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-Stimulated Dendritic Cells in NK Cell Activation: Impairment in Chronic Hepatitis C Virus Infection 1
* Department of Molecular Therapeutics, Osaka University Graduate School of Medicine, Osaka, Japan; and
Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98109
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Abstract |
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production, CD69 expression, and K562 cytolytic ability of NK cells when DCs are prestimulated with various maturation stimuli such as IFN-
or LPS. Of interest is the finding that NK cell activation mediated by LPS-stimulated DCs was dependent on IL-12 produced in DC/NK coculture, but that IFN--stimulated DC-mediated activation was not. Alternatively, MHC class I-related chain A and B (MICA/B), ligands for NKG2D activating receptor, were found to be induced on DCs upon IFN- stimulation and to be responsible for the NK activation because mAb-mediated masking of MICA/B as well as inhibition of direct cell-to-cell contact using transwell insert completely abolished DC-dependent NK cell activation by IFN-. Finally, DCs recovered from chronic hepatitis C virus-infected patients showed defects in the induction of MICA/B and impaired ability to activate NK cells in response to IFN- stimulation. These findings suggested that MICA/B induction on DCs may be one of the mechanisms by which IFN- activates NK cells; this impairment might affect IFN- responsiveness in hepatitis C virus infection. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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It is well known that NK cell activation is regulated by several cytokines such as IL-2, IL-12, IL-18, and IFN- (9). Whereas IL-2 is secreted from polarized effector Th1 CD4+ T cells, the others are predominantly produced by DCs. Soluble factors such as IL-12 and IL-18 (4, 10) were suggested to be partially involved in DC-mediated activation of NK cells. In contrast, IL-12 was shown to be dispensable under some experimental conditions (5, 7).
Recent advances in research on NK cytotoxicity have established that NK cell effector functions are regulated by a balance of inhibitory and activating signals transmitted by membrane receptors that recognize ligands on the cell surface of potential target cells (11, 12). NK cells express receptors for MHC class I which, upon ligation, inhibit NK cell-mediated cytolytic activity. NK cells also express several activating receptors, such as NKG2D and a group of natural cytotoxicity receptors including NKp46, NKp30, and NKp44. Whereas viral proteins such as hemagglutinins of influenza viruses were identified as the ligand for NKp46 and NKp44 (13, 14), human ligands for NKG2D were recently found to be stress-inducible MHC class I-related chain A and B (MICA/B) (15, 16). MICA/B are predominantly expressed in the gastrointestinal epithelium and tumor cells of epithelial origin (17, 18). Recent reports have demonstrated that Mycobacterium tuberculosis-infected DCs (19) also express MICA/B. These observations led us to investigate the possibility that MICA/B are induced on DCs under certain conditions and function as activator ligands for NK cells. With regard to direct interaction of NK cells and DCs, it should be noted that recent reports have identified NKp30 (6) and/or NKp46 (20) as critical cell surface molecules by which activated NK cells kill DCs. In addition, the adhesion molecule LFA-1 was also reported to be involved in NK cytolysis of DCs (21).
In this study, we used an in vitro coculture system to investigate whether MICA/B are induced on DCs and are involved in NK activation by DCs after exposure to various maturation stimuli. We found that IFN- specifically induces MICA/B expression on DCs and thereby endows them with the ability to activate NK cells, as evidenced by an increase in IFN- production, CD69 expression, and cytolytic ability against K562 cells. We also found that induction of MICA/B is virtually absent on DCs derived from patients with chronic hepatitis C virus (HCV) infection, suggesting that this impairment might play a certain role in HCV infection.
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Materials and Methods |
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Ten healthy volunteers and 15 patients with chronic hepatitis C were enrolled in this study after informed consent had been obtained. Ten of 15 HCV-infected patients had previously received one or two rounds of IFN- therapy but had not achieved sustained viral eradication, and the others had not received IFN- therapy. They were histologically diagnosed as having mild or moderate chronic hepatitis and did not display any evidence of other types of liver diseases.
