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The Journal of Immunology, 2008, 181, 6872 -6881
Copyright © 2008 by The American Association of Immunologists, Inc.
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Antiviral-Activated Dendritic Cells: A Paracrine-Induced Response State1

Antonio V. Bordería2,*, Boris M. Hartmann2,{dagger}, Ana Fernandez-Sesma*, Thomas M. Moran* and Stuart C. Sealfon3,{dagger},{ddagger}

* Department of Microbiology, {dagger} Department of Neurology, and {ddagger} Center for Translational Systems Biology, Mount Sinai School of Medicine, New York, NY


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Infection of immature dendritic cells (DCs) by virus stimulates their maturation into APC. Infected DCs can also expose uninfected DCs to a panoply of cytokines/chemokines via paracrine signaling. Mathematical modeling suggests that a high rate of paracrine signaling is likely to occur among DCs located in three-dimensional space. Relatively little is known about how secreted factors modify the early response to virus infection. We used a transwell experimental system that allows passage of secreted factors, but not direct contact, between virus-infected DCs and uninfected DCs to investigate paracrine signaling responses. Paracrine signaling from infected DCs induced an antiviral-primed DC state distinct from that of mature virus-infected DCs that we refer to as antiviral-activated DCs (AVDCs). AVDCs had increased surface MHC class II and CD86 levels, but in contrast to virus-infected DCs, their MHC class I levels were unchanged. Imaging flow cytometry showed that AVDCs had an increased rate of phagocytosis compared with naive DCs. Experiments with IFN-β cytokine indicated that it may be responsible for CD86, but not MHC class II regulation in AVDCs. Both IFN-inducible and IFN-independent genes are up-regulated in AVDCs. Notably, AVDCs are relatively resistant to virus infection in comparison to naive DCs and achieve accelerated and augmented levels of costimulatory molecule expression with virus infection. AVDCs show a distinct antiviral-primed state of DC maturation mediated by DC paracrine signaling. Although further in vivo study is needed, the characteristics of the AVDC suggest that it is well suited to play a role in the early innate-adaptive transition of the immune system.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Dendritic cells (DCs)4 are recognized as a key bridge between the innate and adaptive immune responses (1). A key event in the development of adaptive immunity upon exposure to infection is the maturation of DCs into APCs that instruct lymphocytes to generate responses to specific Ags. Activated DCs efficiently stimulate both innate immune cells, including NK cells (2) and NKT cells (3) as well as key components of adaptive immunity including naive (4) and memory (5) B cells and T cells (1). Thus, DCs are important both for innate immunity as well as for various elements of adaptive immunity (6).

Our study focuses on DC activation by virus infection, using Newcastle disease virus (NDV), a RNA paramyxovirus that has been demonstrated to be a good model for immune activation (7). DC maturation is stimulated by detection of various pathogen-associated molecular patterns (1) that are characteristic of bacteria, fungi, protozoa, or viruses. DCs recognize virus infection either by TLRs or by TLR-independent intracellular viral product detectors such as RIG-I (8) and/or MDA5 (9, 10). Virus recognition activates a signaling cascade involving different cellular factors (IFN regulatory factor 3, NF-{kappa}B, c-jun), causing the expression of type I IFNs and other inflammatory response genes including TNF-{alpha} and IL-6. The first type I IFN to be produced and secreted is IFN-β (11), which signals either in an autocrine or paracrine manner through the IFN receptor (IFNAR) and activates the JAK-STAT pathway (12). This signaling cascade further amplifies the initial response and creates an antiviral state in adjacent cells that renders them resistant to infection.

Maturation is a complex process, which includes changes in morphology, loss of endocytic/phagocytic receptors, up-regulation of costimulatory molecules, such as CD86, translocation of MHC compartments to the surface, and secretion of cytokines and chemokines (13) that attract, differentiate, and polarize other immune effector cells (6). Secretion of chemokines occurs in coordinated waves according to the type of immune cells that need to be attracted and activated (13). One late component associated with maturation is the migration of the DCs to the secondary lymphoid organs (14), where they interact with the naive T and B cells. This activation of Ag-specific T cells by mature DCs is a major aspect of the initiation of adaptive immunity.

The secretion of the different cytokines and chemokines affects other immune cells, including immature DCs, by paracrine signaling. Consequently, some DCs might be exposed to both cytokines and microbial products (11), whereas others only to inflammatory cytokines. Autocrine signaling is regarded as an important mechanism for virus- triggered DC maturation. Integrodifferential modeling of IFN trajectories suggests that ~3% of IFN-β interacts with the DCs that produce it (15).

We investigated the effects of paracrine signaling by DCs on the response state of DCs that are not infected by virus using NDV, which is detected primarily through the cytosolic RIG-I molecule (16). To study paracrine effects, we used a Transwell system which is composed of two chambers separated by a membrane that allows soluble components such as cytokines and chemokines to diffuse between chambers, but prohibits direct contact between the cells placed in different chambers. DCs infected with NDV and naive noninfected DCs were placed in the upper and lower chamber, respectively. The culture was left for 18 h, allowing the infected DCs to initiate cytokine and chemokine secretion. We found that the naive DCs exposed to the specific cytokine/chemokine secretions released by infected DCs enter a partially activated state in which they are relatively resistant to virus infection and primed to generate a more rapid and enhanced response to virus infection.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Differentiation of DCs

All human research protocols for this work have been reviewed and approved by the Institutional Review Board of the Mount Sinai School of Medicine. Monocyte-derived DCs were obtained from healthy human blood donors following a standard protocol described elsewhere (7). All experiments were replicated using cells obtained from different donors. Briefly, human PBMC were isolated from buffy coats by Ficoll density gradient centrifugation (Histopaque; Sigma-Aldrich) at 1450 rpm and CD14+ monocytes were immunomagnetically purified by using a MACS CD14 isolation kit (Miltenyi Biotec). Monocytes were then differentiated into naive DCs by 5- to 6-day incubation at 37°C and 5% CO2 in DC growth medium, which contains RPMI 1640 medium (Invitrogen/Life Technologies) supplemented with 10% FCS (HyClone), 2 mM l-glutamine, 100 U/ml penicillin, and 100 g/ml streptomycin (Invitrogen), 500 U/ml human GM-CSF (PeproTech), and 1000 U/ml human IL-4 (PeproTech).

