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Departments of Internal Medicine
*
I and
II, University Clinic Tübingen, Tübingen, Germany; and
Department of Molecular Virology, Max-Planck-Institute for Biochemistry, Martinsried, Germany
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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In light of vaccination approaches either against infectious diseases or cancer, the process of Ag loading to DCs is a key step (9, 10). For this purpose, DCs can be pulsed with synthetic peptides derived from the Ag of interest (11); however, the application of antigenic peptides is limited for use in patients who express a defined specific HLA haplotype. Moreover, using MHC class I-restricted peptides ignores the important role of MHC class II-restricted Th cells in initiating and maintaining an effective immune response.
As an alternative strategy not requiring previous knowledge of 1) the patient’s HLA type or 2) the relevant T cell peptide epitopes for inducing specific immune responses, the methodology of therapeutic gene transfer can be used. In this context, DCs can either be transfected with RNA or DNA coding for defined Ags or proteins or be transduced with recombinant viruses being used as vectors, such as retrovirus, lentivirus, HSV, or adenovirus (reviewed in Ref.12). However, any manipulation of DCs by an application of a viral vector requires a detailed characterization of the virus/DC interaction. One of the most important advantages using gene transfer approaches in this context is the generation of potent Ag-specific T cell responses that are directed against multiple epitopes (13).
During the last few years, the prototype paramyxovirus Sendai virus (SeV; murine parainfluenza virus type I) has emerged as a new gene transfer system (reviewed in Ref.14). Extraordinary features of SeV are a remarkably brief contact time that is necessary for cellular uptake (15, 16), a strong but adjustable expression of foreign genes (17), a cell cycle-independent transduction of target cells (18), and an exclusively cytoplasmic localized replication cycle without any risk for chromosomal integration. Furthermore, SeV has not been linked to any disease in humans (19). These favorable basic vector characteristics, together with the recent observation that SeV is able to replicate nonproductively in bone marrow-derived mouse DCs, inducing DC maturation (20), suggested SeV as an interesting candidate for gene transfer approaches into human-derived DCs.
Until now, no purposeful infection of human DCs by SeV has been reported. In contrast, MeV, which constitutes a paramyxovirus closely related to SeV, is known to convey an efficient infection of human-derived DCs, followed by DC maturation, and also to induce a severe impairment of DC functions (21, 22). These features clearly exclude MeV as a possible vector for genetic DC manipulation, whereas comparable data on a possible interference with DC functions in the course of SeV infections are still missing.
Here, we show that SeV is able to efficiently infect monocyte-derived human DCs (moDCs). However, SeV wild-type infection rendered moDC insensitive to LPS-induced stimulation, induced DC apoptosis, and severely impaired the allostimulatory activity of moDCs. Interestingly, using various recombinant SeV vectors devoid of single viral genes, we were able to identify the SeV matrix (M) protein as a key component in moDC functional impairment after viral infection. Consequently, use of M-deficient SeV vectors preserved the allostimulatory activity in SeV-infected moDCs despite an efficient expression of all other virally encoded genes.
These findings identify M-deficient SeV vectors as a highly potent new tool for the genetic manipulation of DCs and suggest the possibility to use SeV vectors for the generation of Ag-specific T cell responses being directed against multiple epitopes.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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A recombinant SeV coding for the reporter gene gfp by an insertion between the leader sequence and the start of the viral n gene in the wild-type genome was grown in 9-day-old embryonated chicken eggs as described previously (23, 24). The generation and amplification of single gene-deleted SeV particles SeV-
M-GFP, SeV-F-GFP, and SeV-HN-GFP was performed as described recently (25).
Cell isolation and DC generation from adherent PBMCs
Generation of moDCs was performed as described previously (26). In brief, PBMCs were isolated by Ficoll-Paque (Life Technologies) density gradient centrifugation of heparinized blood obtained from buffy coat preparations of healthy volunteers from the blood bank of the University Clinic Tübingen. Cells were seeded (1 x 107 cells/3 ml/well) into 6-well plates (Costar) in RP10 media (RPMI 1640 supplemented with 10% heat-inactivated FCS, 2 mM L-glutamine, 50 µM 2-ME, 100 U/ml penicillin, and 100 µg/ml streptomycin). After 2 h of incubation at 37°C, nonadherent cells were removed, and the adherent blood monocytes were cultured in RP10 medium supplemented with the following cytokines: human recombinant GM-CSF (Leukomax, 100 ng/ml; Novartis Pharmaceuticals), IL-4 (1000 IU/ml; Genzyme), and for mature DCs with TNF-
(10 ng/ml; Genzyme). The phenotype of DCs was analyzed by flow cytometry after 7 days of culture.
