Side-by-Side Comparison of Lentivirally Transduced and mRNA-Electroporated Dendritic Cells: Implications for Cancer Immunotherapy Protocols

ARTICLE doi:10.1016/j.ymthe.2004.07.017

Side-by-Side Comparison of Lentivirally Transduced andmRNA-Electroporated Dendritic Cells: Implications for

Cancer Immunotherapy Protocols

Melissa Dullaers,1,* Karine Breckpot,1,* Sonja Van Meirvenne,1 Aude Bonehill,1

Sandra Tuyaerts,1 Annelies Michiels,1 Lieven Straetman,1 Carlo Heirman,1

Catherine De Greef,1 Pierre Van Der Bruggen,2 and Kris Thielemans1,y

1Laboratory of Molecular and Cellular Therapy, Department of Physiology–Immunology, Medical School of the Vrije Universiteit Brussel, Laarbeeklaan 103/E,

1090 Brussels, Belgium2Ludwig Institute for Cancer Research, Brussels Branch, Avenue Hippocrate 74, UCL 74.59, 1200 Brussels, Belgium

*These authors contributed equally to this work.

yTo whom correspondence and reprint requests should be addressed. Fax: +32 2 477 45 68. E-mail: [email protected].

Available online 14 August 2004

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The use of tumor antigen-loaded dendritic cells (DC) is one of the most promising approaches toinducing a tumor-specific immune response. We compared electroporation of mRNA to lentiviraltransduction for the delivery of tumor antigens to human monocyte-derived and murine bonemarrow-derived DC. Both lentiviral transduction and mRNA electroporation induced eGFPexpression in on average 81% of human DC. For murine DC, eGFP mRNA electroporation (62%)proved to be more efficient than lentiviral transduction (47%). When we used tNGFR as a transgenewe observed lentiviral pseudotransduction that overestimated lentiviral efficiency. Neither genetransfer method had an adverse effect on viability, phenotype, or allostimulatory capacity of eitherhuman or murine DC. Yet, the mRNA-electroporated DC showed a reduced production of IL-12p70compared to their lentivirally transduced and unmodified counterparts. Human Ii80MAGE-A3-modified DC and murine Ii80tOVA-modified DC were able to present antigenic epitopes in thecontext of MHC class I and class II. Both types of modified murine DC were able to induce OVA-specific cytotoxic T cells in vivo; however, the mRNA-electroporated DC were less potent. Our dataindicate that this may be related to their impaired IL-12 production.

Key Words: dendritic cell, lentivirus, mRNA electroporation, antigen presentation, IL-12,immunotherapy

CTION variables that needs to be addressed is the
INTRODU
Many cancer vaccine strategies are based on the fact thatvarious tumor-associated target antigens can be deliveredin an immunogenic fashion and induce successful andsafe anti-tumor cell-mediated immunity. A key player inthese strategies is the dendritic cell (DC), the professionalantigen-presenting cell of the immune system, fullyequipped to prime naive T cells [1,2]. The ability togenerate human DC from monocyte precursors providesan abundant source of these cells [3–6]. Dendritic cell-based tumor vaccines have already been translated fromthe laboratory to the clinic. Early phase I clinical trialssupport the idea that DC presenting tumor-associatedantigens can initiate an anti-tumor response in patients,leading in some cases to partial or complete regression ofthe tumor [7–11]. Nevertheless, there is a great need forfurther optimization of the cellular vaccines. One of the

efficientantigen loading of DC.

Dendritic cells can be pulsed with antigenic peptidesor proteins, charged with tumor lysates or apoptoticbodies, or genetically modified with DNA or mRNAencoding the antigen of interest. Genetic modificationresults in endogenous processing of peptides and pro-vides long-lasting expression of a spectrum of peptides.Thus, delivery of an entire tumor antigen overcomes theneed to identify immunogenic peptides that match thepatientTs HLA haplotype and increases the range ofpeptides presented. Another major argument in favor ofgenetic modification is the possibility of attachingtargeting signals derived from the invariant chain (Ii) orLAMP to the tumor antigen [12,13] to achieve processingof antigenic epitopes in the HLA class II compartment,resulting in class II presentation.

MOLECULAR THERAPY Vol. 10, No. 4, October 2004

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IG. 1. Flow-cytometric analysis of transduction efficiency of human DC.

uman DC were either electroporated with eGFP mRNA on day 6 or

ansduced with Lenti-eGFP at an m.o.i. of 15 on day 3. The cells were

atured on day 6 and evaluated by FACS for eGFP expression on day 7. The

data shown are representative of three experiments.

ARTICLEdoi:10.1016/j.ymthe.2004.07.017

Efficient gene delivery to human DC has beenachieved with a variety of viral vectors, such asadenovirus [14,15], adeno-associated virus [16], oncor-etrovirus [17,18], vaccinia virus [19,20], and lentivirus.Lentiviral vectors are efficient gene delivery vectorsthat can transduce nondividing, monocyte-derived DC,as well as murine bone marrow-derived DC, withoutharming phenotype nor function [21–27]. These viralvectors are attractive for their application in immuno-therapy since they provide high gene expression inboth dividing and nondividing cells. Preexistingimmunity to lentiviral vectors is rare, which allowsrepeated-immunization strategies [26]. Furthermore,the second-generation lentiviral vectors contain aseries of safety features placing them among the safestviral gene delivery vehicles currently available[23,25,27–34].

Recently an efficient nonviral method, i.e., mRNAelectroporation, has been developed. The electroporationof DC with mRNA has been described as an efficientmethod to load the DC with tumor antigens [35–41].Although this method results in short-term expression ofthe transgene, it is an attractive approach with clinicalperspectives because safety issues and problems withimmunogenicity of vehicle-derived proteins are reducedto a minimum.

In this study, we performed a side-by-side compar-ison of mRNA electroporation and lentiviral trans-duction of DC and show that both strategies resultedin comparable numbers of gene-modified cells forhuman DC, while for murine DC, mRNA electropora-tion yielded more gene-modified cells than lentiviraltransduction. We further studied the effects of bothmodification strategies on DC viability, morphology,phenotype, allostimulatory capacity, cytokine produc-tion, and antigen presentation. The only significantdifference we observed was a reduced IL-12p70 pro-duction after mRNA electroporation. Although weshow that both types of modified DC present antigenicepitopes in the context of MHC class I and class II tothe same extent, we observed that the mRNA-electro-porated DC were less potent in inducing an immuneresponse in vivo, which we attribute to their impairedIL-12 production.