Generation of monocyte-derived DCs from PBMCs
Monocyte-derived DCs were generated from peripheral venous blood of healthy volunteers or chronic hepatitis C patients. PBMCs isolated by Ficoll Hypaque density centrifugation were resuspended with 60% Percoll (Sigma-Aldrich, St. Louis, MO) solution, layered 45% and 34% Percoll, and then centrifuged at 2400 x g for 45 min. The light density fraction floating on the middle layer, which contained highly purified monocytes, was seeded in 24-well culture plates at a density of 5.0 x 105/well (22). After 45 min of incubation at 37°C, nonadherent cells were removed and the adherent cells were cultured in Iscove’s modified Eagle’s medium (Life Technologies, Gaithersburg, MD) containing 10% FCS, 10 U/ml penicillin/streptomycin, 2 mM L-glutamine, 1% nonessential amino acids, and supplemented with GM-CSF (1000 U/ml; kindly provided by Kirin Brewery, Gunma, Japan) and IL-4 (500 U/ml; Strathmann Biotech, Hannover, Germany). At day 6, DCs were stimulated for 24 h using the following agents: IFN- (range of concentration, 1–1000 U/ml; kindly provided by Sumitomo Pharmaceutical, Osaka, Japan), LPS (final concentration, 10 µg/ml; Sigma-Aldrich), and poly(I:C) (final concentration, 12.5 µg/ml; Sigma-Aldrich). DCs were also cocultured with 50 µg/ml mitomycin C-treated fibroblast cell line L cells transfected with CD40 ligand (CD40L; kindly provided by Dr. S. Egawa, Tohoku University Graduate School of Medicine, Sendai, Japan) at the ratio of 1:1 for 24 h.
Flow cytometric analysis of DCs
DCs (5 x 105/ml) were washed and resuspended in PBS containing 1% BSA and 0.05% NaN3 and incubated with a series of mAbs at 4°C for 30 min. The following mAbs were used for immunofluorescent staining: anti-CD86, HLA-DR (BD PharMingen, San Diego, CA), CD83, and DC-lysosome-associated membrane protein (LAMP) (Coulter-Immunotech, Marseille, France) and MICA/B (6D4) (23). For MICA/B staining, the cells were then washed and incubated at 4°C for 30 min in FITC-labeled goat anti-mouse IgG (Coulter-Immunotech) as a second-step Ab. The cells were then washed twice and fixed with 2% paraformaldehyde solution. The cells were analyzed by flow cytometric analysis using a FACScan system (BD PharMingen), and data analysis was performed using CellQuest software.
DC/NK coculture
Allogeneic NK cells in this study were isolated from healthy volunteers’ PBMCs and labeled with the enrichment Ab cocktails for NK cells, and then with magnetic colloid according to the product inserts supplied by StemCell Technologies (Vancouver, BC, Canada). More than 90% of the cells expressed CD56 on their cell surface. Enriched NK cells were cultured in 24-well culture plates (5.0 x 105/well) in RPMI 1640 medium supplemented with 10% FCS and 10 U/ml penicillin/streptomycin. After washing with PBS two times, DCs prestimulated with or without maturation stimuli were added at a concentration of 1.0 x 105/ml in RPMI 1640 medium in the same wells. In some experiments, a transwell insert was also used to prevent direct contact of NK cells and DCs in coculture systems. In these transwell systems, the soluble factors could freely pass through a microporous polycarbon membrane (0.4 µm). NK cells were plated in the upper wells, and DCs were seeded in the lower wells at the same final concentrations given above. As controls, NK cells or DCs alone were plated in 24-well culture plates. In some experiments, anti-MICA/B mAb 6D4 or anti-IL-12 neutralizing Ab (R&D Systems, Minneapolis, MN) was added at the beginning of the DC/NK coculture.