Virus preparation and viral infection

The recombinant Hitchner strain of NDV (rNDV/B1) and NDV/(GFP) were generated in Prof. P. Palese’s laboratory (Mount Sinai School of Medicine, New York, NY) (17, 18) and grown in 9-day embryonated chicken eggs as described previously (17). Recombinant NDV-red fluorescent protein (RFP) was obtained from Prof. A. Garcia-Sastre’s laboratory (Mount Sinai School of Medicine, New York, NY) (19) and grown and titrated similarly to NDV/B1 and NDV-GFP. NDV viruses were titrated by immunofluorescence 18 h after infection of Vero cell plates using mAbs specific for NDV-HN protein generated by the Mount Sinai Hybridoma Core Facility followed by addition of anti-mouse IgG-FITC and visualization using fluorescent microscopy. For infection of naive DCs, NDV stocks were appropriately diluted in DMEM and added directly into pelleted DCs at a multiplicity of infection of 1 (7, 20). After incubation for 40 min at 37°C, fresh DC growth medium (without GM-CSF and IL-4) was added back to the infected cells (1 x 106 cells/ml) for the remainder of the infection. Naive noninfected DCs underwent the same experimental procedure as infected DCs in the absence of virus to ensure that mechanical manipulations could not be responsible for differences in experimental readouts.

Generation of antiviral-activated DCs (AVDCs)

AVDCs were generated by using a Transwell system. The Transwell system consists of an upper and a lower chamber separated by a 0.4-µm polyethylene terephtalate membrane (Millipore) that allows diffusion of cytokines and chemokines through the membrane but avoids the interaction of the cells in both chambers. To generate the AVDCs, naive DCs were infected as described above. After the 40-min incubation, the cells were washed with PBS and cultured in the Transwell system. Infected and noninfected DCs were allocated in the upper and lower chamber, respectively. Two independent wells were set up with infected or naive noninfected DCs as positive and negative controls. The cultures were incubated at 37°C in 5% CO2 for 18 h. All cells were then washed in PBS and harvested for flow cytometry analysis and RNA isolation. The supernatant was kept at –20°C for ELISA analysis of cytokines/chemokines.

Proteinase K treatment

Supernatants derived from NDV-infected DCs for 18 h were treated with proteinase K (MP Biomedicals) for 2 h at 37°C to digest cytokines and chemokines. The proteinase K was then inactivated with a brief heat treatment. Proteinase K-treated and nontreated supernatants were placed in the upper compartment of the Transwell system with naive DCs in the lower compartment to mimic the condition during AVDC generation. We incubated cells for 18 h at 37°C. Naive DCs in the lower compartment were then stained for the maturation markers MHC class I (MHC-I), MHC class II (MHC-II), and CD86.

Culture of human lung fibroblast

Lung epithelial fibroblasts were obtained from Lonza and cultured according to the supplier’s instructions. Briefly, cells were passed when confluence was 70–80% using fibroblast basal medium supplemented with human fibroblast growth factor β, insulin, FBS, and gentamicin/amphotericin B. To perform experiments in the Transwell system, 3 x 105 cells were seeded 8 h before treatment in the Transwell system with DC medium and left at 37°C in the incubator. Fibroblasts were infected with NDV at a multiplicity of infection of 1 and left in the incubator for 40 min. Cells were then washed with PBS and fresh DC medium was added to the culture. Naive DCs were added in the upper compartment and left in the Transwell system for 18 h in the incubator.

IFN-β treatment

DCs were generated as described previously and IFN-β (PBL IFN Source) was added to the culture in a concentration of 2000 U/ml. The culture was left for 18 h in incubation as described before. After the incubation, the cells were washed with PBS and harvested for flow cytometry analysis.

Quantitative RT-PCR

Viral and host RNA expression levels were quantified by real-time RT-PCR. RNA was isolated from cells using a Qiagen Micro RNeasy kit according to the manufacturer’s protocol. cDNA was synthesized from total RNA with AffinityScript MultiTemp RT (Stratagene) with oligo(dT)18 as primer. For real-time PCR, PlatinumTaq DNA polymerase (Invitrogen) and a SYBR Green (Molecular Probes)-containing buffer were used. The real-time PCR was performed using a thermocycler (ABI7900HT; Applied Biosystems) as previously described (21). The RNA levels for the housekeeping genes ribosomal protein S11 (Rps11), tubulin (Tuba), and ß-actin (Actb) were also assayed in all samples to be used as an internal controls. mRNA measurements were normalized using a robust global normalization algorithm. All control crossing threshold (Ct) values were corrected by the median difference in all samples from Actb. All samples were then normalized by the difference from the median Ct of the three corrected control gene Ct levels in each sample, with the value converted to a nominal copy number per cell by assuming 2500 Actb mRNA molecules per cell and an amplification efficiency of 93% for all reactions. The primer sequences used for the assays were: NDV-HN sense, 5'-GACAATGCTTGATGGTGAAC-3' and antisense, 5'-CAATGCTGAGACAATAGGTC-3'; NDV-NP sense, 5IFN-ß sense, 5'-GTCAGAGTGGAAATCCTAAG-3' and antisense, 5'-ACAGCATCTGCTGGTTGAAG-3'; IFN-{alpha}1 sense, 5'-CTGAATGACTTGGAAGCCTG-3' and antisense, 5'-ATTTCTGCTCTGACAACCTC-3'; PKR sense, 5'-TTGTACCACAAGAGAGAGTG-3' and antisense, 5'-AGTGCTGTCCCTCAAGACTC-3'; OAS-1 sense, 5'-TTTGATGCCCTGGGTCAGTT-3' and antisense, 5'-GTGCTTGACTAGGCGGATGA-3'; TNF-{alpha} sense, 5'-GAGGAAGGCCTAAGGTCCAC-3' and antisense, 5'-AGTGAAGTGCTGGCAACCAC-3'; RIG-I sense, 5'-AAAGCCTTGGCATGTTACAC-3' and antisense, 5'-GGCTTGGGATGTGGTCTACT-3'; RANTES sense, 5'-AAGCTCCTGTGAGGGGTTGA-3' and antisense, 5'-TTGCCAGGGCTCTGTGACCA-3'; IL-6 sense, 5'-CTGAGGTGCCCATGCTACAT-3' and antisense, 5'-AATGCCAGCCTGCTGACGAA-3'; IP-10 sense, 5'-TCCCATCACTTCCCTACATG-3' and antisense, 5'-TGAAGCAGGGTCAGAACATC-3'; MXA sense, 5'-CGTGGTGATTTAGCAGGAAG-3' and antisense, 5'-TGCAAGGTGGAGCGATTCTG-3'; Rps11 sense, 5'-CGAGGGCACCTACATAGACA-3' and antisense, 5'-GAGATAGTCCCGGCGGATGA-3'; Actb sense, 5'-GCCTCAACACCTCAAACCAC-3' and antisense, 5'-CCACAGCTGAGAGGGAAATC-3'; and Tuba sense, 5'-AGCGCCCAACCTACACTAAC-3' and antisense, 5'-GGGAAGTGGATGCGAGGGTA-3'.

