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The Journal of Immunology, 2004, 172: 6649-6657.
Copyright © 2004 by The American Association of Immunologists

Messenger RNA-Electroporated Dendritic Cells Presenting MAGE-A3 Simultaneously in HLA Class I and Class II Molecules1

Aude Bonehill*, Carlo Heirman*, Sandra Tuyaerts*, Annelies Michiels*, Karine Breckpot*, Francis Brasseur{dagger}, Yi Zhang{dagger}, Pierre van der Bruggen{dagger} and Kris Thielemans2,*

* Laboratory of Molecular and Cellular Therapy, Department of Physiology-Immunology, Medical School of the Vrije Universiteit Brussel, Brussels, Belgium; and {dagger} Ludwig Institute for Cancer Research, Brussels, Belgium


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
An optimal anticancer vaccine probably requires the cooperation of both CD4+ Th cells and CD8+ CTLs. A promising tool in cancer immunotherapy is, therefore, the genetic modification of dendritic cells (DCs) by introducing the coding region of a tumor Ag, of which the antigenic peptides will be presented in both HLA class I and class II molecules. This can be achieved by linking the tumor Ag to the HLA class II-targeting sequence of an endosomal or lysosomal protein. In this study we compared the efficiency of the targeting signals of invariant chain, lysosome-associated membrane protein-1 (LAMP1) and DC-LAMP. Human DCs were electroporated before or after maturation with mRNA encoding unmodified enhanced green fluorescent protein (eGFP) or eGFP linked to various targeting signals. The lysosomal degradation inhibitor chloroquine was added, and eGFP expression was evaluated at different time points after electroporation. DCs were also electroporated with unmodified MAGE-A3 or MAGE-A3 linked to the targeting signals, and the presentation of MAGE-A3-derived epitopes in the context of HLA class I and class II molecules was investigated. Our data suggest that proteins linked to the different targeting signals are targeted to the lysosomes and are indeed presented in the context of HLA class I and class II molecules, but with different efficiencies. Proteins linked to the LAMP1 or DC-LAMP signal are more efficiently presented than proteins linked to the invariant chain-targeting signal. Furthermore, DCs electroporated after maturation are more efficient in Ag presentation than DCs electroporated before maturation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
T cells play an essential role in inhibiting tumor growth and eradicating cancer cells. Tumor cells express HLA class I, but most often not class II molecules and CD8+ T cells, recognizing target Ags in an HLA class I-restricted fashion, can lyse tumor cells directly and destroy large tumor masses in vivo (1, 2). Therefore, most attention has been given to the role and the induction of CD8+ CTLs in cancer immunotherapy. Clinical trials using HLA class I-restricted tumor-associated Ags (TAAs)3 have shown some evidence of therapeutic effects on inhibiting tumor growth, demonstrating the potential and feasibility of cancer immunotherapy using TAAs recognized by CD8+ CTLs (3, 4, 5). However, the overall immune responses were too weak and transient to eradicate cancer cells in most patients who received immunization. One possible reason for this failure is the lack of tumor-specific CD4+ T cell responses. Indeed, increasing evidence indicates that CD4+ Th cells play a central role in orchestrating an effective antitumor response (6, 7, 8). Th cells are needed for the initiation of the immune response by the conditioning of the APCs via CD40 ligation by CD40 ligand (9, 10, 11). They are also necessary for the maintenance of CTL function and proliferation. Furthermore, they inhibit tumor growth in the absence of CD8+ CTLs by the recruitment of other effector cells, such as macrophages and eosinophils (12, 13). More recently, it has been shown that CD4+ T cell help is required for the generation of a functional CD8+ T cell memory response (14, 15, 16, 17, 18). These findings revived interest in incorporating HLA class II-restricted TAAs that can stimulate CD4+ T cells in tumor vaccines. Human TAAs recognized by CD4+ T cells are now being identified with increasing frequency (19, 20, 21, 22, 23, 24, 25, 26, 27).

DCs are professional APCs, characterized by their ability to stimulate naive and resting memory T lymphocytes. Therefore, these cells are the ideal candidates to act as vectors for immunotherapy of various diseases. In an immature stage, DCs phagocytose and process Ag. They carry information about invading pathogens to the T cell zones of the lymphoid organs. During this migration, the DCs differentiate from an immature to a mature state, with an increased T cell stimulatory capacity. Mature DCs present Ags to CD4+ T cells, and the resulting stimulated CD4+ T cells can then superactivate the DCs to stimulate CD8+ CTLs (28, 29, 30, 31).

Various strategies have been explored over the past few years to obtain presentation of both HLA class I- and class II-restricted, TAA-derived epitopes. The identification of the immunodominant peptides within the TAAs has made it possible to load DCs with high levels of synthetic HLA class I and II peptides (32, 33, 34). However, this approach has some disadvantages, such as the short half-life of the HLA/peptide complexes and the dependence on the knowledge of the HLA haplotype of each patient. Alternatively, DCs can be pulsed with intact protein. Another possibility is manipulating the HLA class II presentation pathway. The major pathway for HLA class II presentation requires endocytosis of exogenous Ags by APCs, followed by protein degradation to peptides in endocytic and lysosomal compartments (35, 36). Invariant chain and lysosome-associated membrane protein-1 (LAMP1)/DC.LAMP are transmembrane proteins that are predominantly localized in endosomes and lysosomes, respectively. The cytoplasmic domains of these proteins contain specific targeting signals that mediate their translocation to these specific compartments. Several groups have reported the use of chimeric constructs encoding Ags linked to the transmembrane domains of Ii (37, 38, 39) or LAMP1 (38, 40, 41, 42), with the aim of targeting these Ags to the HLA class II processing pathway.