Cytolytic assay
Target cells (K562) labeled with 51Cr were incubated in the NK/DC cocultures (with or without transwell systems) or single cultures for 4 h at various E:T ratios. The supernatants were obtained after the incubation and subjected to gamma counting. The maximum or spontaneous release was defined as counts from samples incubated with 5% Triton X-100 or medium alone, respectively. Cytolytic activity was calculated with the following formula: % lysis = (experimental release - spontaneous release) x 100/(maximum release - spontaneous release). The spontaneous release in all assays was <20% of the maximum release.
Analysis of intracellular IFN- and CD69 expression in NK cells cocultured with DCs
Early activation marker CD69 as well as intracellular IFN- expression in NK cells in the presence of DCs was examined using flow cytometry. NK cells (5 x 105) cocultered with DCs (1 x 105) treated with IFN- (1000 U/ml) or LPS (10 µg/ml) for 24 h in 24-well plates were stained with FITC-labeled CD69 and PE-labeled CD56 for 30 min at 4°C. Intracellular staining was performed as described previously (4, 24). In brief, NK cells cultured with DCs were performed with pretreatment of 10 ng/ml PMA plus 1 µM ionomycin (Sigma-Aldrich) in the presence of 1 µl/ml GolgiPlug (BD PharMingen) for 4 h at 37°C. The PMA/ionomycin pretreatment in our condition facilitates detection of intracellular IFN- without affecting levels of baseline staining. At the end of the incubation period, NK cells were stained with PE-labeled CD56 mAb for 30 min at 4°C. Cells were then fixed and permeabilized with Cytofix/Cytoperm buffer (BD PharMingen) for 15 min at room temperature. Permeabilized cells were stained with FITC-labeled anti-IFN- mAb (mouse IgG1) or isotype-matched control IgG. The stained cells were analyzed by flow cytometry.
Analysis of MICA and MICB mRNA expression in DCs by RT-PCR
The presence of MICA/B mRNA in DCs was examined by RT-PCR. One microgram of total RNA extracted using ISOGEN (Nippon Gene, Toyama, Japan) was added to 80 pmol of random primers (Takara Shuzo, Shiga, Japan) and 10 mmol/L of each deoxynucleotide triphosphate, incubated at 65°C for 5 min and quickly chilled on ice. The mixture was combined with 50 mM Tris-HCl, 75 mM KCl, 10 mM DTT, 3 mM MgCl2, and 100 U of Moloney murine leukemia virus reverse transcriptase (Life Technologies) and incubated at 37°C for 50 min. The reaction was terminated by heating to 70°C for 15 min. The resulting cDNA was used in the reaction mixture containing 10 pmol of each upstream MIC sense primer (5'-ACACCCAGCAGTGGGGGGAT-3') and downstream MICA antisense primer (5'-GCAGGGAATTGAATCCCAGCT-3') or MICB antisense primer (5'-AGCAGTCGTGAGTTTGCCCAC-3'), 10 mM Tris-HCl, 50 mM KCl, 1.5 mM MgCl2, 2.5 mM each deoxynucleotide triphosphate, and 2.5 U of Taq DNA polymerase (Takara Shuzo). Each primer pair described above was designed to span at least one intron of the corresponding gene. The amplification protocol included a denaturation step at 95°C for 5 min, followed by 35 cycles of 95°C for 60 s for denaturation, 56°C for 60 s for annealing, and 72°C for 90 s for extension. As a control for the integrity of total RNA, primers specific for G3PDH, sense primer (5'-GCCACCCAGAAGACTGTGGATGGC-3'), and antisense primer (5'-CATGTAGGCCATGAGGTCCACCAC-3') were used.
Measurements of cytokine production of DCs
IFN-, IL-12 p70, and IL-18 in the culture supernatant were determined using commercially available ELISA kits according to the manufacturer’s instructions (Endogen, Woburn, MA).
