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. 2006 Feb;80(4):1826–1836. doi: 10.1128/JVI.80.4.1826-1836.2006

Pulmonary Immunity to Viral Infection: Adenovirus Infection of Lung Dendritic Cells Renders T Cells Nonresponsive to Interleukin-2

Allison T Thiele 1, Tina L Sumpter 1, Joanna A Walker 1, Qi Xu 1, Cheong-Hee Chang 1, Robert L Bacallao 2,4, Rajesh Kher 2,4, David S Wilkes 1,3,5,*
PMCID: PMC1367134  PMID: 16439539

Abstract

Adenovirus (Ad) infection has been identified as predisposing hosts to the development of pulmonary disease through unknown mechanisms. Lung dendritic cells (DCs) are vital for initiating pulmonary immune responses; however, the effects of Ad infection on primary lung DC have not been studied. In contrast to the effects on bone marrow- and monocyte-derived DCs, the current study shows that Ad infection of murine BALB/c lung DCs in vitro and in vivo suppresses DC-induced T-cell proliferation. The effect of Ad on DCs was not due to a downregulation of major histocompatibility complex or costimulatory molecules. Analysis of the production of interleukin-12 (IL-12), alpha interferon (IFN-α), and IFN-γ by the Ad-infected DCs shows no significant differences over noninfected control lung DCs. Ad-induced suppression was not due to a deficiency of IL-2 or other DC-secreted factors and was dependent on viral protein synthesis, as UV irradiation of Ad abrogated the suppressive effect. Results suggest that Ad-infected DCs induce T cells to be nonresponsive to IL-2 during primary coculture, as the addition of IL-2 in secondary cultures recovered T-cell proliferation. In vivo studies supported in vitro results showing that Ad infection resulted in lung T cells with decreased proliferative ability. This study demonstrates that Ad infection induces local immunoincompetence by altering DC-T-cell interactions.

Adenovirus (Ad) infections are common yearly occurrences for many children and adults (24). Although these infections are usually resolved (38), Ad can establish a latent infection (29, 38). In the immunosuppressed, Ad infections can lead to fatal pneumonias, but serious complications affect immunocompetent individuals as well. Ad infection may lead to a predisposition to secondary bacterial infections and has also been associated with the development of chronic lung diseases such as chronic obstructive pulmonary disease (9, 19, 28, 34, 47). Although this link is well established, the mechanisms underlying this phenomenon are not understood. Ad encodes many proteins that function to allow the virus to evade detection by the immune system and prevent antiviral immune responses (5, 7, 19, 29). In addition, it seems likely that Ad, similar to other viruses, can modulate the function of the dendritic cells (DCs) and effector T cells of the immune system.

DCs are the most powerful antigen-presenting cells (APCs) (3). The effects of viruses on DCs are dependent on the both the type of virus and the type of DC. For example, measles and vaccinia virus infection of human monocyte-derived DCs (MODCs) induces apoptosis and thereby decreases T-cell responses (15, 17). Lymphocytic choriomeningitis virus infection blocks maturation and APC function in splenic DCs (37). The majority of studies examining Ad infection in DCs utilized immature bone marrow (BM) DCs and MODCs. These studies show that in vitro Ad infection induces maturation and enhances their function as APCs (20, 23, 26, 27, 31, 33, 36, 38). In studies of pulmonary immunity, intranasal infection of mice with influenza A and respiratory syncytial virus induced lung DC expansion and maturation (6, 11). Studies reporting that Ad lung infection may be a risk factor for later onset of pulmonary disease suggest that Ad may induce a state of local immunoincompetence. Although Ad is the most common respiratory viral pathogen, there are no prior reports of Ad-induced effects on primary lung DCs or responding lung T cells.

To determine the effects of Ad infection on the local pulmonary immune system, we infected lung DCs with Ad vectors as a model of Ad infection. Our studies demonstrate that lung DCs are susceptible to Ad infection in vitro and in vivo. In contrast to BMDCs and MODCs, lung DCs do not mature upon Ad infection and do not stimulate greater T-cell proliferation. Ad-infected lung DCs cause T cells to become nonresponsive to interleukin-2 (IL-2) in primary coculture, though these T cells are not suppressive upon addition to a fresh mixed leukocyte reaction (MLR) culture. Proliferative ability of the T cells was recovered in secondary culture in the presence of exogenous IL-2. Collectively, these data provide a potential mechanism for Ad-induced disease following pulmonary infection.

MATERIALS AND METHODS

Animals.

Female BALB/c (I-Ad and H-2d) and C57BL/6 (I-Ab and H-2b) mice (Harlan, Indianapolis, IN), aged 6 to 8 weeks, were used in all studies. Mice were housed in a specific pathogen-free environment at the Laboratory Animal Research Center (Indiana University School of Medicine [IUSM]) and given food and water ad libitum. All animal studies were approved by the Institutional Animal Care and Use Committee at IUSM.

Viruses.

Replication-deficient (ΔE1/E3) Ad serotype 5 viruses were obtained from the University of Pittsburgh Vector Core (Pittsburgh, PA). Ad viruses used either had no gene insert (AdNull) or contained the transgene for murine tumor necrosis factor alpha (AdTNF-α) or human IL-15 (AdhuIL-15). UV inactivation of Ad was performed by exposing the virus to UV light (254 nm) for 30 min prior to infection. UV inactivation of Ad gene expression was confirmed by infecting follicular skin DCs with UV-irradiated Ad vector encoding enhanced green fluorescent protein (AdEGFP) or untreated AdEGFP and visualizing under a fluorescent microscope (data not shown). UV irradiation abrogated GFP transgene expression in follicular skin DCs.

Ad infection.

In vitro Ad infection of lung DCs and BMDCs (multiplicity of infection [MOI] of 100, 350, or 700) was performed in 12-well tissue culture plates or 15-ml conicals in a volume of 100 μl of RPMI 1640 medium for 2 h. After cells were washed to remove any unattached virus, they were resuspended in complete medium ([cRPMI]; 10% fetal calf serum [FCS; HyClone, Logan, UT], RPMI 1640 medium, 400 mM l-glutamine, 100 U of penicillin/streptomycin, 5 × 10−7 M 2-mercaptoethanol [2-ME; Invitrogen, Carlsbad, CA]) and incubated for 15 h to allow transgene expression. For in vivo infection, dose-response studies were undertaken in which BALB/c mice were intratracheally (i.t.) instilled with 1 × 106 to 1 × 108 PFU of Ad in a 100-μl total volume of 1× phosphate-buffered saline (PBS) to determine the optimal dose. All other in vivo infections were performed using 1 × 108 PFU/mouse.