Recently, we and others have reported a novel approach to load DCs with TAA by using a nonviral gene delivery system. In vitro transcribed mRNA encoding a TAA is introduced into the DCs via electroporation (43, 44, 45, 46). This is a very attractive method, as mRNA-loaded DCs have been proven to be able to stimulate the immune system in vitro and in vivo (43, 46, 47, 48, 49, 50, 51). Furthermore, it is a very safe tool for clinical trials, as mRNA is not immunogenic, has a relatively short half-life, and lacks the potential to integrate into the host genome (52, 53).

In this study we compared the efficiency of the HLA class II-targeting sequences of Ii, LAMP1, and DC.LAMP. As mentioned above, the Ii and LAMP1 sorting signals have already been used to target proteins to the HLA class II compartments. We report the use of a novel sorting signal, namely, the transmembrane and cytoplasmic domains of DC.LAMP, which is a protein specific for mature DCs (54), predominantly localized in lysosomes.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Genetic constructs

The pGEM-enhanced green fluorescent protein (pGEM-eGFP) vector (55) was provided by Dr. E. Gilboa (Duke University Medical Center, Durham, NC). This plasmid contains the eGFP cDNA, flanked by the 5'- and 3'-untranslated regions of the Xenopus laevis {beta}-globin gene and a poly-A tail. At the 3' end of the poly-A tail, unique NotI and SpeI sites are present to allow linearization of the plasmids before in vitro transcription. A bacteriophage T7 promotor allows the in vitro generation of mRNA. A unique NcoI site at the 5' end of the eGFP cDNA and unique BamHI and EcoRI sites at the 3' end, were used to construct the different eGFP and MAGE-A3 cDNA-containing plasmids.

The pGEM-MAGE-A3 plasmid was cloned by replacing eGFP with MAGE-A3 cDNA. The MAGE-A3 gene was amplified from the plasmid pTZ18R-MAGE-A3 with the following primers: Mage3 sense, 5'-CCCCCATGGNcoICTCTTGAGCAGAGGATC-3' (underlined sequences represent restriction sites); and Mage3 antisense, 5'-GGGAGATCTBglIITCACTCTTCCCCCTCTCTCAAAAC-3'. During this PCR, an NcoI site containing a start codon is inserted at the 5' extremity of the cDNA, and a BglII site preceded by a stop codon is inserted at the 3' extremity.

For the pGEM-Ii80-MAGE-A3 construct, eGFP was replaced by the 80 N-terminal residues of Ii, amplified from IipSV51L (provided by Dr. J. Pieters, University of Basel, Basel, Switzerland). The primers used in this PCR were: Ii80 sense, 5'-TTTCCATGGNcoIATGACCAGCGCGAC-3'; and Ii80 antisense, 5'-TTTTGGATCCBamHIGGAAGCTTCATGCGCAGGTTC-3'. This PCR adds an NcoI site spanning the start codon at the 5' end and a BamHI site at the 3' end. MAGE-A3 cDNA was inserted as a BglII-BglII fragment into the BamHI-linearized pGEM-Ii80 plasmid. Therefore, MAGE-A3 was amplified with the following primers: Mage3 sense bis, 5'-GGGAGATCTBglIITGAGCAGAGGAGTCAGCAC-3'; and Mage3 antisense. During this PCR, a BglII site was incorporated at both extremities of the MAGE-A3 gene. The 5' BglII site was in-frame with the BamHI site of the 3' end of Ii80. The 3' BglII site was preceded by a stop codon. In parallel, eGFP cDNA was amplified with the following primers: eGFP sense, 5'-GGGGATCCBamHIGGTGAGCAAGGGCGAGG-3'; and eGFP antisense, 5'-GGGGGATCCBamHIGGGCCCGCGGTACC3', generating BamHI sites at both the 5' and 3' ends of the cDNA. This fragment was cloned in-frame behind the Ii80-encoding cDNA, resulting in the pGEM-Ii80-eGFP plasmid.

