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Journal of Virology, September 2000, p. 8003-8010, Vol. 74, No. 17
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

CD40 Ligand-Dependent Activation of Cytotoxic T Lymphocytes by Adeno-Associated Virus Vectors In Vivo: Role of Immature Dendritic Cells

Yi Zhang, Narendra Chirmule, Guang-ping Gao, and James Wilson*

Institute for Human Gene Therapy and Departments of Medicine and of Molecular and Cellular Engineering, University of Pennsylvania, and The Wistar Institute, Philadelphia, Pennsylvania 19104

Received 22 February 2000/Accepted 19 May 2000


    ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Recombinant adeno-associated virus type 2 (rAAV) is being explored as a vector for gene therapy because of its broad host range, good safety profile, and persistent transgene expression in vivo. However, accumulating evidence indicates that administration of AAV vector may initiate a detectable cellular and humoral immune response to its transduced neo-antigen in vivo. To elucidate the cellular basis of the AAV-mediated immune response, C57BL/6 mouse bone marrow-derived immature and mature dendritic cells (DCs) were infected with AAV encoding beta -galactosidase (AAV-lacZ) and adoptively transferred into mice that had received an intramuscular injection of AAV-lacZ 10 days earlier. Unexpectedly, C57BL/6 mice but not CD40 ligand-deficient (CD40L-/-) mice adoptively transferred with AAV-lacZ-infected immature DCs developed a beta -galactosidase-specific cytotoxic T-lymphocyte (CTL) response that markedly diminished AAV-lacZ-transduced gene expression in muscle fibers. In contrast, adoptive transfer of AAV-lacZ-infected mature DCs failed to elicit a similar CTL response in vivo. Our findings indicate, for the first time, that immature DCs may be able to elicit a CD40L-dependent T-cell immunity to markedly diminish AAV-lacZ transduced gene expression in vivo when a sufficient number of DCs capturing rAAV vector and/or its transduced gene products is recruited.


    INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

The ability of a vector and its transduced gene to persist is one of the important goals for gene therapy. Vectors that are highly efficient at delivering genes in a tissue-specific manner, without inducing strong cellular immune response, are of great interest for gene therapy. Adeno-associated virus type 2 (AAV) has become an attractive tool for gene therapy due to its broad host range, excellent safety profile, and durable transgene expression in infected hosts (7, 12, 13, 19, 20, 37). Intramuscular injection of AAV vectors does not stimulate a cellular immune response to highly expressed neoantigenic transgene products in immunocompetent mice (12, 19, 37), whereas other vector systems expressing the identical transgene, such as adenovirus (40) and naked DNA (36), do. These studies illustrate the role of the AAV vector in modulating (or avoiding) immune responses to the transgene through an unknown mechanism.

Accumulating evidence indicates that the lack of destructive cellular immunity by AAV vectors may depend on the transgene involved and the route of administration (8, 21, 22). In contrast to the long-term expression of beta -galactosidase (beta Gal) in muscles of mice, it has been found that inoculation of AAV encoding beta Gal (AAV-lacZ) into the brains of BALB/c mice induced beta Gal expression during the first 2 months and that this expression decreased gradually by 4 months. Moreover, repeated administration of AAV-lacZ vector into the brains of mice resulted in a loss of beta Gal expression in the original injection sites in 2 of 6 animals (21). Intramuscular injection of AAV vector encoding herpes simplex virus type 2 glycoproteins B and D (AAV-gB and AAV-gD) induces both humoral and cellular immune responses to these antigens (22). More recently, it has been demonstrated that C57BL/6 mice injected via the intraperitoneal, intravenous, or subcutaneous route with AAV encoding ovalbumin (AAV-ova) developed potent ovalbumin-specific cytotoxic T-lymphocyte (CTL) response as well as anti-ovalbumin antibodies. In contrast, mice intramuscularly injected with AAV-ova developed a humoral response to the virus and the transgene product but minimal ovalbumin-specific CTL (8). All these data challenge the claims that AAV vectors are nonimmunogenic. However, the cellular basis of AAV vector-mediated immune response in vivo remains to be elucidated.

