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Early Adenoviral Gene Expression Mediates Immunosuppression by Transduced Dendritic Cell (DC): Implications for Immunotherapy Using Genetically Modified DC¹

Andrea Tuettenberg^*, Helmut Jonuleit^*, Thomas Tüting^*,, Jürgen Brück^*, Volker Biermann, Stefan Kochanek^,, Jürgen Knop^* and Alexander H. Enk^2,*

^* Department of Dermatology, J. Gutenberg-University, Mainz, Germany; Department of Dermatology, Bonn, Germany; Center for Molecular Medicine, Köln, Germany; and Division of Gene Therapy, University of Ulm, Ulm, Germany

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Long-lasting, high-level gene expression in the absence of a toxic or inflammatory response to viral Ags is necessary for the successful application of genetically modified dendritic cell (DC). We previously demonstrated that efficient transduction of mature DC using E1E3 adenoviruses suppressed their stimulatory capacity for T cells. The current study was designed to investigate in more detail the suppressive effect of Ad-DC. We demonstrate that immunosuppression is not mediated by alterations in the T cell phenotype or cytokine profiles released by stimulated T cells. Also DC phenotypes are not affected. However, we demonstrate a cell cycle arrest of the T cell population stimulated by adenovirally transduced DC. Surprisingly, only freshly transduced DC are perturbed in their stimulatory capacity. Experiments using cycloheximide to block early intracellular viral gene expression showed that viral genes expressed in DC are responsible for this transient immunosuppression. In agreement with these findings, high-capacity (gutless) Ad-vectors that differ in viral gene expression from conventional E1E3 adenovirus are suitable for an efficient transduction of human DC. DC transduced with gutless Ad-vectors showed a high allostimulatory capacity for CD4⁺ and CD8⁺ T cells. Thus, the immunosuppressive effect of E1E3 Ad-transduced mature DC seems to be the result of early viral gene expression in DC that can be prevented using gutless Ad-vectors for transduction. These results have important implications for the use of genetically modified DC for therapeutic application.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Dendritic cells (DC)³ are professional APC capable of stimulating primary T cell-mediated immune responses (1, 2). Immunization with Ag-bearing DC efficiently primes CD4⁺ and CD8⁺ T cells resulting in a protective immunity against tumors and infectious agents (3, 4, 5, 6, 7). As Ag-loaded DC seem to be ideal vehicles for the immunotherapy of human tumor patients, a number of laboratories are currently investigating different loading strategies for cultured DC (3, 4, 5, 6, 7, 8, 9, 10, 11, 12). Direct transduction of immunostimulatory DC with DNA coding for tumor Ags has several advantages when compared with loading of DC with peptides and proteins including sustained intracellular expression of entire protein Ags and concurrent processing of multiple peptide epitopes on both MHC class I and II molecules. In recent studies, recombinant replication-deficient E1-deleted adenoviral vectors have been used to achieve strong gene expression in cultured murine or human DC (7, 9, 10, 11, 12). In mice, adenovirally transduced DC (Ad-DC) are capable of inducing tumor Ag-specific CTL in vivo associated with protection against tumor challenge (7, 9, 10, 11). In contrast, our previous results showed that efficient transduction of monocyte-derived mature CD83⁺ DC using recombinant, replication-deficient adenovirus suppressed their T cell stimulatory capacity (13). The effect was dependent on the viral dose used for transduction and correlated with transduction efficiency. Transwell experiments suggested that direct cell contact is important to suppress T cell proliferation. These findings are of significance when using Ad-DC for genetic immunization, as DC to T cell interactions determine the quality of the resulting immune response. Therefore, we analyzed in more detail the mechanisms responsible for the suppressed stimulatory capacity of Ad-DC.

Gutless adenoviral vectors (also called high capacity or helper-dependent) have some advantages over earlier generation Ad-vectors lacking the E1 domain. All viral coding sequences are deleted from the vector genome. Thus, viral proteins are not expressed by the vector resulting in reduced toxicity and reduction of unexpected adverse events. As the size and nature of the vector genome in Ad-vectors may have significant functional consequences following gene transfer we analyzed whether the use of gutless Ad-vectors for transduction of human DC helped to circumvent suppression of T cell proliferation.