For the cloning of pGEM-sig-LAMP1, the plasmid pCMV-sig-LAMP1, provided by Dr. Hwu (Johns Hopkins University, Baltimore, MD), was used as a template for PCR. The signal sequence of LAMP1 (sig) was amplified with primers: sig sense, 5'-CCCCATGGNcoICGGCCCCCGGC3'; and sig antisense, 5'-GGGGGATCCBamHITCAAAGAGTGCTGA-3', adding an NcoI site spanning the start codon at the 5' end and a BamHI site. LAMP1 was amplified with the following primers: LAMP1 sense, 5'-GGGGGATCCBamHITACAACATGTTGATCCCC-3'; and LAMP1 antisense, 5'-GGGAGATCTCTAGATGGTCTGATAGCCGGC-3', adding a BamHI site at the 5' end and a stop codon at the 3' end. PCR products were cloned into the pCR2.1 vector, and subsequently, sig was cloned as a BamHI-BamHI fragment into pCR2.1-LAMP1, linearized with BamHI. This cloning step resulted in pCR2.1-sig-LAMP1 with a BamHI site between the sig- and LAMP1-encoding cDNA. The sig-LAMP1 sequence was then excised with NcoI and EcoRI and cloned into the pGEM-eGFP vector, digested with the same enzymes, resulting in the pGEM-sig-LAMP1 plasmid. MAGE-A3 cDNA was amplified with primers: Mage3 sense bis, and Mage3 antisense bis (5'-CCCAGATCTBglIITCCTCTTCCCCCTCTCTC-3'), generating BglII sites at both 5' and 3' ends of the cDNA. This fragment was cloned in-frame between sig and LAMP1 in pGEM-sig-LAMP1, resulting in the pGEM-sig-MAGE-A3-LAMP1 plasmid. In parallel, eGFP was cloned in-frame between sig and LAMP1 as a BamHI-BamHI fragment, resulting in the pGEM-sig-eGFP-LAMP1 plasmid.

For the cloning of pGEM-sig-DC.LAMP, cDNA extracted from mature human DCs was used as a template for PCR to amplify DCLAMP. The primers used in this PCR were: DC.LAMP sense, 5'-CACAGGATCCBamHICTCGTCTGACTACACAATTGTG-3'; and DCLAMP antisense, 5'-CACAAGATCTBglIITTAGATTCTCTGGTATCCAGATC-3'. This PCR adds a BamHI site at the 5' end and a stop codon at the 3' end. The pGEM-sig-MAGE-A3-DC.LAMP and pGEM-sig-eGFP-DC.LAMP plasmids were cloned similarly to pGEM-sig-MAGE-A3-LAMP1 and pGEM-sig-eGFP-LAMP1, respectively.

Fig. 1 shows a schematic representation of the pGEM vector (Fig. 1A) and the different eGFP (Fig. 1B) and MAGE-A3 constructs (Fig. 1C).



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  		FIGURE 1. Schematic representation of the pGEM-eGFP plasmid (A). The T7 promotor, 5'-untranslated region (5'UTR), 3'UTR, A64 stretch, and the unique SpeI and NotI sites are shown. Schematic representation of the various eGFP (B) and MAGE-A3 (C) constructs is presented. The different HLA class II-targeting sequences and the HLA-A1- and HLA-DP4-restricted MAGE-A3 epitopes are shown.

 
Generation of monocyte-derived DCs

PBMCs were used as a source of DC precursors and were isolated from buffy coat preparations or leukapheresis products. DCs were generated in tissue culture flasks (Falcon; BD Biosciences, San Jose, CA) or in a closed culture system using double-tray Cell Factories (Nunc, Naperville, IL) as described previously by Tuyaerts et al. (56). Briefly, on day 0 PBMCs were plated at a density of 5 x 106 cells/ml in RPMI 1640 medium (Invitrogen, Paisley, U.K.) supplemented with 1% human AB (huAB; PAA Laboratories, Linz, Austria). The cells were left for 2 h to allow plastic adherence of the monocytes. Nonadherent cells were removed by washing, and the adherent cells were cultured in RPMI 1640 medium supplemented with 1% huAB, 800 U/ml GM-CSF (Leucomax; Novartis, Basel, Switzerland), and 100 U/ml IL-4 (BruCells, Brussels, Belgium). On days 2 and 4, medium containing the cytokine amount of day 0 was added to the DC culture.

In vitro transcription of capped mRNA

Before in vitro mRNA synthesis, the pGEM-plasmids were linearized. The plasmids pGEM-eGFP, pGEM-sig-eGFP-LAMP1, pGEM-sig-eGFP-DC.LAMP, pGEM-sig-MAGE-A3-LAMP1 and pGEM-sig-MAGE-A3-DC.LAMP were linearized with SpeI, pGEM-Ii80-eGFP, pGEM-MAGE-A3 and pGEM-Ii80-MAGE-A3 with NotI. The in vitro transcription was performed with T7 polymerase according to the manufacturer’s instructions (mMESSAGE mMACHINE Ultra T7 Kit; Ambion, Austin, TX). The mRNA length, concentration and purity were evaluated with the Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA), using the RNA 6000 Nano LabChip Kit (Agilent Technologies) according to the manufacturer’s instructions. Data analysis was performed with 2100 Bioanalyzer software (Agilent Technologies).

Electroporation of DCs

Four or 8 million DCs were electroporated with 20 or 40 µg in vitro transcribed mRNA, respectively. Before electroporation, the DCs were washed twice, first with RPMI 1640 medium without supplements and secondly with Optimix Solution A (EQUIBIO, Ashford, U.K.). After the second wash step, the DCs were resuspended in a final volume of 200 µl of Optimix Solution B (EQUIBIO) containing the mRNA. Electroporation was performed in a 4-mm gap electroporation cuvette using the EQUIBIO Easyject Plus apparatus. The following conditions were used for electroporation: voltage, 300 V; capacitance, 150 µF; and resistance, 99 {Omega}, resulting in a pulse time of ~5 ms. Immediately after electroporation, the DCs were diluted in RPMI 1640 medium supplemented with 1% huAB, 800 U/ml GM-CSF, and 100 U/ml IL-4 to a final density of 5 x 105 cells/ml.