Flow cytometry analysis

Cells were fixed with 1.5% paraformaldehyde (Sigma-Aldrich), washed with FACS staining buffer (Beckman Coulter), and stained with mAbs for MHC-I, MHC-II, and CD86. NDV-GFP- or NDV-RFP-infected cells were stained with Abs for MHC-I, MHC-II, and CD86 (Beckman Coulter). Cells were assayed on an LSRII flow cytometer (Beckman Coulter) and data were analyzed with the FlowJo software (Tree Star). To provide higher throughput and reduce cell requirements per assay, we used a modified bar-coding method previously described by Nolan et al. (22). Briefly cells were fixed, then stained in DMSO containing different combinations of 0, 0.3, 1, 4, or 15 µg/ml Pacific Blue-NHS, 0, 1.25, 5, or 20 µg/ml Alexa Fluor 350-NHS, and 0, 4, or 20 µg/ml Alexa Fluor 750-NHS for 15 min at 20–25°C (see Fig. 9A). Fluorescence minus 1 controls were obtained by staining naive DCs with all fluorochromes studied excluding the fluorochrome of interest, conjugated Ab against CD45 (23).

Imaging flow cytometry analysis of phagocytosis and morphology

Imaging flow cytometry was used to compare the morphology and phagocytosis levels of AVDCs and naive DCs. To detect phagocytosis, 1-µm 488-nm fluorescence-labeled latex microspheres (Polysciences) at a concentration of 50 beads/cell were cocultured for 2 h at 37°C with each cell type. Single-cell images were acquired using extended depth field imaging distortion to identify beads in different focal planes within a cell. The numbers of beads incorporated by cells were quantified in the images captured using image analysis software (IDEAS Software; Amnis). The distributions were compared using the Kolmogorov-Smirnoff technique.

To measure the cellular morphology, AVDCs, naive DCs, and infected DCs were labeled with a live membrane dye, benzoxazolium, 3-octadecyl-2-[3-(3-octadecyl-2(3H)-benzoxazolylidene)-1-propenyl]-, perchlorate (DiO), following the manufacturer’s protocol (Invitrogen). Samples were then individually acquired by imaging flow cytometry. To analyze the morphology, an algorithm described by Haralick et al. (24), which describes the homogeneity of an given image, was used (IDEAS Software; Amnis).

Multiplex ELISA

Four different cytokines/chemokines (IL-6, IFN-{gamma}-inducible protein 10 (IP-10), TNF-{alpha}, and IFN{alpha}) concentrations were assayed in the culture medium. To minimize the supernatant volume to assay, a Beadlyte Human Multiplex ELISA analysis (Millipore) was used per the manufacturer’s instructions. Briefly, 100 µl from each compartment per well was incubated in a 96-well filter polyvinylidene difluoride 1.2-µm plate specially designed to retain cytokines/chemokines, with a mixture of anti-cytokine IgG-conjugated beads for the different cytokines/chemokines assayed. After 2 h of incubation, the plate was filtered and washed three times with assay solution (PBS (pH 7.4) containing 1% BSA, 0.05% Tween 20, and 0.05% sodium azide). The washes were followed by a 1.5-h incubation with biotin-conjugated anti-cytokine IgG. After assay solution washing, streptavidin-PE was added followed by addition after 30 min of stop solution (0.2% (v/v) formaldehyde in PBS (pH 7.4). The plate was then filtered and each well was resuspended in 125 µl of assay buffer and read in a Luminex 100 machine.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Effectiveness of Transwell chambers to study paracrine signaling

To isolate the effects of direct infection by virus from that of paracrine signaling on naive DCs, we used a Transwell culture system. Naive, immature DCs were infected with NDV and immediately placed in the upper chamber. Noninfected naive DCs were placed in the lower chamber. The chambers were partitioned by a 0.4-µm pore membrane that allowed the diffusion of cytokines and chemokines (Fig. 1). After incubation, cells in each chamber were recovered and analyzed for their surface marker and gene expression patterns.


Figure 1
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  		FIGURE 1. Experimental setup for the generation of AVDCs.

 
DCs do not support the productive infection of NDV (25). To ascertain whether viral particles could infect cells in the lower chamber during these experiments, we performed control experiments with a recombinant NDV containing a GFP-tagged HN protein (17). Infection of cells was assayed by flow cytometry. When NDV-GFP-infected cells were incubated in the upper chamber for 18 h, GFP fluorescence was detected only in cells from the upper, but not the lower chamber, indicating that the experimental system effectively separated infected and uninfected DCs (Fig. 2).


Figure 2
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  		FIGURE 2. Expression of viral GFP protein and maturation markers in infected DCs, AVDCs, and noninfected DCs. Histograms show fluorescence intensity of cells for the viral GFP-tagged protein NDV-HN and the fluorochrome-conjugated Abs for CD86, HLA-ABC (MHC-I), and HLA-DR (MHC-II). Blue histograms indicate noninfected DC controls. Red histograms indicate infected DCs in the Transwell system (A), AVDCs (B), and infected DCs as positive control (C). The data shown are representative of three different experiments using three different donors that showed similar results.

 
As a further test of the isolation of virus infection, the induction of the NDV-HN and NDV-NP viral genes and virus-induced type I IFN genes were compared in membrane-separated cocultured cells using real-time PCR assays. Both viral genes were present in virus-infected DCs from the upper chamber and were undetectable in noninfected control DCs and in DCs coincubated in the lower chamber (Fig. 3). Viral components lead to activation of type I IFN via TLR activation. If uninfected cells were exposed to viral components that passed through the membrane, they would be expected to show induction of type I IFNs. Therefore, we also assayed the expression of IFN-β and IFN-{alpha}1 as a sensitive assay of exposure to very small amounts of virions, viral RNA, debris, or other small particles that might cross the membrane (26). Using a sensitive real-time PCR assay, we found that both IFNs were up-regulated in virus-infected DCs from the upper chamber, but were not induced in naive cells incubated in the lower chamber (Fig. 3). The results of flow cytometry and real-time PCR indicate that the culture system completely eliminates direct virus infection of uninfected, membrane-isolated cocultured cells. To test further the possibility that viral components such as RNA caused the surface marker up-regulation pattern observed (see below), we tested the effect of proteinase K digestion of supernatants from infected DCs on its capacity to affect naive DCs. Proteinase K- treated supernatant did not up-regulate the surface markers of DCs in the other Transwell chamber. Supernatants from infected DCs that were not digested with proteinase K induced a similar surface marker pattern to that seen in AVDCs in the Transwell system (supplementary Fig. S1).5 Thus, any responses of the uninfected DCs in the lower chamber were the results solely of paracrine signaling initiated by infected DCs in the upper chamber.