RESULTS

Efficient and Reproducible Transgene Delivery toHuman Monocyte-Derived DC by LentiviralTransduction and mRNA ElectroporationWe used the reporter gene eGFP to evaluate genetransfer, in terms of both percentage transduced DCand level of transgene expression (mean fluorescenceintensity (MFI) per DC). We compared the electropora-tion of monocyte-derived human DC with in vitro-transcribed mRNA to the transduction of these DC with



lentiviral vectors. Both mRNA-electroporated and lenti-virally transduced DC were matured on day 6, respec-tively 4 h and 3 days after transgene delivery, using acytokine cocktail containing IL-1h, IL-6, TNF-a, andPGE2. We performed analysis on day 7. We obtainedcomparable modification efficiencies: 81 F 5% of themRNA-electroporated and 81 F 9% (n = 3) of thelentivirally transduced cells were eGFP positive (Fig. 1).The MFI of lentivirally transduced DC (1399 F 344) wason average four times higher than that of mRNA-electro-porated DC (352 F 134; n = 3). Similar results wereobtained when tNGFR was used as a reporter gene (resultsnot shown).

Discrepancy Between eGFP Gene Transfer and tNGFRGene Transfer in Murine Bone Marrow-Derived DC:Indications for tNGFR PseudotransductionmRNA electroporation and lentiviral transduction ofmurine bone marrow-derived DC, using tNGFR as atransgene, resulted in respectively 39.7 F 5 and 83.0 F5% tNGFR-positive cells (n = 3). However, when wetried to confirm these results using eGFP as a trans-gene, we observed a totally different outcome: 61.6 F6% of the mRNA-electroporated and 47.3 F 12% (n = 5)of the lentivirally transduced cells were eGFP positive(Fig. 2A).

When we followed the transduction efficiency intime (Fig. 2B), tNGFR efficiency after lentiviral trans-duction declined toward 30% on average, while eGFPefficiency gradually increased toward 65% on average.

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FIG. 2. Transduction efficiency of murine DC and tNGFR-related pseudotransduction. (A) Murine DC were either electroporated with mRNA or lentivirally

transduced on day 7 and subsequently matured. The cells were evaluated by FACS for transgene expression on day 9. Upper 3 plots: nonmodified DC; middle 3

plots: mRNA electroporated DC; lower 3 plots: lentivirally transduced DC. (B) Kinetics of tNGFR and eGFP expression after mRNA electroporation or lentivira

transduction of murine DC. Bars depict transduction efficiency and lines mean fluorescence intensity (MFI). (C) Lentiviral particles were produced by transfection

of 293T with pMD.G, pCMVDR8.9, pHRVtrip eGFP SIN and a eukaryotic vector encoding tNGFR (pREP8), or pHRVtrip tNGFR SIN and a eukaryotic vector encoding

eGFP (peGFP). These lentiviral supernatants were used to infect 293T cells to follow transduction and pseudotransduction in time. The numbers indicate the

percentage of eGFP+/tNGFR+ double-positive cells that are hence pseudotransduced. The data shown are representative of three experiments.


After mRNA electroporation, however, both eGFP andtNGFR efficiency gradually declined with time, which isto be expected considering the half-life of mRNAmolecules.

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We hypothesized that this discrepancy betweentNGFR and eGFP transduction efficiency after lentiviraltransduction could be caused by pseudotransductiondue to producer cell-derived tNGFR in the viral

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membrane that is transferred to the infected cells. Totest this hypothesis, we produced eGFP-encodinglentiviral particles from producer cells that weresimultaneously transfected with a nonviral eukaryoticexpression vector carrying tNGFR and vice versa. Weused these lentiviral supernatants to transduce 293Tcells. We followed the transduction efficiency of bothtNGFR and eGFP and the pseudotransduction efficien-

FIG. 3. Allostimulatory capacity and cytokine/chemokine secretion. (A, B) For the

indicated ratios. Proliferation was measured using [3H]thymidine incorporation

independent experiments. (C) To measure cytokine and chemokine secretion,

supernatant of the activated DC was collected after 24 h of coculture and the cyto

are representative of three independent experiments. (D) To measure IL-12 p

supernatant of the activated DC was collected 24 h after activation and the IL

representative of three independent experiments. Statistical analysis was perform



cies of the corresponding nonvirally delivered trans-gene. We observed pseudotransduction of nonviraltNGFR delivered by eGFP-encoding lentiviruses onday 1 after transduction (37.5%) that decreased withtime and almost disappeared by day 3 after trans-duction. We observed no pseudotransduction with non-viral eGFP delivered by tNGFR-encoding lentiviruses(Fig. 2C).

allo-MLR, mature DC were cocultured for 4 days with allogeneic T cells at the

(represented in counts per minute). The data are representative of three

mature human DC were activated by coculture with 3T6CD40L cells. The

kine/chemokine content was measured using sandwich ELISA. The data shown

roduction, mature murine DC were activated through LPS stimulation. The

-12p70 content was measured using a sandwich ELISA. The data shown are

ed using a paired Student t test and significant P values are mentioned.

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Neither mRNA Electroporation nor LentiviralTransduction Has an Adverse Effect on theImmunophenotype and Function of DC; However,mRNA-Electroporated DC Show an ImpairedIL-12p70 secretionWe investigated the effects of lentiviral transduction andmRNA electroporation on the mature DC viability,phenotype, and function. We performed analyses onday 7 human DC and day 9 murine DC. The viability ofboth types of modified DC was not altered compared tounmodified DC as observed with trypan blue andpropidium iodide staining (data not shown).

To analyze the immunophenotype of the DC, weperformed flow-cytometric analyses for HLA class II,CD80, CD83, CD86, and CD14 for human DC andMHC class II, CD11c, CD80, CD86, and CD40 for murineDC. Both human DC matured in a cytokine cocktailcontaining IL-1h, IL-6, TNF-a, and PGE2 and murine DCmatured with LPS showed a mature phenotype (HLA classII+++, CD80+, CD83+, and CD86++ and MHC class II+,CD11c+, CD80++, CD86++, and CD40++, respectively)without remarkable differences between unmodified,lentivirally transduced, and mRNA-electroporated DC(data not shown).