We demonstrate that immunosuppression is not mediated by alterations in the T cell phenotype or cytokine profiles released by stimulated T cells. Also DC phenotypes are not affected. However, we demonstrate a cell cycle arrest of the T cell population stimulated by Ad-DC. Interestingly, the reduced allostimulatory capacity only occured when DC were used within 24 h after transduction for stimulation of T cells, whereas DC used after 72 h induced normal T cell proliferation. Reduced allostimulatory capacity of DC was not detectable after blocking of viral gene expression in the transduced cell population using cycloheximide. DC transduced with gutless Ad-vectors devoid of all viral coding regions also did not show any impaired T cell stimulatory capacity. Thus, the impaired stimulatory capacity of E1E3 Ad-transduced mature DC seems to be the result of early viral gene and protein expression in transduced DC that can be prevented using gutless Ad-vectors for transduction. These results have important implications that should be taken into account when using genetically modified DC for therapeutic application.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Culture media and cytokines

X-VIVO-15 supplemented with 1% autologous plasma was used for the generation of mature CD83⁺ DC. Allogeneic MLR and alloreactive T cell lines were cultured in X-VIVO 15 (all media from BioWhittaker Europe, Verviers, Belgium) and expanded using X-VIVO 15 supplemented with 10 U/ml IL-2 (Chiron/Behring, Marburg, Germany).

Final concentrations of human recombinant cytokines used in this study were: GM-CSF 800 U/ml (Leukomax; Novartis Pharmaceuticals, Basel, Switzerland), 1000 U/ml IL-4 and IL-6, IL-1, and 10 ng/ml TNF- (all from Strathmann Biotech, Hannover, Germany), and PGE₂ (Minprostin; Pharmacia-Upjohn, Heppenheim, Germany) 1 µg/ml.

Antibodies

The following Abs were used for immunostaining: mouse IgG: CD80 (MAB104), CD86 (IT2.2), CD83 (HB15A) (Coulter/Immunotech, Hamburg, Germany); mouse and rat specific isotypes (Coulter/Immunotech). Conjugated secondary reagents: FITC-conjugated goat-anti-mouse-IgG, PE-conjugated goat-anti-rat IgG (Jackson Immunoresearch Laboratories, West Grove, PA). Staining of immunomagnetically sorted T cells was performed with: FITC- or PE-conjugated CD4, CD25, CD28, CD40-L, CD69, CD70, CD95, CTLA-4 (Immunotech), ICOS (kindly provided by Dr. R. Kroczek, Robert Koch Institute, Berlin, Germany), and FITC- and PE-conjugated mouse-IgG isotypes (Immunotech).

Generation of mature CD83⁺ DC

DC were generated from PBMC obtained from leukapheresis products as previously described (14). Briefly, PBMC were incubated in 6-well plates (Corning/Costar, Bodenheim, Germany,) at 10⁷ cells/3 ml/well in X-VIVO-15 containing 1% autologous plasma for 30–40 min (37°C, 5% CO₂). Nonadherent cells were rinsed off and the remaining cells were further cultured in X-VIVO-15, 800 U/ml GM-CSF, and 1000 U/ml IL-4. Cells were fed on day 3 (+/-1 ml) with X-VIVO-15, 1600 U/ml GM-CSF, and 1000 U/ml IL-4. On day 5, nonadherent immature DC were rinsed off the plates and transferred to fresh 6-well plates in X-VIVO-15, 800 U/ml GM-CSF, and 1000 U/ml IL-4. On day 6, 10 ng/ml IL-1, 10 ng/ml TNF-, 1000 U/ml IL-6, and 1 µg/ml PGE₂ was added to the cultures for terminal maturation (15). Mature, nonadherent DC were harvested on day 7 or 8.