DC isolation.

Lung DCs were isolated as previously described (42). Briefly, murine lungs were obtained after bronchoalveolar lavage (BAL) using 1× PBS and lung perfusion with saline. Lungs were minced and digested with a collagenase D (Roche, Indianapolis, IN)-DNase I (Sigma, St. Louis, MO) solution for 90 min at 37°C, followed by filtration through steel mesh and nylon wool or a 70-μm-pore-size cell strainer to remove debris. Lung mononuclear cells (LMNCs) were then separated by a Percoll (Amersham, Piscataway, NJ) density gradient (1.03/1.075). Magnetic cell sorting (MACS) selection with CD11c microbeads (Miltenyi Biotec, Auburn, CA) was performed on LMNCs to obtain DCs (>70% purity) as reported previously (42, 43). The viability of the DCs exceeded 95% in all studies.

BMDCs were generated as previously described (21) with modifications. First, BM was flushed out of the femurs and tibias of BALB/c mice with DC wash (RPMI medium with 50 μM 2-ME and 1% FCS), strained, and washed before red blood cells were lysed with hemolysis buffer (1.66% NH4Cl in water, pH 7.2). Cells were then washed and resuspended for 1 h at 37°C in the following complement mix: monoclonal antibodies (Abs) ID3 (CD19), M5/114 (major histocompatibility complex class II-APC [MHC-II-APC]), TIB105, and TIB210 (CD8) at 1 μg/ml plus the Ab supernatants RL172 (CD4) and guinea pig complement diluted 1/20. After 4 washes in DC wash, cells were resuspended at 1 × 106 cells/ml in DC growth medium (RPMI with 50 μM 2-ME, 5% FCS, 20 μg/ml gentamicin [G418], and 10 ng/ml recombinant murine granulocyte-macrophage colony-stimulating factor) to be cultured in 24-well tissue culture plates. Medium was replaced at days 2 and 5. Immature DCs were harvested on day 5.

Detection of Ad infection in lung DCs.

Noninfected and Ad-infected lung DCs were affixed onto ProbeOn Plus slides by cytospinning and fixed with 4% paraformaldehyde for 30 min at room temperature. Fixation reactions were quenched with 100 mM NH4Cl dissolved in PBS. Cells were heat treated at 110°C for 10 min and permeabilized with 0.5% Triton X-100. Samples were washed with PBS and then dried at 110°C for 15 min. PCR was performed under conditions described previously (18) by melting at 94°C for 2 min, annealing at 55°C for 1 min, and extending at 72°C for 1 min for 35 cycles. The E2A primers used were primer 1 (5′-[Cy5]CGG AAT TCC AAC AGA GGA TAA AAA GCA AGA CC-3′) and primer 2 (5′-[Cy5]CGG AAT TCA AGG CCA GCT GCT TGT CCG CTC GG-3′). Slides were washed twice with PBS, fixed with 0.25% paraformaldehyde, and stained with Hoechst 34258. Images were collected with a Zeiss 510 confocal laser scanning microscope equipped with a UV and visible argon lasers and two HeNe lasers. All images were processed equivalently to allow for direct comparison. Individual cells were identified by positive nuclear staining with Hoechst 34258. Infected cells were identified by positive perinuclear staining for Cy5. Noninfected cells were identified by the absence of Cy5 staining. The frequency of Ad-infection was determined by averaging the quantity of infected (E2A+) relative to noninfected (E2A−) cells by 400 cells on six high-powered fields.

The frequency of infected cells was also quantified by flow cytometry to determine the percentage of DCs expressing human IL-15 after in vitro infection with AdhuIL-15 (MOI of 350) per intracellular cytokine staining protocol including isotype control Abs (BD-Pharmingen).

MLRs.

Infected and noninfected BALB/c lung DCs or BMDCs (I-Ad and H-2d) were γ-irradiated (2,000 rads) for use as stimulators. C57BL/6 (I-Ab and H-2b) splenic T cells to be used as responders were isolated by MACS selection of splenocytes, following red blood cell lysis using NH4Cl lysing buffer, with CD90 microbeads (Miltenyi Biotech) (>98% purity and > 98% viability). A total of 3 × 105 T cells/well were cocultured in various ratios with lung DCs in a flat-bottom 96-well plate in a final volume of 200 μl. All DC:T-cell ratios were determined at least in triplicate. Plates were incubated for a total of 66 h, with 0.5 μCi/well [3H]thymidine added 18 h prior to the end of the assay. MLR cultures were harvested using a cell harvester (Brandel, Gaithersburg, MD), and [3H]thymidine incorporation was counted using a liquid scintillation counter (Beckman, Fullerton, CA) and reported as counts per minute (cpm) of thymidine incorporation.

DC-conditioned medium.

Immature lung DCs isolated from noninfected or in vivo Ad-infected BALB/c mice were cultured for 24 h in cRPMI medium (1 × 106 cells/ml) in tissue culture dishes. After 24 h, the medium was drawn off and stored at −20°C. Increasing amounts of DC-conditioned medium were added to an MLR culture using noninfected BALB/c lung DCs and CD90+ C57BL/6 splenic T cells at a ratio of 0.3:1.

Flow cytometry.

Lung DCs or BMDCs were stained with fluorescent Abs for 30 min on ice following a 10-min Fc blocking step with purified anti-CD16/32 (Pharmingen, San Diego, CA). Cells were washed twice, fixed, and resuspended in a 1% paraformaldehyde solution and analyzed with a FACScan flow cytometer (Becton Dickinson, Franklin Lakes, NJ). The Abs used were α-CD16/CD32, phycoerythrin (PE)-conjugated CD80, CD86, CD40, I-Ad, H-2d, and biotin-OX40L plus streptavidin-PE along with isotype controls (Pharmingen). For intracellular cytokine staining, freshly isolated lung DCs were resuspended at 2 × 106 cell/ml in cRPMI medium and incubated at 37°C for 4 h in the presence of Golgi-Plug (BD Biosciences) (1 μl/2 × 106 cells) to trap all cytokines produced inside the cells and prevent cytokine secretion into the medium. Cells were then fixed and stained for the presence of cytokines using biotin-conjugated IL-12, gamma interferon (IFN-γ), IL-10 plus streptavidin-PE (Pharmingen), and rat anti-IFN-α (clone F18; Monosan, Uden, The Netherlands) plus fluorescein isothiocyanate-conjugated anti-rat immunoglobulin G1 (Pharmingen). In parallel studies intracellular human IL-15 expression was determined in the murine lung DCs per a protocol for intracellular cytokine detection (BD-Pharmingen).