Peptide pulsing of DCs

The lyophilized synthetic peptides corresponding to the MAGE-A3 epitope presented in the context of HLA-A1 (M3.A1 peptide, aa 167–176, sequence EVDPIGHLY) and HLA-DP4 (M3.DP4 peptide, aa 243–258, sequence KKLLTQHFVQENYLEY) were dissolved in 10 mM acetic acid and 10% DMSO to a final concentration of 2 mg/ml and stored at –20°C. For peptide loading, DCs were diluted to a final density of 2 x 106 cells/ml in RPMI 1640 medium without supplements containing 30 µg/ml M3.A1 or M3.DP4 peptide and incubated for 4 h at 37°C. Subsequently, the cells were washed and resuspended to a final density of 5 x 105 cells/ml in RPMI 1640 medium, supplemented with 1% huAB, 800 U/ml GM-CSF, and 100 U/ml IL-4.

Maturation of DCs

DCs were matured at a cell density of 2.5 x 105 cells/ml with a maturation mixture containing 100 U/ml IL-1{beta} (made in-house), 1000 U/ml IL-6 (made in-house), 100 U/ml TNF-{alpha} (PeproTech, London, U.K.), and 1 µg/ml PGE2 (Sigma-Aldrich, St. Louis, MO).

Quantitative PCR

Total RNA was extracted from electroporated DCs; melanoma cell lines MZ2-MEL3.0, LB373-MEL, and SK-MEL-23; and myeloma cell lines EJM, L363, and U266 using a commercial kit (SV Total RNA Isolation System; Promega, Madison, WI), according to the manufacturer’s protocol. Total RNA (250 ng) was reverse transcribed with the Superscript First-Strand Synthesis System for RT-PCR kit (Invitrogen), according to the manufacturer’s protocol. Expression of MAGE-A3 (GenBank accession no. U03735) was measured by quantitative PCR, based on TaqMan methodology, using the ABI PRISM 7700 Sequence Detection System (Applied Biosystems, Warrington, U.K.). The sequences of the primers and the probe (Applied Biosystems) were as follows: sense primer, 5'-GTCGTCGGAAATTGGCAGTAT-3'; antisense primer, 5'-GCAGGTGGCAAAGATGTACAA-3'; probe, 5'-6FAM-AAAGCTTCCAGTTCCTT-MGB-NFQ-3'. PCR was performed using the quantitative PCR Core Kit without dUTP reagents (Eurogentec, Seraing, Belgium), according to the manufacturer’s instructions. PCR amplification reactions (25 µl) were prepared using 1 µl of cDNA (1/20th of the RT reaction) and with the quantitative PCR Core Kit without dUTP reagents (Eurogentec), according to the manufacturer’s instructions. Thermal conditions were 10 min at 95°C and 35 cycles of 15 s at 95°C and 1 min at 62°C. Quantification of the samples was achieved by extrapolation from a standard curve of four serial dilution points of MAGE-A3 cDNA (107–104 copies/reaction). Samples and standard dilution points were assayed in triplicate. Normalization of the samples was achieved by dividing the copy number of MAGE-A3 by that of the reference gene, {beta}-actin. Copy numbers of {beta}-actin were determined using the Endogenous Control Pre-Developed Assay Reagents (human {beta}-actin) and the TaqMan Universal PCR Master Mix 2x, according to the manufacturer’s instructions (Applied Biosystems). Quantification of the samples was again achieved by extrapolation from a standard curve of four serial dilution points of {beta}-actin cDNA (107–104 copies/reaction). Standard curves for {beta}-actin and MAGE-A3 were closely similar (slope, –3.34 to –3.41; y-intercept, ~40.9; fit, 0.99). The SD of the normalized MAGE-A3 values was calculated from the SD of the MAGE-A3 and the {beta}-actin values using the following formula:

where cv is the SD/mean value (as described in the Sequence Detection System User Bulletin 2; Applied Biosystems).

Inhibition assays

To assess the intracellular mechanisms of Ag processing, 50 µM chloroquine (Sigma-Aldrich) was added to the DCs immediately after electroporation. The eGFP expression was analyzed at different time points after electroporation.

Immunophenotyping of DCs

All stainings were performed on ice in PBS-BSA-NaN3 and were preceded by blocking of FcRs with 10% normal goat serum. To analyze the expression of surface molecules on the cell surface of the DCs, the following mAbs were used: CD80, CD83, CD86, HLA-ABC (all from BD PharMingen, San Diego, CA), and HLA-DP (purified from clone B7/21; a gift from Dr. J. Arroyo, Universidad Complutense, Madrid, Spain). The HLA-DP Ab was biotin labeled and detected through streptavidin-PE (BD PharMingen) binding. The HLA-ABC Ab was FITC conjugated. All other Abs were PE conjugated. Isotype-matched Abs (BD PharMingen) were used as controls. Fluorescence analysis was performed with a FACSCalibur flow cytometer (BD Biosciences) using CellQuest software (BD Biosciences).

