CD83 human dendritic cells transfected with tumor peptide ... - Nature

© 2000 Nature America, Inc. 0929-1903/00/$15.00/⫹0 www.nature.com/cgt

CD83ⴙ human dendritic cells transfected with tumor peptide cDNA by electroporation induce specific T-cell responses: A potential tool for gene immunotherapy Sabine Lohmann, Katharina Galle, Ju ¨rgen Knop, and Alexander H. Enk Department of Dermatology, Johannes Gutenberg-University, Mainz, Germany. Dendritic cells (DC) are the most potent immunostimulatory cells, with the capacity to induce primary T-cell responses. Functional autologous DC can be generated from fetal calf serum-free peripheral blood mononuclear cells in the presence of interleukin-4 and granulocyte-macrophage colony-stimulating factor and are stimulated with a defined cytokine cocktail for terminal maturation. We were able to establish a nonviral transfection protocol for these DC by electroporation. Using enhanced green fluorescent protein as a reporter gene, we achieved transfection efficiencies of up to 10%. FACScan analyses revealed a stable phenotype, and the expression of major histocompatibility complex class II and CD83 was not affected by the transfection conditions used. Like their untransfected counterparts, DC that were functionally transfected with green fluorescent protein were potent inducers of allogeneic T cells. To assess whether cDNAs transfected into DC are functionally expressed, human tyrosinase cDNA was transfected into DC. Tyrosinase-transfected DC, but not controls, resulted in antigen-specific tumor necrosis factor-␣ release of the tyrosinase-specific cytolytic T-cell clone IVSB. Taken together, the data show that genuine (CD83⫹) mature DC can be transfected using a nonviral method, and that the DC retain their functionality. These DC are ideal candidates for immunotherapy (e.g., cancer therapy). Cancer Gene Therapy (2000) 7, 605– 614

Key words: Human dendritic cells; transfection; tumor antigen; tyrosinase; gene immunotherapy; biological therapy of cancer.

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endritic cells (DC) are the most potent antigen (Ag)-presenting cells (APCs) of the immune system, with the capacity to activate resting naive T cells most efficiently and to initiate primary immune responses. Due to their functional properties, DC are ideal adjuvants for immunotherapeutic strategies using DC pulsed with tumor-associated Ags (TAA) for vaccination.1,2 Recently several TAA have been characterized, and they are expressed by the tumor cells. TAA peptides in association with major histocompatibility complex (MHC) are presented on the tumor cell surface, are recognized by Ag-specific T cells, and provide a potential target for immunotherapeutic vaccination approaches.3–5 In various murine studies, DC pulsed with TAA peptide can induce Ag-specific antitumoral responses in vivo.6,7 There are even promising reports about clinical trials in which patients with B-cell lymphoma were successfully vaccinated using autologous Ag-pulsed DC.8 Recently, a clinical pilot study was published that reports on the successful vaccination of melanoma patients with peptide- or tumor lysate-pulsed DC.9 The use of genetically engineered DC as tumor vaccines combines the advantages of immunotherapy with Received July 16, 1999; accepted November 24, 1999. Address correspondence and reprint requests to Dr. Sabine Lohmann, Department of Dermatology, Laboratory 161, Johannes Gutenberg-University, Langenbeckstr. 1, D-55131 Mainz, Germany.

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those of gene therapy. The application of peptide-pulsed DC is limited by the human histocompatibility leukocyte Ag (HLA) restriction of characterized TAA epitopes.3 Using recombinant techniques, the full-length protein Ag is expressed in the genetically engineered APCs and is accessible to the cellular Ag-processing machinery. Therefore, the repertoire of the epitopes presented in context with MHC class I is not limited to known epitopes. As recent studies of genetic immunization have shown, presentation of MHC class II-associated molecules also occurs, leading to the simultaneous stimulation of cytotoxic T-lymphocyte (CTL) and T helper (Th) responses.10 As the stability of the heterotrimeric MHC class I ␤2-microglobulin peptide complex is relatively low and its turnover is rapid, the transfection and stable expression of the TAA, which are permanently processed, provides a further advantage compared with peptide-pulsed DC.11,12 Several reports have dealt with the genetic engineering of murine DC using retroviral or adenoviral transduction approaches. Brossart et al13 induced Ag-specific CTL activation after adenovirus-mediated transfection of DC. Song et al14 were able to elicit protective and therapeutic antitumoral immunity by genetic modification of DC. Immunizing mice with adenovirally transduced DC expressing MART-1 resulted in a class I-restricted MART-1-specific CTL response.15 Retrovirally transduced DC are therapeutically effective against es605