Our previous study demonstrated that adoptive transfer of dendritic cells (DCs) infected with adenovirus (Ad)-lacZ vector leads to immune-mediated elimination of AAV-lacZ-transduced gene expression in muscle fibers in immune competent mice (17). This study underscored the critical role of vector-transduced DCs in initiating cellular immune responses. DCs are antigen-presenting cells that specialize in initiating T-cell immunity, including CTLs that kill virus-infected targets. DCs normally reside in tissues in an immature form with the capacity to capture antigen. After antigen capture, and in response to inflammatory stimuli, immature DCs switch to a T-cell-stimulatory mode to initiate cellular immunity (2, 4, 11, 23, 26, 28, 31, 34). However, it remains to be determined if immature DCs take up AAV vector and play a role in modulating the immune response to the AAV-carried transduced gene in vivo.

To investigate the mechanism of the AAV vector-mediated cellular immune response, purified immature and mature DCs that were generated from murine bone marrow (BM) hematopoietic progenitor cells (HPCs) (41, 42) were infected with either AAV-lacZ or Ad-lacZ and adoptively transferred into C57BL/6 and CD40 ligand-deficient (CD40L-/-) mice. We show that immature DCs can take up AAV-lacZ vector and initiate a CD40L-dependent T-cell immunity in vivo to the AAV-transduced gene in muscle fibers.


    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Animals. C57BL/6 mice were purchased from Jackson Laboratory (Bar Harbor, Maine). CD40L-/- mice were purchased from Jackson Laboratory and maintained in the Animals Facility of Wistar Institute. CD40L-/- mice have been backcrossed to the background of C57BL/6 mouse strain. In this study, 4- to 5-week-old mice were used.

Production and purification of rAAV. Recombinant AAV (rAAV) expressing lacZ (AAV-lacZ) or green fluorescent protein (GFP) (AAV-GFP) were generated by the 293 triple-transfection method. The cytomegalovirus promoter drives the expression of lacZ or GFP in these vectors. Briefly, for triple transfection of 293 cells, the cis-acting plasmid pAAVCMVLacZ was derived from psub201 and contains a lacZ minigene in place of the AAV rep and cap genes. The functions of rep and cap were provided by the trans-acting plasmid pTrans-600 trans (38). Ad helper functions were supplemented by pAdDelta F6, a plasmid construct carrying all essential Ad helper genes (38). Transfection was carried out using the standard calcium phosphate coprecipitation method (38).

rAAV vector preparations used in this study were purified by the CsCl gradient centrifugation process as described earlier (12). The genome titers of vector preparations were determined by the real-time quantitative PCR method, and the transducing titers of vector preparations were assayed on 84-31 cells, as described elsewhere (14).

Intramuscular injections. Mice were anesthetized with ketamine-xylazine (70 and 10 mg/kg of body weight, respectively). AAV-lacZ (1011 genomes/mouse) was injected into the tibialis anterior in a volume of 25 µl after a small incision was made to expose the muscle. The incision was closed with Vicryl suture. The muscle was harvested on day 28 after adoptive transfer of various DCs by placing the tissue on OCT embedding compound (Sakura Finetek U.S.A., Inc.), freezing it in nitrogen-cooled isopentane for 7 s, and transferring it to dry ice. Frozen sections were analyzed for beta Gal activity by 5-bromo-4-chloro-3-indolyl-beta -D-galactopyranoside (X-Gal) histochemistry as previously described (12, 17, 40). beta Gal-positive fibers were counted in cross-sections of muscle, and the number was calculated as a percentage using an image analyzer (Phase Three Imaging System; Microphot FXA, Nikon, and Mavigraph, Sony).

Generation of immature and mature DCs from murine BM HPCs. BM was obtained by aspiration from the femurs and tibias of 8- to 10-week-old female C57BL/6 mice as previously described (41, 42). BM mononuclear cells were separated by centrifugation on Histopaque-1077 (Sigma Chemical Co., St. Louis, Mo.). These cells were stained with a biotinylated anti-c-kit antibody (Pharmingen, San Diego, Calif.) and stained with streptavidin-conjugated microbeads (Miltenyi Biotech, Auburn, Calif.). c-kit+ HPCs were isolated using the magnetic cell-sorting system as instructed by the manufacturer (Miltenyi Biotech). The purity of these c-kit+ HPCs was consistently greater than 95% as revealed by an immunofluorescence analysis.