To determine the immunofunction of the maturegene-modified DC, we evaluated their capacity to stim-ulate allogeneic T cells and their capacity to producecytokines and chemokines upon activation (CD40 liga-tion for human DC and LPS stimulation for murine DC).We observed no differences between unmodified, mRNA-electroporated, or lentivirally transduced DC in allo-MLR(Figs. 3A and 3B). We could not detect IL-4, IFN-a, or IFN-g secretion by activated human DC. Secretion of IL-6,

FIG. 4. Quantification of mRNA content. (A) Human DC were either electroporate

an m.o.i. of 15 on day 3. (B) Murine DC were either electroporated with Ii80tOVA

were harvested for mRNA extraction 24, 72, and 120 h after the electroporation.

performed. cDNA quality and quantity were assessed in RT-PCR for the houseke

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TNF-a, MIP-1a, MIP-1h, MIP-3h, and PARC did not differsignificantly between unmodified, mRNA-electroporated,or lentivirally transduced DC (Fig. 3C). However, weobserved a reduced IL-12p70 production upon activationby mRNA-electroporated DC compared to unmodified orlentivirally transduced DC. Both unmodified and lentivi-rally transduced DC produced a large amount of IL-12 afteractivation (respectively, 365 F 73 and 355 F 54 pg/2 �105 cells/ml for human DC and 363 F 152 and 550 F268 pg/106 cells/ml for murine DC), whereas the mRNA-electroporated DC produced approximately 10 times lessIL-12 (35 F 15 pg/2 � 105 cells/ml for human and 52.5 F40 pg/106 cells/ml for murine DC; n = 3) (Figs. 3C and 3D).This difference was statistically significant with P = 0.006for human DC and P = 0.005 for murine DC betweenmRNA-electroporated and lentivirally transduced DC,while the difference between lentivirally transduced andunmodified DC was not significant.

Transgene mRNA Content after mRNA Electroporationand Lentiviral TransductionWe performed a semiquantitative PCR to assess theMAGE-A3 mRNA and the OVA mRNA content in electro-porated or lentivirally transduced human DC and murineDC, respectively (Figs. 4A and 4B). We compared thesemiquantitative RT-PCRs for MAGE-A3 and OVA to theh-actin RT-PCR at 24, 72, and 120 h after maturation. Inboth human and murine DC, the mRNA content in themRNA-electroporated DC was the strongest 24 h aftermaturation and declined gradually with time. The mRNAcontent in the lentivirally transduced DC remained atapproximately the same level with time, compared to theh-actin mRNA content.

d with Ii80MAGE-A3 mRNA on day 6 or transduced with Lenti-Ii80MAGE-A3 a

mRNA or transduced with Lenti-Ii80tOVA at an m.o.i. of 15 on day 7. The cell

cDNA was synthesized and a semiquantitative PCR for MAGE-A3 and OVA wa

eping gene h-actin.


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In Vitro Antigen Presentation by LentivirallyTransduced and mRNA-Electroporated DCTo elicit an effective immune response, gene-modifiedDC should be able to present antigenic peptides in thecontext of both MHC class I and class II.

We assessed the capacity of Ii80MAGE-A3-modifiedHLA-A1 human DC and HLA-DP0401 human DC topresent MAGE-A3-derived epitopes to respectively theCD8+ T cell clone ESBI684 and the CD4+ T cell cloneR12-C9. For that purpose, we cocultured mature day 7and day 10, unmodified, lentivirally transduced ormRNA-electroporated DC for 24 h with the correspond-ing T cells. The IFN-g content of the culture super-natant served as a measure of antigen presentation. Asa reference for IFN-g production by the T cells, weused mature DC loaded with the peptide EVDPIGHLYor with the peptide TQHFVQENYLEY, presented respec-

FIG. 5. In vitro antigen presentation of human DC. Day 7 or day 10 unmodifi

cocultured with the MAGE-A3-specific CD4+ (R12-C9) and CD8+ (ESBI684) T ce

presentation of fresh DC, (B) antigen presentation of frozen and thawed DC. Th



tively in the context of HLA-A1 and HLA-DP0401. Bothlentivirally and mRNA-electroporated, Ii80MAGE-A3-modified DC were able to present antigenic epitopesin the context of HLA class I and HLA class II.Lentivirally transduced DC were more potent andinduced approximately two times as much IFN-gsecretion by the specific T cells as mRNA-electropo-rated DC and this difference was statistically signifi-cant. We observed no significant difference inpresentation capacity between day 7 (4 days posttrans-duction) and day 10 (7 days posttransduction) modi-fied DC (Fig. 5A).

Since most cancer immunotherapy protocols includeseveral rounds of vaccination, it is essential thatmodified DC can be frozen and thawed without losingtheir T cell stimulatory capacity. Therefore we repeatedthe in vitro antigen presentation assays with thawed DC.

ed, MAGE-A3 mRNA electroporated or Lenti-MAGE-A3-transduced DC were

ll clones for 24 h. The IFN-g content was measured in ELISA. (A) The antigen

e data shown are representative of three independent experiments.

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The obtained results indicate that both Ii80MAGE-A3-electroporated and lentivirally transduced DC retaintheir capacity to present antigenic epitopes in the con-text of HLA class I and class II after freezing and thawing(Fig. 5B).

We tested Ii80tOVA mRNA-electroporated and pHRVIi80tOVA-transduced murine DC for antigen presenta-tion of OVA-derived peptides to the OVA-specific T cellhybridomas RF33.70 (class I) and MF2.2D9 (class II), this24, 48, 72, and 96 h after electroporation or trans-duction. Both lentivirally and mRNA Ii80tOVA-modifiedDC were able to present antigenic epitopes in thecontext of MHC class I and class II. We observed noremarkable differences in presentation kinetics betweenboth types of modified DC. The antigen presentationwas at maximum 24 and 48 h after the modification andgradually declined afterward. Overall, mRNA-electropo-rated DC were slightly more potent than lentivirallytransduced DC (Figs. 6A and 6B).

FIG. 6. In vitro antigen presentation kinetics of murine DC. (A, B) Unmodified

Ii80tOVA mRNA-electroporated or Lenti-Ii80tOVA-transduced DC were cocul

tured with TT hybridomas that recognize OVA-specific peptides in MHC class

(RF33.70) and class II (MF2.2D9). The IL-2 secretion of these hybridomas wa

measured as proliferation of the IL-2-dependent CTLL-2 cell line (represented

in counts per minute). The data shown are representative of three

independent experiments.

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In vivo Priming of Ovalbumin-Specific Cytotoxic TCells by Lentivirally Transduced andmRNA-Electroporated Murine DC: Important Role ofIL-12 in VivoThe ultimate goal is to use the gene-modified DC as avaccine to induce an immune response against the trans-gene in vivo. Therefore, we compared lentivirally trans-duced and mRNA-electroporated DC in terms of theircapacity to induce an immune response in vivo against thesurrogate tumor antigen OVA. We immunized mice witha single injection of unmodified, Ii80tOVA mRNA-electro-porated or Lenti-Ii80tOVA-transduced murine DC. Sevendays postimmunization, we collected the draining lymphnode cells, restimulated them with E.G7-OVA cells, and 5days later tested them for lysis of EL4 and E.G7-OVA in astandard 51Cr-release assay.