RT-PCR.

Total RNA was isolated from cells using Tri-Reagent with a polyacryl carrier (Molecular Research Center, Cincinnati, OH). cDNA was prepared by reverse transcribing 1.0 to 2.0 μg of RNA with an iScript cDNA Synthesis Kit (Bio-Rad, Hercules, CA) following the manufacturer's instructions. PCR was then carried out by amplifying 2.0 to 4.0 μl of cDNA with Taq Mastermix (QIAGEN, Valencia, CA). The primers used were as follows: for human indoleamine 2,3-dioxygenase (IDO), 5′-ACT ACA AGA ATG GCA CAC GC-3′ and 5′-TTG CAC AGA CAC AGT CTG CA-3′; for β-actin, 5′-TCT TTT GTT TTG GCG CTT CT-3′ and 5′-CAA CAC CTC AAC CCA CTC CT-3′. PCR conditions for IDO detection were as previously described (16). Reverse transcription-PCR (RT-PCR) for β-actin was performed in parallel to IDO detection. RT-PCR products were resolved on a 1% ethidium bromide precast gel (Bio-Rad) and visualized using an AlphaImager (AlphaInnotech, San Leandro, CA).

Proteomics.

Noninfected and in vivo Ad-infected lung DCs were isolated and analyzed for differential protein expression using methods described previously (12). Briefly, cell extracts were prepared for two-dimensional (2-D) gel electrophoresis, and the 2-D gels were imaged using a Fluor-S MAX MultiImager system (Bio-Rad) and analyzed using PDQuest software (version 7.0; Bio-Rad). Protein spots with twofold or greater differences between groups in three replicate assays were excised using a PROTEAN 2-D spot cutter (Bio-Rad), processed robotically using a MassPREP Workstation (Perkin-Elmer, Wellesley, MA), and analyzed with a matrix-assisted laser desorption ionization-time of flight mass spectrophotometer (Micromass, Manchester, United Kingdom). Measured peptide mass profiles were then compared with the theoretical peptide masses using the ProFound search engine and NCBI database for protein identity.

T-cell suppressor and IL-2 assays.

BALB/c in vivo Ad-infected and noninfected lung DCs (4.5 × 105) were cocultured with 1.5 × 106 C57BL/6 splenic CD90+ T cells for 66 h in a 24-well plate (total volume, 1.0 ml/well). Cells were collected, and the T cells were reisolated using CD90 magnetic microbeads. For the suppressor assay, the reisolated T cells (putative regulators) were cocultured in increasing ratios with 5 × 104 BALB/c CD90− splenocytes (as stimulators) and 5 × 104 C57BL/6 CD90+ splenic T cells (as responders) in a 96-well U-bottom plate. All conditions were set up in triplicate. The secondary coculture was incubated for a total of 66 h, and T-cell proliferation was measured by [3H]thymidine incorporation for the last 18 h of coculture as described above. In the anergy assay, IL-2 (50 U/ml) and/or anti-CD3 (0.5 μg/ml) was added to 5 × 104 of the reisolated T cells in a 96-well U-bottom plate with a total volume of 200 μl/well. Each condition was set up in triplicate wells. Cells were cultured for 25 h, with T-cell proliferation being measured by [3H]thymidine incorporation for the last 6 h of incubation. Plates were harvested using a Basic 96 Harvester cell harvester (Skatron Instruments, Molecular Devices, Sunnyvale, CA), and filters were counted using a Wallac 1450 Microbeta Plus liquid scintillation counter (Perkin Elmer, Boston, MA). Proliferation was reported as a stimulation index that refers to multiples of proliferation in T cells induced by stimulators compared to proliferation in T cells alone (32).

Lung T-cell proliferation assay.

CD90+ T cells were positively selected by MACS isolation from the CD11c− fraction of noninfected and in vivo Ad-infected LMNCs. A total of 5 × 104 lung T cells were cultured with increasing amounts of γ-irradiated (2,000 rads) CD90− BALB/c splenocytes and 0.5 μg/ml anti-CD3 (clone 145-2C11) in a U-bottom 96-well plate. All coculture conditions were performed in triplicate wells. The coculture was incubated for a total of 72 h, and T-cell proliferation was measured by the incorporation of [3H]thymidine (0.5 μCi/well) for the last 6 h of culture. Assays were harvested using a Basic 96 Harvester, and filters were counted with a Wallac 1450 Microbeta Plus liquid scintillation counter. The purity and viability of lung T cells exceeded 98%.

Statistics.

Significance between groups was determined by performing a two-way analysis of variance with a Tukey post hoc analysis using Systat 8.0 (Point Richmond, CA), GraphPad Prism 4.0 (GraphPad Inc., San Diego, CA), or Microsoft Excel (Microsoft Corp., Redmond, WA).

RESULTS

Ad infection of lung DCs.

The ability of Ad to infect many cell types including BMDCs and MODCs has been well described (8, 13, 20, 22, 25). However, to the best of our knowledge, there are no prior reports of Ad infecting primary lung DCs. Many experiments examining BMDCs and MODCs utilized an Ad vector expressing a fluorescent transgene to confirm cell infection (13, 20, 22, 25). Our initial studies utilized a similar approach, but the autofluorescence that is characteristic of lung DCs prevented reliable detection of Ad vectors expressing either EGFP or enhanced blue fluorescent protein. Therefore, to demonstrate the ability of Ad to infect lung DCs in vitro and in vivo, we performed in situ PCR to detect the Ad E2A gene in lung DCs. As shown in Fig. 1, Ad E2A was detected in lung DCs following in vitro AdNull infection with MOIs of 350 and 700 (MOI of 350 shown; data not shown for an MOI of 700). Quantitation of Ad infection revealed that 25.4% of DCs were infected at an MOI of 350. In addition, Ad E2A was also found in lung DCs following in vivo Ad infection by i.t. instillation of 1 × 108 PFU of AdNull. In addition, we determined the frequency of infection using AdhuIL-15 vectors, followed by intracellular detection of human IL-15 by flow cytometry. Human IL-15 was detected in 18.6% of lung DCs after infection with vectors containing this human IL-15 transgene (MOI of 350). These results show that lung DCs can be infected with Ad both in vitro and in vivo, and the frequency of infection as detected by E2A gene expression is similar to that reported by Timares et al., who examined the frequency of Ad infection in BMDCs.