Infection of moDCs
moDCs were washed once with X-VIVO 20 media (BioWhittaker). Infection with the indicated viral particles was performed either at a multiplicity of infection (MOI) of 1 or 2 in a total volume of 300 µl of X-VIVO 20. Cells were washed twice with RP10 media and cultured in RP10 media with supplements (see above). In the indicated experiments, 2 µg/ml bacterial LPS from Salmonella typhimurium (Sigma-Aldrich) were added for the last 24 h of culturing.
Immunostaining
Cell staining was performed using FITC- or PE-conjugated mouse mAbs against CD86, CD40 (BD Pharmingen), CD80, HLA-DR, CD14 (Becton Dickinson), CD83 (Coulter-Immunotech), and CD1a (OKT6; Ortho Diagnostics). Appropriate mouse IgG isotypes were used as controls (Becton Dickinson). The samples were analyzed on a FACSCalibur (BD Biosciences).
Nuclear extracts
moDC pellets were washed in 1 ml of ice-cold buffer A (10 mM HEPES (pH 7.9), 1.5 mM MgCl, 10 mM KCl, 0.5 mM PMSF, and 1 mM DTT) and incubated for 10 min on ice in 1 ml of buffer A plus 0.4% Igepal CA-630 (Sigma-Aldrich). The cell membranes obtained were centrifuged at 750 x g for 5 min. Pellets were resuspended in 200 µl of buffer B (20 mM HEPES (pH 7.9), 0.42 M NaCl, 1.5 mM MgCl, 0.2 mM EDTA, 0.5 mM PMSF, and 1 mM DTT), and nuclei were mechanically lysed for 2 h at 4°C. Cell debris were pelleted for 15 min at 7500 x g, and supernatant was recovered and stored at –70°C until use. Proteinase inhibitors (aprotinin and leupeptin; Sigma-Aldrich) were added to the buffers just before use.
PAGE and Western blotting for detection of RelB protein
Protein concentration of nuclear extracts were determined using a bicinchoninic acid assay (Pierce). Twenty micrograms of total protein were separated on a 12% polyacrylamide gel, blotted on a polyvinylidene difluoride membrane, and probed with a polyclonal rabbit RelB Ab (C-19; Santa Cruz Biotechnology) and a monoclonal mouse c-Rel Ab (B-6; Santa Cruz Biotechnology). Bands were visualized by ECL staining (Amersham Biosciences).
Detection of apoptosis
After the indicated time periods, moDCs were washed and fixed with 4% buffered paraformaldehyde (Merck). Leakage of fragmented DNA from apoptotic nuclei was measured as described previously (27). Cell nuclei were prepared by lysing cells in hypotonic lysis buffer (0.1% sodium citrate, 0.1% Triton X-100, and 50 mg/ml propidium iodide) and immediately analyzed by flow cytometry (FACSCalibur). Nuclei containing hypodiploid DNA were considered to be apoptotic.
Mixed lymphocyte reaction
moDCs either infected or uninfected were cultured for 1 day. moDCs were washed, and different numbers were incubated with 105 allogeneic PBMCs in a flat 96-well plate for 5 days before the addition of BrdU was performed for the last 18 h at 37°C. Thereafter, labeled cells were harvested, and proliferation was assessed using an ELISA (Sigma-Aldrich), which allows quantitation of BrdU-labeled DNA. Experiments addressing a possible influence of IL-10 or IL-12 were performed in the presence of 1 µg/ml anti-human IL-10R or 10 ng/ml rIL-12, both from R&D Systems.
Cytokine analysis
Supernatants from infected, LPS-stimulated, or uninfected moDCs were frozen at –20°C and subsequently analyzed by ELISAs for contents of IL-6, IL-10, IL-12, or TNF- (all from Beckman Coulter), according to the manufacturer’s instructions.