Fig. 7A shows that unmodified DC did not generateCTL against EL4 or E.G7-OVA. Both lentivirally trans-duced and mRNA-electroporated DC induced OVA-spe-cific CTL, because they were able to lyse EG.7-OVA anddid not lyse EL4. However, the CTL response induced byIi80tOVA mRNA-electroporated DC was consistentlyweaker, with a mean specific lysis of 24.4% F 10.7 usingan E/T ratio of 100, while lentivirally transduced DCinduced a CTL response with a mean specific lysis of53.5% F 11.5. The average lyses of the Lenti group weresignificantly higher than those of the mRNA group at E/Tratios from 100/1 to 6/1 (all P b 0.05).

We wanted to investigate the possibility that mRNA-electroporated DC are inferior CTL inducers due toimpaired trafficking to the lymph nodes. For thatpurpose, we performed RT-PCR for OVA on lymph nodesisolated 18 h after injection of unmodified, IitOVAmRNA-electroporated, and Lenti-IitOVA-transduced DC.The PCR we performed has only qualitative value. Wedetected OVA amplification in both lymph nodes frommice injected with IitOVA mRNA and Lenti-IitOVA DC(Fig. 7B) and conclude that both types of OVA-modifiedDC did reach the draining lymph nodes.

Because the only difference we could detect in vitrobetween lentivirally transduced and mRNA-electropo-rated DC was the IL-12p70 impairment of the mRNADC, we investigated the role of IL-12 in the in vivo CTLassay. Therefore we did the same experiment with DCthat were retrovirally transduced to overexpress IL-12p70and subsequently mRNA electroporated or lentivirallytransduced to express OVA (further referred to as IL-12Lenti-OVA or IL-12 mRNA-OVA DC). These DC secretedover 2500 pg/ml IL-12p70/106 cells/ml, which is aboutfive times the amount of normal activated DC. IL-12Lenti-OVA DC induced a mean E.G7-OVA lysis of 57.0 F5.8%, which is not different from normal lentivirallytransduced DC. IL-12 mRNA-OVA DC, however, induceda mean E.G7-OVA lysis of 59.5 F 4.6%, which issignificantly higher than normal mRNA-electroporatedDC ( P = 0.04). These data indicate that IL-12p70



FIG. 7. In vivo CTL assay. (A) Mice were subcutaneously immunized with 105 unmodified or OVA gene-modified DC. After 7 days, the lymph node cells were

isolated and restimulated for 5 days with E.G7-OVA cells. A chromium-release assay was performed to evaluate the cytotoxic activity against EL4 and E.G7-OVA

cells at the indicated E/T ratios. The lysis of EL4 and E.G7-OVA cells induced by CTL generated from mice immunized with unmodified DC, Ii80tOVA mRNA-

electroporated DC, and Lenti-Ii80tOVA-transduced DC is shown. Statistical analysis was performed using a paired Student t test. The lyses of the Lenti group are

significantly higher than those of the mRNA group at E/T ratios ranging from 100/1 to 6/1 (all P b 0.05). (B) 18 h after injection of 106 DC, lymph nodes were

isolated. RNA was extracted and cDNA synthesized. RT-PCR for OVA was performed. Lane 1, unmodified DC; lanes 2 and 3, Lenti-OVA DC; lanes 4 and 5, mRNA

OVA DC. (C) In vivo CTL assay with retro-IL-12-transduced DC, subsequently lentivirally transduced or mRNA electroporated.


production plays an important role in the induction of acellular immune response in vivo.

DISCUSSION

The use of gene-modified DC for cancer immunotherapyis already well documented [7–10]. DC therapy could alsobe applied for HIV and other infectious diseases [44,45].The ideal system for genetic modification of DC shouldresult in presentation of transgene-derived antigenicpeptides in both MHC class I and class II, withoutaltering the mature phenotype and function of the celland thus resulting in the induction of transgene-specificCTL and T helper cells.

We have performed a side-by-side comparison ofmRNA electroporation and lentiviral transduction ofhuman DC in vitro and of murine DC in vitro and in vivo.Both gene transfer systems were optimized in our lab[24,40,41].

The first striking observation was the different behav-ior of human and murine DC. Human DC displayedequal transduction efficiencies (81%) with a four timeshigher eGFP expression after lentiviral transductioncompared to mRNA electroporation. Murine DC, how-ever, showed a discrepancy in transduction efficienciesbetween eGFP and tNGFR gene transfer. Where the Lenti-tNGFR transduction (83%) was more efficient than the



tNGFR electroporation (40%), the eGFP electroporation(62%) was more efficient than the Lenti-eGFP trans-duction (47%). Taking into account the OVA mRNAexpression and antigen presentation, the eGFP results aremore likely to be accurate.

Furthermore, when we followed the expression intime, we observed that tNGFR expression after lentiviraltransduction declined quickly, whereas eGFP expressionincreased with time. These observations point toward akind of pseudotransduction related to tNGFR. Wehypothesized that Lenti-tNGFR transduction is overesti-mated shortly after transduction because of fusion withempty viral particles that contain producer cell-derivedmembrane-bound tNGFR. We have tested this hypothesisusing Lenti-eGFP that was produced from producer cellsthat were simultaneously transfected with a nonviraltNGFR plasmid. We did observe tNGFR-related pseudo-transduction until approximately 48 h after transduction.These data confirm our hypothesis that part of the tNGFRefficiency that is observed early after transduction is dueto transfer of the protein rather than to stable trans-duction. Gallardo et al. and Liu et al. have reported asimilar pseudotransduction that occurs only when usingVSV-G-pseudotyped retroviral particles [46,47]. Theyascribe the phenomenon as being related to the concen-tration of the particles using ultracentrifugation, whichwould promote copurification of the transgene protein.

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This pseudotransduction phenomenon is observedonly with murine DC and not with human DC. Thismust be related to the fact that human DC are muchmore permissive to transduction than murine DC, giventhe high rates of both lentiviral transduction and mRNAelectroporation.

The higher efficiency of eGFP electroporation com-pared to tNGFR electroporation could be due to accumu-lation of eGFP. With a half-life of more than 24 h, eGFPhas been shown to accumulate even at low expressionlevels [48,49].

Both lentiviral transduction and mRNA electropora-tion of human DC prove thus to be more efficient thanmodification of murine DC. This is probably due to thedifference in the DC populations that are used: thehuman DC are generated from CD14+-derived precursorsselected through MACS sorting, while the murine DC arebone marrow derived. We have reported that CD14-positive selection renders human monocytes in a state ofpartial activation that makes them more permissive tolentiviral transduction than their adherence purifiedcounterparts [55].

