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International Immunology Advance Access originally published online on September 18, 2007
International Immunology 2007 19(10):1145-1155; doi:10.1093/intimm/dxm073
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Induction of NKG2D ligands on human dendritic cells by TLR ligand stimulation and RNA virus infection

Takashi Ebihara1,2, Hisayo Masuda3,4, Takashi Akazawa3, Masashi Shingai1, Hideaki Kikuta2,5, Tadashi Ariga2, Misako Matsumoto1,3 and Tsukasa Seya1,3

1 Departments of Microbiology and Immunology
2 Department of Pediatrics, Hokkaido University Graduate School of Medicine, Kita-15, Nishi-7, Kita-ku, Sapporo 060-8638, Japan
3 Department of Immunology, Osaka Medical Center for Cancer, Higashinari-ku, Osaka 537-8511, Japan
4 Present address: Department of Molecular Immunogy, Nara Institute for Science and Technology, Ikoma, Nara 631-0101, Japan
5 Present address: Toei Hospital, Kita-41, Higashi-6, Higashi-ku, Sapporo 007-0841, Japan

Correspondence to: T. Seya; E-mail: seya-tu{at}med.hokudai.ac.jp


   Abstract
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 Abstract
Introduction
 Methods
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 Discussion
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Monocyte-derived dendritic cells (mDCs) and NK cells are reciprocally activate via cytokines and cell–cell contact. Although seven human NKG2D ligands (NKG2DLs), UL16-binding proteins (ULBP) 1, 2, 3 and 4, retinoic acid early transcript 1G (RAET1G) and MHC class I-related chains A and B, have been reported, the differential distribution and roles of these ligands in the maturation of human mDCs have not been elucidated. In the present study, we produced polyclonal antibodies (pAbs) directed against human ULBP1, 2 and 3. All these ULBPs were detected on human mDCs when probed by the pAbs, although their expression profiles were different. We next investigated what kinds of Toll-like receptor agonists and RNA viruses [influenza virus, human respiratory syncytial virus (RSV), measles virus and hepatitis C virus (HCV)] induced the expression of NKG2DLs on mDCs. ULBP1 was up-regulated on mDCs in response to LPS or infection with RSV. The expression of ULBP2 was induced by LPS and poly I:C, indicating that the TIR-containing adapter molecule-1 (TIR domain-containing adaptor-inducing IFN) pathway is associated with ULBP2 induction. Although infection with HCV did not cause up-regulation of NKG2DLs, other RNA virus infections and poly I:C promoted expression of ULBP2 and RAET1G in an IFN-{alpha}/ß-independent manner. Finally, the over-expression of ULBP1 and 2 on mDCs facilitated NK cell proliferation and IFN-{gamma} production through a mDC–NK cell interaction in the presence of IL-2. Hence, the results reflect the important role of NKG2DLs on human mDCs in mDC-mediated NK cell activation.

Keywords: dendritic cells, human, NK activation, Toll-like receptors, ULBP, viral infection


   Introduction
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 Abstract
 Introduction
Methods
 Results
 Discussion
 Funding
 References
 
NK cells are specialized lymphocytes in the innate immune system that are able to sense and eliminate pathogen-infected cells and tumor cells in the absence of antigen-specific recognition and clonal expansion (1). The functions of NK cells are regulated by a balance of activating and inhibiting signals. NK cells receive inhibitory signals via MHC class I-specific surface receptors, such as killer Ig-like inhibitory receptors and CD94–NKG2A heterodimers. NK cells also have an array of stimulatory receptors, including natural cytotoxicity receptors (NKp46, NKp30 and NKp44), NKG2D and other orphan receptors (1). Of these activating receptors, NKG2D is the best-characterized receptor. In humans, NKG2D is expressed on CD8+-{alpha} and -ß cells as well as NK cells. Although NKG2D on CD8+-{alpha} and -ß cells is thought to serve as a co-simulatory molecule for antigen recognition, signals through NKG2D on NK cells promote NK cell activities, such as proliferation, cytotoxicity and IFN-{gamma} production (2, 3).