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tablished pulmonary metastases.16 For the transfection of human DC, several protocols exist that use retroviral infections of proliferating DC progenitors.17,18 Many groups transduced CD34⫹ cells using retroviral gene transfer methods.19,20 Reeves et al21 also used retroviral transduction with tumor-associated MART-1 Ag and were able to raise antitumoral CTLs from autologous quiescent T cells. Interleukin-4 (IL-4) and granulocytemacrophage colony-stimulating factor (GM-CSF) cultured human DC were transduced with high efficiency using an adenovirus.22 Alijagic et al23 were the first to report on a DC transfection protocol using a nonviral method. These authors used cationic liposomes to transfect immature DC derived from peripheral blood mononuclear cells (PBMC) with GM-CSF and IL-4 culture. Transfected DC were able to induce Ag-specific T-cell activation. Two publications exist for a nonviral transfection approach using a gene gun for gene transfer into DC. Antitumoral immunity in vivo was elicited using a murine human papillomavirus model.24 Furthermore, human monocyte-derived DC were shown to express melanoma Ags and to induce primary cytotoxic T-cell responses in vitro after particle bombardment.25 There is no report on the transfection of mature CD83⫹ DC using a nonviral protocol. We report here on a nonviral transfection protocol using electroporation as a gene transfer method to generate transgenic fully mature human DC. The cells generated express the DC marker molecule CD83⫹ and are functionally highly potent. They express the transfected melanocyte-restricted TAA tyrosinase and present the processed peptide Ags together with HLA-A2 on the cell surface. In vitro assays showed that the cells are potent activators of allogeneic T cells and are able to induce tyrosinase-specific CTL responses in vitro. The strategy used combines the efforts of DCmediated immunotherapy with gene therapy. As we use a nonviral transfection protocol and generate the DC using fetal calf serum (FCS)-free culture conditions, this genetically engineered DC may be a very save and useful anti-cancer vaccine for application in clinical trials. MATERIALS AND METHODS

Preparation and differentiation of blood-derived DC Whole blood from healthy HLA-A2⫹ donors was heparinized; PBMC were separated by Ficoll gradient. T and B cells were depleted using immunomagnetic beads coated with monoclonal antibodies (mAbs) for CD2 and CD19 (Dynal, Oslo, Norway). The depleted cell fraction was cultured according to the recently published two-step protocol.26 In brief, the cells were cultured in X-VIVO-15 medium (BioWhittaker, Walkersville, Md) supplemented with 2% autologous plasma, 1000 U/mL recombinant human IL-4 (rhIL-4) (PBH, Hannover, Germany), and 1600 U/mL recombinant human GM-CSF (Leukomax; Sandoz, Basel, Switzerland). On culture day 7, nonadherent cells were harvested, resuspended in fresh complete culture medium as described above, and additionally stimulated with a cytokine cocktail for stable maturation of the DC containing rhIL-␤ (10 ng/mL), recombinant human tumor

necrosis factor-␣ (TNF-␣) (10 ng/mL, PBH), rhIL-6 (1000 U/mL ⬇ 5 ng/mL; R&D Systems, Wiesbaden, Germany), and prostaglandin E2 (1 ␮g/mL, Minprostin; Upjohn, Heppenheim, Germany). All cell cultures were maintained in a watersaturated atmosphere with 5% CO2 at 37°C. Fluorescenceactivated cell sorter (FACS) analysis or functional tests were performed on day 9 of DC culture.