Real-time quantitative RT-PCR
RNA was extracted using NucleoSpin RNAII (Macherey-Nagel); contaminating DNA was digested according to the manufacturer’s protocol, and the remaining RNA was dissolved in water and stored at –70°C. First-strand cDNA was prepared from total RNA by reverse transcription using Superscript RNase H– Reverse Transcriptase (Invitrogen Life Technologies) and oligomers dT16–18 (specific for mRNA species) or the primer ld55 (specific for the leader region of the SeV genome; 5'-TAGGGTCAAAGTATCCACCCTGAAGAG-3'). Gene expression was measured in the ABI Prism 7700 Sequence Detection System (Applied Biosystems). PCR primers (Biomers.net) were selected with Primer software (Applied Biosystems) to result in amplicons <450 bp to enhance efficiency of PCR amplification. The GFP primers were as follows: forward, 5'-TAAACGGCCACAAGTTCAGCGTGT-3'; reverse, 5'-TTACTTGACAGCTCGTCCATGCC-3'. Human tubulin primers were used as endogenous controls: forward, 5'-ATGCCAAGCGTGCCTTTGTTCACTGGTAC-3'; reverse, 5'-GAAATTCTGGGAGCATGACATGCTGCAG-3'. The resulting PCR products were cloned directly into the vector pCR 2.1-TOPO by using the TOPO TA Cloning kit (Invitrogen Life Technologies) and subsequently sequenced to confirm primer specificity. The specificity of each PCR product was confirmed by agarose gel electrophoresis and dissociation curve analysis. Amplification of specimens and serial dilutions of the amplicon standards was conducted in a total volume of 20 µl of SyberGreen Master Mix (Applied Biosystems) at optimized concentrations. Standard curves were generated for each gene, and the amplification was found to be 90–100% efficient. The threshold cycle (CT) at which the fluorescence passes the fixed threshold is inversely proportional with the starting amount of a specific RNA in the sample. Relative quantitation of gene expression was determined with the use of the comparative CT method as suggested by the manufacturer. All results are displayed relative to tubulin (differences between CT of GFP and tubulin (dCT)). Low dCT values denote a small difference between the signals of GFP and the housekeeping gene tubulin (i.e., high expression levels of GFP).
Statistical analysis
Two-way ANOVA with Bonferroni’s post hoc test was performed using GraphPad Prism version 4.00 (GraphPad). Significant difference was considered for p < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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A direct infection of moDCs by SeV has not yet been investigated. Therefore, SeV particles expressing 1) all viral genes (wild-type situation) together with the 2) GFP reporter gene (SeV-GFP) were used in different amounts for incubation with immature (GM-CSF- and IL-4-treated) and mature (GM-CSF-, IL-4-, and TNF--treated) moDCs. Twenty-four hours later, these moDCs were screened for a possible infection by fluorescence microscopy, and the proportion of infected cells was determined by FACS analysis. Interestingly, SeV-GFP particles were able to efficiently infect both immature as well as mature moDCs. Depending on the amount of incoming virus particles (MOI 1 or 2) the fraction of GFP-positive cells was higher in mature moDCs (56 or 82%) (Fig. 1, C and D) than in immature moDCs (47 or 58%) (Fig. 1, A and B). The increased number of GFP-positive cells using mature moDCs reflects a different level of susceptibility to SeV infection of immature and mature cells that cannot be explained by an increased level of particle endocytosis, because mature DCs are known to completely lose their endocytotic activity. To exclude an interference of LPS activation of moDCs and SeV infection, an additional experiment was performed by infecting LPS-stimulated moDCs (Fig. 1, E and F).
