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,
* Pulmonary and Critical Care Medicine,
Microbiology, Immunology and Molecular Genetics, and
Jonsson Comprehensive Cancer Center, University of California, Los Angeles, School of Medicine, Los Angeles, CA 90095
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Early-transcribed AdV genes are involved in viral replication and also down-regulate MHC expression and Ag presentation (11). To diminish virulence and immunosuppressive effects, replication-incompetent AdV vectors lacking the E1 and/or E3 genes have been used in most DC vaccine models (1, 5, 6, 12, 13). However, cells transduced with these vectors still express viral proteins and host responses to them have been shown to modify AdV therapy in animal models (14, 15). In contrast, helper-dependent AdV (Hd-AdV), a so-called gutless vector, lacks viral coding regions in the final infectious particle (16, 17). When injected in vivo into the mouse, Hd-AdV vectors produce a higher level and longer-lasting transgene expression than that associated with first- or second-generation AdV vectors (18, 19). These features correlate with a reduced antiviral immune response. It is unclear whether the same effects will occur in humans (20, 21), but the use of Hd-AdV vector to modify DCs might allow high-level transgene expression but limit unwanted stimulation of host antiviral T cell responses.
In this study, human monocyte-derived DCs were infected with either
conventional replication-deficient E1-deleted AdV (E1-AdV) or an Hd-AdV
carrying the same marker transgene. DCs transduced by these two vectors
were directly compared for their expression of the transgene product
(
-galactosidase (-gal)) and their ability to stimulate antiviral
T cells. Hd-AdV proved to be 50- to 275-fold more efficient than E1-AdV
at expressing -gal protein. However, both vectors stimulated the
same frequency of AdV-specific T cells, suggesting that pre-existing
antiviral immunity is directed primarily against the viral capsid and
not viral genes transcribed de novo. Our results suggest that host
responses to AdV vectors, including Hd-AdV, should be carefully
considered for their impact on human DC-based immunotherapy.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Complete medium consisted of RPMI 1640 (Irvine Scientific, Santa
Ana, CA) supplemented with 10% heat-inactivated human AB serum (Omega
Scientific, Tarzana, CA), 10 mM HEPES (Calbiochem, La Jolla, CA), 100
U/ml penicillin, 100 µg/ml streptomycin, and 0.25 µg/ml fungizone
(Life Technologies, Rockville, MD). Abs used for DC phenotyping
included fluorochrome-conjugated anti-HLA-DR (BD Biosciences, San
Jose, CA) and anti-CD86 (BD PharMingen, San Diego, CA). Abs used to
purify CD3+ T cells (anti-CD14,
anti-CD16, and anti-CD19) and in vitro-cultured DCs
(anti-CD3, anti-CD19, and anti-CD56) were obtained from BD
PharMingen (no azide, low endotoxin). Fluorochrome-conjugated Abs
against human CD4, CD8, TNF-
, and IFN-
were obtained from BD
Biosciences. Sheep anti-mouse Ig-conjugated immunomagnetic beads
were obtained from Dynal (M450 Dynabeads; Lake Success, NY).
AdV vectors
A purified E1-AdV type 5 containing the green-fluorescent
protein (GFP) reporter gene under control of a CMV enhancer/promoter
(E1-AdV/GFP) was kindly provided by Dr. A. J. Berk (Molecular
Biology Institute, University of California, Los Angeles, CA). Viral
stocks of recombinant E1-AdV type 5 that either lacked a reporter
construct (E1-AdV/RR5) or contained the Escherichia coli
lacZ reporter gene under control of a CMV enhancer/promoter
(E1-AdV/-gal) were propagated in 293 cells, purified by CsCl
gradient ultracentrifugation, dialyzed, and stored at -80°C as
previously described (2). An Hd-AdV type 5 vector
containing the Lac-Z reporter gene under control of the same
CMV enhancer/promoter (Hd-AdV/-gal) was constructed using an
E1-deleted helper virus with lox sites flanking the packaging signals
(AdLC8cLuc; Merck, Whitehouse Station, NJ) and a 293 cell line
expressing Cre recombinase (293-Cre4 cells; Microbix, Toronto, Canada)
as previously described (16, 17). The titer of
Hd-AdV/-gal was determined to be 2.1 x
109 blue-forming units (BFU)/ml using 293 cells
according to an established protocol (22), and
contamination by helper virus was determined to be <0.01% by standard
plaque assay. Stock preparations for E1-AdV/-gal and Hd-AdV/-gal
were also evaluated by spectrophotometry at 260 nm to determine viral
particle number by the following equation: particle number =
(OD260) x (1.1 x
1012). In some experiments, transcription of
viral genes and transgenes was disrupted by placing vectors into
uncovered 96-well microtiter plates and irradiating with a UV
cross-linker set to deliver between 0.4 and 6
J/cm2 of UV energy at 254 nm (FB-UVXL 1000;
Fisher Scientific, Pittsburgh, PA).