Statistical analysis
Data demonstrated in Fig. 7 were expressed as the mean and SD and compared using two sample t tests with Welch’s correction. Differences were considered significant when the p value was <0.01.
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Results |
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After being cultured in the presence of GM-CSF and IL-4 for 6 days, peripheral monocytes from healthy donors displayed a typical phenotype characterizing immature DCs with low to intermediate expression of CD83, CD86, and HLA-DR and no expression of DC-LAMP. After further stimulation by LPS, CD40L-transfected L cells, or poly(I:C) for 24 h, they obtained maturation phenotypes with high CD83, CD86, HLA-DR, and DC-LAMP. When immature DCs were cultured with 1000 U/ml of IFN- for 24 h, their phenotypes were characterized by high CD86, HLA-DR, and DC-LAMP, but low CD83 expression (Fig. 1A). To examine whether DCs can activate NK cells, allogeneic NK cells derived from peripheral blood of healthy human donors were cocultured for 24 h with DCs stimulated with or without maturation stimuli and then subjected to analysis of IFN- production, CD69 expression, and cytolytic ability against K562 cells. NK cells cocultured with LPS-, poly(I:C)-, CD40L-, or IFN--treated DCs increased cytolytic activity against K562 cells in comparison with NK cells cultured without DCs. In contrast, immature DCs had no effect on the enhancement of K562 cytolysis by NK cells. Although IFN- is a well-known activator for NK cells, 24-h treatment of NK cells with IFN- (1000 U/ml) did not augment NK cytolysis against K562 cells in our experimental conditions (Fig. 1B). Also, it should be noted that DCs stimulated with LPS, poly(I:C), CD40L, or IFN- displayed no cytolytic activity against K562 (data not shown). Flow cytometric analysis showed that IFN- production as well as CD69 expression of NK cells clearly increased upon coculture with LPS-, poly(I:C)-, CD40L-, or IFN--stimulated DCs, but did not do so efficiently upon coculture with immature DCs (Fig. 1C). These results indicated that DCs gain the ability to activate NK cells after various maturation stimuli.
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-stimulated DCs
Expression of MICA/B on DCs after various stimuli for 24 h was analyzed by flow cytometry (Fig. 2A). Cell surface expression of MICA/B was not detected on immature DCs. LPS, poly(I:C), and CD40L stimulation did not have any effect on MICA/B expression. In contrast, IFN- stimulation substantially induced MICA/B expression on DCs. Additional experiments showed that IFN- induced MICA/B on DCs in a dose- and time-dependent manner (Fig. 2B). RT-PCR analysis revealed that, upon IFN- treatment, both transcripts of MICA and MICB genes were induced in DCs (Fig. 2C). Furthermore, addition of neutralizing mAb against IFN-/
receptor abolished the induction of MICA/B on DCs by IFN- (Fig. 2, C and D), indicating that MICA/B induction by IFN- is regulated in receptor-mediated signal transduction mediated by IFN- stimulation.
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-stimulated DCs
To examine whether MICA/B specifically induced by IFN- are involved in NK cell activities, mAb 6D4, which can mask 1 and 2 extracellular domain of MICA/B (17), was incubated during the NK/DC coculture. The incubation of mAb 6D4 during coculture clearly inhibited K562 cytolysis of NK cells stimulated by IFN--treated DCs, whereas the same treatment had little effect on cytolytic activity of NK cells stimulated by LPS-treated DCs not expressing MICA/B (Fig. 3A). In addition, IFN- production and CD69 expression of NK cells cocultured with IFN--stimulated DCs were substantially decreased when mAb 6D4 was incubated in NK/DC coculture, whereas the incubation of mAb 6D4 did not affect NK cells cocultured with LPS-stimulated DCs (Fig. 3B). We also measured IFN- production in the supernatant of NK/DC coculture without the prestimulation of PMA/ionomycin. The ELISA result indicated that substantial levels of IFN- were produced in NK/DC coculture when DCs were stimulated with IFN- or LPS. The incubation of mAb 6D4 during the cuculture completely suppressed the IFN- production in NK/IFN--stimulated DC coculture, but it did not in NK/LPS-stimulated DC coculture (Fig. 3C). Taken together, these results indicated that MICA/B induced on DCs by IFN- are responsible for the activation of NK cells.