Transfection of DC A total of 2.5 ⫻ 106 DC were transfected with several cDNAs by electroporation (EPI 2500; Dr. Fischer, Intergen, Heidelberg, Germany) using a 0.4-mm electroporation cuvette and 10 –20 ␮g of plasmid DNA. The cells were pulsed at 500 V for 2 milliseconds. Plasmid preparation was performed using a Endofree Plasmid preparation kit (Qiagen, Hilden, Germany). The method was established using the vector pEGFP-C1 (Clontech, Palo Alto, Calif) by analyzing the expression of the reporter gene enhanced green fluorescent protein (EGFP) by flow cytometry to quantify the transfection efficiencies. For functional tests, the cells were transfected with the tyrosinase cDNA clone 123.B2 (a kind gift of Dr. Thomas Wo ¨lfel, Department of Hematology, Johannes Gutenberg University Hospital of Mainz, Mainz, Germany), which is inserted into the eukaryotic expression vector pcDNAI/Amp (Invitrogen, San Diego, Calif) or with the vector pcDNAI/Amp alone as a negative control.

Flow cytometric analysis On day 9 of culture, DC were harvested for analysis by FACS. After washing the cells twice with phosphate-buffered saline-2% FCS, indirect immunofluorescence staining was performed using the following mAbs: CD58 (AICID58; Immunotech, Hamburg, Germany), CD83 (Immunotech), CD80 (MAB104; Immunotech), CD86 (IT2.2; PharMingen, Hamburg, Germany), HLA-DR (Serotec/Camon, Wiesbaden, Germany), HLA-A2 (BB7.2 hybridoma purchased from the American Type Culture Collection, Manassas, Va), and mouse and rat immunoglobulin G (IgG) subclass-specific isotypes (Immunotech). As secondary reagents, 5-([4,6-dichlorotriazin-2yl]amino)fluorescein-conjugated goat anti-rat IgG (Jackson Immunoresearch Laboratories, West Grove, Penn) and phycoerythrin-conjugated donkey anti-mouse IgG (Jackson Immunoresearch Laboratories) were used. A total of 10,000 cells were analyzed by flow cytometry, using a life gate to exclude dead cells as well as cell debris that showed lower forward and side scatter properties. Dot blots of live cells were blotted using FACScan Lysis II software (Becton Dickinson, Mountain View, Calif).

RNA isolation and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis The expression of the transfected cDNAs on RNA level was monitored by RT-PCR analysis. Therefore, 1–5 ⫻ 105 cells were harvested for RNA extraction within each transfection experiment. The RNA was extracted using the HighPure RNA Isolation kit (Roche Diagnostics GmbH, Mannheim, Germany) according to the manufacturer’s instructions. The RNA isolation protocol includes a deoxyribonuclease step to remove contaminating DNA. For cDNA synthesis and specific amplification, we used the Titan One-Tube RT-PCR system (Roche Diagnostics GmbH, Mannheim, Germany) mainly according to the product description. In brief, each reaction was performed with 100 –200 ng of total RNA and 2.5 mM MgCl2 in a 50-␮L volume with the tyrosinase-specific primers HTYR1

Cancer Gene Therapy, Vol 7, No 4, 2000


(TTG GCA GAT TGT CTG TAG CC) and HTYR2 (AGG CAT TGT GCA TGC TGC TT), as published by Brossart et al.27 As a internal positive control, we analyzed the expression of the housekeeping gene ␤-actin using the following primers: 5⬘-GAG CGG GAA ATC GTG CGT GAC ATT and 3⬘-GGA GGT AGT TTC GTG GAT GCC. Reverse transcription was performed for 30 minutes at 50°C followed by 5 minutes at 94°C to inactivate the enzyme. PCR conditions were as follows: 30 cycles at 94°C for 1 minute, at 65°C for 1.5 minutes, and at 72°C for 2 minutes.

Allogeneic mixed lymphocyte reaction (MLR) The ability of various DC to stimulate CD4⫹ T cells was assessed by MLR. DC (transfected and nontransfected) were prepared as described previously. DC were plated in serial dilutions in 96-well, flat-bottom microtiter plates starting with 1 ⫻ 104 cells/well and were cocultured with 1 ⫻ 105 CD4⫹ T cells/well. T lymphocytes were prepared from human blood by Ficoll gradient, enriched by nylon wool columns, and additionally positively selected using immunomagnetic beads coated with mAb for CD4 (MACS Systems, Palo Alto, Calif) according to the manufacturer’s instructions.