Recombinant adenoviruses, gutless adenoviral vectors, and transduction of DC

Recombinant E1-substituted, E3-deleted Ad-vectors encoding green fluorescent protein (Ad-EGFP), gp100 (Ad-gp100), or MelanA/MART-1 (Ad-MelanA) were constructed through Cre-lox mediated recombination (16), propagated on CRE8 or 293 cells and purified by cesium chloride gradient density centrifugation and subsequent dialysis according to standard protocols. The EGFP-expressing high-capacity AdFK7 vector (17) was rescued as previously described. Briefly, AdFK7 was coinfected in 293cre66 cells with helper virus Ad5LC8cluc at a multiplicity of infection of five. After complete cytopathic effect, the infected cells were harvested and lysed by freeze thaw cycles to release the virus. Aliquots of the crude vector lysate were serially passaged through 293cre66 cells to increase the titer. In both adenoviral vectors the expression of EGFP is driven by the CMV promoter. The yield of AdFK7 as well as of E1-substituted, E3-deleted conventional Ad-vector after cesium chloride density centrifugation was determined by slotblot assay (18) and was found to be 2 x 10¹⁰ infectious units per milliliter at a ratio of total particles to infectious particles of 20.

All adenoviral vectors were added to DC cultures on day 6, 6 h after stimulation with the cytokine mixture at a multiplicity of infection (MOI) of 300 (i.e., 3 x 10⁸ infectious particles/10⁶ DC/well). After overnight culture, DC were washed and used for T cell stimulation.

Preparation and stimulation of T cells

CD4⁺ or CD8⁺ T lymphocytes were sorted from PBMC using MACS beads (Miltenyi Biotec, Bergisch-Gladbach, Germany). A total of 2 x 10⁵ CD4⁺ or CD8⁺ T lymphocytes were stimulated with titrated numbers of allogeneic Ad-treated or untreated human DC in 96-well plates for 4 days plus 16 h in the presence of [³H]thymidine (37 kBq/well, Amersham, Braunschweig, Germany). To investigate the long term influence of Ad-DC on T cell proliferation, alloreactive T cell lines were established as follows: 1 x 10⁵ allogeneic, untreated, or Ad-treated DC (24 h after transduction) were used for stimulation of 1 x 10⁶ CD4⁺ or CD8⁺ T cells. Four to 5 days after primary stimulation, activated alloreactive T cells were expanded using IL-2 (10 U/ml). T cells were fed every other day and restimulated 14 days after primary stimulation using either Ad-treated or untreated DC. Restimulations were repeated every 8–10 days. Twenty-four to 48 h after stimulation activated T cells were analyzed by FACS staining concerning their phenotype and cytokine production induced.

Inhibition of protein synthesis or protein export of viral proteins

For the inhibition of viral de novo protein synthesis, DC were transduced with vector and treated either directly or three hours after viral incubation with cycloheximide (10 µg/ml) for 12 h to block viral gene and protein expression. Subsequently, DC treated with cycloheximide were analyzed by FACS staining and their stimulatory capacity was investigated by proliferation assays using alloreactive CD4⁺ or CD8⁺ T cells as mentioned above.

Flow cytometric analysis

For phenotyping of DC and T cells, cells were washed in cold PBS/human serume albumin (HSA) (5) 0.5% and incubated for 20 min at 4°C with each mAb (5 µg/ml). After washing with cold PBS/HSA the cells were incubated with FITC- and PE-conjugated second-step mAb for 20 min at 4°C, washed three times, and analyzed by flow cytometry (FACSCalibur, CellQuest software, BD Biosciences, Mountain View, CA), data being collected on 5,000–10,000 viable cells.

Intracellular FACS staining

For intracellular analysis of cytokine production anti-IFN--FITC, anti-IL-2-PE, anti-IL-4-PE, anti-IL-10-PE, and PE/FITC-conjugated isotypic mAb were used according to manufacturer’s instructions (BD PharMingen, San Diego, CA). Briefly, monensin was added 3–4 h after restimulation to the activated T cells. After overnight incubation, cells were collected, washed, fixed, permeabilized, and stained with 0.5 µg of the cytokine-specific mAb.