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Previous studies showed that viral infections may interfere with the phenotype or function of DCs. To determine the influence of SeV in this context, immature moDCs were infected with SeV-GFP (MOI 1), and FACS analysis of typical cell-surface markers was performed. As a result, some up-regulation of the surface markers CD80 and HLA-DR could be found, whereas a down-regulation of MHC class I molecules occurred (Fig. 2A). CD1a and CD86 revealed no relevant changes in their cell-surface expression. It is well known that the phenotype of DCs depends on the maturation status of these cells. A powerful mediator of DC maturation is bacterial LPS, which induces a rapid change of molecules displayed at the DC surface. Therefore, we next analyzed whether a SeV infection interferes with the capacity of moDCs to adequately respond to LPS. For this purpose, moDCs were first infected with SeV-GFP (MOI 1). Twenty-four hours later, moDC cultures were incubated with LPS for an additional 24-h time period, and surface markers were subsequently analyzed by FACS analysis. In contrast to noninfected moDCs (Fig. 2B, right column), SeV-infected moDCs did not show a characteristic LPS-induced maturation pattern (Fig. 2B, left column), represented by an inadequate increase in the expression of CD80, HLA-DR, CD40, CD83, CD86, and MHC class I molecules. These observations led to the assumption that SeV infections of DCs could severely impair the capacity of T cell stimulation.
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To this end, we tested moDC stimulatory activity to induce T cell proliferation in an allogeneic MLR. moDCs were infected with SeV-GFP and 24 h later were cocultured with allogeneic PBMCs for four additional days, followed by a 16-h pulse, after which the incorporation of BrdU was measured. Compared with uninfected moDCs, SeV-GFP infection severely impaired the T cell stimulatory capacity (Fig. 3). This functional impairment could be due to severe cellular alterations (as observed for the expression of MHC and costimulating molecules) generally induced in the course of SeV wild-type infections. This could be mediated by defined viral proteins interfering with a proper DC function (as it has been shown, for example, for HSV-1) (28).
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Recombinant viral particles, harboring deletions of single viral genes, can be used to investigate the contribution of the deleted genes to an observed biological phenomenon. In this context, single gene-deleted recombinant SeV particles can be used to test whether a specific SeV-encoded protein interferes with the function of infected moDCs. However, a direct comparison between different viral particles is only possible if these particles are able to infect moDCs with a similar efficacy. Thus, we first infected immature and mature moDCs with the following recombinant SeV particles (MOI 1 or MOI 2), analyzing the percentage of infected DCs by FACS analysis (Table I, DCs incubated with IL-4/GM-CSF or IL-4/GM-CSF/TNF-): SeV missing the gene for the viral attachment protein hemagglutinin-neuraminidase HN (SeV-HN-GFP), the fusion protein F (SeV-F-GFP), or the matrix protein M (SeV-M-GFP). Interestingly, taking the SDs into account, all single gene-deleted particles demonstrated comparable infection rates to the SeV wild-type setting (SeV-GFP), even when using LPS-stimulated moDCs, thereby permitting a direct comparison between the different SeV particles used.
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) known to play an important role in the Ag presentation and T cell stimulatory capacity of DCs. Interestingly, TNF- levels were increased in all supernatants of virally infected cultures in a similar manner (Fig. 4A, left). However, for IL-6 SeV-HN-GFP and SeV-M-GFP, infection led to an increased amount in the supernatant compared with SeV-GFP, SeV-F-GFP, or uninfected control cells (Fig. 4A, right).
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). In contrast to the SeV wild-type situation (SeV-GFP), SeV-F-GFP and SeV-M-GFP showed an increased release of both IL-6 and IL-12 (Fig. 4B). The amount of secreted IL-10 and TNF- revealed only minor differences between the differentially infected moDC cultures (Fig. 4B). However, compared with uninfected control cells, the secretion of IL-10 was elevated in all infected DC cultures, which could contribute to a reduced MLR activity, as shown in Fig. 3. Together, these observations implied a different reaction pattern of moDCs to the different SeV-derived particles used in this study. Therefore, an additional characterization of infected moDCs was performed.