Purified c-kit+ HPCs were incubated as previously described (41, 42) for 6 days in Iscove's modified Dulbecco's medium (IMDM; Gibco, Rockville, Md.) supplemented with 10% fetal bovine serum, 5 × 10-5 M 2-mercaptoethanol, penicillin G (100 µ/ml), and streptomycin (100 µg/ml) in the presence of granulocyte-monocyte colony-stimulating factor (GM-CSF), stem cell factor (SCF), Flt-3 ligand (Flt-3L), and tumor necrosis factor alpha (TNF-alpha ). All the recombinant murine cytokines were purchased from R&D Systems, Inc., Minneapolis, Minn.). For preparation of immature DCs, the 6-day-old cultures were harvested and stained with anti-CD11c antibody-conjugated microbeads (Miltenyi Biotech) to magnetically sort CD11c+ immature DCs. The purity of the sorted immature DCs was consistently greater than 98% as analyzed by immunofluorescence staining. Mature DCs were generated from the purified CD11c+ immature DCs by stimulation with GM-CSF plus TNF-alpha for an additional 3 days.

Adoptive transfer of DNA viral vector-infected DCs. All recipient C57BL/6 mice and CD40L-/- mice were injected with AAV-lacZ in the left tibialis anterior on day 1. As described in Table 1, 5 × 105 DCs generated from C57BL/6 mice and infected with different vectors were subcutaneously injected into the right lower quadrant of the ventral abdominal wall on day 10. All DCs infected with various DNA viral vectors were completely washed with phosphate-buffered saline (PBS) more than eight times before adoptive transfer.

                              
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  		TABLE 1.   Overview of the experimental strategya

As shown in Table 1, DCs were suspended in 400 µl of serum-free IMDM and infected separately with AAV-lacZ (2 × 104 genomes/cell), AAV-GFP (2 × 104 genomes/cell), or Ad-lacZ (2 × 103 genomes/cell) at 37°C for 2 h and recultured in the presence of GM-CSF plus TNF-alpha for an additional 3 days to induce maturation. These DCs infected ex vivo with various DNA viral vectors were adoptively transferred into recipient mice (group 3 [Ad-lacZ-infected immature DCs], group 4 [AAV-lacZ-infected immature DCs], and group 6 [AAV-GFP-infected immature DCs]). In group 5, mature DCs were infected with AAV-lacZ and directly transferred into the recipient mice. Control mice were injected with PBS (group 1) or with mature DCs (group 2) or immature DCs (group 7) that were not infected by any viral vector. The left tibialis anterior was harvested 28 days later, after adoptive transfer of various DCs, cryosectioned, and stained for beta Gal activity. The abbreviations in Table 1 refer to the recipient mice adoptively transferred with or without various DCs infected with different DNA viral vectors unless otherwise indicated.

Immunofluorescence analysis. Immunofluorescence analyses were performed as previously described (41, 42). All the monoclonal antibodies (MAb) and other reagents for immunostaining were obtained from Pharmingen (San Diego, Calif.) unless otherwise indicated. In two-color analyses, the cultured immature DCs were stained with fluorescein isothiocynanate (FITC)-conjugated anti-CD86, anti-CD40, anti-Ia, and anti-major histocompatibility complex class I and phycoerythrin (PE)-conjugated anti-CD11c. In some experiments, the cultured cells were stained with PE-conjugated anti-CD86 and anti-Ia and FITC-conjugated anti-CD40. For intracellular staining to determine beta Gal expression, cells were fixed and permeabilized using the Cytofix/Cytoperm Plus kit (Pharmigen) and then subjected to biotinylated mouse anti-beta Gal and FITC-conjugated streptavidin staining. The instrument compensation was set in each experiment using single- and/or two-color stained samples.

T-cell assays. Lymphocytes were isolated from the spleens and regional lymph node of mice 28 days after adoptive transfer of various DCs and prepared for T-cell assays. CTL assays were performed as previously described (12, 17) by restimulating the single-cell suspension with beta Gal (20 µg/ml; Sigma) for 5 days at 5 × 106 cells/ml. These cells were assayed on MC57 target cells at different effector-to-target-cell ratios (starting at 6.25:1) in a 6-h 51Cr release assay. As target cells, MC57 cells were infected with Ad-lacZ (100 genomes/cell) for 16 h or a beta Gal-expressing cell line (established by transducing MC57 cells with pLj-lacZ retrovirus) was used (17). For cytokine enzyme-linked immunosorbent assays (ELISA), the lymphocytes (2 × 106 cells/ml) were cultured in the presence of interleukin-2 (IL-2) (50 U/ml; R&D Inc.) and restimulated with beta Gal (20 µg/ml) for 72 h. The supernatants were harvested and analyzed for the secretion of gamma interferon (IFN-gamma ) and IL-10 by using an ELISA kit as recommended by the manufacturer (Pharmingen).