Cytokine ELISA and NO production

Commercially available ELISA specific for the human cytokines IFN-, IL-4, IL-2, IL-12p70, and IL-10 (BD PharMingen) were used as indicated by the manufacturer. Detection limits: 15.6 pg/ml. The production of NO was measured by formation of the stable decomposition product nitrite in cell-free supernatants (50 µl) after mixing with an equal volume of the Griess reagent (19).

Cell cycle analysis

Cell cycle analysis of each T cell population was performed by determination of the DNA content using propidium iodide staining. After stimulation with mature transduced or untransduced DC, CD4⁺ T cells were washed in PBS and fixed in 70% ethanol for 2 h at -20°C. After incubation, cells were treated with PBS containing 1% glucose, 2 mg/ml RNase, and 0.05 mg/ml propidium iodide (all from Sigma-Aldrich, Deisenhofen, Germany) for 30 min at room temperature. DNA content of stained cells was analyzed by FACScan (BD Biosciences).

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Transduction with recombinant adenovirus does not affect the phenotype and cytokine production of mature DC

The addition of purified E1/E3 deleted vector to the DC culture at an MOI of 300 resulted in a transduction efficiency of >50% without significant cytotoxic effects. The expression levels of surface molecules like CD80/86 and CD83 were similar to those of untransduced DC (Fig. 1). Ad-DC even showed higher levels of IL-12p70 secretion already 24 h after transduction and lasting up to 72 h, whereas IL-10 was not detectable (Table I). To exclude inhibitory effects provided by the presence of NO following adenoviral transduction of DC, cell-free supernatants of untreated and Ad-transduced DC were compared. NO production was not detectable following adenoviral transduction of DC (Table I). Thus, typical morphological and phenotypical characteristics of DC were not affected following adenoviral transduction and did not contribute to their reduced allostimulatory capacity.

View larger version (41K): [in this window] [in a new window]   FIGURE 1. Transduction with recombinant adenovirus does not affect the phenotype of mature DC. Mature DC were either transduced with a recombinant human Ad encoding for EGFP or left untreated. Twenty-four hours after transduction, DC were stained with PE-conjugated mAbs as indicated. Flow cytometric analysis was performed to simultaneously detect the expression of the indicated surface markers on green fluorescent DC. Results of a representative experiment of three are presented.

Next we analyzed the stimulatory capacity of Ad-DC compared with untransduced DC. Therefore, alloreactive CD4⁺ or CD8⁺ T cells were stimulated either with untransduced or with Ad-DC. In proliferation assays we could demonstrate that proliferation of CD4⁺ and CD8⁺ T cells was suppressed at high DC to T cell ratios using Ad-DC (Fig. 2). As stimulation of T cells using Ad-DC resulted in an impaired T cell proliferation, we next performed cell cycle analysis. T cells were either cocultured with untransduced or with Ad-DC (Fig. 3). After stimulation, the DNA content of each T cell population was determined by propidium iodide staining. As shown in Fig. 3, T cells stimulated using Ad-DC showed a significant arrest in the G1-phase (p 0.001, Student’s t test) with a significantly lower number of cells in the S-phase (p = 0.001) when compared with T cells stimulated with untransduced DC (G2 p = 0.07).

View larger version (14K): [in this window] [in a new window]   FIGURE 2. Adenoviral transduction of mature DC results in an impaired allostimulatory capacity for CD4+ and CD8+ T cells. Mature DC were transduced on day 6, using Ad-vectors encoding for EGFP at an MOI of 300 or left untreated. Allogeneic DC at day 7 were used as stimulators for CD4+ and CD8+ T cells at different DC to T cell ratios. Incorporation of [3H]thymidine was assessed for the final 16 h at the end of 5 days. Results of a representative experiment of seven are presented.

View larger version (20K): [in this window] [in a new window]   FIGURE 3. The inhibitory effect of Ad-DC on the proliferation of alloreactive T cells results in a cell cycle arrest of the stimulated T cell population. DC were transduced on day 6, using Ad-vectors encoding for EGFP at an MOI of 300 or left untreated. Allogeneic DC at day 7 were used as stimulators for CD4+ T cells at a DC to T cell ratio of 1:20. After stimulation, the DNA content of each T cell population was determined by propidium iodide staining. Results of a representative experiment of four are presented.