Culture of the tyrosinase-specific CTL clone IVSB The tyrosinase-specific CTL clone IVSB was kindly provided by Dr. Thomas Wo ¨lfel. The CTL clone IVSB was maintained according to the protocol published by Wo ¨lfel et al.28 Briefly, IVSB was restimulated weekly with irradiated autologous melanoma cells (SK29-Mel-1) and allogeneic feeder cells (AK-EBV-B) in RPMI 1640 medium supplemented with 10% human sera, 25 U/mL rhIL-2 (Proleukin; Chiron, Ratingen, Germany) and 5 U/mL rhIL-4 (PBH).

Induction of tyrosinase Ag-specific T-cell activation using tyrosinase-transfected DC Activation of the tyrosinase-specific CTL clone IVSB is Agspecific and results in the release of TNF-␣. To assess whether the cDNAs transfected into the DC are functionally expressed and processed for Ag presentation, cocultures of DC and the tyrosinase-specific CTL clone IVSB were performed. Therefore, the various DC populations (untreated or transfected as described above) were harvested at day 9 of culture and plated in serial dilutions in 96-well, flat-bottom plates starting with 2.5 ⫻ 104 cells/well in RPMI 1640 medium supplemented with 10% FCS (Seromed, Mu ¨nchen, Germany), 1% L-glutamine (200 mM; Life Technologies, Paisley, UK) 1% penicillinstreptomycin (Sigma, Deisenhofen, Germany), and 25 U/mL IL-2 (Chiron). Positive controls with tyrosinase peptide epitope 368 –376 (50 ␮g/mL) and a respective negative control using a non-sense peptide (50 ␮g/mL) were performed within each experiment (data not shown). The CTL clone IVSB was added at a concentration 2.5 ⫻ 104 cells/well in RPMI 1640 as mentioned above. The test was incubated in a water-saturated atmosphere with 5% CO2 at 37°C for 24 – 48 hours.

Analysis of TNF-␣ release in culture supernatants using a bioassay The supernatants of the cocultures were harvested after 24 – 48 hours of incubation. The TNF-␣ content of the supernatants was determined by its cytolytic effect on the mouse sarcoma cell line WEHI 164 clone 13 (WEHI-13). Therefore, 5 ⫻ 104 WEHI-13 cells/well were plated in a 96-well, flat-bottom plate in 50 ␮L of RPMI 1640 supplemented with 10% FCS (Se-


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romed), 1% L-glutamine (200 mM, Life Technologies) 1% penicillin-streptomycin (Sigma), and 1 ␮g/mL actinomycin D (Sigma). The amount of released TNF-␣ was estimated by comparing its effect on WEHI-13 with a TNF-␣ concentration standard (1000 ng/mL to 119 pg/mL; PBH). A total of 50 ␮L of supernatant was added per well, and the TNF bioassay was incubated for 24 hours at 5% CO2 at 37°C. After incubation overnight, 50 ␮L of 3-(4,5-dimethylthiazol-2-yl)-2,5-dimethyltetrazolium bromide solution (2.5 mg/mL; Merck, Darmstadt, Germany) was added per well, and the test was incubated for an additional 2 hours. Finally the cells were lysed by 100 ␮L of dimethylformamide-sodium dodecyl sulfate per well and analyzed with an enzyme-linked immunosorbent assay reader at 570 nm and 690 nm as a reference.

RESULTS

A total of 10% of the mature CD83⫹ DC population showed expression of the reporter gene EGFP after transfection by electroporation. A total of 2.5 ⫻ 106 DC were transfected using 10 ␮g of plasmid DNA as described in Materials and Methods. To monitor our transfection efficiencies, we assessed the expression of the reporter gene EGFP, which was measured by FACS analysis in the FL1 channel. FACScan analysis was performed using a life gate, which represented the mature CD83⫹ DC population. Expression of the EGFP protein was detectable in every transfection experiment performed. Approximately 10% of the mature CD83⫹ DC were positive for EGFP. As shown in Figure 1, we established a transfection protocol using electroporation; this protocol enabled us to achieve transfection efficiencies of up to 10% of the mature DC population (Fig 1). As a negative control, we used DC transfected with irrelevant plasmids (pcDNAI, pcDNAIII, 123.B2). No background fluorescence was detectable. EGFP expression was usually analyzed at 72 hours after gene transfer. Kinetic studies performed showed a very stable expression of the transgene. Even at 5 days posttransfection, EGFP expression was detectable with comparable intensity as on day 1 of the experiment (data not shown).