    RESULTS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Interaction of AAV-lacZ vector ex vivo with murine BM-derived DCs. Culture of murine BM HPCs with GM-CSF, SCF, and TNF-alpha generated immature DCs with monocyte-like morphology by day 6 (Fig. 1A). These highly purified immature DCs, which were magnetically sorted using anti-CD11c antibody-conjugated microbeads, could differentiate into mature DCs, characterized by typical DC morphology (Fig. 1A) and increased expression of Ia, CD86, and CD40 (Fig. 1B), in response to GM-CSF plus TNF-alpha . These purified immature DCs were separately infected with Ad-lacZ (2 × 103 genomes/cell), AAV-lacZ (2 × 104 genomes/cell), or PBS (mock infection) at 37°C for 2 h and recultured in the presence of GM-CSF or GM-CSF plus TNF-alpha for an additional 3 days. Infection of immature DCs with Ad-lacZ moderately enhanced the expression of Ia, CD86, and CD40 antigens in the presence of either GM-CSF (Fig. 1C, column 1) or GM-CSF plus TNF-alpha (Fig. 1C, column 2), suggesting that Ad-lacZ may be able to augment the maturation of BM-derived immature DCs. However, infection of immature DCs with AAV-lacZ failed to affect the expression of Ia, Cd86, and CD40 on immature DCs under the same conditions (Fig. 1C). These data indicate that the inability of AAV vectors to induce the maturation of immature DCs may contribute to the mechanism of attenuated cellular responses by these vectors.



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  		FIG. 1.   Interaction of AAV-lacZ with BM-derived immature DCs. (A) Immature DCs were generated from BM HPCs stimulated with GM-CSF, SCF, Flt-3L, plus TNF-alpha on day 6 and purified by magnetic-cell sorting using anti-CD11c-conjugated microbeads. CD11c+ immature DCs were induced to differentiate into mature DCs with GM-CSF plus TNF-alpha for an additional 3 days. Giemsa staining shows the typical morphology of immature and mature DCs. Magnification, ×304. (B to D) These highly purified immature DCs and their mature counterparts were stained with FITC-conjugated anti-CD40, anti-CD86, and anti-Ia and subjected to flow cytometry analyses (B). CD11c+ immature DCs were suspended in 400 µl of serum-free IMDM, infected with Ad-lacZ and AAV-lacZ for 2 h at an infectious activity of 2 × 103 and 2 × 104 genomes/cell, respectively, and recultured in the presence of GM-CSF or GM-CSF plus TNF-alpha for an additional 3 days. The cells were stained with PE-conjugated anti-Ia and anti-CD86 and FITC-conjugated anti-CD40 and subjected to flow cytometry analyses (C). Immature and mature DCs were infected separately with Ad-lacZ, AAV-lacZ, or AAV-lacZ plus wild-type Ad (100 genomes/cell), as indicated in panel C, cultured in the presence of GM-CSF plus TNF-alpha for 3 days, and subjected to intracellular staining with anti-beta Gal MAb and flow cytometry analyses (D). The x-axes of the histograms in panels B to D show a log scale of the fluorescence intensity of the tested antigens, and the y axes show a linear scale of the number of cells with a given fluorescence intensity. (E) The infected immature DCs were cytocentrifuged for X-Gal histochemistry staining. Magnification, ×122. The blue color indicates beta Gal-positive cells.