Figure 3
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  		FIGURE 3. Quantitative RT-PCR of RNA expression for virus and type I IFN genes in infected control DCs (Inf-DCs), infected DCs from the Transwell cultures, AVDCs, and noninfected naive DCs (N-DCs) genes to viral infection. The results shown are averages of three independent experiments performed using different donors.

 
Effects of DC paracrine signaling on maturation markers

Several cellular surface proteins have been described as maturation markers of DCs, including CD86, MHC-I, and MHC-II (6). There is an increase in the density of these markers in the cellular membrane of the DCs upon activation and maturation. In the coculture Transwell system, upper chamber-infected DCs and the positive control DCs showed a comparable up-regulation of MHC-I, MHC-II, and CD86 molecules (Fig. 2, A and C), indicating the maturation of those cells. Interestingly, lower chamber DCs in the coculture Transwell system also showed an up-regulation of MHC-II and CD86 molecules, while MHC-I remained at levels equivalent to the noninfected control DC levels (Fig. 2B).

To determine whether the changes in naive DCs induced by infected DCs were unique to infected DCs, we studied the effects of secreted factors from NDV-infected primary lung fibroblasts on naive DCs using the Transwell system. Our results showed that the presence of infected primary lung fibroblasts instead of infected DCs in the same Transwell system did not induce a comparable surface marker expression pattern in the naive DCs (supplementary Fig. S2). Furthermore, noninfected control DCs did not up-regulate any of the surface markers, even if cells were maintained in the Transwell system for periods up to 48 h (supplementary Fig. S2). These results indicate that the experimental manipulations alone were not affecting maturation marker up-regulation. Our results suggest that paracrine signaling alone between infected and uninfected DCs produces an increase of some DC maturation markers, leading to a unique DC state that differs both from naive DCs and from fully matured virus-infected DCs.

Levels of paracrine cytokines

We assayed cytokines that could contribute to paracrine activation of DCs. Factors secreted by DCs for which DCs expressed receptors include IL-6, TNF-{alpha}, IP-10, and type I IFN. Measurement of the levels of these cytokines in upper and lower chambers by ELISA during a coculture experiment indicated that high levels of all four cytokines were achieved in the lower chamber (Fig. 4).


Figure 4
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  		FIGURE 4. Multiplex ELISA for IL-6, IP-10, TNF-{alpha}, and IFN{alpha} levels in supernatants from the chambers containing infected DCs, AVDCs, and noninfected naive DCs (N-DCs) genes to viral infection, all in the Transwell system. The results shown are averages of three independent experiments performed using different donors. Inf-DCs, Infected DCs.

 
Gene program response in AVDCs

To further understand the characteristics of the paracrine-generated AVDC state, the regulation of a variety of genes was assayed by real-time PCR in AVDCs, NDV-infected DCs, and naive DCs. Paracrine and autocrine IFN acting at the type I IFN receptor activates a number of genes through JAK-STAT signaling, including protein kinase R (PKR), 2'-5'-oligoadenylate synthetase (OAS), myxovirus resistance A (MxA), and IP-10. Consonant with the detection of high levels of IFN-{alpha} in the medium by cytokine assays (Fig. 4), AVDCs showed robust induction of these IFN-activated genes (Fig. 5). OAS and MxA showed the same levels in the lower chamber as in infected DCs. Although PKR and IP-10 were lower than in infected DCs, they were still significantly induced in comparison with noninfected DCs. IFN is a key paracrine signaling factor during virus infection and has long been recognized to generate complex transcriptional responses (7, 26). However the state of AVDCs appears to result from additional factors besides IFN, because genes such as TNF-{alpha}, which are not inducible by IFN are up-regulated in AVDCs (Fig. 5). The hypothesis that additional secreted factors contribute to the AVDC state was further supported by comparing the pattern of maturation markers in AVDCs and in IFN-β-treated naive DCs. The induction of the costimulatory molecule CD86 was similar in AVDCs and IFN-exposed DCs, suggesting that this regulatory event may result from IFN signaling. However, the increased expression of MHC-II was seen only in AVDCs (Fig. 6). Overall, the gene response patterns and maturation marker analysis indicate that type I IFN signaling represents only part of the paracrine environment necessary for the generation of AVDCs.


Figure 5
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  		FIGURE 5. Quantitative RT-PCR of antiviral and inflammatory gene expression in infected control DCs (Inf-DCs), infected DCs from the Transwell cultures, AVDCs, and noninfected naive DCs (N-DCs) genes to viral infection. The results shown are averages of three independent experiments performed using different donors.

 

Figure 6
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  		FIGURE 6. Flow cytometry analysis studying the involvement of IFN-β signaling in the generation of AVDCs. CD86, MHC-I, and MHC-II were assayed and the results are shown in the different columns. Blue indicates treatment of naive DCs with IFN-β cytokine, green indicates AVDCs, and red-infected DCs as control The data shown are representative of three different experiments using three different donors that showed similar results.

 
Quantification of AVDC morphology and rate of phagocytosis

The morphology of AVDCs and naive DCs was compared using imaging flow cytometry. Cells were stained with a live-cell membrane-localized dye, DiO, and morphology was measured using the texture analysis algorithm developed by Haralick et al. (24) as implemented in IDEAS software (see Materials and Methods). By this method, the spatial relationships between the texture features and the pixel values in an image were measured, and an H homogeneity mean and a SD value were obtained for each set of cells. The H homogeneity is a measure of the average shape of a cell. Representative raw images are shown in Fig. 7A. These analyses reveal that the average morphology of AVDCs is different from that of naive DCs. AVDCs showed an increase in textural homogeneity compared with naive cells that was comparable to the level seen in infected DCs (Fig. 7B).


Figure 7
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  		FIGURE 7. Homogeneity test to show differences in morphological shapes. A, Representative micrographs of naive DCs, AVDCs, and infected control DCs (Inf-DCs) captured using an imaging flow cytometry. B, Histogram of the homogeneity value of naive DCs, AVDCs, and infected DCs. ***, p < 0.0001 by Mann-Whitney U test in comparison with naive DCs. The results shown are averages of three independent experiments performed using different donors. N-DCs, Noninfected DCs.

