cell survival was determined 24 and 48 h after transfection. In these experiments, mature DC were frozen 24 h after mRNA electroporation and transgene expression and cell survival was determined 6 and 24 h after thawing (Table 2; Figure 1b). Six hours after thawing, mature DC cultures appeared to survive the freezing and have a similar, but significantly lower, number of EGFP+ cells (63% vs 71%, P = 0.033) and a similar MFI level of EGFP+ cells as compared to non-frozen control 2 cultures (P = 0.5183). Mature DC survived the thawing procedure better than frozen immature DC (64% cell death for immature DC vs 25% for mature DC after 24 h of culture, P = 0.00004). This is in concordance with a previous recent report suggesting that mature DC are superior to immature DC for cryopreservation.9

Table 2 Transgene expression in cryopreserved mRNA- electroporated DC
Figure 1
figure1

FCM analysis of transgene expression in immature and mature DC after EGFP mRNA electroporation in non-frozen controls and after thawing of cryopreserved samples. The dot plots show EGFP fluorescence on the x-axis and ethidium bromide staining on the y-axis. Analysis was performed on cells exhibiting a large forward scatter and large side scatter profile, in order to allow exclusion of contaminating autologous lymphocytes. Percentage of dead cells (upper left corner), viable EGFP+ cells (lower right corner) and viable EGFP cells (lower left corner) is indicated based on the number of dots in the quadrant analysis. (a) Dot plots show analysis of non-frozen iMo-DC 24 h after mRNA electroporation (left), and of mRNA-electroporated iMo-DC 6 h after thawing (middle) and 24 hours (right) after thawing. (b) Dot plots show analysis of non-frozen mMo-DC 24 h after mRNA electroporation (left),and of mRNA-electroporated mMo-DC 6 h after thawing (middle) and 24 h (right) after thawing. EP, electroporation.

Phenotypical properties of cryopreserved mRNA-electroporated DC

To determine whether the mRNA electroporation and the freezing cycle have an influence on the phenotypical properties of mRNA-electroporated DC, we performed flow cytometric analysis for characteristic DC markers including CD1a, HLA-DR, CD80 and CD86. Immunophenotyping of immature and mature EGFP+ cells was performed 24 h after thawing. As controls, immature and mature non-transfected and non-frozen mRNA-electroporated DC were used for phenotyping. In general, DC were cultured for 2 days after day 6, with or without a cryopreservation period at day 7. As shown by the dot plot analysis (Figure 2), iMo-DC undergo maturation at 48 hours after mRNA electroporation as demonstrated by an upregulation of HLA-DR, CD80 and CD86 (Figure 2a and b). Thawed DC have the same upregulation of HLA-DR and CD80, but have lower levels of CD86 (Figure 2c). This is probably caused by the fact that frozen immature DC are dying 24 h after thawing. Immature Mo-DC responded well to the maturation cocktail as seen by the upregulation of HLA-DR, CD80 and CD86 in mMo-DC as compared with the expression levels in iMo-DC (Figure 2a and d). The combination of mRNA electroporation and a maturation stimulus seems to be even more potent in maturing DC, as this combination results in a high level of HLA-DR, CD80 and CD86 expression (Figure 2e). Frozen mature DC that were electroporated showed a similar high level of maturation marker expression after thawing (Figure 2f).