Single gene-deleted SeV constructs revealed a reduced rate of apoptotic moDC cell death
A premature death of virally infected moDCs could be an additional reason of a reduced allostimulatory activity. We and others reported previously that SeV wild type is able to induce apoptosis in viral host cells (27, 29, 30). Therefore, we next investigated whether infected moDCs underwent apoptotic cell death. Mature moDCs were either infected with SeV-GFP or one of the single gene-deleted viral recombinants (MOI 2) and analyzed apoptotic cell death induction by FACS analysis at two different time points (48 and 72 h after infection). Compared with uninfected control moDCs, SeV-GFP infection (wild-type situation) led to a profound increase in the rate of apoptotic cells (4 vs 13% after 48 h and 14 vs 25% after 72 h) (Fig. 5A). Interestingly, the single gene-deleted construct SeV-M-GFP showed the lowest rate of apoptotic moDCs compared with all other virally infected cultures and differed only slightly from uninfected control cells (7 vs 4% after 48 h and 14 vs 14% after 72 h) (Fig. 5A). These results clearly show that gene deletions in the viral genome alter the response and fate of moDCs to SeV infection.
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To further investigate the contribution of single SeV proteins for an impairment of moDC function, mature moDCs were infected with either one of the different recombinant constructs (MOI 2) before their use in an alloreactive MLR. Surprisingly, SeV-M-GFP-infected moDCs induced a strong allostimulatory activity that nearly reached the activity of uninfected control moDCs (Fig. 6, A and B). Furthermore, the deletion of either the F or HN gene also led to a partial reconstitution, but not in the same manner as the deletion of M (Fig. 6, A and B). These results demonstrate that either one of the viral-encoded proteins, M, F, or HN, contributes to the functional impairment of virally infected moDCs; however, the deletion of the viral M gene led to an almost complete reconstitution of the T cell stimulatory ability of infected DCs.
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M-GFP-infected moDCs could be a differential regulation of cytokines such as IL-10 or IL-12. To explore a possible impact of these two cytokines in more detail, additional alloreactive MLR experiments were performed comparing the influence of moDC infection with full-length SeV-GFP (Fig. 6C, gray bars) with SeV-M-GFP (Fig. 6C, black bars). First, modulation by IL-10 secretion was blocked by adding an Ab against the IL-10 receptor (IL-10R), and in a second approach, IL-12 was added to the culture medium (IL-12). As a result, neither blocking the IL-10 receptor nor adding IL-12 to the culture medium was able to revert the observed impairment of the allostimulatory activity by full-length SeV-GFP. In contrast, as shown in Fig. 6A, infection with SeV-M-GFP particles (Fig. 6C, black bars) did not lead to a significant impairment of the allostimulatory activity compared with uninfected control cells (Fig. 6C, white bars). In conclusion, a deregulation of a single cytokine such as IL-10 or IL-12 seems not to be responsible for the observed effects.
From a virological point of view, the deletion of a single viral gene theoretically could alter the transcription of other virally encoded genes substantially. Therefore, we next compared the transcription of the virally encoded GFP gene in SeV-GFP- and SeV-M-GFP-infected moDCs by quantitative PCR analysis (Fig. 7). GFP-specific signals were related to tubulin as an internal control (dCT). Because the dCT value is inversely proportional to the number of specific mRNA within a given sample, high dCT values represent less copy numbers of GFP compared with tubulin; vice versa, low dCT values represent high detection levels of specific RNA species compared with tubulin. As a result, mRNA of GFP is slightly enhanced in SeV-M-GFP-infected moDCs (Fig. 7, filled bar on the left) compared with SeV-GFP-infected cells (Fig. 7, open bar on the left), whereas the number of viral genomes is clearly higher in SeV-GFP-infected cells (Fig. 7, open bar on the right) compared with SeV-M-GFP-infected moDCs (Fig. 7, filled bar on the right). In conclusion, although the influence of the different number of viral genomes within infected moDCs is unclear, we could not detect a substantial difference in the transcription rate of GFP within SeV-GFP- and SeV-M-GFP-infected moDCs.
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B family of transcription factors are important for the differentiation and function of DCs (31, 32, 33, 34). Therefore, we analyzed SeV-infected moDCs as described previously (35, 36) but found that the nuclear localization of the RelB and c-Rel proteins was not affected in the infected DC populations (Fig. 8), suggesting that the observed impairment after SeV infection is not mediated by inhibition of NF-B signaling pathways.