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-stimulated DCs, NK cells were cultured with DCs together or separately by a transwell system for 24 h. NK cell cytolytic ability significantly increased when NK cells were cultured together with IFN--stimulated DCs, but not when the cells were separated by a 0.4-µm porous membrane. In contrast, LPS-, poly(I:C)-, or CD40L-stimulated DCs enhanced NK cell cytolytic ability, even if the cells were separated in transwells (Fig. 4A and data not shown). Similarly, the levels of CD69 expression and intracellular IFN- production of NK cells cultured with IFN--stimulated DCs in transwells were apparently lower than those of NK cells cocultured without porous membrane. Coculture in transwells did not affect CD69 expression and intracellular IFN- production of NK cells cultured with LPS-, poly(I:C)-, or CD40L-stimulated DCs (Fig. 4B and data not shown). These results indicated that intimate cell-to-cell contact is required for NK cell activation mediated by IFN--stimulated DCs, but not for that mediated by LPS-, poly(I:C)-, or CD40L-stimulated DCs.
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Because it was previously reported that IL-12 and IL-18 were partially involved in DC-induced NK cell activation under some experimental conditions (4, 10), we measured IL-12 p70 as well as IL-18 in the supernatant of NK/DC coculture by ELISA. IFN-stimulated DCs did not produce any detectable amounts of IL-12 p70 cocultured with or without NK cells. In contrast, DCs could produce IL-12 p70 in the presence of LPS stimulation; this production was markedly increased when LPS-stimulated DCs were cultured with NK cells for 24 h (Fig. 5A). Although the data are not shown, IL-18 could not be detected under any culture conditions. To examine the role of IL-12 in NK activation, we treated with recombinant IL-12 at the concentration of 1 ng/ml or 10 ng/ml for 24 h, and then subjected to the K562 cytolytic assay. As shown in Fig. 5B, NK cell activity was clearly up-regulated by IL-12 treatment. More importantly, the addition of anti-IL-12 neutralizing Ab during NK/DC coculture substantially suppressed K562 cytolytic ability of NK cells cocultured with LPS-stimulated DCs, but not of those cocultured with IFN--stimulated DCs (Fig. 5C). The treatment also suppressed IFN- expression of NK cells cocultured with LPS-stimulated DCs, but not of those cocultured with IFN--stimulated DCs (Fig. 5D). These results indicated that IL-12 produced in the LPS-stimulated DC/NK coculture serves as an important soluble factor involved in activation of NK cells.
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stimulation is impaired in patients with chronic HCV infection
We and others previously reported that there is functional impairment of DCs derived from chronic HCV-infected patients (HCV-DCs) (25, 26, 27). These studies suggested that diminished allostimulatory capacity and impaired maturation of TNF--stimulated DCs may be correlated with reduced Th responses and impaired virus clearance. However, it remained to be examined whether functional impairment of DCs also occurs during the interaction with NK cells. Therefore, we examined whether HCV-DCs can activate NK cells in terms of K562 cytolytic ability as well as IFN- and CD69 expression. NK cells exhibited increased cytolytic ability when cocultured with HCV-DCs stimulated with LPS. In marked contrast, IFN--treated HCV-DCs did not augment NK cytolytic ability (Fig. 6A). In addition, HCV-DCs, upon stimulation with LPS, enhanced the expression of intracellular IFN- as well as CD69 in NK cells, but this ability was virtually absent when they were stimulated with IFN- (Fig. 6B). Phenotypical analysis of HCV-DCs revealed that IFN- or LPS stimulation clearly enhanced expression of CD86, HLA-DR, and DC-LAMP at similar levels observed in DCs from healthy donors. However, more importantly, induction of MICA/B was strongly impaired in HCV-DCs (Fig. 6C). Fig. 7 summarizes the expression of MICA/B on HCV-DCs as well as DCs from healthy donors after stimulation of IFN-. The results indicated that induction of MICA/B was significantly impaired in HCV-DCs.