FIG. 1.

FIG. 1.

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Visualization of Ad infection of lung DCs. In situ PCR for Ad E2A was performed to detect Ad infection in noninfected and Ad-infected lung DCs. All cells were stained with Hoechst stain to identify nuclei. (A) Noninfected lung DCs. (B) No primer control. (C) In vitro AdNull-infected (MOI of 350) lung DCs. (D) In vivo AdNull-infected lung DCs (1 × 108 PFU).

In vitro Ad infection suppresses the function of lung DCs.

To determine the effects of Ad infection on primary lung DC APC function, immature BALB/c lung DCs were infected in vitro with AdNull (MOIs of 350 and 700) and then cocultured in increasing ratios with allogeneic C57BL/6 splenic CD90+ T cells to measure the ability of DCs to stimulate T-cell proliferation. With increasing DC:T-cell ratios, noninfected lung DCs induce a dose-dependent increase in their ability to stimulate T-cell proliferation (Fig. 2A). In contrast, infection with AdNull suppressed DC-induced T-cell proliferation. This difference reached significance at DC:T-cell ratios of 0.3:1 for MOIs of 350 and 700.

FIG. 2.

FIG. 2.

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APC function of in vitro Ad-infected lung DCs versus BMDCs. Following a 2-h in vitro infection and 15-h incubation period, Ad-infected and noninfected immature lung or BMDCs were γ-irradiated and cocultured in the indicated ratios with 3 × 105 CD90+ splenic T cells from C57BL/6 mice in an MLR culture as described in Materials and Methods. T-cell proliferation was measured by adding [3H]thymidine 18 h prior to the end of the 66-h assay. Results shown are representative of at least three independent experiments, and data are reported as means ± standard deviations of [3H]thymidine incorporation in replicate wells. (A) AdNull- and AdTNF-α-infected lung DCs at MOIs of 350 and 500. Counts of DCs alone did not exceed 362 cpm. (B) AdNull- and AdTNF-α-infected BMDCs infected at MOIs of 100 and 350. Counts of DCs alone were under 225 cpm. *, P < 0.001; †, P < 0.05.

Our results showing that Ad infection of lung DCs suppresses their function as APCs contrasts with other studies showing that Ad infection of BMDCs and MO-derived DCs enhances their APC ability (20, 26, 33, 35, 36, 44) or has no effect (13, 50). To confirm previous studies, we performed an MLR using Ad-infected immature BALB/c BMDCs (Fig. 2B). BMDCs infected with AdNull stimulated T cells as well as, or better than, noninfected controls (at an MOI of 100, DC:T-cell ratio of 0.3:1; P < 0.001).

TNF-α is one of the most potent inducers of DC maturation and function (3, 10). Therefore, we next determined if TNF-α could induce maturation and thereby overcome DC-induced suppression. TNF-α was overexpressed in DCs via AdTNF-α. Surprisingly, TNF-α supplied by infecting DCs with AdTNF-α did not enhance the APC ability of in vitro infected lung DCs (Fig. 2C). Again, at the higher DC:T-cell ratio of 0.3:1, AdTNF-α-infected lung DCs were significantly decreased in their APC function as measured by T-cell proliferation compared to noninfected controls. In vitro infection of BMDCs with AdTNF-α at MOIs of 100 and 350 significantly enhanced APC function compared to noninfected controls at ratios of 0.075:1 (MOI of 350 only), 0.15:1, and 0.3:1 (P < 0.001) (Fig. 2D). BMDCs infected with AdNull and AdTNF-α at an MOI of 700 were also better T-cell stimulators than noninfected controls (data not shown). In agreement with published data, Ad-infected BMDCs are as good or better APCs than noninfected controls. In addition, the data suggest that Ad-induced suppression of DC function is unique to lung DCs.

In vivo Ad infection of lung DCs suppresses their APC function.

Next, we investigated whether Ad infection induced suppression of DC function in vivo. Forty-eight hours after i.t. instillation with 1 × 108 PFU of AdNull, BALB/c lungs were harvested for DC isolation. Dose-response studies determined optimal transgene expression without inducing adverse effects in the lung (data not shown). These immature lung DCs were then cocultured in an MLR culture with allogeneic CD90+ splenic T cells from C57BL/6 mice. Noninfected DCs induced T-cell proliferation in a dose-dependent manner (Fig. 3). However, lung DCs isolated from AdNull-infected mice had a decreased ability to stimulate T cells at DC:T-cell ratios higher than 0.075:1. These results show that the ability of lung DCs to stimulate T-cell responses, and therefore initiate pulmonary immune responses, is diminished following infection with Ad. Decreased T-cell stimulation was not the result of poor cell viability in Ad-infected lung DCs. Viability as determined by trypan blue exclusion was >90% and comparable across groups.

FIG. 3.

FIG. 3.

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APC function of in vivo Ad-infected lung DCs. Immature lung DCs isolated from BALB/c mice either not infected or i.t. instilled with 1 × 108 PFU of Ad for 48 h were set up in an MLR culture as described above. Data shown are representative of four independent experiments using DCs isolated from the lungs of 10 mice/group. APC function was significantly suppressed in AdNull-infected lung DCs compared to noninfected controls (at ratios of 0.15:1, 0.3:1, and 0.5:1). *, P < 0.001. Counts of DCs only were <300 cpm.

Ad infection does not affect phenotype of lung DCs.