 
The rates of phagocytosis of naive DCs and AVDCs were also compared using high-resolution imaging flow cytometry. Both cells types were incubated for 2 h with 1-µm 488-nm fluorescence-labeled latex microspheres. The number of particles taken up by each cell was quantified in approximately several thousand cells in each group using automated image analysis (Fig. 8A). In three independent experiments using cells from different donors, AVDCs showed significantly higher rates of phagocytosis.


Figure 8
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  		FIGURE 8. Phagocytosis of microspheres. A, Representative images showing examples of cells containing between one and seven beads imaged using enhanced depth of field-equipped imaging flow cytometry. B, Distribution of number of microspheres counted in naive DCs (n = 2000) and AVDCs (n = 2700). The distributions are statistically different (p < 10–13). The data shown are representative of three different experiments using cells from three different donors that showed similar results. N-DCs, Noninfected DCs.

 
AVDCs resist viral infection and showed enhanced generation of maturation markers by infection

We next investigated whether AVDCs differ from naive DCs in their response to direct virus infection by NDV. For this purpose, AVDCs generated by coculturing with NDV-GFP-infected DCs for 18 h using the Transwell system and naive DCs were exposed to a recombinant NDV virus expressing RFP (NDV-RFP) (19). The level of RFP signal generated over 12 h was assayed by flow cytometry, as a reflection of the generation of intracellular viral protein within the DCs following direct infection. To allow accurate comparison of the level of signal in different samples, a bar-coding flow cytometry approach was used (22). In this approach, each sample was first labeled with a characteristic pattern of three deconvolution dyes (Fig. 9A) and then the 60 mixed samples were labeled with fluorescently labeled surface marker Abs. Detection of virus protein expression and surface marker expression in each sample was then assayed in a single multispectral flow cytometry run. This analysis showed that AVDCs generated much lower levels of viral protein with infection than naive DCs (Fig. 9B). These results indicate that, in comparison to naive DCs, the AVDCs showed a relatively virus-resistant state.


Figure 9
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  		FIGURE 9. AVDCs resist viral infection. A, Bar coding for flow cytometry analysis. Cells were stained with different combinations of 0, 0.3, 1, 4, or 15 µg/ml Pacific Blue-NHS, 0, 1.25, 5 or 20 µg/ml Alexa Fluor 350-NHS, and 0, 4, or 20 µg/ml Alexa Fluor 750-NHS to enable 60 different conditions in one FACS run. B, Time course results for NDV-RFP expression following infection in naive DCs and AVDCs. C, Time course for expression of MHC-I, MHC-II, and CD86 surface markers in the same cells studied in A and B. The results shown are representative of two independent experiments using cells from different donors.

 
Interestingly, AVDCs also expressed much higher levels of maturation markers than naive DCs following virus infection. Time course analysis by flow cytometry showed that the levels of CD86, MHC-I, and MHC-II were dramatically higher in infected AVDCs at all time points than in infected naive DCs (Fig. 9C). Notably, this enhanced activation state included both the CD86 and MHC-II markers that were increased in AVDCs without infection as well as the MHC-I signal that was unchanged in AVDCs before virus infection. These results indicate that the paracrine signaling environment cause the DCs to enter a state in which they are relatively resistant to infection by virus and their maturation response to infection is greatly enhanced.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
These experiments reveal that paracrine signals from virus-infected cultured human DCs act on naive uninfected DCs to induce an activated antiviral state. This AVDC state is characterized by basal up-regulation of CD86 and MHC-II cell surface markers, induction of a variety of antiviral response genes, altered morphology, and increased basal phagocytosis. The antiviral state of these AVDCs is evident from their relative resistance to virus infection and their enhanced rate and level of maturation marker expression following infection. These features of the AVDC are well suited to facilitate the development of adaptive immunity to virus infection.

The Transwell experimental system with which we characterized the AVDC, while used in various studies of immune cell differentiation (27), has not previously been used to study paracrine signaling among immune cells. The pore size of the polyethylene terephtalate membranes present in our experiments would not be expected to restrict virus particles or components. NDV does not cause productive viral infection in DCs (24). Nevertheless, we tested whether the Transwell culture system might effectively separate the paracrine-stimulated naive DCs from the effects occurring with direct encounter with virus. Using sensitive assays for expression of fluorescent viral protein and stimulation of virus infection-dependent gene expression (viral proteins and type I IFN induction), we demonstrated that the change in state seen in the AVDC results solely from the transmission of secreted factors and that AVDCs do not show any signs of infection by the virus or exposure to viral components.

Since the discovery of the effects of type I IFN more than 50 years ago, these cytokines have been well studied as inducers of a variety of antiviral responses that promote the development of DC maturation (28). We have shown previously that NDV infection of human DCs results in a strong activation pattern, including production of type I IFN, TNF-{alpha}, and other proinflammatory cytokines as well as IFN-inducible genes (7). We find that NDV-infected DCs, located in the upper chamber of the Transwell system, also secrete TNF-{alpha}, IL-6, and IP-10, which have receptors present on DCs and are therefore also potential paracrine factors (see Fig. 4). Several previous studies have considered the role of uninfected or "bystander" DCs in the response to virus infection (28, 29, 30). However, the effects of paracrine signaling on these bystander cells has either not been evaluated or attributed entirely to type I IFN. Therefore, we were interested in determining the role of type I IFN in generating the AVDC state that we have identified. We found that only some of the characteristics of AVDCs can be attributable to type I IFN signaling, indicating that the state of these cells reflects more complex, multifactor paracrine signals. The identification of which combinations and concentrations of factors are responsible for generating the AVDC state is an interesting subject for further investigation.

Pretreatment of immature DCs and/or DC precursors with single cytokines that are secreted either by DCs or other immune cells has been reported to lead to distinct cellular phenotypes. The effects of type I IFN (31, 32, 33), thymic stromal lymphopoietin (34, 35), TNF-{alpha} (36), IL-10 (37, 38), IFN-{gamma} (39), and IL-15 (6, 40, 41, 42) have all been reported. During infection of a host, the extracellular environment of the DCs in the infected tissue in vivo contains multiple cytokines and chemokines and the response state of the uninfected DCs is likely to result from more than a single factor. Understanding the mechanisms underlying the effects of the integration of multiple signals has been the object of several experimental and computational systems biology studies (43, 44, 45). Recently, we have begun to develop models to describe paracrine and autocrine signaling among DCs located in three-dimensional spaces (15). Understanding the mechanisms by which AVDCs are generated is facilitated by studies integrating mathematical simulation with cytokine interaction studies.