We had already demonstrated that stimulation with Ad-DC resulted in a suppressed T cell proliferation and a cell cycle arrest. To investigate whether the adenoviral transduction of DC also influenced the cytokine production of T cells, alloreactive CD4⁺, and CD8⁺ T cell lines were established. After every restimulation, T cells were analyzed with regard to their proliferation and the phenotype and cytokine production induced. Whereas proliferation of CD4⁺ and CD8⁺ T cells was suppressed at high DC to T cell ratios using Ad-DC (Fig. 2), we could not detect an altered cytokine production of CD4⁺ and CD8⁺ T cells when compared with T cells stimulated with untreated DC. The analysis of the cytokine profiles by intracellular FACS staining revealed an even increased population of IFN- and IL-2 producing T cells after restimulation with Ad-DC (Fig. 4A). IL-4 or IL-10 production was not detectable in any of the cultures.

View larger version (21K): [in this window] [in a new window]   FIGURE 4. Adenoviral transduction of DC has no effect on the cytokines and phenotype of alloreactive T cells. A, To investigate the cytokine profiles induced, CD4+ T cells were stimulated and restimulated with either untreated or Ad-DC. Three to 4 h after restimulation, monensin was added overnight. The next day, intracellular staining of the cytokines indicated was performed using PE/FITC-conjugated mAb. B, After stimulation, T cells were collected and stained with PE-/FITC-conjugated mAbs to determine their phenotype as indicated. Expression of surface molecules after stimulation with untreated DC or Ad-DC is demonstrated.

Adenoviral transduction of DC results in a transient suppression of their T cell stimulatory capacity

To investigate the kinetics of the altered immunostimulatory properties of mature DC after transduction, Ad-DC were used at different time points after transduction for the stimulation of alloreactive T cells. DC were transduced with Ad-EGFP, collected 24, 48, or 72 h after transduction and used for stimulation of alloreactive CD8⁺ or CD4⁺ T cells. Twenty-four hours after transduction, Ad-DC showed an inhibited stimulatory capacity compared with untransduced DC (Fig. 5A). Interestingly, this effect decreased with time after adenoviral transfection of DC. Forty-eight and 72 h after viral transduction, the allostimulatory capacity of Ad-DC was similar to the one of untreated cells independent of the DC to T cell ratios (Fig. 5A). However, DC still showed a strong transgene expression even 72 h after transduction (Fig. 5B). Taken together, these results demonstrate that the inhibitory effect of Ad-transduced DC on the proliferation of T cells is a transient phenomenon that decreases with time after transduction.

View larger version (31K): [in this window] [in a new window]   FIGURE 5. The inhibitory effect of adenovirus on the stimulatory capacity of DC is transient and decreases with time. A, Mature DC were transduced on day 6 using Ad-EGFP at an MOI of 300 or left untreated. Cultured cells were collected 24, 48, or 72 h after viral transduction and coincubated with allogeneic T cells at various DC to T cell ratios. Incorporation of [3H]thymidine was assessed for the final 16 h at the end of 5 days. B, Percentage of transduced DC was determined in parallel 24, 48, or 72 h after viral transduction. Results of a representative experiment of 10 are presented.

To evaluate whether the immunosuppressive effect of Ad-DC was mediated by viral genes and proteins expressed in Ad-transduced cells, DC were treated with the protein synthesis inhibitor cycloheximide immediately after transduction. When treating immature DC with cycloheximide as described before (20), we could efficiently inhibit the up-regulation of CD83, showing that cycloheximide efficiently blocks de novo protein synthesis (Fig. 6A). As shown in Fig. 6B, the addition of cycloheximide to Ad-DC restored their immunostimulatory capacity comparable to untransduced DC. The low allostimulatory capacity of cycloheximide-treated, Ad-transduced and untransduced DC can be explained by the inhibited up-regulation of the costimulatory molecules CD80 and CD86 and cytokines following the treatment with cycloheximide (data not shown). These findings indicate that viral gene expression and thus de novo viral protein synthesis may be necessary for the impaired allostimulatory capacity of Ad-DC.