The viability, phenotype, and allostimulatory capacityof neither mRNA-electroporated nor lentivirally trans-duced DC were hampered compared to mature unmodi-fied DC. Secretion of the cytokines IL-6 and TNF-a andthe chemokines MIP-1a, MIP-1h, MIP-3h, and PARC afteractivation of human DC did not significantly differbetween unmodified, mRNA-electroporated, and lentivir-ally transduced DC. However, when evaluating the IL-12p70 production upon activation, we observed that themRNA-electroporated DC produced 10 times less IL-12p70 than the unmodified and lentivirally transducedDC, both for human and for mouse DC. This reducedcapacity to produce IL-12 is partly due to the electricpulse, since the DC that received the pulse withoutmRNA also showed a decreased IL-12 secretion, however,to a lesser extent [41].

This reduced IL-12 production could have importantconsequences for the induction of an optimal immuneresponse in vivo since IL-12 plays a critical role in thepolarization of the immune response toward a Th1response. The presence or absence of IL-12 has also beendescribed to play a critical role in inducing tolerance oractivation [50,51]. In addition to the activating effect onnaive CD8+ T cells, IL-12 influences CD4+ T cells tomediate CTL-independent suppression of tumor growthin vivo [52] and provides a survival signal to CD4+ andCD8+ T cells in vivo, inhibiting TCR-induced T cell deathby down regulating Fas ligand and up regulating FLIPs[53].

The semiquantitative RT-PCR for MAGE-A3 and OVAshowed that the mRNA content after electroporationdeclined, while after lentiviral transduction the contentremained about the same until 120 h after maturation.This was expected, because after electroporation, the

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mRNA source is limited and is progressively degraded,while after lentiviral transduction, there is a continuoustranscription of the integrated sequences.

Given the lower protein expression after mRNAelectroporation, it was no surprise that the mRNA-electroporated human DC were less potent in stimulatingIFN-g production by MAGE-A3-specific CD4+ and CD8+ Tcell clones. However, the difference was not that pro-nounced, with the lentivirally transduced DC being atbest only two times stronger than the mRNA-electro-porated DC. In addition, both gene-modified DC werestill able to stimulate the T cell clones at day 10 (3 dayspostmaturation) to the same extent as on day 7 (1 daypostmaturation). This implies that the antigen presenta-tion of transgenes is still efficient 3 days after mRNAelectroporation, while the protein expression starts todiminish 2–3 days after electroporation [40,41].

Freezing and thawing of the Ii80MAGE-A3 mRNA-electroporated and Lenti-Ii80MAGE-A3-transduced DCdid not affect their T-cell-stimulatory capacity. This isan important criterion, since most cancer immunother-apy protocols include several rounds of vaccination.

In vitro antigen presentation assays with OVA-modi-fied murine DC showed no remarkable differences inpresentation kinetics between the lentivirally transducedand the mRNA-electroporated DC. The fact that evenlentivirally transduced DC lose their antigen presentationcapacity gradually with time although the mRNA contentremains high could be a result of immune responsetermination. DC are programmed for apoptosis once theyare activated. In an initial phase inflammatory stimulisuch as LPS and TNF-a up regulate genes involved ininitiation and amplification of the immune response. Inthe second phase, the immune response is terminated:the cells shut down protein synthesis and die of apoptosis8–9 days after activation [54].

The ultimate goal of DC therapy is to use the gene-modified DC as a vaccine to induce an immune responseagainst the transgene in vivo. A variety of factors, whichcannot all be studied with in vitro models, will determinethe outcome. We used ovalbumin as a surrogate antigento investigate the capacity of both types of modified DCto induce an immune response in vivo. We observed thatboth mRNA-electroporated and lentivirally transducedDC were capable of inducing an OVA-specific CTLresponse. However, the CTL response generated withmRNA-electroporated DC was consistently weaker thanthat generated with lentivirally transduced DC. Toexamine the possibility that these weaker responses aredue to impaired trafficking to the draining lymph nodes,we performed RT-PCR for OVA on lymph node RNA. Inboth mRNA-DC-injected and Lenti-DC-injected lymphnodes the OVA signal could be amplified. Thus bothtypes of DC reached the lymph nodes. Although this PCRis not quantitative, it indicates that poor traffickingcannot explain the weaker CTL responses induced by




mRNA DC. We repeated the in vivo CTL induction withDC that were retrovirally transduced to overexpress IL-12p70 and noticed that these IL-12 mRNA DC were ableto induce the same extent of lysis as the IL-12 Lenti DC ornormal Lenti DC. This is a strong indication that mRNA-electroporated DC are weaker CTL inducers, due to theirimpaired IL-12p70 production, despite the fact that theyare as potent as the lentivirally transduced DC in antigenpresentation in vitro.

We can conclude that both mRNA electroporation andlentiviral transduction are efficient transgene deliverystrategies for DC that do not affect viability nor pheno-type. Although the mRNA-electroporated DC showedstrongly reduced IL-12p70 production, both types ofgene-modified DC were able to process and presentantigenic epitopes in vitro, even after freezing andthawing. However, the IL-12 impairment of the mRNA-electroporated DC is at least partly responsible for lowerin vivo responses. Given the ease and safety of themethod, which make it an interesting tool for modifica-tion of DC for clinical purposes, attempts to restore the IL-12 production should be considered. This could be easilydone by coelectroporation of the antigen with IL-12p70.

Our results suggest that lentiviral transduction is themost powerful of the two systems tested in vivo. However,there are still some obstacles to overcome before thelentiviral system is ready to be used in clinical trialsbecause the currently used virus production methods arevery labor intensive and the achieved yields and puritiesdo not yet meet the demands for use in the clinic. Theease of producing large amounts of in vitro-transcribedmRNA is the major advantage of mRNA electroporationand as a consequence mRNA-electroporated DC arealready being used in small-scale clinical pilot studies.

MATERIALS AND METHODS

Mice and cell lines. Six- to eight-week-old female C57Bl/6 mice were

purchased from Harlan (Ad Horst, The Netherlands). Animals were

maintained according to the institutional guidelines.

The EL4 and E.G7-OVA tumor cell lines, TT hybridoma cell lines

RF33.70 and MF2.2D9, IL-2-sensitive cell line CTLL-2, 293T cells, and 3T6

and 3T6-huCD40L cells were previously described [24].

The human MAGE-A3-specific T cell clones, ISBE684 and R12-C9,

which recognize MAGE-A3-derived peptides, respectively EVDPIGHLY in

the context of HLA-A1 and TQHFVQENYLEY in the context of HLA-

DP0401, were previously described [42,43].

Generation of dendritic cells. Human monocyte-derived DC and murine

bone marrow-derived DC were generated as described earlier [24].