Preparation and transduction of human monocyte-derived DCs
PBMCs were obtained from healthy normal donors by density-gradient centrifugation and DCs were prepared by culturing the adherent fraction with 800 U/ml GM-CSF (Immunex, Seattle, WA) and 500 U/ml IL-4 (R&D Systems, Minneapolis, MN) at 37°C as previously described (23). DCs were recovered on day 5 by vigorous rinsing and were purified by negative depletion using a mAb mixture (anti-CD3, anti-CD19, and anti-CD56) and anti-mouse Ig-conjugated immunomagnetic beads (23). A total of 3–6 x 105 DCs were suspended in 200 µl of complete medium and mixed with the appropriate titer of AdV suspended in 100 µl of PBS with Ca2+/Mg2+. After a 2-h incubation at 37°C, complete medium containing GM-CSF and IL-4 was added and cells were cultured for another 48 h. Cell recovery and viability were determined by hemocytometer counts using trypan blue exclusion before use in subsequent experiments. For some experiments, cells from a human breast cancer cell line (MCF-7; American Type Culture Collection, Manassas, VA) were used as targets for AdV transduction according to the same protocol.
Assessment of transgene expression
Expression of -gal was evaluated after 48 h by FACS
analysis or by luminescence assay. For FACS, 1 x
105 cells were suspended in staining buffer (PBS
containing 4% FCS, 10 mM HEPES, pH 7.2) and incubated for 5 min at
37°C with 2 mM di--D-galactopyranoside (FDG; Molecular
Probes, Eugene, OR) as described by the manufacturer. Anti-human HLA-DR
PerCP was added and cells were incubated another 5 min at 37°C. The
reaction was stopped by the addition of ice-cold staining buffer
containing 1 mM phenylethyl -D-thiogalactopyranoside,
and cells were analyzed using a FACSCalibur flow cytometer and
CellQuest software (BD Biosciences). DCs were identified on the basis
of their size and HLA-DR expression and their -gal content was
determined by fluorescence of metabolized FDG. For more sensitive
quantitation, 1 x 105 transduced cells were
suspended in lysate buffer (100 mM potassium phosphate, pH 7.4, 10 µM
DTT) and subjected to three rounds of freeze/thaw. A sample of lysate
was mixed with -gal reaction buffer (Clontech, Palo Alto, CA) and
enzyme activity was measured after 30 min at room temperature using a
luminometer (model BG-1; GEM Biomedical, Sparks, NV). Results are
reported in light units. Cells transduced with E1-AdV/GFP were examined
by FACS analysis for GFP-related fluorescence and results are reported
as mean fluorescence intensity.
PCR and RT-PCR
Genomic DNA was extracted from control and transduced DCs using
a Genomic Prep kit (Amersham Pharmacia, Piscataway, NJ). Total RNA was
extracted by the TRIzol method (Life Technologies). For PCR, 0.01 µg
of DNA was used as a template and amplified with 25 cycles consisting
of 45 s at 92°C, 45 s at 50°C, and 45 s at 72°C.