Figure 2
figure2

Representative example of phenotypical analysis of non-frozen and frozen mRNA-electroporated immature and mature DC. Dot plots show FCM analysis of PE-labeled monoclonal antibodies directed against typical DC markers including CD1a, HLA-DR, CD80 and CD86 (y-axis). As controls to set quadrants, isotype-matched antibodies and a PE-labeled monoclonal CD14 antibody was used. Analysis of DC markers was done on viable EGFP cells in control samples and on viable EGFP+ cells in mRNA-electroporated DC as shown by the EGFP fluorescence on the x-axis. (a) iMo-DC on day 8 of culture, (b) EGFP+ iMo-DC after mRNA electroporation on day 6, followed by 48 h of culture. (c) EGFP+ iMo-DC after mRNA electroporation on day 6, culture for 18 h, cryopreservation, thawing and culture for 24 h. (d) iMo-DC that have been stimulated for 48 h with a maturation cocktail after day 6. (e) EGFP+ iMo-DC after mRNA electroporation on day 6 and stimulation for 48 h with the maturation cocktail. (f) EGFP+iMo-DC after mRNA electroporation on day 6 and culture for 24 h with a maturation cocktail, cryopreservation, thawing and culture for 24 h in the presence of the maturation cocktail. In general, phenotyping was performed after 2 days of culture, with or without a frozen interval (that was not counted), following day 6 of the Mo-DC culture.

Stimulatory capacity of cryopreserved mRNA-electroporated DC

To show that cryopreserved mRNA-electroporated mature DC retain their stimulatory capacity after thawing, we examined in an influenza in vitro model system whether they could stimulate antigen-specific T cells upon coculture with PBMC. In these experiments, influenza matrix protein M1 mRNA-electroporated thawed mature DC were cocultured with fresh PBMC. No additional cytokines were added during the culture. After 6 days of culture, the primed PBMC were restimulated with T2 cells pulsed with a MHC class I-restricted influenza matrix protein M1 peptide (T2/M1), and IFN-γ secretion was determined after 6 h by ELISA (Figure 3). Upon restimulation with peptide-pulsed T2 cells, the activated T cells in the primed PBMC culture produced IFN-γ against the immunodominant M1 matrix protein peptide. The specificity of this activation is shown by only background IFN-γ production of the primed PBMC culture against unpulsed T2 cells (P = 0.00063; T2/M1 vs T2). To show that these cultured PBMC were antigen specifically stimulated during the 6 day culture, the same experiment was done with fresh PBMC. After coculture with either T2 cells or T2 cells pulsed with the peptide, no IFN-γ production was detected above background level (Figure 3).

Figure 3
figure3

Stimulatory capacity of cryopreserved mRNA-electroporated mature DC. Cryopreserved matrix protein M1 mRNA-electroporated mature DC were used as stimulators for PBMC during a 6 day coculture. Primed PBMC were then stimulated with T2 cells, pulsed with an MHC class I-restricted M1 immunodominant epitope (T2/M1), during a 6 hour coculture. Antigen-specific T cells in the primed PBMC culture were detected as shown by positive IFN-γ production. As controls, unpulsed T2 cells were used as stimulators and fresh PBMC as responders. Results are shown as mean ± standard error of three individual experiments.

Stimulatory capacity of non-cryopreserved vs cryopreserved mRNA-electroporated mature DC

To compare the stimulatory capacity of non-cryopreserved and cryopreserved mRNA-electroporated mature DC, we examined in an influenza model system whether there was a difference in activation capacity of autologous antigen-specific T cells upon coculture with PBMC. In these experiments, immature DC were electroporated with M1 matrix protein mRNA followed by 24 h maturation. Next, these mature DC were either directly used for stimulation of PBMC or cryopreserved for 1 week. During a 7 day coculture of DC (non-cryopreserved or cryopreserved) with PBMC, no additional cytokines were added. After 7 days of culture, CD8+ cells were isolated and restimulated with T2 cells pulsed with a MHC class I-restricted influenza matrix protein M1 peptide (T2/M1). IFN-γ secretion was determined after 6 h by ELISA (Figure 4). Upon restimulation with peptide-pulsed T2 cells, the activated CD8+ cells produced IFN-γ against the immunodominant M1 matrix protein peptide. The specificity of this activation was shown by only background IFN-γ production of the isolated CD8+ cells against T2 cells pulsed with an irrelevant E7 peptide (T2/E7). (T2/M1 vs T2/E7 comparisons: P values 0.0006 and 0.0055, for non-cryopreserved and cryopreserved mature DC, respectively.) By comparing the level of specific IFN-γ secretion, there was no significant reduction (P = 0.078) in the activation capacity of cryopreserved vs non-cryopreserved DC (Figure 4).