Paracrine and autocrine signaling are recognized to contribute to the maturation of DCs following TLR stimulation (42, 46, 47, 48, 49). Spörri and Reis e Sousa (49) found that paracrine signaling cannot substitute for contact for TLR-mediated contact with pathogen components in generating fully activated DCs. To our knowledge, the investigation of the role of paracrine signaling in modulating the DC response to subsequent encounter with virus, a sequence likely to occur during actual infection, has not been previously investigated.

During the early stages of infection, only a few cells are infected. In the case of respiratory virus, inhalation of only a few droplets containing virus particles can be sufficient to induce a successful infection, and virus transmission usually involves few viral particles (50). Thus, early in the infection, only a few DCs are likely to come into contact with virus or with virus-infected tissue. Therefore, we hypothesize that paracrine signaling events capable of inducing the generation of AVDCs are likely to play an important role in the early response stages of the immune system to virus infection (Fig. 10). Infection of primary lung fibroblasts was not able to generate AVDCs (supplementary Fig. S2), indicating that the AVDC is not generated by exposure to secreted factors from any cell type. Pathogenic viruses express immune antagonists that prevent the activation of innate immune responses in infected cells (51, 52, 53). AVDCs could be able to respond even in the face of a viral antagonist due to their heightened activated state. Results obtained in cultured cells can be extrapolated only cautiously to the intact organism. However, characterizing the state of these cells and the mechanisms underlying their induction is important to set the stage for investigating the presence and role of AVDCs using in vivo models.


Figure 10
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  		FIGURE 10. Schematic of the possible role of AVDCs in developing adaptive immunity. The paracrine signals released by DCs that are first infected by virus generate AVDCs that are primed to resist virus and to develop into APCs.

 
In conclusion, the characteristics of the AVDCs suggest that they are in an advanced state of readiness, but also capable of resisting the negative effects of the virus infection. In the case of human host viruses, the formation of AVDCs may provide an important process to generate APCs capable of overcoming the effect of viral protein inhibitors on the maturation of DCs. AVDCs were relatively resistant to virus infection and showed induction of several genes, including RIG-I, PKR, and OAS, that promote virus resistance. More importantly for their proposed role in the transition from innate to adaptive immunity, they showed an accelerated rate of maturation when infected and an increased level of expression of maturation markers needed for productive interaction with T cells. The increased phagocytosis rate and increased morphological texture of AVDCs also make them suitable for heightened antiviral surveillance and response.


   Acknowledgments
 
We thank Mathieu Coppey and Stas Y. Shvartsman for fruitful discussions. We also thank Gary Nolan for organizing the advanced flow cytometry workshop. We thank Peter Palese, Adolfo Garcia-Sastre, and Luis Martinez-Sobrido for generously providing the viral chimeras and Esther Rhee and Nada Marianovic for technical assistance. We thank the Mount Sinai flow cytometry and the Microarray, PCR, and Bioinformatics Shared Research Facilities for assistance with these studies.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by Contract HHSN266200500021C and Grant U19 AI06231 from the National Institute of Allergy and Infectious Diseases. Back

2 A.V.B. and B.M.H. contributed equally to this work. Back

3 Address correspondence and reprint requests to Stuart C. Sealfon, Department of Neurology and Center for Translational Systems Biology, Mount Sinai School of Medicine, 1 Gustave L. Levy Place, New York, NY 10029. E-mail address: stuart.sealfon{at}mssm.edu Back

4 Abbreviations used in this paper: DC, dendritic cell; NDV, Newcastle disease virus; AVDC, antiviral-activated DC; MHC-I/II, MHC class I/II; Ct, crossing threshold; DiO, benzoxazolium, 3-octadecyl-2-[3-(3-octadecyl-2(3H)-benzoxazolylidene)-1-propenyl] perchlorate; IP-10, IFN-{gamma}-inducible protein 10; RFP, red fluorescent protein; PKR, protein kinase R; OAS, 2'-5'-oligoadenylate synthetase; MxA, myxovirus resistance A. Back