View larger version (21K): [in this window] [in a new window]   FIGURE 6. The inhibitory effect of Ad-DC on the proliferation of alloreactive T cells is dependent on de novo synthesis of viral proteins. A, To block de novo protein synthesis, immature DC were treated shortly after stimulation with the cytokine mixture on day 6 with cycloheximide. Twenty-four to 36 h later, surface expression of CD83 was assessed. Fluorescence intensities of mature DC, immature DC and immature DC + mixture + cycloheximide are presented. B, Next, DC were treated after transduction with cycloheximide. Twenty-four hours after transduction, allogeneic DC were used for the stimulation of CD4+ T cells at different DC to T cell ratios. [3H]thymidine was added for the final 16 h at the end of 5 days. Results of a representative experiment of three are presented.

To determine whether an interaction of viral gene expression and the resulting expression of viral proteins with DC is responsible for their reduced allostimulatory capacity, we transduced DC using gutless Ad-vectors at an MOI of 300. In general, DC were not altered in their morphology, phenotype, viability and cytokine production when compared with untransduced or E1E3 Ad-transduced DC (data not shown). When using gutless Ad-transduced DC for the stimulation of alloreactive CD4⁺ and CD8⁺ T cells, no immunosuppressive effect was detectable (Fig. 7) compared with untransduced DC. These data demonstrate that interaction of viral gene expression and viral proteins with DC structures may be responsible for the immunosuppressive effects on DC-T cell cocultures.

View larger version (15K): [in this window] [in a new window]   FIGURE 7. Gutless Ad-vectors do not suppress the T cell stimulatory properties of DC. DC were transduced on day 6, using E1E3 or gutless adenoviral vectors encoding EGFP at an MOI of 300 or left untreated. DC at day 7 were used as stimulators for allogeneic CD4+ and CD8+ T cells at different DC to T cell ratios. Incorporation of [3H]thymidine was assessed for the final 16 h at the end of 5 days. Results of a representative experiment of seven are presented.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

In agreement with our data, it has been described that transduction of DC using viral systems like the measles virus, herpes simplex virus, rhinovirus, vaccinia virus, or influenza virus leads to DC with an inhibited T cell stimulatory ability (21, 22, 23, 24, 25, 26, 27). Distinct mechanisms including down-regulation of CD83 (22), massive alteration of viability (24, 25) as well as a modified cytokine pattern including inhibition of IL-12 and induction of IL-10 or TGF- synthesis have been described (23, 24, 26). All of these inhibitory effects could be explained by an inhibition of DC maturation by viral products. In contrast, we and other investigators demonstrated that recombinant Ad efficiently transduced mature DC without changing their morphology, phenotype, or maturation state (28). In addition, increased induction of apoptosis in virus-treated DC and T cells could not be detected when compared with untreated DC (26, 27).

Several investigators working with replication competent influenza virus, rhinovirus, and measles virus (25, 26, 29) demonstrated that virus-infected DC could no longer act as stimulatory cells in allogeneic MLR at high DC to T cell ratios. These viruses possess glycoproteins that are expressed on the surface of the infected cells and are able to transduce an inhibitory transmembrane signal to the responding T cell population. The interaction of T cells with these viral glycoproteins subsequently leads to an arrest of their proliferation. After transduction using Ad-vectors it is possible that viral proteins expressed in DC are responsible for the transmission of negative signals toward T cells. These viral proteins might induce a cell cycle arrest because the treatment of Ad-DC with the protein synthesis inhibitor cycloheximide prevented the inhibitory effects of virus transduction. In agreement with this hypothesis, gutless-Ad vectors, missing all viral coding sequences, did not suppress T cell stimulatory properties of transduced DC.

In agreement with others (30, 31), we could not detect alterations in the expression of activation markers or an impaired cytokine profile of T cells activated in the presence of Ad-DC. However, T cells stimulated with Ad-DC showed an inhibited proliferation and a cell cycle arrest in the G1 phase. Induction of a cell cycle arrest in responding T cells has been described is a well known viral escape mechanism for other viruses as well.