MHC and costimulatory molecules are necessary for proper stimulation of T cells, (3), and decreased expression of these molecules on the cell surface could account for the diminished APC ability of Ad-infected lung DCs. To investigate this possibility, in vivo infected lung DCs were examined by flow cytometry for the levels of MHC-I and -II, as well as the costimulatory molecules CD80, CD86, CD40, and OX40L. Flow cytometry analysis of noninfected and AdNull-infected lung DCs showed no significant differences in cell surface expression of MHC-I and -II molecules or costimulatory molecules CD80, CD86, CD40, and OX40L (Fig. 4 and Table 1). Ad infection of BMDCs upregulated expression of MHC-I (62.1 ± 1.3%) compared to noninfected cells (46.7 ± 0.8; P < 0.02, n = 3). However, Ad infection did not affect expression of CD80, CD86, or MHC-II compared to noninfected cells (P > 0.05; n = 3). Based on these data, Ad infection did not cause a loss of antigen-presenting or costimulatory molecules on the cell surface. Interestingly, overexpression of TNF-α did not alter expression of the same molecules (Table 1), and it had a variable effect on DC function (data not shown). As such, altered maturation status did not account for the diminished ability of Ad-infected lung DCs to stimulate T cells.

FIG. 4.

FIG. 4.

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Expression of MHC and costimulatory molecules does not change on lung DCs following Ad infection. Flow cytometry analysis compares the phenotypes of noninfected (filled black area), in vivo AdNull-infected (solid gray line), and AdTNF-α-infected (dotted gray line) lung DCs. At 48 h postinfection lungs were harvested and lung DCs were isolated. Cells were stained with Abs for MHC-I and -II and for CD80, CD86, CD40, and OX40L following an Fc blocking step. These are representative histograms from three replicates. Values are given in Table 1.

TABLE 1.

Phenotype comparison of noninfected versus Ad-infected lung DCs

Molecule Avg mean fluorescence intensity (± SD) in lung DCsa
Noninfected Infected with:
AdNull AdTNF-α
MHC-I 101.9 ± 12.5 98.6 ± 28.8 116.9 ± 10.4
MHC-II 41.3 ± 2.3 39.7 ± 4.6 41.2 ± 8.3
CD80 22.3 ± 3.8 23.3 ± 3.6 23.5 ± 2.5
CD86 19.9 ± 4.6 19.5 ± 8.5 23.6 ± 3
CD40 21 ± 12.9 20 ± 10.6 15.7 ± 2.9
OX40L 29.5 ± 12.5 26.9 ± 10.9 25 ± 5.5
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a

Values are from three replicate experiments.

Ad-infected lung DCs do not produce greater levels of cytokines.

Studies showing that Ad infection of BMDCs increases their APC function have demonstrated that these Ad-infected DCs also produce greater amounts of the cytokine IL-12 (20, 26, 33). Alterations in the balance between stimulatory cytokines such as IL-12 and antiviral cytokines such as IFN-α and IFN-γ and inhibitory cytokines such as IL-10 may account for the decrease in Ad-infected DC stimulation of T-cell proliferation. To examine this possibility, noninfected and in vivo AdNull-infected immature DCs were isolated from BALB/c mice and stained for intracellular IL-12, IFN-α, IFN-γ, and IL-10. The results show that noninfected and AdNull-infected lung DCs have the same number of IL-12-, IFN-α-, IFN-γ-, and IL-10-producing cells (Fig. 5A and Table 2). In addition, we assayed cytokine protein levels in noninfected versus in vivo AdNull-infected mouse BAL specimens (6 mice/group) by cytometric bead array. No differences were found between AdNull-infected and noninfected mice with respect to the levels of IL-12p70, IL-10, IL-6, TNF-α, or MCP-1. No IFN-γ was detected in the BAL specimen of either group (data not shown). This coincides with our intracellular cytokine data showing no differences between AdNull-infected and noninfected lung DCs in their production of cytokines. To determine if the decrease in T-cell proliferation during coculture with Ad-infected DCs was due to a lack of sufficient amounts of IL-2 for T-cell priming, DC-T-cell coculture supernatants were assayed for levels of IL-2. As shown in Fig. 5B, there was no significant difference between the IL-2 levels in supernatants from noninfected DC-T-cell cocultures and Ad-infected DC-T-cell cocultures. Indeed, there was a trend toward higher IL-2 in the Ad-infected groups.

FIG. 5.

FIG. 5.

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Ad-infected lung DCs produce cytokines similar to noninfected lung DCs. (A) Lung DCs were isolated from noninfected and 48-h in vivo Ad-infected BALB/c mice and treated with Golgi-Plug for 4 h to trap cytokines produced inside the cell. Cells were then fixed and intracellularly stained for IFN-α, IL-12, IFN-γ, and IL-10. Representative histograms show cytokine staining (black line overlay) compared to secondary antibody alone (filled gray area). Values from replicate experiments are given in Table 2. (B) IL-2 levels in DC-T-cell coculture supernatants. Noninfected and Ad-infected BALB/c lung DCs were cocultured with C57BL/6 splenic CD90+ T cells at a ratio of 0.3:1. After 66 h, supernatants were collected and stored until assayed. IL-2 levels were determined by cytokine enzyme-linked immunosorbent assay. Bars represent the average IL-2 levels for three independent experiments ± standard deviation.

TABLE 2.

Comparison of levels of cytokine production from noninfected and Ad-infected lung DCs

Cytokine Avg percent gated cells (± SEM) in lung DCsa
Noninfected AdNull-infected
IFN-α 3.3 ± 1.2 2.6 ± 1.2
IL-12 6.6 ± 1.1 6.5 ± 2.2
IFN-γ 4.6 ± 0.9 4.1 ± 2
IL-10 9.7 ± 2.4 10 ± 3.5
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a

Values are from 4 replicate experiments.

Ad-infected DCs do not produce soluble factors that suppress T-cell proliferation.

Recently, our laboratory demonstrated that lung DCs can suppress T-cell proliferation by the constitutive expression of the enzyme IDO (42). IDO catabolizes the essential amino acid tryptophan, which T cells require for expansion (30). Viral infection can also induce IDO expression in lung tissue (49). RT-PCR analysis of IDO mRNA shows no difference between Ad-infected and noninfected lung DCs with respect to mRNA levels (Fig. 6A) as determined by densitometry (data not shown). IDO protein levels were also similar in noninfected and Ad-infected lung DCs as determined by intracellular IDO staining for flow cytometry (data not shown). These results suggest that the lack of T-cell proliferation in MLR cultures following coculture with Ad-infected lung DCs is not due to suppression of T-cell proliferation by IDO.