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M-GFP 24 h after infection (MOI 2) of mature moDCs. Consistent with the changes in moDC function (Fig. 6), a substantial increase in the expression of CD1a, CD83, CD86, and MHC class I molecules could be found in SeV-M-GFP-infected moDCs but not in SeV-GFP (Fig. 9). Compared with other deletion mutants, SeV-M-GFP-infected moDCs had a higher expression of CD86, HLA-DR, and HLA class I molecules (Table II), thus further explaining the results obtained in the MLR assay.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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An infection of DCs has been reported for three other SeV-related members of the paramyxovirus family: parainfluenzavirus type 3 (38), respiratory syncytial virus (RSV) (39), and MeV (7, 8). Notably, all of these SeV-related viruses do induce a maturation of virally infected DCs, a premature apoptotic death of DCs, and an interference with the capacity of allogeneic T cell stimulation (7, 38, 39, 40). The underlying mechanisms for these phenomena in parainfluenzavirus type 3- or RSV-infected DCs have not yet been revealed in detail. For MeV, it has been postulated that apoptosis induction in DCs results from Fas-mediated signaling (41), and the functional impairments in the allostimulatory properties were associated with an inhibition of IL-12 production by infected DCs (42) or a TRAIL-mediated T cell depletion (43). Interestingly, the MeV surface glycoproteins hemagglutinin (H) and fusion (F) proteins, which both are synthesized and presented by infected DCs, might play a decisive role in the interference with DC functions by inducing fusion events between DCs and T cells or by inhibiting T cell proliferation through surface contact-dependent negative signaling (44).
Our results for SeV infection of moDCs now demonstrate that the viral glyoproteins HN and F as well as the matrix protein M are key components in the observed functional impairment of DCs. In a situation in which all of these viral proteins were present (SeV-GFP infection of DCs; wild-type situation), a strong impairment of the allostimulatory DC properties could be observed (Fig. 3), together with a decrease in the production of IL-12 (Fig. 4B). For MeV infection, fusion events between DCs themselves or between DCs and surrounding T cells triggered by the presence of the viral proteins H and F on the DC surface have been suggested as one major mechanism for the interference with proper DC functions (8). In contrast to these observations, in our setting, we could exclude the involvement of fusion events by choosing culture conditions that definitively excluded any activation of SeV F proteins due to a lack of trypsin supplementation in the culture media.
In several experiments, we could detect a weaker GFP signal in SeV-M-GFP-infected moDCs compared with all other viral particles. The explanation of this phenomenon remains elusive because we could not detect a reduced infection rate (Table I) nor a reduced transcription efficacy of GFP within SeV-M-GFP-infected moDCs (Fig. 7). This issue will have to be addressed in an additional study focusing on the contribution of the M protein to viral transcription and replication efficacies in detail, because it has been investigated previously for the related paramyxovirus rabies in a cell type different to moDCs (45).
Nevertheless, both surface proteins HN and F contributed to the reduced allostimulatory activity, which could be demonstrated by a partial functional improvement in MLR assays by using recombinant SeV that is either genetically devoid of HN (SeV-HN-GFP) or F (SeV-F-GFP). Until now, the adverse affect of F and HN on the stimulatory capacity of DCs has not been elucidated. For MeV, an alteration in the cytokine secretion profile of infected DCs, such as a decrease in IL-12 or an increase in IL-10 secretion, is thought to skew T cell responses (42). In our study, a decrease in IL-12 secretion was most prominent after infection of moDCs with SeV particles resembling the wild-type situation or being devoid of the viral HN protein. No difference could be found between either SeV-F-GFP- or SeV-M-GFP-infected DCs. Concerning IL-10 secretion, the amount of cytokine release into the supernatant was found to be similar for all different viral particles. Moreover, adding IL-10R Abs or IL-12 to the culture medium, we could not detect a significant reversion of the full-length SeV-mediated impairment in the MLR activity. Together, we could not identify single cytokines playing a major role in this process.