For example, it has been demonstrated, that stimulation of T cells with freshly transduced DC using measles virus also leads to a reduced proliferation of T cells and to an arrest in the G1 phase of the cell cycle. Furthermore, human primary airway cells showed an arrest in the G2-phase of cell cycle as a direct consequence of infection with a replication-defective E1 or E1E3 Ad vector (30). The massive reduction of T cell proliferation following stimulation with measles virus transduced DC is caused by release of viral particles and subsequent infection of the cocultured T cells (32, 33). Nevertheless, a release of viral particles and transfection of cocultured T cells could not be observed for Ad-vectors. The impaired cell cycle progression of airway cells after infection with Ad-vectors involved proteins encoded by E4 sequences that remained within the E1 or E1E3 Ad vector. In contrast, transduction of DC with gutless Ad-vectors, deficient of E4 proteins, maintained the T cell stimulatory properties of DC and prevented the cell cycle arrest of cocultured T cells. These results suggest that proteins encoded by E4 sequences and transiently produced by Ad-DC are responsible for the cell cycle arrest of responding T cells.

Other groups working with Ad-DC have not observed the reduced allostimulatory capacity of transduced DC. Roth et al. (34) compared the use of E1-deleted vs gutless adenoviral vectors in DC and observed increased T cell proliferation following transduction of DC with both types of virus without any difference between the adenoviral vectors. Several explanations are feasible. Ad-DC are often compared with immature DC (34, 35, 36) that show a much lower allostimulatory capacity than the mature DC used for stimulation in our study. Furthermore, many groups used Ad-transduced DC two days after transduction as stimulator APC (28, 34). However, the strong suppressive effect of Ad-transduction using E1-deleted vectors is transient and detectable only 24–48 h after transduction of DC. These data might explain the controversial observations.

Taken together, our findings are important for the use of Ad-DC in genetic immunization strategies and vaccine production. Despite their intact morphology and phenotype, Ad-DC not only failed to stimulate but even actively inhibited T cell proliferation and cell cycle progression by as yet undefined mechanisms. The fact that inhibitory signals of Ad are delivered early after Ad-transduction of DC resulting in a marked suppression of their stimulatory capacity indicates that genetically modified DC-vaccines should be applicated 48–72 h after transduction. At this time point, Ad-DC are able to induce normal T cell proliferation comparable to untransduced DC. Furthermore, the number of Ad-DC for vaccination has to be adapted to the delivery methods used. Intravenous, intranodal, or intradermally application (37) may lead to different, high, or low DC to T cell ratios in the draining lymph nodes resulting in either suppression or activation of immune responses within 24 h posttransduction. Yet, the optimal DC number that is necessary for a DC to T cell ratio that leads to activation and efficient induction of tumor-specific T cells even early after transduction still has to be defined in vivo.

Alternatively, gutless Ad-vectors, highly efficient to transduce human mature DC without alteration of their T cell stimulatory properties, can be used for therapeutic gene transfer. The findings that conventional Ad-vectors alone but not gutless Ad-vectors critically alter DC immune functions have important implications for the design of immunotherapy strategies using vector-based gene transfer to modulate immunity.

	Acknowledgments

 
We thank A. Kandemir, L. Paragnik, and P. Hölter for excellent technical assistance and K. Steinbrink for help with the cell cycle analysis and critical reading of the manuscript.

	Footnotes

 
¹ This work was supported by the German Science Foundation (Grant No. SFB-432 to A.E.).

² Address correspondence and reprint requests to: Dr. Alexander H. Enk, Department of Dermatology, J. Gutenberg-University, Langenbeckstrasse 1, D-55101 Mainz, Germany. E-mail address: enk{at}hautklinik.klinik.uni-mainz.de

³ Abbreviations used in this paper: DC; dendritic cell; Ad-DC; adenovirally transduced DC; EGFP, encoding green fluorescent protein; HAS, human serume albumine; MOI, multiplicity of infection.

Received for publication August 8, 2003. Accepted for publication November 25, 2003.

	References

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