Transfected DC show the morphology of mature DC The morphology of mature DC (culture day 9), either untransfected or transfected with EGFP (or other plasmids, data not shown), was analyzed by comparative morphology studies using a microscope with a temperated work table (Axiovert 135; Carl-Zeiss, Frankfurt, Germany). Figure 2 demonstrates that the selected transfection conditions did not affect or inhibit the maturation of the DC progenitors. Using the established transfection protocol, a homogeneous fully mature DC population was generated. The transfected DC showed the same stable morphology as the nontransfected controls, with the characteristic nonadherent-round shaped cell body with cytoplasmatic veils. By establishing the transfection conditions for the DC, it became evident that it was very important to use lipopolysaccharide-free plasmid preparations, to titrate DNA amounts and to

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Figure 1. Expression of EGFP by mature CD83⫹ DC after gene transfer by electroporation. The expression of EGFP was analyzed by flow cytometry. Non-sense transfected controls (123. B2) were used as negative controls. FACS analysis was performed using a life gate representing the mature CD83⫹ DC population. As shown in the FACS diagram, 10% of mature CD83⫹ DC express significant amounts of the protein, which correlates with a transfection efficiency of 10%.

select a suitable vector that may correlate with the differences in the CpG motifs as discussed later.

Electroporation-mediated gene transfer does not alter DC phenotype To assess whether transfection conditions would alter the phenotype of DC, we performed FACS analyses. Using the established transfection protocol, we were able to generate transfected DC that showed a high level of MHC class II expression (Fig 3). Furthermore, the expression of MHC class I molecules that present Ags to CTLs was not modified by electroporation-mediated gene transfer according to our protocol. As DC are the most potent immunostimulatory cells, with the capacity to induce primary immune responses, they express co-

stimulatory molecules such as B7.1 and B7.2 (CD80, CD86) as well as leukocyte function-associated Ag-3 (CD58). The expression pattern of these costimulatory molecules remained unaffected by the transfection conditions chosen. Furthermore, we analyzed the expression of the DC marker molecule CD83. This molecule was equally expressed after gene transfer. In summary, we were able to generate functionally competent CD83⫹transfected DC.

Transfected DC are as potent inducers of T-cell activation as their untransfected counterparts The DC generated by gene transfer are highly mature, as shown by morphological and phenotypical characterization. We subsequently wanted to analyze their functional

Figure 2. Transfected DC show the morphology of mature DC. Comparative morphology studies of mature DC (culture day 9) (untransfected versus transfected) with EGFP revealed no influence of the transfection conditions on the maturation process of the DC. The photos were taken at ⫻100 magnification.



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Figure 3. Transfection of DC with cDNAs shows an unaltered phenotype under the chosen transfection conditions. DC were transfected as described previously and harvested on day 9 of culture. Indirect immunofluorescence staining was performed, and the expression pattern was analyzed by flow cytometry. Results are presented as dot blot diagrams.

competence with regard to inducing T-cell responses. Therefore, we analyzed their functional properties for T-cell activation in comparison with untransfected controls by MLR. Cocultures were set up starting with 1 ⫻ 104 DC/well in serial dilutions together with 1 ⫻ 105 CD4⫹ T cells/well. After 4 days, T-cell proliferation was analyzed by [3H]thymidine deoxyribose incorporation. Our data revealed that transfected DC are at least as potent inducers of T-cell activation as their untransfected counterparts, as shown by MLR (Fig 4).


Tyrosinase mRNA is expressed in transfected DC Expression of the transfected cDNAs in DC was monitored by RT-PCR analysis, using tyrosinase-specific primers and an RT-PCR protocol as described above. RNA was extracted from several DC transfections (pEGFP, hIL-15pcDNAIII, 123.B2pcDNAI/Amp) and an untreated negative control. After PCR amplification, we detected a specific product of 284 bp in the tyrosinase (123.B2pcDNAI/Amp)-transfected samples (Fig 5). The expression of ␤-actin was monitored in parallel to verify

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Figure 4. Transfected DC are very potent inducers of T-cell activation. DC were prepared as described previously, and transfected DC as well as untransfected controls were harvested on day 9 of culture. After extensive washing, DC (1 ⫻ 104 cells/well) were cocultured with allogeneic CD4⫹ T cells (1 ⫻ 105 cells/well) for 4 days. T-cell proliferation was quantified by [3H]thymidine deoxyribose incorporation during the last 16 hours of culture. The results are the mean counts per minute of triplicate experiments.

the quality of the RNA preparation. Every RNA sample showed a specific ␤-actin signal at 221 bp. Taken together, these results indicate that the transfected cDNAs are expressed on the mRNA level.