Lentiviral transduction of dendritic cells. The multiple attenuated

packaging plasmid pCMVDR8.9 and the VSV-G-encoding plasmid

pMD.G were a kind gift from Dr. D. Trono (University of Geneva,

Switzerland). The transfer vectors pHRVtrip CMVeGFP SIN (pHRVeGFP),

HRVtripCMVIi80MAGE-A3-Ires-tNGFR SIN (pHRVIi80MAGE-A3), and

pHRVtripCMVIi80tOVA-Ires-tNGFR SIN (pHRVIi80tOVA) were previously

described [24].

The preparation, concentration, and titration of the lentivirus stocks

and the transduction of human and murine DC were performed as

previously described [24].



For the pseudotransduction experiments, lentiviral particles were

produced by transfection of 293T cells with pMD.G, pCMVDR8.9,

pHRVtrip eGFP SIN and a eukaryotic vector encoding tNGFR (pREP8),

or pHRVtrip tNGFR SIN and a eukaryotic vector encoding eGFP (peGFP-

C1) that does not contain a packaging signal. These lentiviral super-

natants were used to infect 293T cells to follow transduction and

pseudotransduction in time.

mRNA electroporation of DC. The vectors pGEM4Z/eGFP/64A and

pGEM4Z/muIi80tOVA/64A were previously described [41].

The plasmid pGEM4ZIi80MAGE-A3/64A was based on the plasmid

pGEM4Z/eGFP/64A. The Ii80 fragment was generated by PCR, which

introduced a BglII site at the 5V end and a BamHI site at the 3V end of the

Ii80 fragment. This PCR product was cloned into the BamHI-linearized

pGEM4Z/64A, resulting in pGEM4Z/Ii80/64A. To obtain the plasmid

pGEM4Z/Ii80MAGE-A3/64A, a BglII–BglII MAGE-A3 fragment was cloned

into the BamHI-linearized pGEM4Z/Ii80/64A vector.

Prior to in vitro transcription, the pGEM plasmids were linearized

with SpeI or NotI. In vitro RNA transcription was performed using the T7

RNA polymerase and ARCA (anti-reverse cap analogue) (Ambion mMES-

SAGE mMACHINE T7ultra Kit; Austin, TX, USA) as described previously

[40].

The mRNA electroporation of human and murine DC was previously

described by, respectively, Tuyaerts et al. [41] and Van Meirvenne et al.

[40]. Briefly, human DC were harvested on day 6 and washed once in

serum-free medium and once in Optimix solution A (EQUIBIO, Kent, UK).

Cells were adjusted to a final cell density of 20 � 106 DC/ml in Optimix

solution B (EQUIBIO). Murine DC were harvested on day 7 and washed

three times with phosphate-buffered saline. Cells were resuspended in

OptiMEM to a cell density of 20 � 106 DC/ml. Two hundred microliters of

both human and murine cell suspensions, mixed with 20 Ag of mRNA,

was subsequently transferred to a 4-mm-gap electroporation cuvette for

immediate electroporation using the EQUIBIO Easyject Plus apparatus.

Electroporation conditions were voltage of 300 V, capacitance of 150 AF,

resistance of 99 ohms resulting in a pulse time of 5–6 ms. Immediately

after electroporation, the DC were transferred into their respective culture

medium.

Cryopreservation of human dendritic cells. Dendritic cells were frozen in

cryotubes in 1 ml 20% albumin solution with 10% DMSO (Sigma–

Aldrich) at 1–5 � 106 DC per vial. The DC were slowly frozen to �808Cusing a cryofreezing container (Cryo 18C freezing container, rate of

cooling �18C/min; Nalgene, Hereford, UK) and subsequently stored in

liquid nitrogen until use. Thawing of the cryopreserved DC was

performed in a 378C water bath until small ice crystals were visible. Cold

HankTs balanced salt solution (Invitrogen) was added dropwise. The cells

were centrifuged in a precooled centrifuge (48C) and afterward resus-

pended in 5 ml of prewarmed X-VIVO 15 medium, supplemented with

1% huAB serum. After a resting period of 15 min, the cell viability was

determined using trypan blue.

Phenotypical and functional analysis of the DC. Flow cytometry and

allo-MLR were performed to evaluate phenotype, transgene expression,

and transgene function as described previously [24]. Human DC were

tested for their capacity to secrete cytokines and chemokines after

activation through CD40 ligation (as described in [6]). ELISAs for the

human cytokines IL-4 (Biosource), IL-6, TNF-a (eBioscience), IL-12p70,

IFN-a (Bender MedSystems), and IFN-g (PeProtech) and for the human

chemokines MIP-1a, MIP-1h, MIP-3h, and PARC (all from R&D Systems)

were performed conforming the manufacturerTs instructions. Murine DC

were tested for their IL-12p70 secretion after activation through LPS,

using the mIL-12p70 ELISA kit from eBioscience.

Semiquantitive RT-PCR. The extraction of total RNA, the synthesis of

cDNA, and the RT-PCR for the housekeeping gene h-actin and the

transgenes MAGE-A3 and ovalbumin were previously described [24].

In vitro antigen presentation assays. The antigen presenting capacity of

gene-modified human DC was assessed through their capacity to induce

IFN-g production by the MAGE-A3-specific CD4+ T cell clone R12-C9 and

CD8+ T cell clone ISBE684 as described previously [24].

777


The antigen presenting capacity of gene-modified murine DC was

measured as their capacity to induce IL-2 production by the OVA-specific

TT hybridomas RF33.70 (class I) and MF2.2D9 (class II) as described

previously [24]. The IL-2 production was detected as proliferation of the

IL-2-dependent cell line CTLL-2.

In vivo CTL induction. Day 7 murine DC were either pHRVIi80tOVA

transduced or Ii80tOVA mRNA electroporated and matured in the

presence of 100 ng/ml LPS for 4 h. Six- to eight-week-old C57Bl/6 mice

received one subcutaneous injection into the footpad with 1 � 105

Ii80tOVA-modified DC (four animals/group). Seven days after immuni-

zation, draining popliteal lymph nodes were isolated. The lymph node

responder cells were restimulated in vitro for 5 days with mitomycin

C-treated E.G7-OVA cells (ovalbumin-transfected EL4 cells) at an T cell/

stimulator ratio of 1/2. After restimulation, the cultures were tested for

their cytolytic activity against EL4 and E.G7-OVA cells in a standard51Cr-release assay, as described earlier [18]. To assess the influence of

the IL-12 on the in vivo outcome, the same experiment was done with

DC that were retrovirally transduced on days 2 and 3 with pMFG mIL-

12 (as described in [56]) and subsequently lentivirally transduced or

mRNA electroporated to express OVA. The production of retroviral

supernatants and the retroviral transduction of DC were described

previously [18].