5 The online version of this article contains supplemental material. Back

Received for publication April 2, 2008. Accepted for publication September 5, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Reis e Sousa, C.. 2004. Activation of dendritic cells: translating innate into adaptive immunity. Curr. Opin. Immunol. 16: 21-25. [Medline]
  2. Fernandez, N. C., A. Lozier, C. Flament, P. Ricciardi-Castagnoli, D. Bellet, M. Suter, M. Perricaudet, T. Tursz, E. Maraskovsky, L. Zitvogel. 1999. Dendritic cells directly trigger NK cell functions: cross-talk relevant in innate anti-tumor immune responses in vivo. Nat. Med. 5: 405-411. [Medline]
  3. Kadowaki, N., S. Antonenko, S. Ho, M. C. Rissoan, V. Soumelis, S. A. Porcelli, L. L. Lanier, Y. J. Liu. 2001. Distinct cytokine profiles of neonatal natural killer T cells after expansion with subsets of dendritic cells. J. Exp. Med. 193: 1221-1226. [Abstract/Free Full Text]
  4. Caux, C., C. Massacrier, B. Vanbervliet, B. Dubois, I. Durand, M. Cella, A. Lanzavecchia, J. Banchereau. 1997. CD34+ hematopoietic progenitors from human cord blood differentiate along two independent dendritic cell pathways in response to granulocyte-macrophage colony-stimulating factor plus tumor necrosis factor {alpha}: II. Functional analysis. Blood 90: 1458-1470. [Abstract/Free Full Text]
  5. Jego, G., A. K. Palucka, J. P. Blanck, C. Chalouni, V. Pascual, J. Banchereau. 2003. Plasmacytoid dendritic cells induce plasma cell differentiation through type I interferon and interleukin 6. Immunity 19: 225-234. [Medline]
  6. Ueno, H., E. Klechevsky, R. Morita, C. Aspord, T. Cao, T. Matsui, T. Di Pucchio, J. Connolly, J. W. Fay, V. Pascual, et al 2007. Dendritic cell subsets in health and disease. Immunol. Rev. 219: 118-142. [Medline]
  7. Fernandez-Sesma, A., S. Marukian, B. J. Ebersole, D. Kaminski, M. S. Park, T. Yuen, S. C. Sealfon, A. Garcia-Sastre, T. M. Moran. 2006. Influenza virus evades innate and adaptive immunity via the NS1 protein. J. Virol. 80: 6295-6304. [Abstract/Free Full Text]
  8. Yoneyama, M., M. Kikuchi, T. Natsukawa, N. Shinobu, T. Imaizumi, M. Miyagishi, K. Taira, S. Akira, T. Fujita. 2004. The RNA helicase RIG-I has an essential function in double-stranded RNA-induced innate antiviral responses. Nat. Immunol. 5: 730-737. [Medline]
  9. Hornung, V., J. Ellegast, S. Kim, K. Brzozka, A. Jung, H. Kato, H. Poeck, S. Akira, K. K. Conzelmann, M. Schlee, et al 2006. 5'-Triphosphate RNA is the ligand for RIG-I. Science 314: 994-997. [Abstract/Free Full Text]
  10. Pichlmair, A., O. Schulz, C. P. Tan, T. I. Naslund, P. Liljestrom, F. Weber, C. Reis e Sousa. 2006. RIG-I-mediated antiviral responses to single-stranded RNA bearing 5'-phosphates. Science 314: 997-1001. [Abstract/Free Full Text]
  11. Macagno, A., G. Napolitani, A. Lanzavecchia, F. Sallusto. 2007. Duration, combination and timing: the signal integration model of dendritic cell activation. Trends Immunol. 28: 227-233. [Medline]
  12. Murray, P. J.. 2007. The JAK-STAT signaling pathway: input and output integration. J. Immunol. 178: 2623-2629. [Abstract/Free Full Text]
  13. Reis e Sousa, C.. 2006. Dendritic cells in a mature age. Nat. Rev. Immunol. 6: 476-483. [Medline]
  14. Mellman, I., R. M. Steinman. 2001. Dendritic cells: specialized and regulated antigen processing machines. Cell 106: 255-258. [Medline]
  15. Coppey, M., A. M. Berezhkovskii, S. C. Sealfon, S. Y. Shvartsman. 2007. Time and length scales of autocrine signals in three dimensions. Biophys. J. 93: 1917-1922. [Medline]
  16. Kato, H., S. Sato, M. Yoneyama, M. Yamamoto, S. Uematsu, K. Matsui, T. Tsujimura, K. Takeda, T. Fujita, O. Takeuchi, S. Akira. 2005. Cell type-specific involvement of RIG-I in antiviral response. Immunity 23: 19-28. [Medline]
  17. Park, M. S., A. Garcia-Sastre, J. F. Cros, C. F. Basler, P. Palese. 2003. Newcastle disease virus V protein is a determinant of host range restriction. J. Virol. 77: 9522-9532. [Abstract/Free Full Text]
  18. Park, M. S., M. L. Shaw, J. Munoz-Jordan, J. F. Cros, T. Nakaya, N. Bouvier, P. Palese, A. Garcia-Sastre, C. F. Basler. 2003. Newcastle disease virus (NDV)-based assay demonstrates interferon-antagonist activity for the NDV V protein and the Nipah virus V, W, and C proteins. J. Virol. 77: 1501-1511. [Medline]
  19. Mibayashi, M., L. Martinez-Sobrido, Y. M. Loo, W. B. Cardenas, M. Gale, Jr, A. Garcia-Sastre. 2007. Inhibition of retinoic acid-inducible gene I-mediated induction of β interferon by the NS1 protein of influenza A virus. J. Virol. 81: 514-524. [Abstract/Free Full Text]
  20. Lopez, C. B., A. Garcia-Sastre, B. R. Williams, T. M. Moran. 2003. Type I interferon induction pathway, but not released interferon, participates in the maturation of dendritic cells induced by negative-strand RNA viruses. J. Infect. Dis. 187: 1126-1136. [Medline]
  21. Yuen, T., E. Wurmbach, R. L. Pfeffer, B. J. Ebersole, S. C. Sealfon. 2002. Accuracy and calibration of commercial oligonucleotide and custom cDNA microarrays. Nucleic Acids Res. 30: e48[Abstract/Free Full Text]
  22. Krutzik, P. O., G. P. Nolan. 2006. Fluorescent cell barcoding in flow cytometry allows high-throughput drug screening and signaling profiling. Nat. Methods 3: 361-368. [Medline]
  23. Perfetto, S. P., D. Ambrozak, R. Nguyen, P. Chattopadhyay, M. Roederer. 2006. Quality assurance for polychromatic flow cytometry. Nat. Protoc. 1: 1522-1530. [Medline]
  24. Haralick, R. M., K. Shanmugan, I. Dinstein. 1973. Textural features for image classification: IEEE transactions on systems, man, and cybernetics. SMC 3: 610-621.
  25. Lopez, C. B., A. Fernandez-Sesma, S. M. Czelusniak, J. L. Schulman, T. M. Moran. 2000. A mouse model for immunization with ex vivo virus-infected dendritic cells. Cell. Immunol. 206: 107-115. [Medline]
  26. Perry, A. K., G. Chen, D. Zheng, H. Tang, G. Cheng. 2005. The host type I interferon response to viral and bacterial infections. Cell Res. 15: 407-422. [Medline]
  27. Randolph, G. J., S. Beaulieu, M. Pope, I. Sugawara, L. Hoffman, R. M. Steinman, W. A. Muller. 1998. A physiologic function for p-glycoprotein (MDR-1) during the migration of dendritic cells from skin via afferent lymphatic vessels. Proc. Natl. Acad. Sci. USA 95: 6924-6929. [Abstract/Free Full Text]