For RT-PCR, 1 µg of total RNA was used to synthesize cDNA with a cDNA
Cycle Kit (Invitrogen, Carlsbad, CA), and 1 µl of the reverse
transcriptase product was used as a template to perform PCR as
described above. -Gal was detected by amplifying the fragment from
2884 bp to 3366 bp as the primer sequence, and GAPDH was detected by
amplifying the full-length cDNA. PCR or RT-PCR products were resolved
on a 1.2% agarose gel and visualized with a UVP Image Store 7500 (UVP,
Upland, CA). Expression of the GAPDH gene was used to control for
loading, and signal intensities were quantified by densitometry
using NIH Image 1.62 software to obtain a -gal:GAPDH ratio. Using
this approach, serial dilutions of genomic DNA or total mRNA
demonstrated a linear relationship between starting copy number and
measured signal intensity over a ninefold range of dilutions.
Proliferation assay
T cells were purified from PBMCs by labeling with anti-human
CD14, CD16, and CD19 mAb and depleting labeled cells with M450
Dynabeads. Control DCs, or DCs transduced with the various AdV
constructs, were cocultured with purified T cells from the same donor
at a 1:20 ratio in 96-well round-bottom plates. After 5 days of
culture, cells were pulsed with 1.25 µCi/well
[3H]thymidine (Amersham Pharmacia) and
harvested the next day onto glass fiber strips using a semiautomated
cell harvester (Brandel, Gaithersburg, MD). Proliferation, as
determined by [3H]thymidine uptake, was
measured by scintillation counting and reported as the average cpm for
five replicate wells. In some experiments, mAb against CD86 or
2-microglobulin (10 µg/ml) were added at the
start of the assay to block Ag-specific or CD8-specific T cell
proliferation, respectively (23).
Detection of AdV-specific CD4+ and CD8+ T cells
DCs were transduced with either E1-AdV/-gal or Hd-AdV/-gal
(125 multiplicity of infection (MOI)) and 1 day later were activated
with 1 µg/ml CD40 ligand (generously provided by Immunex) and 500
U/ml IFN- (PeproTech, Rocky Hills, NJ) for an additional 24 h.
Two days after transduction, DCs were washed and used to stimulate
autologous T cells at a 1:20 ratio in round-bottom 96-well plates
containing complete medium supplemented with 10 IU/ml rIL-2 (Chiron,
Emeryville, CA) and 2 ng/ml rIL-7 (Biosource International, Camarillo,
CA). After 1 wk of stimulation, T cells were collected, washed, and
challenged for 5 h at a 1:10 ratio using either autologous control
DCs (no AdV vector) or DCs transduced with E1-AdV/RR5 (250 MOI). Cells
were cultured in fresh medium containing 1 ng/ml IL-12 (PeproTech) and
3 µg/ml anti-CD28 Ab to maximize cytokine expression (BD
PharMingen). Brefeldin A (Sigma-Aldrich, St. Louis, MO) was added at 10
µg/ml after the first hour, and intracellular staining for IFN-
and TNF- was performed according to the manufacturer’s protocol (BD
Biosciences). Cells were counterstained with fluorochrome-conjugated
anti-CD4 and anti-CD8 mAb and fixed in 1% paraformaldehyde.
Flow cytometry data were obtained using a FACSCalibur flow cytometer
and CellQuest software.
Chromium release assay for detection of AdV-specific CTLs
Purified CD3+ T cells were stimulated in vitro at a 20:1 ratio with either autologous Hd-AdV-transduced DCs or E1-AdV-transduced DCs using the same protocol as described above for the induction of AdV-specific CD4+ and CD8+ T cells. After 1 wk in culture, T cells were collected, washed, and used as effector cells in a 4-h chromium release assay. HLA-matched M202 cells that had been transduced 18 h previously with E1-AdV/RR5 at a MOI of 40 were loaded for 1 h with 100 µCi of [51Cr] and used as target cells at a final concentration of 2.5 x 103 targets/well in U-bottom 96-well microtiter plates. Sufficient effector T cells were added to produce E:T ratios of 50:1, 25:1, and 12.5:1 in a total volume of 200 µl of complete medium. After a 4-h culture at 37°C, plates were centrifuged and the amount of chromium released by cell lysis was determined by removing 100 µl of supernatant from each well and counting in a gamma counter. Spontaneous release of chromium was obtained from wells containing only target cells, and the maximal release of chromium was determined from wells lysed with 0.5% Nonidet P-40 detergent. Cytotoxic activity was expressed by the following formula: % lysis = 100 x [(experimental release - spontaneous release)/(maximal release - spontaneous release)]. Assay wells were run in triplicate and results are presented as the mean ± SD.