Figure 4
figure4

Stimulatory capacity of non-cryopreserved vs cryopreserved mRNA-electroporated mature DC. M1 matrix protein mRNA-electroporated non-cryopreserved (NON-CRYO) and cryopreserved (CRYO) mature DC were used as stimulators for PBMC during a 7 day coculture. Isolated CD8+ cells from the primed PBMC cultures were then stimulated with T2 cells, pulsed with an MHC class I-restricted M1 immunodominant epitope (T2/M1), during a 6 h coculture. Antigen-specific T cells in the primed PBMC culture were detected as shown by positive IFN-γ production. As control, T2 cells pulsed with an irrelevant E7 peptide (T2/E7) were used as stimulators. Results are shown as mean ± standard deviation of three individual experiments.

Conclusions

In general, for cryopreservation of mRNA-electroporated DC to be used for in vitro purposes, we propose the following: (1) mRNA electroporation of DC in an immature state; (2) maturation of DC during 24 h with a maturation cocktail (TNF-α + PGE2 + IL-1 + IL-6); and (3) cryopreservation of maturated DC in 90% FCS + 10% DMSO. This cryopreservation protocol provides viable high transgene-expressing mature DC after thawing. As we previously described a protocol for mRNA-electroporation of DC cultured from monocytes in the presence of autologous human plasma, here we show that DC cultured in the presence of FCS can also be efficiently loaded by mRNA electroporation. Optimization experiments for culture of influenza-specific T cells showed that DC, cultured in serum-free medium, in autologous plasma, or in FCS, were all able to induce a similar strong specific response against an influenza M1 matrix protein peptide when using peptide-pulsed DC as stimulators of PBMC (data not shown). In our hands, the only difference with the use of FCS for culture of DC as compared to the culture in autologous plasma, is a slight maturation observed after the electroporation of mRNA. However, this is not a serious problem since mature DC are expected to be more potent in stimulatory capacity.12 Moreover, in our hands, this was not associated with non-specific T cell response in vitro. In addition, we provide functional evidence, demonstrating that cryopreserved mRNA-electroporated mature DC are inducing a specific in vitro immune response against influenza antigens. Furthermore, there was no significant reduction in stimulatory capacity of non-cryopreserved vs cryopreserved DC. Finally, for in vitro experiments, where the generation of DC or culture of cytotoxic T cells are still routinely being performed in the presence of FCS,3,13 we conclude that this cryopreservation method is a simple and useful method for continuous availability of antigen-loaded DC in laboratory experiments. However, for the cryopreservation of DC for clinical use, it will be mandatory to use serum-free or autologous plasma conditions. These approaches are currently under investigation.

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Acknowledgements

This work was supported by grant Nos 3.0109.96, G.0157.99 and G.0313.01 of the Fund for Scientific Research – Flanders, Belgium (FWO), by grants of the Scientific Committee of the Fortis Bank (FB)-financed Cancer Research and of the Belgian Federation against Cancer (BFK). PP, NC, AVD are holders of a PhD fellowship from the Flemish Institute for Science and Technology (IWT). VFIVT is a postdoctoral fellow of the FWO.

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Correspondence to ZN Berneman.

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Ponsaerts, P., Van Tendeloo, V., Cools, N. et al. mRNA-electroporated mature dendritic cells retain transgene expression, phenotypical properties and stimulatory capacity after cryopreservation. Leukemia 16, 1324–1330 (2002). https://doi.org/10.1038/sj.leu.2402511

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Keywords

  • dendritic cells
  • cryopreservation
  • mRNA electroporation
  • immunotherapy
  • influenza
  • gene transfer

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