Cryopreservation of DCs

DCs were frozen in cryotubes at 1–5 x 106 DCs/tube in 1 ml of huAB with 10% DMSO and 2% glucose. The DCs were slowly frozen to –80°C using a cryofreezing container (Cryo 1°C freezing container; rate of cooling, –1°C/min; Nalgene, Hereford, U.K.) and were subsequently stored in liquid nitrogen until use. DCs were thawed in a 37°C water bath until small ice crystals were visible. Cold HBSS (Invitrogen) was added dropwise, and the cells were pelleted in a precooled centrifuge (4°C). The thawed DCs were resuspended in 5 ml of prewarmed RPMI 1640 medium supplemented with 1% huAB. Cell viability was determined with trypan blue.

T cell clones

Two MAGE-A3-specific T cell clones were used. Clone R12-C9 is HLA-DP4 (HLA-DPB1*0401) restricted and specific for the MAGE-A3 epitope aa 243–258 (23). Clone ESBI 648 is HLA-A1 restricted and specific for the MAGE-A3 epitope aa 167–176 (57). The cells, which will be referred to as M3.DP4- or M3.A1-specific T cells, were cultured in IMDM (Invitrogen) supplemented with 10% huAB, asparagine-arginine-glutamine (AAG; Invitrogen), penicillin-streptomycin (Invitrogen), 50 U/ml IL-2 (PeproTech), and 5 ng/ml IL-7 (PeproTech). The M3.A1-specific T cells were restimulated weekly; the M3.DP4-specific T cells were restimulated every 2 wk. The cells were restimulated with irradiated allogeneic LG2 EBV-B cells (1.5 x 106/24-well plate) as feeder cells and MZ2 melanoma cells preincubated with synthetic peptide (1 x 105/24-well plate) as stimulator cells.

Ag presentation assays

To investigate the T cell stimulatory capacity of the DCs, 2 x 104 electroporated or peptide-pulsed DCs were cocultured with 5000 M3.DP4- or M3.A1-specific T cells in 200 µl of IMDM supplemented with 10% huAB, AAG, penicillin-streptomycin, and 25 U/ml IL-2. Each coculture was performed in triplicate in round-bottom microwells. After 20 h of coculture, the supernatant was assessed for the presence of IFN-{gamma} by ELISA using a commercially available kit (Human IFN-{gamma} Cytoset; BioSource International, Camarillo, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
As HLA class II sorting signals, we used the transmembrane and cytoplasmic domains of invariant chain, LAMP1 and DC.LAMP. For Ii and LAMP1, the targeting sequences were chosen on the basis of previously described sequences (37, 38, 39, 40, 41, 42). For the sorting signal of DC.LAMP, we used a Kyte Doolittle hydrophobicity plot to determine the positions of the transmembrane and cytoplasmic domains. Fig. 2 shows the portion of the DC.LAMP protein used as a sorting signal.



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  		FIGURE 2. Kyte Doolittle hydrophobicity plot of the DC.LAMP protein. The cytoplasmic and transmembrane domains, used as targeting signal, are boxed.

 
The quality of the in vitro-transcribed mRNA was analyzed before electroporation with the Agilent 2100 Bioanalyzer. This method is more accurate than the traditionally used spectrophotometry and gel electrophoresis. Based on an electropherogram (Fig. 3A), the mRNA concentration was calculated, and the samples were checked for mRNA impurities or degradation. A gel-like image (Fig. 3B) allowed us to analyze the length of the mRNA derived from the different pGEM constructs. Only mRNA preparations without any contamination or degradation were used for electroporation.



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  		FIGURE 3. Quality control of the in vitro-transcribed mRNA. An electropherogram of MAGE-A3 mRNA, representative of all mRNA samples (A) and a gel-like image of mRNA derived from all pGEM-MAGE-A3 constructs (B) are shown. This gel-like image is representative of the pGEM-eGFP constructs (relatively).

 
It is important that the mRNAs encoding the different MAGE-A3 genes are efficiently electroporated into the DCs. We conducted a quantitative PCR analysis for MAGE-A3 on cDNA derived from RNA extracted from DCs electroporated with the different MAGE-A3 mRNAs before or after maturation (Table I). Our data show that the DCs electroporated with the various MAGE-A3 mRNAs contain a high mRNA copy number and that the differences in electroporation efficiencies are rather small. The efficiencies of mRNA electroporation in mature and immature DCs seem equivalent. Nonelectroporated, immature or mature DCs did not express MAGE-A3 mRNA (results not shown). Several melanoma and myeloma cell lines were evaluated for their MAGE-A3 mRNA expression level (Table II). In comparison with the electroporated DCs, the tumor cells expressed only a small fraction of the amount of MAGE-A3 mRNA found in the electroporated DCs. This indicates that these electroporated DCs contain an amount of MAGE-A3 mRNA exceeding the physiological levels found in tumor cells.


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  		Table I. Quantitative PCR for MAGE-A3 on electroporated DCsa

 

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  		Table II. Quantitative PCR for MAGE-A3 on tumor cell linesa

 
We analyzed the immunophenotype of immature and mature DCs (Fig. 4). On day 5 of DC culture, half the DCs were isolated and matured with the cytokine mixture. Twenty-four hours later, the cells were electroporated and cultured in maturation medium for another 24 h. The remainder of the immature DCs were electroporated on day 6 of DC culture. Four hours after electroporation, they were matured with the cytokine mixture for 24 h. DCs cultured in the presence of the cytokine mixture showed a fully mature phenotype (up-regulation of CD80, CD83, CD86, and HLA classes I and II), whereas DCs cultured without the cytokine mix remained immature. DCs that had been cultured in maturation medium for 48 h (24 h before and 24 h after electroporation), showed a slightly higher expression level of the different maturation markers than DCs that had been cultured in maturation medium for 24 h. The viabilities of the DCs electroporated before and after a 24-h culture in the inflammatory cytokine mixture were similar (>90%).