Statistical analysis
Data for each group are represented as the average value ±
SD for the indicated number of replicate determinations. Differences
between groups were determined by two-tailed Student’s t
tests with significant differences documented at p values of
0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Hd-AdV/-gal does not replicate independently in 293 cells, and
therefore titers for it and E1-AdV/-gal were based on detection of
-gal+293 cells (BFU) and not on plaque-forming
activity. Viral preparations were also examined for OD260
to determine the number of intact viral particles, the values of which
correlated directly with the measured BFU. Their capacity to express
-gal protein in human monocyte-derived DCs was evaluated at MOIs of
125, 250, and 500 BFU (Fig. 1
A). These MOIs had minimal
effects on DC recovery or viability, whereas MOIs in excess of 1000
were observed to be cytopathic. DCs transduced with Hd-AdV expressed
higher -gal enzyme activity under every condition with an average
150-fold increase over E1-AdV at the 500 MOI (range, 52- to 275-fold
increase). Flow cytometry of FDG-loaded cells confirmed enhanced
transgene expression at the single-cell level (Fig. 1B).
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To determine whether higher -gal expression was the result of
more efficient transduction (vector loading) or a higher level of
transcription, genomic DNA and total mRNA were extracted from control
and transduced DCs and were analyzed by semiquantitative PCR (Fig. 2A) and RT-PCR (Fig. 2B), respectively. When transduced using the same BFU,
identical levels of lacZ transgene were detected from DCs
exposed to either vector. In contrast, levels of mRNA encoding for
-gal were always higher in cells exposed to the Hd-AdV. These
results suggest that both vectors exhibit equal transduction
efficiencies, but that higher transcriptional activity and/or mRNA
stability accounts for the higher -gal levels in cells exposed to
Hd-AdV.
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To evaluate the impact of viral transduction on T cell
stimulation, DCs were transduced with E1-AdV/-gal at various MOIs
and were cocultured with purified autologous T cells. -Gal
expression was used as a measure of viral transduction efficiency (Fig. 3A) and resulting T cell
proliferation was measured by radiolabeled thymidine incorporation
(Fig. 3B). Both -gal activity and T cell proliferation
increased in a concentration-dependent manner as the vector MOI was
increased. To determine whether this effect was specific for the
-gal insert or related to the viral components of the vector,
experiments were repeated using an E1-AdV/GFP (Fig. 3, C and
D) or E1-AdV/RR5 (data not shown). The same dose-dependent
increase in proliferation was observed, suggesting that transduced DCs
process and present viral Ags and stimulate the proliferation of
viral-reactive T cells. T cell proliferation was blocked by the
addition of anti-CD86 mAb, consistent with this hypothesis, and the
addition of mAb against 2-microglobulin
partially reduced T cell proliferation, suggesting proliferative
responses by both CD4+ and
CD8+ T cells (data not shown).