To further investigate the role of DCs in AAV gene transfer, immature and mature DCs were separately infected with AAV-lacZ, Ad-lacZ, AAV-lacZ plus wild-type Ad or PBS (mock infection) for 72 h and then subjected to anti-beta Gal MAb staining for flow cytometry analyses. In contrast to the efficient transduction of Ad-lacZ in immature DCs (Fig. 1D, top right panel), infection of immature DCs with AAV-lacZ alone induced only minimal beta Gal expression (top left panel). Coinfection of immature DCs with AAV-lacZ and wild-type Ad substantially enhanced lacZ gene expression in the immature DCs (top left panel), and this was further confirmed by X-Gal histochemistry staining (Fig. 1E). However, infection of mature DCs with either AAV-lacZ or AAV-lacZ plus wild-type Ad failed to induce detectable beta Gal expression (Fig. 1D, bottom left panel). Interestingly, beta Gal expression was also significantly reduced in Ad-lacZ infected mature DCs (bottom right panel). These results suggest that immature DCs can take up AAV-lacZ and that the maturation of these cells may markedly reduce their susceptibility to the infection of AAV-lacZ.

Adoptive transfer of AAV-lacZ-infected immature DCs can significantly diminish AAV-lacZ-transduced muscle fibers in C57BL/6 mice. Adoptive transfer of various DCs infected with AAV and Ad vectors was performed to investigate the role of immature DCs in modulating the immune response in vivo (Table 1). In the first set of experiments, immature DCs were infected with either AAV-lacZ or Ad-lacZ. After being cultured in vitro in the presence of GM-CSF plus TNF-alpha for 3 days, these cells were adoptively transferred into C57BL/6 mice that had been injected with AAV-lacZ vector 10 days before (Table 1 and Fig. 2A). The mice were sacrificed 28 days later, and their muscles were isolated for X-Gal histochemistry staining. Consistent with our previous observation (12, 17), intramuscular injection of AAV-lacZ vector resulted in long-term expression of beta Gal in muscle fibers (Fig. 2A), with no activation of CTL in response to beta Gal antigen (Fig. 3A). Adoptive transfer of Ad-lacZ-infected immature DCs, but not mock-infected ones, completely eliminated AAV-lacZ-transduced muscle fibers with significant infiltration of inflammatory cells (Fig. 2). This elimination was associated with activation of Ad- and beta Gal-specific CTLs (Fig. 3A). Unexpectedly, adoptive transfer of AAV-lacZ-infected immature DCs markedly diminished beta Gal expression in AAV-lacZ-transduced muscle fibers (Fig. 2A and B) and was associated with a modest beta Gal-specific CTL response (Fig. 3A and B). In contrast, adoptive transfer of AAV-GFP-infected immature DCs failed to eliminate AAV-lacZ-transduced muscle fibers (Fig. 2C, panel e). These observations suggest that the immune response initiated by AAV-lacZ-infected immature DCs was specific for the lacZ gene product and not for the capsid protein of the vector.


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  		FIG. 2.   Impact of adoptive transfer of various DCs infected with AAV-lacZ and Ad-lacZ on AAV-lacZ-transduced muscle fibers of wild-type C57BL/6 and CD40L-/- mice. All mice were injected with AAV-lacZ in the left tibialis anterior on day 1. Then 5 × 105 of various DCs, generated from C57BL/6 mice and infected with different vectors as described in Materials and Methods and Table 1, were subcutaneously injected in the right lower quadrant of the ventral abdominal wall on day 10. (A and C) Representative macrographs of X-Gal histochemical stains of the left tibialis anterior that were harvested on day 28 after adoptive transfer of various DCs are presented. (B) beta Gal-positive fibers were counted in cross-sections of muscle, and the number was calculated as a percentage. The data represent the mean and standard deviation of the percentage of beta Gal-positive fibers from at least four mice. * P < 0.05 compared with the mice without adoptive transfer of virus-infected DCs. The abbreviation of each group is indicated in Table 1 and shows the recipient animals adoptively transferred with various DCs infected with distinct vectors. Magnification, ×45 (A) or ×40 (C).


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  		FIG. 3.   Induction of the CTL response to beta Gal in C57BL/6 mice after adoptive transfer of AAV-lacZ-infected DCs. C57BL/6 (A and B) or CD40L-/- (C) mice were first intramuscularly injected with AAV-lacZ and then given various DCs, infected with AAV-lacZ or Ad-lacZ, by adoptive transfer (Table 1). The lymphocytes isolated from the spleen were restimulated with Ad-lacZ in vitro for 5 days and analyzed for specific lysis using either Ad-lacZ-infected MC 57 (A and C) or pLj-beta Gal (B) transgenic cells as syngeneic target cells. All recipient mice were injected with AAV-lacZ. Results of one representative experiment of at least two are shown.