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  		FIGURE 4. Phenotype of immature and mature DCs. DCs were electroporated on day 6 of DC culture and were matured with the cytokine mixture 24 h before or 4 h after electroporation. The DCs were cultured for an additional 24 h, after which they were immunophenotyped by flow cytometry (results represented as black lines). DCs stained with isotype-matched Abs are represented as thin gray lines. This figure is representative of three independent experiments.

 
We assessed whether proteins linked to the various HLA class II pathway-targeting sequences were efficiently targeted to and degraded in the HLA class II-processing compartments. DCs were electroporated before or after maturation with the different eGFP mRNAs, and immediately after electroporation, the lysosomal degradation inhibitor chloroquine was added to the DC culture. At different time points (6–48 h) after electroporation, eGFP expression by DCs cultured with or without chloroquine was determined (Fig. 5). Six hours after electroporation, no significant difference in eGFP expression was observed between nontreated and chloroquine-treated DCs. After 24 h, eGFP linked to a targeting signal is almost completely degraded in nontreated DCs, whereas it remains detectable in chloroquine-treated DCs until 48 h after electroporation. The degradation of unlinked eGFP was not inhibited by addition of chloroquine. This suggests that the eGFP protein is transported to and degraded in the HLA class II-processing compartments when linked to a targeting sequence. eGFP linked to the LAMP1 or DC.LAMP sorting signal is highly expressed in DCs electroporated both before and after maturation. When it is linked to the Ii-targeting domain, the expression level is lower in both nontreated and chloroquine-treated DCs, especially when electroporated before maturation.



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  		FIGURE 5. Degradation of the eGFP protein in the HLA class II compartments when linked to the various sorting signals. DCs were electroporated with the various eGFP mRNAs before or after maturation. Chloroquine was added to the DC culture, and eGFP expression was analyzed between 6 and 48 h later. Nonelectroporated DCs are shown as gray dotted lines, nonchloroquine-treated DCs are shown as full gray lines, and chloroquine-treated DCs are shown as full black lines. This figure is representative of three independent experiments.

 
We investigated whether DCs electroporated with mRNA encoding the different MAGE-A3 constructs were able to stimulate an HLA class I- and an HLA class II-restricted T cell clone. Therefore, immature and mature DCs were electroporated on day 6 of DC culture. Four hours after electroporation, the immature DCs were matured with the cytokine mixture. Twenty-four hours after electroporation, all DCs were frozen and stored in liquid nitrogen. After thawing, the DCs were cocultured with MAGE-A3-specific, HLA-A1-restricted (Fig. 6A) and HLA-DP4-restricted (Fig. 6B) T cells for another 24 h. eGFP-electroporated DCs were used as a negative control; similar results were obtained with nonelectroporated DCs (results not shown). IFN-{gamma} released in the supernatant during the 24-h coculture was measured by ELISA. In general, MAGE-A3-electroporated DCs can stimulate HLA class II-restricted, M3.DP4-specific T cells, only when MAGE-A3 is linked to a sorting signal. Alternatively, MAGE-A3 has to not be linked to a targeting sequence to activate HLA class I-restricted, M3.A1-specific T cells. Our results also show that the HLA class I- and class II-restricted, MAGE-A3-specific epitopes are presented by the DCs with different efficiencies, depending on the targeting signal and the maturation state of the DCs when electroporated. Indeed, when MAGE-A3 is linked to the Ii-derived sorting signal, the electroporated DCs are less efficient in stimulating the M3.DP4 specific T cells than when MAGE-A3 is linked to the LAMP1- or DC.LAMP-targeting sequence. This trend is also observed for M3.A1-specific T cells. Even more, linking MAGE-A3 to a sorting signal enhances HLA class I presentation. Furthermore, our data clearly indicate that the T cell stimulatory capacity of DCs is greatly enhanced when the DCs are electroporated at a mature state instead of before maturation.



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  		FIGURE 6. T cell stimulatory capacity of DCs electroporated before or after maturation with mRNA encoding the various MAGE-A3 constructs. IFN-{gamma} production by M3.A1-restricted (A) and M3.DP4-restricted (B) T cells is shown. The results are shown as the mean ± SD and are representative of three independent experiments.

 
The T cell stimulatory capacity of DCs electroporated with the different MAGE-A3 constructs was compared with the T cell stimulatory capacity of peptide-pulsed DCs. On day 5 of DC culture, the cells were induced to mature in the presence of the inflammatory cytokine mixture. The next day the nonelectroporated and matured DCs were pulsed with 30 µg/ml M3.A1 or M3.DP4 peptide, mimicking the situation used in clinical studies (34). After 4 h of peptide pulsing, the DCs were frozen and stored in liquid nitrogen. After thawing, the DCs were cocultured with M3.A1- and M3.DP4-specific T cells for another 24 h. IFN-{gamma} released in the supernatant during this coculture was measured by ELISA (Table III). These data show that up to ~80% of the T cell stimulatory capacity of peptide-pulsed DCs can be reached by mRNA electroporated DCs.