Figure 5. Tyrosinase mRNA is expressed after electroporationmediated gene transfer in DC. RNA was extracted from several DC transfections (pEGFP, hIL-15pcDNAIII, 123.B2pcDNAI/Amp) and an untreated negative control. Afterward, RT-PCR was performed as described in Materials and Methods. During agarose-gel electrophoresis, a tyrosinase-specific PCR product of 284 bp was detectable in the DC preparation transfected with the respective cDNA. As an internal positive control for RNA preparation, ␤-actin was analyzed in parallel using the same samples.

Tyrosinase-transfected DC are able to express the transgene and induce specific TNF-␣ release in a tyrosinase-specific CTL clone To determine whether the transfected tyrosinase cDNA is expressed on the protein level, processed, and presented together with MHC class I molecules, we used the TNF-␣ release by the tyrosinase-specific CTL clone IVSB after peptide-specific activation as a functional read out system. Therefore, non-sense (pcDNAI) and tyrosinase (123.B2)-transfected DC (1 ⫻ 104 cells/well) in serial dilutions were cocultured with the CTL clone IVSB (1 ⫻ 104/well) for 24 – 48 hours. Afterward, supernatants were harvested, and the TNF-␣ release by the tyrosinase peptide-specific CTL clone IVSB was monitored in a TNF-␣ bioassay. Figure 6A shows the microscopic picture after 24 hours of coculture. The pcDNAtransfected controls show viable T cells and DC in the cocultures, whereas almost no DC survived in the cocultures with the tyrosinase-transfected DC. This is due to lysis by the tyrosinase-specific CTL clone IVSB. This finding correlates with the results we obtained by analyzing the TNF-␣ content of the supernatants during the TNF-␣ bioassay (Fig 6B). There were significantly higher amounts of TNF-␣ detectable in cocultures of



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Figure 6. A: Tyrosinase-transfected DC are a target for the tyrosinase-specific CTL clone IVSB. DC were transfected as described previously and harvested at day 9 of culture. DC were cocultured with the tyrosinase-specific CTL clone IVSB for 24 hours. Microscopic analysis showed that the tyrosinase-transfected target cells are recognized by the CTL clone IVSB and lysed, whereas the vector-transfected control cells remain mainly unaffected. B: Tyrosinasetransfected DC are able to induce a tyrosinase-specific T-cell response in vitro. DC were transfected as described previously and harvested at day 9 of culture. DC were cocultured with the tyrosinase-specific CTL clone IVSB for 24 – 48 hours. To estimate T-cell activation, the supernatants of triplicate experiments were analyzed for TNF-␣ secretion using a sensitive bioassay.


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tyrosinase-transfected DC together with the CTL clone IVSB than in comparable non-sense transfected controls.

DISCUSSION The data presented here demonstrate that tyrosinasetransfected human autologous DC express, process, and present the transgenic TAA tyrosinase with HLA-A2 molecules after electroporation-mediated gene transfer. The transfected DC generated express high levels of MHC class II as well as costimulatory molecules and the DC marker CD83. Furthermore, the tyrosinase-transfected DC were able to induce Ag-specific T-cell immune responses in vitro. Thus, we report on the nonviral transfection of fully mature DC that are functionally competent APCs. Until now, there have been several studies using viral transduction protocols for the genetic engineering of DC. The use of viral transduction methods has disadvantages for its application in humans. During retroviral infection, the genomic information is inserted into the genome, bearing the risk of oncogenesis or insertional mutagenesis.29 In addition, only dividing cells are infected by retroviruses; therefore, mature DC are not suitable for this application. With of adenoviral gene delivery systems, both dividing and nondividing cells are transduced. However, recent reports show strong immunogenicity of adenoviral transduction, eliciting massive cellular and humoral responses.30 Adenovirally transduced DC activate CD8⫹ and CD4⫹ T cells, resulting in transient expression of the transgene due to elimination of the cells carrying the transgene.31 Most of the reports mentioned have used monocytederived DC generated in GM-CSF and IL-4. However, the DC populations generated under these conditions do not represent DC with a fully mature and stable phenotype. They express a more macrophage-like phenotype. Under special circumstances, these cells induce tolerance rather than activate CTLs.32–34 Thus far, there has only been one report on the creation of fully mature transgenic DC by retroviral transduction.20 The recently published two-step protocol by Jonuleit et al26 allows the generation of autologous DC derived from the PBMC of tumor patients. The DC population generated is homogeneous with respect to their typical dendritic morphology, expressing high levels of MHC class II, the costimulatory molecules CD80 and CD86 as well as CD58, and most importantly, the DC marker molecule CD83. As these DC are terminally differentiated, they express a stable phenotype, which is very important for clinical applications. DNA-based immunization strategies using naked DNA that is usually applied to the skin seem to be dependent upon the uptake of DNA by skin-derived DC.33,35,36 Again, this emphasizes the important role of