Statistical analysis. Statistical analyses were performed using paired

Student t tests.

ACKNOWLEDGMENTS

We thank Elsy Vaeremans and Margareth Verbuyst for DNA and mRNA

preparation. M.D. was supported by a grant from the Fund for Scientific

Research Flanders (FWO-Vlaanderen) and K.B. by a grant from the Flemish

Institute for Science and Technology (IWT). This work was further supported by

grants to K.T. from the FWO-Vlaanderen, the IWT, the Ministry of Science

(IUAP/PAI), the Fortis Bank, and the Belgische Federatie voor Kankerbestrijding.

RECEIVED FOR PUBLICATION JULY 7, 2004; ACCEPTED JULY 15, 2004.

REFERENCES1. Banchereau, J., et al. (2000). Immunobiology of dendritic cells. Annu. Rev. Immunol. 18:

767 – 811.

2. Banchereau, J., and Steinman, R. M. (1998). Dendritic cells and the control of

immunity. Nature 392: 245 – 252.

3. Thurner, B., et al. (1999). Generation of large numbers of fully mature and stable

dendritic cells from leukapheresis products for clinical application. J. Immunol. Methods

223: 1 – 15.

4. Rouard, H., et al. (2000). Adenoviral transduction of human dclinical gradeT immature

dendritic cells enhances costimulatory molecule expression and T-cell stimulatory

capacity. J. Immunol. Methods. 241: 69 – 81.

5. Suen, Y., et al. (2001). Comparison of monocyte enrichment by immuno-magnetic

depletion or adherence for the clinical-scale generation of DC. Cytotherapy 3: 365 – 375.

6. Tuyaerts, S., et al. (2002). Generation of large numbers of dendritic cells in a closed

system using cell factories. J. Immunol. Methods. 264: 135 – 151.

7. Hsu, F. J., et al. (1996). Vaccination of patients with B-cell lymphoma using autologous

antigen-pulsed dendritic cells. Nat. Med. 2: 52 – 58.

8. Nestle, F. O., et al. (1998). Vaccination of melanoma patients with peptide- or tumor

lysate-pulsed dendritic cells. Nat. Med. 4: 328 – 332.

9. Timmerman, J. M., and Levy, R. (1999). Dendritic cell vaccines for cancer

immunotherapy. Annu. Rev. Med.. 50: 507 – 529.

10. Marten, A., et al. (2002). Therapeutic vaccination against metastatic renal cell

carcinoma by autologous dendritic cells: preclinical results and outcome of a first

clinical phase I/II trial. Cancer Immunol. Immunother. 51: 637 – 644.

11. Krause, S. W., et al. (2002). The treatment of patients with disseminated malignant

melanoma by vaccination with autologous cell hybrids of tumor cells and dendritic

cells. J. Immunother. 25: 421 – 428.

12. Sanderson, S., Frauwirth, K., and Shastri, N. (1995). Expression of endogenous

peptide-major histocompatibility complex class II complexes derived from invariant

chain-antigen fusion proteins. Proc. Natl. Acad. Sci. USA 92: 7217 – 7221.

13. Wu, T. C., et al. (1995). Engineering an intracellular pathway for major histo-

compatibility complex class II presentation of antigens. Proc. Natl. Acad. Sci. USA 92:

11671 – 11675.

14. Dietz, A. B., and Vuk-Pavlovic, S. (1998). High efficiency adenovirus-mediated gene

transfer to human dendritic cells. Blood 91: 392 – 398.

778

15. Arthur, J. F., et al. (1997). A comparison of gene transfer methods in human dendritic

cells. Cancer Gene Ther. 4: 17 – 25.

16. Ponnazhagan, S., Curiel, D. T., Shaw, D. R., Alvarez, R. D., and Siegal, G. P. (2001).

Adeno-associated virus for cancer gene therapy. Cancer Res. 61: 6313 – 6321.

17. Reeves, M. E., Royal, R. E., Lam, J. S., Rosenberg, S. A., and Hwu, P. (1996). Retroviral

transduction of human dendritic cells with a tumor-associated antigen gene. Cancer

Res. 56: 5672 – 5677.

18. De Veerman, M., et al. (1999). Retrovirally transduced bone marrow-derived dendritic

cells require CD4+ T cell help to elicit protective and therapeutic antitumor immunity.

J. Immunol. 162: 144 – 151.

19. Di Nicola, M., et al. (2003). Clinical protocol. Immunization of patients with malignant

melanoma with autologous CD34(+) cell-derived dendritic cells transduced ex vivo

with a recombinant replication-deficient vaccinia vector encoding the human

tyrosinase gene: a phase I trial. Hum. Gene Ther. 14: 1347 – 1360.

20. Di Nicola, M., et al. (1998). Gene transfer into human dendritic antigen-presenting

cells by vaccinia virus and adenovirus vectors. Cancer Gene Ther. 5: 350 – 356.

21. Chinnasamy, N., et al. (2000). Efficient gene transfer to human peripheral blood

monocyte-derived dendritic cells using human immunodeficiency virus type 1-based

lentiviral vectors. Hum. Gene Ther. 11: 1901 – 1909.

22. Gruber, A., Kan-Mitchell, J., Kuhen, K. L., Mukai, T., and Wong-Staal, F. (2000).

Dendritic cells transduced by multiply deleted HIV-1 vectors exhibit normal pheno-

types and functions and elicit an HIV-specific cytotoxic T-lymphocyte response in vitro.

Blood 96: 1327 – 1333.

23. Schroers, R., et al. (2000). Transduction of human PBMC-derived dendritic cells and

macrophages by an HIV-1-based lentiviral vector system. Mol. Ther. 1: 171 – 179.

24. Breckpot, K., et al. (2003). Lentivirally transduced dendritic cells as a tool for cancer

immunotherapy. J. Gene Med. 5: 654 – 667.

25. Dyall, J., Latouche, J. B., Schnell, S., and Sadelain, M. (2001). Lentivirus-transduced

human monocyte-derived dendritic cells efficiently stimulate antigen-specific cytotoxic

T lymphocytes. Blood 97: 114 – 121.

26. Esslinger, C., Romero, P., and MacDonald, H. R. (2002). Efficient transduction of

dendritic cells and induction of a T-cell response by third-generation lentivectors. Hum.

Gene Ther. 13: 1091 – 1100.

27. Firat, H., et al. (2002). Use of a lentiviral flap vector for induction of CTL immunity

against melanoma: perspectives for immunotherapy. J. Gene Med. 4: 38 – 45.

28. Zufferey, R., Nagy, D., Mandel, R. J., Naldini, L., and Trono, D. (1997). Multiply

attenuated lentiviral vector achieves efficient gene delivery in vivo. Nat. Biotechnol. 15:

871 – 875.