FIG. 6.

FIG. 6.

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Ad-infected DCs do not secrete a soluble factor that is mediating the decrease in T-cell proliferation. (A) IDO mRNA levels in noninfected versus Ad-infected lung DCs. Lung DCs were isolated from 48-h in vivo AdNull-infected and noninfected BALB/c mice, and RNA was isolated to determine IDO mRNA expression by RT-PCR. β-actin mRNA is included as a loading control (Ctl). (B) Immature BALB/c lung DCs (pooled from 20 mice) were set up in a coculture with C57BL/6 CD90+ splenic T cells at a ratio of 0.3:1 using increasing amounts of noninfected or Ad-infected lung DC-conditioned medium. Cocultures were incubated for 66 h, adding [3H]thymidine for the last 18 h to measure T-cell proliferation. Values shown are from a representative experiment (of three experiments) and are the mean counts per minute for replicate wells ± standard deviation. The mean count of DCs alone was 630 cpm. DC-conditioned medium from noninfected DCs and AdNull-infected DCs had no effect on T-cell proliferation.

Our next step was to determine if the decrease in T-cell proliferation in the MLR was due to another suppressive soluble factor produced by Ad-infected lung DCs. DC-conditioned medium from either noninfected or AdNull-infected lung DCs was added in increasing amounts to a coculture of noninfected lung DCs and splenic T cells in a 0.3:1 ratio to determine its effects on DC-T-cell interactions and subsequent T-cell proliferation. As shown in Fig. 6B, increasing amounts of either noninfected or AdNull-infected DC-conditioned medium do not significantly affect DC-induced T-cell proliferation. Even when DC-conditioned medium comprises 50% (100 μl) of total culture medium, there is no significant difference between the two groups in T-cell proliferation. These results suggest that Ad-induced suppression of the ability of DCs to stimulate T cells is not due to a soluble factor that they are producing and suggests that this suppression could be mediated through a contact-dependent mechanism. These results are consistent with our data in Fig. 5 showing that AdNull-infected DCs do not secrete differential amounts of cytokines known to help stimulate or decrease T-cell proliferation.

Viral protein synthesis is required for suppression of lung DC function.

Exposure to UV light permits viral entry but prevents expression of the viral genome. Although AdNull is replication deficient, in that it lacks the E1 and E3 regions, a low level of transcription of the remaining viral genes is possible from the E2A transactivator (14). Additionally, Ad vectors similar to those used in these studies have been shown to produce viral proteins after infecting cells and present them at the cell surface (2, 48). Therefore, it is possible that some remaining viral protein could be mediating the suppressive effect through a direct or an indirect mechanism. In order to determine if viral proteins played a role in Ad-induced suppression of lung DC function, AdNull was UV inactivated prior to in vivo infection of BALB/c mice. Results show that there is no significant difference in the ability to stimulate T-cell proliferation between noninfected immature lung DCs and UV-AdNull immature lung DCs (Fig. 7) regardless of the DC:T-cell ratio. This suggests that transcription of a viral gene and the translation of its subsequent protein may be involved in inducing the suppressive effect on lung DC function.

FIG. 7.

FIG. 7.

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UV-inactivation of Ad abrogates suppression of DC function. Lung DCs from in vivo UV-AdNull-infected and noninfected BALB/c mice were isolated and γ-irradiated before being cocultured in an MLR culture with allogeneic CD90+ splenic T cells at the specified ratios. T-cell proliferation was measured by the addition of [3H]thymidine 18 h before harvesting at the end of a 66-h incubation. Graph is a representative experiment (of three experiments) with DCs pooled from 10 mice/group. Data points represent the mean of replicate wells ± standard deviation. Counts of DCs only were <520 cpm.

Proteomic analysis of noninfected versus Ad-infected lung DCs.

We took a proteomics approach to discover any differences in the proteins between noninfected and Ad-infected lung DCs that could account for the differences in function. In particular, we were attempting to identify proteins involved in the antigen processing/presentation pathway that could account for the decrease in T-cell stimulation following Ad infection. Protein spots with a twofold or greater difference in expression between noninfected and Ad-infected DCs in each of three replicate 2-D gels were excised and analyzed for protein identity. Surprisingly, these proteins served primarily a housekeeping function or have no known function in DC-T-cell interactions (Table 3).

TABLE 3.

Identified proteins with differential expression in uninfected versus AdNull-infected lung DCs

Protein name SSP no.a Mass (kDa) Isoelectric point Coverage (%)b Z scorec
Proteins upregulated in AdNull lung DCs
    Limb expression 1 homolog 1304 33.12 5.7 21 2.43
    Hsp member 7 2101 18.67 6.0 25 2.17
    Similar to transcription factor 3208 24.59 5.9 15 1.89
    Porphobilinogen deaminase 4607 38.01 7.0 23 2.43
    RecA-like protein 4611 38.75 7.1 20 2.43
    Myotubularin 5811 70.09 7.4 8 2.43
    Aldolase 1, A isoform 8512 39.79 8.8 33 2.43
Proteins downregulated in AdNull lung DCs
    Unnamed protein product 6508 39.54 8.9 36 2.43
    SYNCRIP; NS1-associated protein 6708 62.75 7.2 13 2.43
    Vrk2 protein 7704 52.91 9.2 27 2.43
    d-glucuronyl C5 epimerase 7710 70.42 9.1 12 2.43
    Spermatid perinuclear RNA-binding protein 6709 71.48 9.1 18 2.43
    Casein kinase 1α1 8501 37.73 9.7 42 2.43
    Aspartyl-tRNA synthetase 4702 57.56 6.1 31 2.43
    Natriuretic peptide receptor-3 5816 60.11 7.0 19 2.43
    Unnamed protein product 6409 31.87 8.6 27 2.43
    Unnamed protein product 6209 23.92 7.9 29 2.14
Proteins found in AdNull lung DCs only
    mK1AA0887 protein 1701 50.97 5.6 27 2.43
    Alcohol dehydrogenase 5 (class III) 4603 40.33 7.0 29 2.43
    Calmodulin-like 4 6101 17.78 7.9 44 2.43
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a

The SSP number is designated by PDQuest software and is used in matching spots for all gels.

b

Coverage is defined as the ratio of the portion of protein sequence covered by matched peptides to the whole length of protein sequence.

c

The Z score is calculated by the ProFound search engine and is an indicator of search result quality. A Z score is estimated when the search result is compared against an estimated random match population and is the distance to the population mean in units of standard deviation. For example, a Z score of 2.326 for a search means that the search is in the 99th percentile. One percent of random matches could yield higher Z scores than this search.