View this table:
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  		Table III. Comparison of the T cell stimulatory capacity of electroporated and peptide-pulsed DCsa

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The main goal of cancer immunotherapy is to generate a strong and persistent tumor-specific immune response. There is consensus that not only CD8+ CTLs, but also CD4+ Th cells, must be induced to achieve this goal (6, 7, 8, 9, 10, 11, 12, 13). Therefore, APCs must present TAA-derived epitopes in the context of both HLA class I and class II molecules. Among the different methods to obtain presentation of both HLA class I- and class II-restricted, TAA-derived epitopes, we chose to link the TAA-encoding information to a HLA class II pathway sorting signal. This strategy offers the advantage that multiple antigenic epitopes of the TAA will be presented. Consequently, this method is not dependent on knowledge of the HLA haplotype of each patient or of the HLA class I- and class II-restricted, TAA-derived epitopes.

In this study we compared the HLA class II trafficking signals of Ii, LAMP1, and a novel signal, the DC.LAMP sorting signal. The HLA class II sorting signal of Ii consists of the 80 N-terminal amino acids of the protein. This sequence encompasses the cytoplasmic and transmembrane domains of Ii. It contains the trafficking signals Leu-Ile and Pro-Met-Leu that are required for transport to the HLA class II-processing compartments (58, 59). This sequence has been shown to induce the highest level of Ag presentation in comparison with several other Ii constructs (37). We have successfully used Ii80 for targeting TAAs in EBV-immortalized B cells (21, 23) and in lentivirally transduced DCs (60). The targeting signals of LAMP1 and DC.LAMP also encompass the cytoplasmic and transmembrane domains of these proteins. At their C termini, these amino acid sequences respectively contain the Tyr-Gln-Thr-Ile and Tyr-Gln-Arg-Ile sequences, whose structures conform to the Tyr-Xaa-Xaa-hydrofobic amino acid motif that is known to mediate cell membrane internalization and lysosomal targeting of several cell surface receptors (61, 62, 63, 64). In contrast with Ii, which is a type II transmembrane protein, LAMP1 and DC.LAMP are type I transmembrane proteins. To translocate these chimeric proteins to the rough endoplasmic reticulum, we added a signal sequence at their N termini.

Viral vectors such as recombinant lentivirus, adenovirus, and vaccinia virus have been reported to be efficient tools for the genetic modification of DCs. Nevertheless, nonviral gene delivery systems are preferable because safety issues and the immunogenicity of vector-encoded Ags are reduced to a minimum. Therefore, we used the recently described mRNA electroporation method to introduce the TAA linked to a HLA class II trafficking signal into DCs.

Various factors that play important roles in the efficiency of this mRNA electroporation method were investigated. First, we analyzed the quality of the in vitro transcribed mRNA with a sensitive method in order to be able to compare the Ag presentation by DCs electroporated with the different constructs. We used the Agilent 2100 Bioanalyzer to analyze the mRNA length, concentration, and overall quality. This method allowed a fast quality control of the mRNA. High quality mRNA could be produced. Secondly, we performed a quantitative RT-PCR analysis for MAGE-A3 on DCs electroporated before or after maturation with the various MAGE-A3 mRNAs. Our data showed that the number of MAGE-A3 mRNA copies is much higher in electroporated DCs than in tumor cell lines endogenously expressing MAGE-A3. As mRNA electroporation confers such a high MAGE-A3 copy number, we can suppose that the number of mRNA copies available for translation will not be a limiting factor for protein synthesis. Thirdly, we investigated whether electroporating DCs induces any phenotypical changes in these cells. FACS analysis showed that electroporation had no effect on the DC’s phenotype or ability to mature in response to a mixture of inflammatory cytokines.

When using HLA class II sorting signals, several questions must be addressed. The first is whether proteins linked to these signals are targeted to the HLA class II-processing compartments. Therefore, we linked the eGFP protein to the different trafficking signals. As eGFP is a fluorescent protein, it is easy to assess its expression by FACS analysis. In untreated DCs, eGFP expression was completely negative after 24 h when eGFP was linked to a targeting signal. When the lysosomal degradation inhibitor chloroquine was added to DCs electroporated with eGFP linked to a HLA class II targeting signal, a clear inhibition of eGFP degradation was noticed after 24 h, lasting up to 48 h after electroporation. The degradation of unlinked eGFP was not inhibited when chloroquine was added. These results suggest that proteins linked to an HLA class II sorting signal are targeted to and degraded in an acidic endosomal or lysosomal compartment. Secondly, to be useful for immunization purposes, the proteins linked to the targeting signal must be processed and presented in the context of HLA class II molecules without losing their presentation in HLA class I molecules. When cocultured with MAGE-A3-specific, HLA class I- or class II-restricted T cells, DCs electroporated with MAGE-A3 linked to the various trafficking signals displayed a high T cell stimulatory capacity in both HLA class II and class I. In comparison with peptide-pulsed DCs, which are frequently used in clinical studies, mRNA-electroporated DCs reached an Ag presentation level ~80% of that reached with peptide-pulsed DCs.