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Discussion |
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, or CD40L, undergo a process of differentiation termed maturation, which is characterized by up-regulation of MHC class I and II, adhesion, and costimulatory molecules. DCs can acquire such shared characteristics but also exhibit stimulus-specific responses (28, 29, 30). In the present study, we demonstrated that all of the stimuli tested consistently enabled DCs to activate NK cells, as evidenced by increased IFN- production, CD69 expression, and cytolysis of K562; however, the underlying mechanisms seem to vary among the stimuli. Indeed, IL-12 produced in DC/NK coculture plays an important role in activation of NK cells when LPS is used to stimulate DCs. In contrast, IFN- stimulation suppresses IL-12 production in DC/NK coculture (Fig. 5A), which is consistent with previous reports (31, 32, 33), and IL-12 is entirely dispensable for activation of NK cells by IFN--stimulated DCs.
In addition to its capacity to suppress IL-12 production, IFN- was reported to be a relatively inefficient stimulus for the maturation of DCs generated from IL-4/GM-CSF-stimulated monocyte (33, 34). In agreement with these observations, IFN--stimulated DCs used in the present study lack the ability to stimulate allogeneic helper T cells in contrast with LPS-stimulated DCs (our unpublished observation) and show only minimal induction of CD83 under our experimental conditions (Fig. 1A). Taken together, IFN- seems to have somehow different capability in activating DCs from other maturation stimuli.
We demonstrated here that MICA/B, not expressed in immature DCs, are induced upon IFN- stimulation and that direct cell-to-cell contact through MICA/B is responsible for NK activation by IFN--stimulated DCs. This MICA/B-dependent NK cell activation represents a distinct property of IFN--stimulated DCs, because other maturation-inducing stimuli tested in this study did not induce MICA/B expression on DCs. It remains unclear why poly(I:C), a known inducer of IFN- (35), did not up-regulate MICA/B expression, but it may be possible that some soluble factors produced by poly(I:C), but not by IFN-, may contribute to the suppression of MICA/B. As a mechanism of induction of cell surface MICA/B expression, it has been previously reported that MICA/B expression can increase to high cell surface levels by heat shock and thus may be under control of heat shock promoter element (17, 23). Our present study reveals that IFN- can up-regulate expression of both transcripts of MICA and MICB through IFN type I receptor, but the precise mechanism of MICA/B induction on DCs should be determined in future study.
It has been generally considered that MICA/B function as activating ligands expressed on potential target cells and work in the effector phase of NK cytotoxicity. However, MICA/B expressed on DCs seem not to be simple target molecules, because mature DCs were relatively resistant to NK-mediated cytolysis (our unpublished data), in agreement with previous reports (36, 37). Therefore, MICA/B induced on DCs may be important for DC-mediated NK cell activation rather than triggering of NK-mediated killing of DCs exposed to IFN-.
Both IFN- and IL-12 are predominantly produced by DCs and have been known to be activating factors for NK cells (9). Our present data show that IFN- itself is not able to efficiently up-regulate NK cytolytic ability and needs DCs as accessory cells (Fig. 1A). In contrast, IL-12 can activate NK cells by itself (Fig. 5B). IFN- is widely used for clinical treatment of certain tumors and viral infections, and its antitumor and antiviral properties involve, at least partly, the ability to activate NK cells (9, 38). Our finding suggests that MICA/B induction on DCs may be one of the important mechanisms by which IFN- activates NK cell-mediated immune responses, although further study will be needed to examine the significance of MICA/B induction on DCs in the in vivo condition. In addition, given the recent findings that pre-DC2 is a main source of IFN- upon virus infection (39, 40, 41, 42), we are currently investigating the possibility that IFN- derived from pre-DC2 induces its MICA/B expression in an autocrine manner.