Ad-infected lung DCs render T cells initially nonresponsive to IL-2 but do not induce regulatory T cells.

One possible mechanism of Ad-induced DC-T-cell interactions could be that Ad-infected DCs induce anergy in responding T cells. To address this question, T cells isolated from Ad-infected DC-T-cell cocultures were restimulated in the presence of αCD3, IL-2, or both. Figure 8A shows that the addition of IL-2 to secondary cultures recovered T-cell proliferation. As IL-2 levels were sufficient in DC-T-cell cocultures (Fig. 5B), this suggests that coculture of T cells with Ad-infected DCs may result in the T cells' becoming nonresponsive to IL-2. Tuettenberg et al. (45) reported that Ad-infected mature CD83+ DCs induced G1 cell cycle arrest in T cells during coculture. To investigate this possibility, T cells previously cocultured with noninfected and Ad-infected lung DCs were stained with propidium iodide and analyzed by flow cytometry for DNA content. The results showed that in our studies the T cells were not in cell cycle arrest (data not shown).

FIG. 8.

FIG. 8.

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Ad-infected lung DCs induce IL-2 nonresponsiveness in T cells. CD90+ T cells were reisolated from primary coculture with noninfected or Ad-infected lung DCs and tested for IL-2 responsiveness and/or regulatory function. (A) Reisolated T cells were treated with IL-2 (50 U/ml) and/or anti-CD3 (0.5 μg/ml) to determine if exogenous IL-2 could rescue T-cell proliferation. Cells were cultured for 25 h, and proliferation was determined by [3H]thymidine incorporation during the last 6 h of culture. Bars represent the mean stimulation index from four replicate experiments ± standard error of the means. (B) Reisolated T cells were added back in increasing amounts in a secondary coculture with γ-irradiated BALB/c APCs (stimulators) and freshly isolated C57BL/6 splenic CD90+ T cells (responders). Cells were cocultured for a total of 66 h, and T-cell proliferation was measured by [3H]thymidine incorporation in the last 18 h of culture. Bars represent mean cell proliferation from replicate wells ± standard error of the mean. The mean count for stimulators alone was 79 cpm.

Viral-induced regulatory T (Treg) cell activity could also explain the inability of T cells to respond to secondary stimulation, as reported by investigators examining other viruses (41, 46). To determine if T cells previously cocultured with Ad-infected lung DCs became regulators, we reisolated T cells from primary DC-T-cell cocultures and added them back in increasing amounts to a secondary MLR culture with fresh stimulators and responders (BALB/c splenic APCs and C57BL/6 splenic T cells) (Fig. 8B). Increasing the quantities of these T cells to MLR cultures did not suppress proliferation of freshly isolated T cells. Further evidence that Ad-infected lung DCs do not mediate suppression of T-cell proliferation by induction of Treg cells is our finding showing that foxp3 transcripts, a marker of Treg cells, were not altered in T cells cultured in the presence of Ad-infected DCs compared to noninfected DCs. Moreover, analysis of CD25 (IL-2 receptor) expression on T cells from primary Ad-infected DC-T-cell cocultures also shows that the inability of these cells to respond to IL-2 present in medium (Fig. 5B) was not due to lack of IL-2 receptor expression (data not shown). Collectively, these studies suggest that Ad-infected DCs render T cells nonresponsive to IL-2 in a contact-dependent manner. This is independent of cell cycle arrest and is not associated with regulatory function.

Ad infection suppresses lung T-cell proliferation in vivo.

The proliferative ability of T cells isolated from the lungs of Ad-infected BALB/c mice was assessed to determine if the trend reported above mimicked the in vivo status of T cells. Lung T cells were cocultured with autologous CD90− APCs and anti-CD3. As shown in Fig. 9, proliferation of lung T cells from Ad-infected mice is significantly decreased compared to T cells isolated from the lungs of noninfected animals. These results suggest that the phenomenon in which Ad-infected lung DCs fail to stimulate T-cell proliferation in vitro may also be occurring in vivo.

FIG. 9.

FIG. 9.

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In vivo Ad infection decreases the proliferation of lung CD90+ T cells. Noninfected and 48-h in vivo Ad-infected BALB/c mice were sacrificed, and their lungs were harvested for T-cell isolation as described in Materials and Methods. Lung T cells were cultured for 72 h with γ-irradiated splenic APCs and anti-CD3 (0.5 μg/ml). Points represent mean proliferation of wells in triplicate ± standard deviation. *, P < 0.001 (at ratios of 0.5:1 and 1:1). The mean cpm of T cells alone was <300.

DISCUSSION

Although Ad tropism for airway epithelium is well documented, this study is the first to report that Ad infects lung DCs and that Ad impairs lung DC function. This effect is likely mediated by a viral protein and mediates IL-2 nonresponsiveness in the responding T cells through a contact-dependent mechanism during primary coculture. Finally, T-cell hyporesponsiveness in vitro was also observed in lung T cells after Ad pulmonary infection in vivo.

In studies using immature BM and MO-derived DCs, Ad infection of the DCs induces maturation. In those studies Ad-infected DCs had increased cell surface expression of MHC, costimulatory and adhesion molecules, and IL-12 production, and enhanced their ability to stimulate T cells (20, 26, 33, 35, 36, 44). We have confirmed that in vitro Ad infection of BMDCs (Fig. 1) and human MODCs (data not shown) enhances APC function in an MLR. Jonuleit et al. reported that mature human MO-derived DCs transduced with Ad have a diminished T-cell stimulatory capacity at high DC:T cell ratios (22), which is mediated by early Ad gene expression (45). These findings showing Ad-induced suppression of DC function are similar to ours, but there are a number of differences between our studies. In contrast to our experiments, DCs in the previous studies were generated from blood monocytes by culture with granulocyte-macrophage colony-stimulating factor and IL-4 and then matured with IL-1β, TNF-α, IL-6, and prostaglandin E2. The current data are the first to report the effects of Ad infection in primary tissue-derived cells without long-term culture or exposure to exogenous recombinant cytokine cocktails. Indeed, DCs derived from these techniques are altered significantly and are phenotypically and functionally distinct relative to primary tissue DCs (43). The maturation status of DCs is key to determining DC function. While the studies from Jonuleit et al. and Tuettenberg et al. studied the effect of Ad infection on mature DCs, our laboratory has shown that freshly isolated lung DCs are immature, as shown by the low-level expression of MHC-II and costimulatory molecules (Fig. 4) (42). Since the primary route of Ad infection is via the lower respiratory tract, then the virus will encounter immature DCs and not mature DCs. Therefore, our studies have strong clinical relevance as they demonstrate the effects of Ad on DC function after infection through the respiratory tract, which is the primary route of virus entry into the body.