Despite a proven influence of HN and/or F on the function of moDCs, the use of M-deficient recombinant SeV particles led to a nearly complete reversion of the allostimulatory capacity of infected DCs compared with uninfected control cells. Therefore, the contribution of HN or F to the functional impairment of SeV-infected DCs could be neglected in settings in which M proteins were not synthesized in DCs. M is considered to be the central organizer of SeV morphogenesis, linking the ribonucleoprotein complex with the surface glycoproteins. Viral assembly takes place on internal cytoplasmic membranes (46), and M is able to bind independently to either the F or the HN glycoprotein (47). Intracellularly, M directly interacts with tubulin and microtubules, and recent work even suggested that the interaction between M and tubulin might have an important role in the regulation of SeV transcription (48). Interestingly, the role of tubulin and the microtubule network in DCs has come into focus recently in the context of DC-activating pattern recognition receptors: TLR2 and TLR4 could not be located on the surface but inside DCs in colocalization with -tubulin, indicating that microtubules serve as transport tracks for TLR vesicles (49). These results also suggested that an intracellular stimulation of TLR2 or TLR4 leads to an activation and maturation of DCs. Depolymerization of the microtubule network disrupted the intracellular expression of TLRs and profoundly inhibited IL-12 production (49). Taking these observations into account, it is tempting to speculate that an intracellular colocalization of M and TLRs could lead to a functional impairment of DCs that can be overcome by using M-deficient SeV particles. For SeV, an interaction with pattern recognition receptors has not yet been described, but for other paramyxoviruses, an interaction of the RSV fusion protein with TLR4 (50) or the MeV H protein with TLR2 (44) has already been found.
Finally, for future gene transfer applications of DCs (51), this work has several important implications. First, SeV vectors can be used for an extraordinary efficient infection of human moDCs. Second, impairment of the allostimulatory capacity of DCs as induced by SeV wild-type infection can be efficiently overcome by using gene-deleted (preferentially M gene-deleted) recombinant SeV vectors. Third, the observation that M gene-deleted recombinant SeV particles did not lead to a profound alteration of DC functions in MLR assays qualifies SeV-M vectors as new, interesting candidates for genetic DC manipulation. In addition to the observations made in this study, SeV-M vectors exhibit the benefit that no viral particles can be released from infected cells (52), which is an important safety issue for possible clinical applications of this vector system in the future.
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Acknowledgments |
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Disclosures |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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Footnotes |
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1 This work was supported by grants from the Deutsche Forschungsgemeinschaft (BI 669/3-2 and BI 669/3-6), the European Commission (FP5, QLK2-CT-2002-01722), and the research program "fortüne" of the medical faculty at the University Clinic Tübingen (fortüne F1281330). 
2 S.A. and M.B. contributed equally to this work.
3 U.M.L. and P.B. share senior authorship.
4 Address correspondence and reprint requests to Dr. Peter Brossart, University Clinic Tübingen, Otfried-Mueller-Strasse 10, D-72076 Tübingen, Germany. E-mail address: Peter.brossart{at}med.uni-tuebingen.de
5 Abbreviations used in this paper: DC, dendritic cell; MeV, measles virus; HSV-1, HSV type 1; HIV-1, HIV type 1; SeV-M-GFP, SeV-F-GFP, and SeV-HN-GFP, Sendai virus with deleted genes coding for matrix (M), fusion (F), and hemagglutinin-neuraminidase (HN) proteins; moDC, monocyte-derived human DC; MOI, multiplicity of infection; CT, threshold cycle; dCT, differences between CT of GFP and tubulin; RSV, respiratory syncytial virus.
Received for publication July 16, 2004. Accepted for publication July 11, 2005.
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References |
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B2 is a negative regulator of dendritic cell function. J. Immunol. 172:752.-756.
B and octamer factors is a hallmark of the generation and maturation of dendritic cells. Blood 95:277.-285. B dependent: coordinate regulation of MHC, co-stimulatory molecules and cytokines. Int. Immunol. 13:675.-683. 
. J. Immunol. 169:1228.-1235.
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), infected with SeV-GFP (MOI 2) 24 h before the addition of PBMCs (SeV-GFP; 
), or incubated with medium alone (no PBMNC; 
). Successful infection was confirmed by GFP expression (81% of GFP-positive cells in FACS analysis). The mean values of OD from the colorimetric evaluation of triplicate experiments and single SDs are shown. Significant differences (p < 0.05) between the assays using SeV-GFP-infected DCs compared with uninfected DCs (no infection; *) or with the control setting without addition of PBMCs (no PBMNC; ++) are indicated.
) or 72 h (B, 
), SeV-
), SeV-