The interesting finding of our study is that DCs derived from HCV-infected patients lacked the ability to express MICA/B in response to IFN-, whereas other surface molecules such as CD86, HLA-DR, and DC-LAMP were normally up-regulated on DCs upon IFN- stimulation in these patients (Fig. 6C). In addition, IFN--stimulated HCV-DCs could not activate NK cells, confirming again the critical role of MICA/B in DC-mediated activation of NK cells. IFN- is constitutively expressed at low levels under physiological conditions and increases in response to viral infection. IFN--based therapy has been established as the most potent therapy for eradicating this virus from chronic HCV patients (43). Although the mechanisms by which IFN- can eradicate HCV have not been fully elucidated, it has been recently recognized that IFN- is capable of augmenting immune responses to efficiently eliminate HCV, and lack of this response may lead to insufficient virus eradication and thus chronically infectious status (44, 45, 46). The present study demonstrates that IFN- cannot induce DC-mediated NK cell activation in chronic HCV patients. This impairment might contribute to the persistence of this viral infection as well as insufficient outcome for IFN- therapy. It is noteworthy that DCs derived from chronic hepatitis B patients expressed MICA/B in response to IFN- stimulation (data not shown), suggesting that impaired MICA/B induction seems to be specific for chronic HCV infection. We have begun investigating whether impaired MICA/B induction on DCs is correlated with the response to IFN-based therapy with a cohort of patients to further examine the functional relevance of MICA/B expression on DCs in chronic HCV infection.
In conclusion, we have demonstrated that MICA/B are induced on DCs by IFN- stimulation and are capable of activating NK cells. This novel mechanism of NK activation by DCs might contribute to initiation and regulation of the innate immune response mediated by IFN-.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Norio Hayashi, Department of Molecular Therapeutics, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan. E-mail address: hayashin{at}moltx.med.osaka-u.ac.jp
3 Abbreviations used in this paper: DC, dendritic cell; MICA/B, MHC class I-related chain A and B; HCV, hepatitis C virus; CD40L, CD40 ligand; LAMP, lysosome-associated membrane protein; HCV-DC, DC derived from chronic HCV-infected patient.
Received for publication July 17, 2002. Accepted for publication November 26, 2002.
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References |
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T cells of MICA and MICB. Proc. Natl. Acad. Sci. USA 96:6879.2V2 cells enhances their antigen-dependent effector function. Immunity 15:83.[Medline]
T cells. Science 279:1737./ inhibition of interleukin 12 and interferon- production in vitro and endogenously during viral infection. Proc. Natl. Acad. Sci. USA 94:634. enhances CD40 ligand-mediated activation of immature monocyte-derived dendric cells. Int. Immunol. 14:367., and TNF effects on antiviral state and NK cell responses during cytomegalovirus infection. J. Immunol. 156:4746.[Abstract]
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V. Makarenkova, A. K. Chakrabarti, J. A. Liberatore, P. Popovic, G. Lu, S. Watkins, and N. L. Vujanovic Dendritic cells and natural killer cells interact via multiple TNF family molecules J. Leukoc. Biol., March 1, 2005; 77(3): 408 - 413. [Abstract] [Full Text] [PDF]
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A. T. Kamath, C. E. Sheasby, and D. F. Tough Dendritic Cells and NK Cells Stimulate Bystander T Cell Activation in Response to TLR Agonists through Secretion of IFN-{alpha}{beta} and IFN-{gamma} J. Immunol., January 15, 2005; 174(2): 767 - 776. [Abstract] [Full Text] [PDF]