  28. Bosnjak, L., M. Miranda-Saksena, D. M. Koelle, R. A. Boadle, C. A. Jones, A. L. Cunningham. 2005. Herpes simplex virus infection of human dendritic cells induces apoptosis and allows cross-presentation via uninfected dendritic cells. J. Immunol. 174: 2220-2227. [Abstract/Free Full Text]
  29. Pollara, G., M. Jones, M. E. Handley, M. Rajpopat, A. Kwan, R. S. Coffin, G. Foster, B. Chain, D. R. Katz. 2004. Herpes simplex virus type-1-induced activation of myeloid dendritic cells: the roles of virus cell interaction and paracrine type I IFN secretion. J. Immunol. 173: 4108-4119. [Abstract/Free Full Text]
  30. Palmer, D. R., P. Sun, C. Celluzzi, J. Bisbing, S. Pang, W. Sun, M. A. Marovich, T. Burgess. 2005. Differential effects of dengue virus on infected and bystander dendritic cells. J. Virol. 79: 2432-2439. [Abstract/Free Full Text]
  31. Santini, S. M., C. Lapenta, M. Logozzi, S. Parlato, M. Spada, T. Di Pucchio, F. Belardelli. 2000. Type I interferon as a powerful adjuvant for monocyte-derived dendritic cell development and activity in vitro and in Hu-PBL-SCID mice. J. Exp. Med. 191: 1777-1788. [Abstract/Free Full Text]
  32. Luft, T., K. C. Pang, E. Thomas, P. Hertzog, D. N. Hart, J. Trapani, J. Cebon. 1998. Type I IFNs enhance the terminal differentiation of dendritic cells. J. Immunol. 161: 1947-1953. [Abstract/Free Full Text]
  33. Blanco, P., A. K. Palucka, M. Gill, V. Pascual, J. Banchereau. 2001. Induction of dendritic cell differentiation by IFN-{alpha} in systemic lupus erythematosus. Science 294: 1540-1543. [Abstract/Free Full Text]
  34. Watanabe, N., S. Hanabuchi, V. Soumelis, W. Yuan, S. Ho, R. de Waal Malefyt, Y. J. Liu. 2004. Human thymic stromal lymphopoietin promotes dendritic cell-mediated CD4+ T cell homeostatic expansion. Nat. Immunol. 5: 426-434. [Medline]
  35. Soumelis, V., P. A. Reche, H. Kanzler, W. Yuan, G. Edward, B. Homey, M. Gilliet, S. Ho, S. Antonenko, A. Lauerma, et al 2002. Human epithelial cells trigger dendritic cell mediated allergic inflammation by producing TSLP. Nat. Immunol. 3: 673-680. [Medline]
  36. Chomarat, P., C. Dantin, L. Bennett, J. Banchereau, A. K. Palucka. 2003. TNF skews monocyte differentiation from macrophages to dendritic cells. J. Immunol. 171: 2262-2269. [Abstract/Free Full Text]
  37. Steinbrink, K., M. Wolfl, H. Jonuleit, J. Knop, A. H. Enk. 1997. Induction of tolerance by IL-10-treated dendritic cells. J. Immunol. 159: 4772-4780. [Abstract]
  38. Sato, K., N. Yamashita, M. Baba, T. Matsuyama. 2003. Regulatory dendritic cells protect mice from murine acute graft-versus-host disease and leukemia relapse. Immunity 18: 367-379. [Medline]
  39. Martin-Fontecha, A., L. L. Thomsen, S. Brett, C. Gerard, M. Lipp, A. Lanzavecchia, F. Sallusto. 2004. Induced recruitment of NK cells to lymph nodes provides IFN-{gamma} for TH1 priming. Nat. Immunol. 5: 1260-1265. [Medline]
  40. Mohamadzadeh, M., F. Berard, G. Essert, C. Chalouni, B. Pulendran, J. Davoust, G. Bridges, A. K. Palucka, J. Banchereau. 2001. Interleukin 15 skews monocyte differentiation into dendritic cells with features of Langerhans cells. J. Exp. Med. 194: 1013-1020. [Abstract/Free Full Text]
  41. Dubsky, P., H. Saito, M. Leogier, C. Dantin, J. E. Connolly, J. Banchereau, A. K. Palucka. 2007. IL-15-induced human DC efficiently prime melanoma-specific naive CD8+ T cells to differentiate into CTL. Eur. J. Immunol. 37: 1678-1690. [Medline]
  42. Mattei, F., G. Schiavoni, F. Belardelli, D. F. Tough. 2001. IL-15 is expressed by dendritic cells in response to type I IFN, double-stranded RNA, or lipopolysaccharide and promotes dendritic cell activation. J. Immunol. 167: 1179-1187. [Abstract/Free Full Text]
  43. Janes, K. A., J. G. Albeck, S. Gaudet, P. K. Sorger, D. A. Lauffenburger, M. B. Yaffe. 2005. A systems model of signaling identifies a molecular basis set for cytokine-induced apoptosis. Science 310: 1646-1653. [Abstract/Free Full Text]
  44. Janes, K. A., M. B. Yaffe. 2006. Data-driven modelling of signal-transduction networks. Nat. Rev. Mol. Cell, Biol. 7: 820-828. [Medline]
  45. Miller-Jensen, K., K. A. Janes, J. S. Brugge, D. A. Lauffenburger. 2007. Common effector processing mediates cell-specific responses to stimuli. Nature 448: 604-608. [Medline]
  46. Hoshino, K., T. Kaisho, T. Iwabe, O. Takeuchi, S. Akira. 2002. Differential involvement of IFN-β in Toll-like receptor-stimulated dendritic cell activation. Int. Immunol. 14: 1225-1231. [Abstract/Free Full Text]
  47. Honda, K., S. Sakaguchi, C. Nakajima, A. Watanabe, H. Yanai, M. Matsumoto, T. Ohteki, T. Kaisho, A. Takaoka, S. Akira, et al 2003. Selective contribution of IFN-{alpha}/β signaling to the maturation of dendritic cells induced by double-stranded RNA or viral infection. Proc. Natl. Acad. Sci. USA 100: 10872-10877. [Abstract/Free Full Text]
  48. Hoebe, K., E. M. Janssen, S. O. Kim, L. Alexopoulou, R. A. Flavell, J. Han, B. Beutler. 2003. Upregulation of costimulatory molecules induced by lipopolysaccharide and double-stranded RNA occurs by Trif-dependent and Trif-independent pathways. Nat. Immunol. 4: 1223-1229. [Medline]
  49. Sporri, R., C. Reis e Sousa. 2005. Inflammatory mediators are insufficient for full dendritic cell activation and promote expansion of CD4+ T cell populations lacking helper function. Nat. Immunol. 6: 163-170. [Medline]
  50. Brankston, G., L. Gitterman, Z. Hirji, C. Lemieux, M. Gardam. 2007. Transmission of influenza A in human beings. Lancet Infect. Dis. 7: 257-265. [Medline]
  51. Garcia-Sastre, A.. 2004. Identification and characterization of viral antagonists of type I interferon in negative-strand RNA viruses. Curr. Top. Microbiol. Immunol. 283: 249-280. [Medline]
  52. Garcia-Sastre, A., C. A. Biron. 2006. Type 1 interferons and the virus-host relationship: a lesson in detente. Science 312: 879-882. [Abstract/Free Full Text]
  53. Alcami, A.. 2003. Viral mimicry of cytokines, chemokines and their receptors. Nat. Rev. Immunol. 3: 36-50. [Medline]




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