DC in eliciting Ag-specific T-cell responses. The results achieved by Ribas et al15 suggest that immunization with DC and TAA elicits a better protective antitumoral response compared with naked DNA or in vivo delivery using an adenoviral vector. One reason for the potent immune responses elicited by the relatively small amounts of Ag expressed by the transfected cells may be the adjuvant properties of DNA vaccines. The immunostimulatory effect of the bacterial DNA is mediated by CpG motifs and by the absence of cytosine methylation. The immunostimulatory sequences induce a Th1 response as they induce interferon-␥, IL-12, and IL-18 secretion by APCs.12 In our DC transfection experiments, we have seen an ISS effect that positively affects DC maturation, depending upon the amount of DNA used and the vector type. The maturation process becomes evident if early DC progenitors are transfected by the development of a mature phenotype and high expression of the DC marker CD83 as well as costimulatory molecules such as CD80 and CD86 (data not shown). For successful transfection, it is very important to titrate the DNA amounts used and to use plasmid preparations with a lipopolysaccharide content of ⬍1.0 European unit/mL. Transfection of DC with full-length TAA cDNAs for vaccination offers several advantages: As the protein is processed endogenously, the presentation is independent of the patient’s haplotype and known antigenic epitopes. Furthermore, the antigenic epitopes are presented on MHC class I and II molecules. This may provide additional helper epitopes for efficient antitumoral responses. Other groups tried to solve this problem using tumor cell extracts to pulse DC.37,38 This approach bears the risk of the presentation of self Ags and consequently the elicitation of autoimmunity. Loading the DC with acid-eluted Ags may be an alternative solution, but this option is limited by the availability of tumor cells for this procedure. Furthermore, acid elution of MHC class I Ags is very problematic, as it has been known for many years that the suppression of MHC class I molecules on the cell surface of tumor cells is a very common mechanism to escape immune surveillance.39,40 As a new method, the use of tumor cellderived RNA for transfection might avoid all of these obstacles.37,41 In conclusion, several aspects favor the use of nonvirally transfected DC in clinical trials: (a) DC-TAA vaccines generated by electroporation are saver for clinical applications compared with viral delivery systems. (b) DC transfected with TAA cDNA are more potent in inducing an Ag-specific immune response compared with peptide-based strategies, as they elicit both CTL and Th responses as well as B-cell antibody responses in vivo. (c) DC transfected with TAA cDNA generate an Ag-specific T-cell response in vivo independent of the patient’s MHC haplotype and characterized TAA epitopes. In the future, cotransfection of DC with cytokine genes and TAA may be an additional tool to modulate the immune response.



ACKNOWLEDGMENTS We thank Drs. Catherine and Thomas Wo ¨lfel for the tyrosinase plasmid and the tyrosinase-specific T-cell clone IVSB. We also thank Alexander Liske and Volker Lennartz for helpful discussions and assistance with the T-cell culture; Ruth Alt for excellent technical assistance; Drs. Gerhard Go ¨llner, Gabriele Mu ¨ller, and Martin Weisser for critical review of the manuscript, and Mrs. Pirch for help with the figures. This work was supported by the Deutsche Forschungsgesellschaft and the Bundesministerium fu ¨r Forschung und Technologie.

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