All Ag presentation assays were performed after cryopreservation of the DCs, mimicking the situation in clinical trials. Efficient induction of an antitumor response will most likely require repeated injections of the vaccine. Moreover, several quality controls must be performed on the vaccine before injecting it into patients, such as control of sterility, functionality, and phenotype. Therefore, it is essential to be able to freeze the DCs without losing their T cell stimulatory capacity.

As immature DCs are considered to be specialized in Ag capture and processing, whereas mature DCs present Ag and have an increased T cell stimulatory capacity, the consensus has been to electroporate DCs before maturation. In this case, the immature DCs still have time to translate the electroporated mRNA into protein and process it into epitopes, which could be presented by the DCs after maturation. Our data, however, indicate that electroporation of DCs after maturation results in a better CD8+ and CD4+ T cell stimulatory capacity. We hypothesize that this might be due to the fact that DCs electroporated after maturation were cultured in maturation medium for 48 h, instead of 24 h for DCs electroporated in an immature state. These DCs express higher levels of HLA molecules and costimulatory molecules, leading to an enhanced presentation of the Ag-derived epitopes and an increased T cell stimulatory capacity. Furthermore, electroporating DCs in a mature state offers several other advantages, such as a higher yield of DCs before electroporation and a higher recuperation after electroporation. Indeed, when DCs are matured before harvesting, more cells are recovered, as one of the characteristics of maturation is their decreased adhesion to plastic. Also, when immature DCs are used for electroporation, many cells will stick to the electroporation cuvette. Overall, DC yields after electroporation and cryopreservation were ~1.7% of the total input PBMCs for DCs electroporated before maturation vs 3.8% for DCs electroporated after maturation (for further details, see also A. Michiels, S. Tuyaerts, A. Bonehill, J. Corthals, K. Breckpot, C Heirman, S. Allard, P. van der Bruggen, and K. Thielemans, manuscript in preparation). Regarding clinical trial settings, these data indicate that the use of DCs electroporated after maturation will result in the use of less material, such as the expensive maturation cytokines and in vitro-transcribed mRNA.

Although we have successfully used the Ii-targeting signal in retrovirally transduced EBV B cells and in lentivirally transduced DCs, our results show that the targeting, degradation, and presentation in both HLA class I and class II molecules are very poor in mRNA-electroporated DCs compared with the LAMP1 and DC.LAMP signals. A possible explanation for this observation is that the in vitro-transcribed mRNA, encoding proteins linked to the Ii-targeting signal, has secondary structures that make it difficult for ribosomes to translate the proteins.

When MAGE-A3 is not linked to a targeting signal, no presentation in HLA class II was observed. In contrast, targeting MAGE-A3 to the HLA class II-processing pathway induces presentation in HLA class I, suggesting that these proteins are efficiently cross-presented.

The reason for the enhanced HLA class I presentation by the DCs electroporated with the chimeric constructs compared with the unmodified MAGE-A3 mRNA is not clear and is the subject of ongoing experiments. Similar results were obtained with the Melan A differentiation Ag (data not shown). The very high level of Ag-encoding mRNA as determined by quantitative PCR analysis might give rise to the translation of abnormal proteins and the increased formation of so-called DRiPs or defective ribosomal products (65). The enhanced peptide presentation after electroporation with the LAMP1- and DC.LAMP-containing mRNA might be due to presentation by recycling HLA class I molecules. Internalized HLA class I molecules can associate with peptides generated in the endosomal pathway (66, 67, 68).

In conclusion, we report in this study on the successful use of a novel targeting signal, namely DC.LAMP. Furthermore, we show that mRNA electroporation of DCs with a TAA linked to an HLA class II sorting signal is a very powerful tool for presenting Ag-derived epitopes in the context of both HLA class I and class II molecules, provided that the efficiency of the sorting signal is carefully investigated for each application.


   Acknowledgments
 
Material from patient R12 (from which clone R12-C9 was derived) was kindly provided by Drs. B. Schuler-Thurner and E. S. Schultz (University Hospital of Erlangen, Erlangen, Germany). We thank Dr. C. De Greef for helpful discussions, and D. Carels, C. Huysmans, L. Hollanders, E. Vaeremans, P. Verbuyst, J. Volkaert, and C. Wildmann for technical assistance.


   Footnotes
 
1 This work was supported by a Ph.D. fellowship from the Flemish Institute for Science and Technology (IWT) (to A.B.). K.T. was supported by grants from the Fund for Scientific Research-Flanders (FWO-Vlaanderen), the Ministry of Science (IUAP/PAI V), the FORTIS Bank, the Belgische Federatie voor Kankerbestrijding, and the Brussels Region. Back

2 Address correspondence and reprint requests to Dr. Kris Thielemans, Laboratory of Molecular and Cellular Therapy, Department of Physiology-Immunology, Medical School of the Vrije Universiteit Brussel, Laarbeeklaan 103/E, 1090 Brussels, Belgium. E-mail address: kris.thielemans{at}vub.ac.be Back

3 Abbreviations used in this paper: TAA, tumor-associated Ag; AAG, asparagine-arginine-glutamine; DC, dendritic cell; eGFP, enhanced green fluorescent protein; huAB, human AB serum; Ii, invariant chain; LAMP1, lysosome-associated membrane protein-1. Back

Received for publication January 21, 2004. Accepted for publication March 31, 2004.


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