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C. Munz, T. Dao, G. Ferlazzo, M. A. de Cos, K. Goodman, and J. W. Young Mature myeloid dendritic cell subsets have distinct roles for activation and viability of circulating human natural killer cells Blood, January 1, 2005; 105(1): 266 - 273. [Abstract] [Full Text] [PDF]
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M. Hansson, A. Romero, F. Thoren, S. Hermodsson, and K. Hellstrand Activation of cytotoxic lymphocytes by interferon-{alpha}: role of oxygen radical-producing mononuclear phagocytes J. Leukoc. Biol., December 1, 2004; 76(6): 1207 - 1213. [Abstract] [Full Text] [PDF]
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 G. Ferlazzo, M. Pack, D. Thomas, C. Paludan, D. Schmid, T. Strowig, G. Bougras, W. A. Muller, L. Moretta, and C. Munz Distinct roles of IL-12 and IL-15 in human natural killer cell activation by dendritic cells from secondary lymphoid organs PNAS, November 23, 2004; 101(47): 16606 - 16611. [Abstract] [Full Text] [PDF]
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 M. J. Smyth, J. Swann, J. M. Kelly, E. Cretney, W. M. Yokoyama, A. Diefenbach, T. J. Sayers, and Y. Hayakawa NKG2D Recognition and Perforin Effector Function Mediate Effective Cytokine Immunotherapy of Cancer J. Exp. Med., November 15, 2004; 200(10): 1325 - 1335. [Abstract] [Full Text] [PDF]
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M. Jinushi, T. Takehara, T. Tatsumi, T. Kanto, T. Miyagi, T. Suzuki, Y. Kanazawa, N. Hiramatsu, and N. Hayashi Negative Regulation of NK Cell Activities by Inhibitory Receptor CD94/NKG2A Leads to Altered NK Cell-Induced Modulation of Dendritic Cell Functions in Chronic Hepatitis C Virus Infection J. Immunol., November 15, 2004; 173(10): 6072 - 6081. [Abstract] [Full Text] [PDF]
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C. Borg, A. Jalil, D. Laderach, K. Maruyama, H. Wakasugi, S. Charrier, B. Ryffel, A. Cambi, C. Figdor, W. Vainchenker, et al. NK cell activation by dendritic cells (DCs) requires the formation of a synapse leading to IL-12 polarization in DCs Blood, November 15, 2004; 104(10): 3267 - 3275. [Abstract] [Full Text] [PDF]
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A. Ahmad and F. Alvarez Role of NK and NKT cells in the immunopathogenesis of HCV-induced hepatitis J. Leukoc. Biol., October 1, 2004; 76(4): 743 - 759. [Abstract] [Full Text] [PDF]
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 J. Siren, T. Sareneva, J. Pirhonen, M. Strengell, V. Veckman, I. Julkunen, and S. Matikainen Cytokine and contact-dependent activation of natural killer cells by influenza A or Sendai virus-infected macrophages J. Gen. Virol., August 1, 2004; 85(8): 2357 - 2364. [Abstract] [Full Text] [PDF]
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 M. Larsson, E. Babcock, A. Grakoui, N. Shoukry, G. Lauer, C. Rice, C. Walker, and N. Bhardwaj Lack of Phenotypic and Functional Impairment in Dendritic Cells from Chimpanzees Chronically Infected with Hepatitis C Virus J. Virol., June 15, 2004; 78(12): 6151 - 6161. [Abstract] [Full Text] [PDF]
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M. Jinushi, T. Takehara, T. Tatsumi, T. Kanto, V. Groh, T. Spies, T. Suzuki, T. Miyagi, and N. Hayashi Autocrine/Paracrine IL-15 That Is Required for Type I IFN-Mediated Dendritic Cell Expression of MHC Class I-Related Chain A and B Is Impaired in Hepatitis C Virus Infection J. Immunol., November 15, 2003; 171(10): 5423 - 5429. [Abstract] [Full Text] [PDF]
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; n = 15) was determined by flow cytometry. Horizontal bars indicate mean and SD.