We have previously reported that AAV-lacZ-transduced splenic mature DCs failed to diminish AAV-lacZ transgene expression in muscle fibers (17). Mature and immature DCs differ in phenotype, migration patterns, T-cell stimulatory function, and antigen uptake and processing capacity (2, 4, 11, 23, 26, 28, 34, 41, 42). The next set of experiments was undertaken to compare the effects of immature and mature DCs. Adoptive transfer of AAV-lacZ-infected mature DCs could not diminish AAV-lacZ-transduced muscle fibers, whereas AAV-lacZ-infected immature DCs did so efficiently (Fig. 2B and C). Consequently, AAV-lacZ infected mature DCs could not induce a significant CTL response to beta Gal antigen (Fig. 3B). These results demonstrate that the differences between immature and mature DCs in initiating an immune response to the AAV-transduced gene may result from the distinct transduction efficiency and differentiation states. It is possible that the susceptibility to AAV-lacZ and the potent capacity of antigen uptake and processing may enable immature DCs to initiate an immune response to diminish AAV-lacZ-transduced gene expression.

Adoptive transfer of AAV-lacZ-infected DCs initiates CD40L-dependent T-cell immunity. Lymphocytes were isolated from the spleens and regional lymph node of the recipient animals, as shown in Table 1, and cultured ex vivo to examine the cytokine release. Intramuscular injection of C57BL/6 mice with AAV-lacZ (Fig. 4A) but not with PBS (data not shown) could induce the production of IL-10, in agreement with our previous studies (12, 17). Adoptive transfer of AAV-lacZ-infected immature DCs further enhanced IL-10 secretion (Fig. 4A) and markedly induced an IFN-gamma response in the recipients (Fig. 4B), implying activation of CD4+ T cells by AAV-lacZ-infected immature DCs. To further elucidate the role of CD4+ T cells, CD40L-/- mice were used as recipients for the next set of experiments. Adoptive transfer of Ad-lacZ or AAV-lacZ-infected immature DCs failed to elicit a CTL response (Fig. 3C), resulting in persistent beta Gal expression in muscles of CD40L-/- mice (Fig. 2A). Since DCs express CD40 but not CD40L molecules (4, 5), adoptive transfer of DCs derived from C57BL/6 mice into CD40L-/- mice cannot supplement any additional CD40L signaling to activate T lymphocytes. These observations show that CD40L-CD40 interactions play a critical role in regulating immature DC-mediated immune responses following AAV-lacZ gene transfer.


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  		FIG. 4.   Adoptive transfer of AAV-lacZ-infected DCs enhances the secretion of IFN-gamma by cultured lymphocytes. The splenic mononuclear cells were isolated from the animals on day 28 after adoptive transfer of various DCs infected or not infected with AAV-lacZ (Table 1). A total of 2 × 106 cells were cultured in medium containing IL-2 (50 µ/ml) and beta Gal (10 µg/ml). The supernatants were collected 3 days later and assessed for IL-10 (A) and IFN-gamma (B) by ELISA. Results are shown as the cytokine levels in pooled splenic mononuclear cells of four mice per group. The abbreviation for each group is described in Table 1 and shows the recipient animals given various DCs, infected with distinct vectors, by adoptive transfer. Results of representative experiment of two are shown.


    DISCUSSION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Reports on immune responses to AAV vector-mediated transgene products have been conflicting. Many laboratories have demonstrated that the AAV vector can evade cellular immunity and induce a durable expression of the AAV-mediated transgene in vivo (7, 12, 13, 19, 20, 37). On the other hand, accumulating evidence indicates that in some circumstances, the AAV vector may initiate a detectable cellular and humoral immune response to the AAV vector-transduced neoantigen in vivo (8, 21, 22). Our findings demonstrate, for the first time, that immature DCs can take up AAV-lacZ and induce a CD40L-dependent T-cell immune response to markedly diminish AAV-lacZ-transduced muscle fibers of immunocompetent mice.