We have also performed functional studies using in vivo infected mature DCs (43). Our functional experiments using in vivo Ad-infected mature DCs showed the same trend toward suppression; however, there were no significant differences compared to noninfected controls (data not shown). These ongoing studies suggest that either mature DCs are more resistant to Ad infection or that the strong expression of MHC and costimulatory molecules characteristic of mature DCs partially rescues Ad-induced suppression of lung DC function.

DC-induced T-cell activation requires two signals: the T-cell receptor recognizing MHC-I and -II molecules and costimulatory molecule recognition of their corresponding ligand (3). Diminished DC function was not caused by a decrease in the cell surface expression of MHC or costimulatory molecules. Another possibility is that phenotypic differences between Ad-infected and noninfected lung DCs would only be apparent following coculture with T cells. Interestingly, the majority of known Ad immunoevasins, whose actions include decreasing MHC-I expression and inhibiting apoptosis and the host antiviral response, are encoded in the E1 and E3 regions (5, 7, 19, 29), which have been deleted from the Ad used in these experiments. Thus, data showing that UV irradiation abrogated Ad-induced suppression suggest that a viral protein may be mediating the suppressive activity in DCs. Studies are being undertaken to determine which specific viral region or protein is mediating the suppression and if this protein disrupts signaling required for T-cell activation and proliferation.

AdNull-infected lung DCs were not found to be producing IDO or producing other soluble factors able to suppress T-cell proliferation. These results were consistent with flow cytometry data showing no difference between Ad-infected and noninfected DCs in their production of IL-12, IFN-α, IFN-γ, and IL-10. Additionally, there were equivalent levels of IL-2 in noninfected and Ad-infected DC-T-cell coculture supernatants, indicating that there was sufficient IL-2 available for T-cell priming. This suggests that the decrease in the ability of DCs to stimulate T-cell proliferation is a contact-dependent phenomenon.

Recent studies have demonstrated a strong link between virus infection and the activation of regulatory T cells, resulting in modulation of the immune response. Feline immunodeficiency virus (46), herpes simplex virus (41), and human immunodeficiency virus (1) increase Treg cell function and suppress the antiviral immune response. In addition, depletion of Treg cells prevents disease progression in the murine AIDS model (4) and exacerbates herpes simplex virus-induced ocular lesions through the increase in IFN-γ-producing T cells that infiltrate the area (40). However, T cells previously cocultured with Ad-infected DCs were not regulatory T cells, as adding them back to a fresh MLR culture did not suppress T-cell proliferation (Fig. 8B). Although T cells cultured with Ad-infected lung DCs were not suppressive in secondary cultures with noninfected T cells and APCs, they were hyporesponsive to stimuli. Further examination revealed that the proliferation of these T cells was recovered by the addition of IL-2 (Fig. 8A). These data suggest that Ad-infected DCs induce nonresponsiveness to IL-2 in responding T cells during primary coculture. Proliferation studies using lung T cells from noninfected and Ad-infected mice showed decreased T-cell proliferation following Ad infection, which correlates with our in vitro findings.

The data reported here may explain the results of another study examining the role of TNF-α in the clearance of bacterial pneumonia. Standiford et al. (39) reported that mice i.t. instilled with a similar replication-deficient Ad vector had increased mortality following a subsequent challenge with Klebsiella pneumoniae than mice infected with Ad containing the murine TNF-α transgene or noninfected controls. In fact, these animals infected with control vector had an increased bacterial load in their lungs and plasma, showing impaired bacterial clearance abilities. While the mechanism of impaired immunity to the pathogen was not a focus of that report, based on data in the current study we hypothesize that the inability to clear the K. pneumonia in the empty Ad vector-treated lungs was due to Ad-induced suppression of DC function.

The findings in this study showing that Ad infection of lung DCs induces a suppression of their APC function both in vitro and in vivo has important implications. First, these results highlight the importance of using primary tissue-derived cells and performing in vivo infections at the site of natural infection to get a clearer picture of disease progression. Our studies suggest that Ad lung infection may lead to a state of local immunoincompetence via infection of DCs. This could be a contributing mechanism through which pulmonary disorders such as postviral pneumonias develop. Secondly, these results must be considered when evaluating Ad vectors as a therapy for lung diseases. Ad vectors are an attractive option, as Ad is tropic to the epithelium. However, as our experiments show, Ad infection of lung DCs suppresses their function as stimulators of immune responses. These data suggest that using Ad vectors for gene therapy in the lung may result in compromises in the patient's pulmonary immunity, possibly increasing the patient's risk to subsequent infection and disease.

Acknowledgments

The authors thank the personnel in the laboratories of Mark Pescovitz, Homer Twigg, and Janice Blum, as well as Pat Smith for technical assistance.

Mass spectrometry (or two-dimensional gel electrophoresis) was provided by the Indiana Centers for Applied Protein Sciences (INCAPS) with support in part from the Indiana Genomics Initiative and the Indiana 21st Century Research and Technology Fund. The Center for Immunobiology and Center of Biologic Microscopy are in part supported by the Indiana Genomics Initiative of Indiana University, which is supported in part by Lilly Endowment, Inc. This work was supported by grants (to D.S.W.) from the National Institutes of Health (HL60797). A.T.T. was supported in part by a postgraduate scholarship from the National Science and Engineering Research Council of Canada (PGSB-242760-2001) and a doctoral research award from the Canadian Institutes of Health Research. T.L.S. was supported by the National Institutes of Health, grant NRSA 1 T32 AI 060519 of the Immunology and Infectious Disease Program at IUSM.

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