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Working under the hypothesis that viral gene products synthesized
de novo act as an important source of viral Ags, we expected that
E1-AdV-transduced DCs would stimulate significantly greater T cell
proliferation than Hd-AdV-transduced DCs. However, both sets of DCs
stimulated the same level of T cell proliferation (Fig. 4). To evaluate this further,
intracellular cytokine staining was used to identify the frequency of
viral-reactive T cells. Fresh T cells were stimulated for 1 wk in vitro
with either Hd-AdV-transduced DCs or E1-AdV-transduced DCs and then
were challenged with either control DCs or DCs transduced with the
E1-AdV/RR5 vector. This approach allowed us to discriminate
viral-specific T cell activation (as measured by challenge with
AdV/RR5-transduced DCs) from nonspecific activation (as measured by
challenge with autologous DCs alone). Intracellular production of
TNF- and IFN- was determined by flow cytometry as a sensitive
measure of Ag-specific activation (Fig. 5). As with the proliferation assay, DCs
transduced with the same MOI of either vector stimulated similar
frequencies of AdV-specific T cells. The response frequencies were
high, consistent with the activation of pre-existing antiviral
immunity. Both CD4+ T cells and
CD8+ T cells responded to E1-AdV/RR5-transduced
DCs, suggesting efficient loading of vector-related Ags into both the
MHC class I and II pathways (Fig. 5). The generation of AdV-specific
cytolytic activity was confirmed using a standard chromium release
assay. Again, both Hd-AdV-transduced DCs and E1-AdV-transduced DCs
resulted in the stimulation of cytolytic activity against
viral-transduced target cells (19.8 ± 3.8% vs 10.4 ± 1.5%
lysis at a 50:1 E:T ratio and 11 ± 1.7% vs 5.1 ± 2.4%
lysis at a 25:1 E:T ratio, respectively). The stimulation of a primary
immune response to -gal (as compared with the memory response to
AdV) was not expected in this short-term assay and no
cytokine-producing T cells or CTLs were detected when challenged with
DCs loaded with -gal protein (data not shown).
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Theoretically, the vector components that Hd-AdV/-gal,
E1-AdV/-gal, and E1-AdV/RR5 share in common are limited to viral
capsid and the noncoding packaging signals. To confirm the role of
preformed viral components as the source of responsible Ags, we
disrupted gene transcription in both the HD-AdV and the E1-AdV vectors
using UV irradiation. To do this, Hd-AdV and E1-AdV were exposed to 6
J/cm2 of UV light before using them to transduce
DCs. This dose was sufficient to disrupt vector-related DNA and reduce
the capacity for gene transcription by 3 logs (Fig. 6A). However, irradiation of
the vectors in this manner had no effect on the capacity for transduced
DCs to stimulate T cell proliferation (Fig. 6B). Because
minimal vector-related transcription was possible under these
conditions, we conclude that T cell responses against the vectors were
most likely directed against the preformed elements contained in their
viral capsids.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The level of -gal protein in DCs transduced by Hd-AdV/-gal
averaged 150-fold higher than that produced by an equal MOI of
E1-AdV/-gal. Our results are consistent with work by Parks et al.
(25), which demonstrated enhanced transgene expression
when A549 lung cancer cells were transduced with Hd-AdV in vitro. By
comparing different Hd-AdV constructs, they concluded that differences
in expression were due to an enhancer effect mediated by noncoding
regions contained in the stuffer DNA. In our studies, we used
semiquantitative PCR and RT-PCR to confirm that both vectors load the
same amount of transgene DNA, but that Hd-AdV produces higher levels of
transgene mRNA. Other approaches for increasing AdV-based transgene
expression in DCs have been reported, but they rely on modifying the
viral capsid to increase viral loading (26, 27). The use
of Hd-AdV to transduce DCs may be advantageous in this respect,
producing high levels of expression but limiting the loading of
viral Ags.
When DCs were loaded with E1-AdV at different MOIs, there was a direct
correlation between viral load and their capacity to stimulate T cell
proliferation and generate AdV-specific T cells. This proliferative
effect was not induced by transgene expression, in that similar
responses were observed with E1-AdV/-gal, E1-AdV/GFP, or E1-AdV/RR5.
Furthermore, it was blocked by the addition of anti-CD86 or
anti-2-microglobulin Ab, suggesting a
specific response by both CD4+ T cells and
CD8+ CTLs to AdV Ags. The generation of
AdV-specific CTLs was confirmed by standard chromium release assays.
Olive et al. (28) recently reported the presence of memory
antiviral T cells in fresh peripheral blood from 20 of 22 randomly
tested donors. Similarly, proliferative responses to AdV-transduced
DCs, but not control DCs, were observed in every one of our subjects.