Previous studies showed that immunization with antigen-bearing DCs efficiently primes both CD4+ helper and CD8+ CTL immunity and leads to protective immunity to infectious agents and tumors (9, 30, 35). Genetically modified DCs, with an Ad vector encoding the beta Gal tumor model antigen or tumor-associated antigen, confer potent protection against a lethal tumor challenge (30). In our studies, adoptive transfer of AAV-lacZ-infected immature DCs into immunocompetent mice markedly diminished AAV-lacZ-transduced gene expression, adding further evidence for the critical role of DCs in eliciting T-cell-mediated immunity. However, the CTL response elicited by adoptive transfer of AAV-lacZ-infected immature DCs failed to completely eliminate AAV-lacZ-transduced muscle fibers. Prolonged CTL responses in vivo to the neotransgene products have also been found in mice given AAV vectors encoding ovalbumin and herpes simplex virus type 2 virus glycoproteins B and D (8, 22), suggesting the production of persistent transgenic antigen stimulation in vivo. This would predict that the decline of AAV-transduced gene expression in vivo, if any, would occur in a chronic way due to the limited activation of T-cell immunity.

However, it remains unknown whether the elimination of AAV-lacZ-transduced muscle might result from shutting down the transgenic expression rather than killing the AAV-lacZ-containing cells. The muscle morphology of the mice given AAV-lacZ-infected immature DCs by adoptive transfer appeared to be fairly intact (Fig. 2A), in spite of the significant reduction of beta Gal activity in muscle fibers. The transgene expression of AAV in permissive cells correlates with the promoter activity and the phosphorylation state of the single-stranded D-sequence-binding protein (25). The previous studies have demonstrated that the cytomegalovirus promoter, which drives AAV-lacZ in our system, can be silenced by several factors in vivo independent of vector systems (10, 15, 16, 18). Although a substantial CTL response that could specifically kill the lacZ transgenic target cells ex vivo was observed from C57BL/6 mice given AAV-lacZ-infected immature DCs by adoptive transfer, we cannot rule out the possibility that other factors, such as cytokines, elicited by adoptive transfer of AAV-lacZ-infected immature DCs might interfere with the transgenic expression of AAV-lacZ in muscle.

Why did ex vivo AAV-lacZ-transduced immature DC, but not direct injection of AAV-lacZ into muscle, induce a cellular immune response? It has been shown that direct intramuscular administration of AAV-lacZ fails to induce significant infiltration of inflammatory cells (7, 12, 19, 20, 37) and to mobilize enough immature DCs into the injected sites to take up AAV-lacZ (17). Moreover, AAV-lacZ could induce a lower level of transduction of immature DCs. The experiments involving adoptive transfer of AAV-lacZ-infected DCs may artificially provide sufficient numbers of DCs bearing AAV-lacZ to initiate beta Gal-specific CTL response, which may not be achieved in mice given intramuscular injections of AAV-lacZ alone. However, this does not exclude the possibility that immature DCs might be recruited to the site to interact in situ with AAV vectors under some particular condition(s) such as delivery pathways and the presence of other inflammatory stimuli mobilizing the immature DCs. The evidence that intravenous injection of mice with AAV-ova can elicit a more potent ovalbumin-specific CTL response than intramuscular injection does (8) implies that immature DCs may be recruited to interact in situ with AAV vectors in the intravenous pathway. These observations suggest that a threshold of AAV vector-transduced immature DCs may control the induction of the T-cell-mediated immune response to the transgene product. It is noted that DCs have recently been shown to take up antigens, not only through direct transduction by virus but also through cross-priming presentation by virus-infected targets (3, 6, 29). This study and other previous reports (8, 17, 21, 22) do not rule out the possibility that DCs might elicit a CTL response to AAV vector-transduced gene products through the cross-priming pathway. Further experiments are under way to address these mechanisms.