29. Dull, T., et al. (1998). A third-generation lentivirus vector with a conditional packaging

system. J. Virol. 72: 8463 – 8471.

30. Miyoshi, H., Blomer, U., Takahashi, M., Gage, F. H., and Verma, I. M. (1998).

Development of a self-inactivating lentivirus vector. J. Virol. 72: 8150 – 8157.

31. Zufferey, R., Donello, J. E., Trono, D., and Hope, T. J. (1999). Woodchuck hepatitis virus

posttranscriptional regulatory element enhances expression of transgenes delivered by

retroviral vectors. J. Virol. 73: 2886 – 2892.

32. Chinnasamy, D., et al. (2000). Lentiviral-mediated gene transfer into human

lymphocytes: role of HIV-1 accessory proteins. Blood 96: 1309 – 1316.

33. Follenzi, A., Ailles, L. E., Bakovic, S., Geuna, M., and Naldini, L. (2000). Gene transfer by

lentiviral vectors is limited by nuclear translocation and rescued by HIV-1 pol

sequences. Nat. Genet. 25: 217 – 222.

34. Zennou, V., et al. (2000). HIV-1 genome nuclear import is mediated by a central DNA

flap. Cell 101: 173 – 185.

35. VanTendeloo, V. F., et al. (1998). Nonviral transfection of distinct types of human

dendritic cells: high-efficiency gene transfer by electroporation into hematopoietic

progenitor- but not monocyte-derived dendritic cells. Gene Ther. 5: 700 – 707.

36. VanTendeloo, V. F., et al. (2001). Highly efficient gene delivery by mRNA electro-

poration in human hematopoietic cells: superiority to lipofection and passive pulsing of

mRNA and to electroporation of plasmid cDNA for tumor antigen loading of dendritic

cells. Blood 98: 49 – 56.

37. Nair, S. K., et al. (1998). Induction of primary carcinoembryonic antigen (CEA)-specific

cytotoxic T lymphocytes in vitro using human dendritic cells transfected with RNA. Nat.

Biotechnol. 16: 364 – 369.

38. Ponsaerts, P., et al. (2002). mRNA-electroporated mature dendritic cells retain trans-

gene expression, phenotypical properties and stimulatory capacity after cryopreserva-

tion. Leukemia 16: 1324 – 1330.

39. Lundqvist, A., et al. (2002). Nonviral and viral gene transfer into different subsets of

human dendritic cells yield comparable efficiency of transfection. J. Immunother. 25:

445 – 454.

40. VanMeirvenne, S., et al. (2002). Efficient genetic modification of murine dendritic cells

by electroporation with mRNA. Cancer Gene Ther. 9: 787 – 797.

41. Tuyaerts, S., et al. (2003). Induction of influenza matrix protein 1 and melanA-specific

T lymphocytes in vitro using mRNA-electroporated dendritic cells. Cancer Gene Ther.

10: 696 – 706.

42. Schultz, E. S., et al. (2000). A MAGE-A3 peptide presented by HLA-DP4 is recognized

on tumor cells by CD4+ cytolytic T lymphocytes. Cancer Res. 60: 6272 – 6275.

43. Schultz, E. S., et al. (2001). A MAGE-3 peptide recognized on HLA-B35 and HLA-A1 by

cytolytic T lymphocytes. Tissue Antigens. 57: 103 – 109.




44. Chassin, D., et al. (1999). Dendritic cells transfected with the nef genes of HIV-1

primary isolates specifically activate cytotoxic T lymphocytes from seropositive

subjects. Eur. J. Immunol. 29: 196 – 202.

45. Andrieu, M., et al. (2001). Downregulation of major histocompatibility class I on

human dendritic cells by HIV Nef impairs antigen presentation to HIV-specific CD8+ T

lymphocytes. AIDS Res. Hum. Retroviruses 17: 1365 – 1370.

46. Gallardo, H. F., Tan, C., Ory, D., and Sadelain, M. (1997). Recombinant retroviruses

pseudotyped with the vesicular stomatitis virus G glycoprotein mediate both stable

gene transfer and pseudotransduction in human peripheral blood lymphocytes. Blood

90: 952 – 957.

47. Liu, M. L., Winther, B. L., and Kay, M. A. (1996). Pseudotransduction of hepatocytes by

using concentrated pseudotyped vesicular stomatitis virus G glycoprotein (VSV-G)-

Moloney murine leukemia virus-derived retrovirus vectors: comparison of VSV-G and

amphotropic vectors for hepatic gene transfer. J. Virol. 70: 2497 – 2502.

48. Chalfie, M., Tu, Y., Euskirchen, G., Ward, W. W., and Prasher, D. C. (1994). Green

fluorescent protein as a marker for gene expression. Science 263: 802 – 805.

49. Corish, P., and Tyler-Smith, C. (1999). Attenuation of green fluorescent protein half-life

in mammalian cells. Protein Eng. 12: 1035 – 1040.



50. Schmitz, M. L., Bacher, S., and Dienz, O. (2003). NF-kappaB activation pathways

induced by T cell costimulation. FASEB J. 17: 2187 – 2193.

51. Rouard, H., et al. (2002). IL-12 secreting dendritic cells are required for optimum

activation of human secondary lymphoid tissue T cells. J. Immunother. 25: 324 – 333.

52. Hess, S. D., et al. (2003). Human CD4+ T cells present within the microenviron-

ment of human lung tumors are mobilized by the local and sustained release of

IL-12 to kill tumors in situ by indirect effects of IFN-gamma. J. Immunol. 170:

400 – 412.

53. Lee, S. W., Park, Y., Yoo, J. K., Choi, S. Y., and Sung, Y. C. (2003). Inhibition of TCR-

induced CD8 T cell death by IL-12: regulation of Fas ligand and cellular FLIP expression

and caspase activation by IL-12. J. Immunol. 170: 2456 – 2460.

54. Granucci, F., Vizzardelli, C., Virzi, E., Rescigno, M., and Ricciardi-Castagnoli, P. (2001).

Transcriptional reprogramming of dendritic cells by differentiation stimuli. Eur.

J. Immunol. 31: 2539 – 2546.

55. Breckpot, et al. (2004). Activation of monocytes via the CD14 receptor leads to the

enhanced lentiviral transduction of immature dendritic cells. Hum. Gene Ther. 15:

562 – 573.

56. Kuipers, et al. (2004). J. Leuk. Biol., (in press).

779

Side-by-Side Comparison of Lentivirally Transduced and mRNA-Electroporated Dendritic Cells: Implications for Cancer Immunotherapy Protocols

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