AdV is a ubiquitous pathogen and both memory T cell responses and
circulating Ab appear to exist in most healthy individuals (28, 29). More importantly, antiviral responses are boosted in a
dose-dependent manner after treatment with E1-AdV in vivo (9, 30). The amount of viral Ags present in DCs is therefore likely
to predict the magnitude of the host antiviral response.
Work by Yang et al. (15) suggested that immune responses to E1-AdV in vivo are directed against viral gene products expressed de novo. Based upon this, many investigators speculated that Hd-AdV would circumvent antiviral immunity. This viewpoint was reinforced by studies in which administration of Hd-AdV to immunocompetent mice produced long-lasting expression with limited toxicity (18). However, significant antiviral responses against Hd-AdV have been detected, and factors other than a lack of antiviral immunity may contribute to the prolonged in vivo expression associated with Hd-AdV (31, 32). Maione et al. (19) reported that injection of Hd-AdV produced the same titer of neutralizing antiviral Ab as did injection of the same amount of E1-AdV. Escape from antiviral immunity was achieved by lowering the dose of Hd-AdV vector. Other work by the same group found that stability of Hd-AdV expression was strain dependent, with long-lasting responses in C57BL/6 mice, but not in BALB/c mice (32). Histology and molecular marker analysis correlated the loss of gene expression with a mononuclear influx, particularly CD8+ cells. It appears from these studies that Hd-AdV can induce an antiviral response depending upon dose, route, and strain-specific factors.
In this report, DCs transduced with an Hd-AdV produced the same degree of antiviral T cell activation as did DCs transduced with E1-AdV. This was confirmed by intracellular cytokine expression, which also demonstrated a balanced response by both CD4+ and CD8+ T cells. Viral Ags were efficiently processed and presented regardless of whether the vector carried viral DNA. This was investigated further by infecting DCs with UV-inactivated vectors. These transcription-incompetent virions produced the same T cell responses as their functional counterparts, confirming that antiviral responses were directed almost exclusively against preformed components of the viral capsid. Our results are in agreement with work by Molinier-Frenkel et al. (21), who determined that CD8+ CTLs from E1-AdV-immunized patients recognize capsid components, and Smith et al. (20), who demonstrated that CTLs resulting from E1-AdV-transduced DCs can lyse infected targets in the absence of viral gene transcription. Fiber knob, penton base, and hexon components all appear capable of eliciting potent human immune responses (21, 33).
We conclude from our studies that Hd-AdV offers an effective approach for achieving high-level transgene expression in human DCs. However, by packaging the transgene into a viral capsid, Hd-AdV still delivers potent viral Ags for processing and presentation by DCs. Although this raises concerns about the generation of antiviral responses, recent studies suggest that this form of antiviral response could have a positive impact on the generation of Ag-specific immunity. Intact E1-AdV, inactivated AdV particles, or even purified hexon proteins were recently shown to enhance Ag-specific responses when administered in conjunction with a peptide vaccine (33). There is likely an optimal balance between the adjuvant and destructive effects of viral Ag presentation. Further in vivo studies are warranted to evaluate and define this balance. If viral immunogenicity needs to be reduced, efforts should be directed at producing vectors that express the highest level of transgene expression but deliver the least amount of viral protein. Hd-AdV may be advantageous in this respect, and transgene expression might be increased even further by introducing additional enhancers/promotors (34). Alternatively, the combined use of E1-AdV and/or Hd-AdV derived from different capsid serotypes may allow repeated administration and still limit humoral and cellular immune responses (35).
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Michael D. Roth, Division of Pulmonary and Critical Care, Department of Medicine, University of California, Los Angeles, School of Medicine, Los Angeles, CA 90095-1690. E-mail address: mroth{at}mednet.ucla.edu
3 Abbreviations used in this paper: DC, dendritic cell; AdV, adenovirus; Hd-AdV, helper-dependent AdV; E1-AdV, E1-deleted AdV; -gal, -galactosidase; GFP, green-fluorescent protein; BFU, blue-forming unit; FDG, di--D-galactopyranoside; MOI, multiplicity of infection.
Received for publication February 12, 2002. Accepted for publication August 6, 2002.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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