A sharp distinction was demonstrated between immature and mature DCs infected with AAV-lacZ in initiating beta Gal-specific CTL response to diminish AAV-lacZ-transduced fibers. Mice given AAV-lacZ-infected mature DCs by adoptive transfer failed to elicit a beta Gal-specific CTL response, whereas adoptive transfer of AAV-lacZ-infected immature DCs induced the generation of CTLs that markedly diminished the AAV-lacZ-transduced muscle fibers. Immature DCs are characterized by high endocytic and phagocytic activity and low expression of accessory signals for T-cell activation (2, 4, 11, 23, 26, 28, 31, 34, 41, 42). Maturation of DCs is always associated with the down-regulation of antigen uptake and the alternative expression of many functional surface molecules (2, 4, 11, 23, 26, 28, 31, 34, 41, 42). In fact, AAV-lacZ-infected immature DCs, but not mature ones, could express a minimal level of beta Gal, which was substantially enhanced by addition of wild-type Ad, suggesting that immature DCs are more susceptible to infection by AAV-lacZ than are mature DCs. The susceptibility of cells to infection by AAV-2 depends on its coreceptors alpha Vbeta 5 integrin and/or fibroblast growth factor receptor 1 and its primary receptor, membrane-associated heparan sulfate proteoglycan (24, 32, 33). In the absence of the coreceptor, for example alpha Vbeta 5 integrin, infection of cells with the AAV-2 vector fails to efficiently induce the transgenic expression (32). Immature DCs express high levels of alpha Vbeta 5 integrin, which mediates the phagocytosis of cells (2), although heparan sulfate proteoglycan and fibroblast growth factor receptor 1 remain to be examined. Interestingly, the expression of alpha Vbeta 5 integrin is significantly decreased during maturation (2); this may account for the reduced susceptibility of mature DCs to AAV-lacZ infection. Further investigation will be focused on elucidating the molecular mechanism of immature DCs taking up AAV-lacZ and processing the vector-transduced gene products.

Does AAV-mediated activation of CTL response require CD4+ T cells? Intramuscular injection of AAV-lacZ induces the production of IL-10 by lymphocytes but not that of IFN-gamma (12, 17). It is reminiscent of Th2-dominated immunity to AAV-lacZ-transduced gene products (1). However, adoptive transfer of AAV-lacZ-infected immature DCs induced the production of IFN-gamma , indicating that DCs preferentially polarized the development of Th1-mediated immunity, which may be responsible for the diminished beta Gal expression in AAV-lacZ-transduced muscle fibers. Activated Th cells express CD40L, and many studies show that its signaling through CD40 plays a critical role in enhancing the function of DCs to elicit cellular immunity (5, 27, 39). CD40L-/- mice given C57BL/6 mouse-derived immature DCs, infected with either AAV-lacZ or Ad-lacZ, by adoptive transfer failed to develop a beta Gal-specific CTL response to diminish AAV-lacZ-transduced muscle fibers. This indicates that vector-transduced DCs may in fact require additional signals in vivo via CD40-CD40L, despite the presence of high levels of surface B7 molecules prior to adoptive transfer (27). Alternatively, activation of third-party antigen-presenting cells via CD40-CD40L would be required to eliciting a T-cell response. It is anticipated that the administration of anti-CD40L and/or anti-CD4 antibodies may effectively block a possible vector-mediated cellular immunity and lead to full recovery of gene expression transduced by AAV vectors.

In summary, our findings demonstrate that the AAV vector might be able to initiate a cellular response to its transduced gene products if sufficient immature DCs capturing the AAV vector and its transduced neoantigens are recruited. We propose that the AAV-carried transgene and the route of administration of the AAV vectors should be taken into the account when AAV vector-based gene therapy is designed for chronic genetic diseases. Moreover, one should consider that the treatment of patients with Ad-based vectors may prevent them from being treated with AAV-2 vectors carrying the same transgene as a result of the memory immune response, when a combination of different vectors carrying the same gene is used in clinical trials of gene therapy.


    ACKNOWLEDGMENTS

We thank the members of the Cell Morphology Core of the Institute for Human Gene Therapy.

This work was supported by grants from the NIH (P30 DK47757-05, P01 AR/NS43648-04, and P01 HL59407-01), the Muscular Dystrophy Association, and Genovo, Inc., a biotechnology company founded by J. Wilson, who has equity in it.


    FOOTNOTES

* Corresponding author. Mailing address: 204 Wistar Institute, 3601 Spruce St., Philadelphia, PA 19104-4268. Phone: (215) 898-3000. Fax: (215) 898-6588. E-mail: wilsonjm{at}mail.med.upenn.edu.


    REFERENCES
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

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Journal of Virology, September 2000, p. 8003-8010, Vol. 74, No. 17
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Copyright © 2000, American Society for Microbiology. All rights reserved.


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