Monocyte-derived dendritic cells (mDCs) are representative of professional antigen-presenting cells and are regarded as sentinels of the adaptive immune system. Immature mDCs recognize a diverse array of pathogen-associated molecular patterns (Pamps) through Toll-like receptors (TLRs) (4). After sensing pathogens, immature mDCs are transformed to the mature form, which presents increased surface levels of MHC proteins and co-stimulatory molecules. This maturation process is required to efficiently prime naive T cells. Furthermore, mDCs are known to promote the activity of NK cells through mDC–NK reciprocal interactions in a variety of settings (5, 6). Bacterially infected or LPS-stimulated mDCs have been shown to augment NK cell activity through cytokines, such as IL-2, IL-12, IL-15, IL-18 and type I IFNs, and through cell–cell contact (7–12). Flagellin-activated mDCs have also been reported to induce NK cell proliferation and IFN-{gamma} production (13). During viral infection, virus-infected Dendritic cells (DCs) play an important role in NK cell activation. Murine cytomegalovirus-infected CD11b+ DCs efficiently activate NK cells in a mouse model (14). In a DC–NK co-culture system, IFN-{gamma} production is mainly dependent on IL-12 and IL-18 released from DCs, while IFN-{alpha}/ß and a NKG2D–NKG2D ligands (NKG2DLs) interaction are required for NK cell cytotoxicity (3–6).

In humans, seven NKG2DLs, UL16-binding proteins (ULBP) 1, 2, 3 and 4, retinoic acid early transcript 1G (RAET1G) and MHC class I-related chains (MIC) A and B, have been reported so far (15–19). Although increasing evidence has shown the importance of a NKG2D–NKG2DLs interaction in DC-mediated NK cell activation, there have been few studies in which the expression of NKG2DLs on human mDCs has been examined. Earlier studies suggested that LPS and IFN-{alpha} induce ULBP1 and MICA and B expression on human mDCs, respectively (10, 20). However, these earlier studies were performed with limited knowledge on human TLR output and the properties of antibodies against NKG2DLs. Therefore, we produced anti-ULBP1, 2 and 3 polyclonal antibodies (pAbs) and examined whether stimulation with Pamps or infection with RNA viruses [influenza virus (Flu) A/PR8/34, human respiratory syncytial virus (RSV), measles virus (MV) and hepatitis C virus (HCV)] influenced the expression of NKG2DLs on human mDCs.

By using our pAbs against ULBPs, we found that not only ULBP1 and MICA/B but also ULBP2 and 3 were expressed on the surface of human mDCs. We also found that stimulation of human mDCs with Pamps or RNA virus infections induced expression of various NKG2DLs, which contributed to NK cell proliferation and IFN-{gamma} production as a consequence of the mDC–NK interaction.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 Funding
 References
 
Cell lines, antibodies, plasmids and reagents
Hep-2, Vero, RK13 (derived from the rabbit kidney) and 293FT cells were maintained in DMEM (Invitrogen, Carlsbad, CA, USA) supplemented with 10% heat-inactivated FCS (JRH Biosciences, Lenexa, KS, USA) and antibiotics. MDCK cells were maintained in MEM (Nissui) supplemented with 5% heat-inactivated FCS and antibiotics. Huh-7.5.1 cells were kindly provided by Francis V. Chisari. Huh-7.5.1 cells were maintained in DMEM supplemented with 10% heat-inactivated FCS, 10 mM Hepes, 2 mM glutamine, 1 mM Na-pyruvate, 0.1 mM non-essential amino acid and antibiotics. Anti-ULBP1 mAb (170818), anti-ULBP2 mAb (165903), anti-ULBP3 mAb (166510, 166514), anti-ULBP1 pAb (AF1380), anti-ULBP2 pAb (AF1298) and anti-human MICA/B mAb (159207) and anti-NKG2D mAb (149810) were obtained from R&D Systems (Minneapolis, MN, USA). Mouse IgG2a and anti-CD56 mAb (MEM188) were purchased from eBioscience (San Diego, CA, USA). A mAb against the hepatitis C core protein (C7-50) was obtained from Affinity BioReagents (Golden, CO, USA). Anti-Flag M2 mAb was obtained from Sigma-Aldrich (St Louis, MO, USA). FITC-conjugated goat anti-mouse IgG F(ab')2 and anti-rabbit IgG F(ab')2, and HRP-conjugated goat anti-rabbit and anti-mouse Igs were obtained from American Qualex (San Clemente, CA, USA). The plasmid pJFH-1 in which HCV cDNA (genotype 2a) was cloned behind a T7 promoter was kindly provided by T. Wakita. cDNAs for ULBP1, 2 and 3 were cloned in our laboratory by reverse transcription (RT)–PCR from mRNA of 293FT. Expression vectors for N-terminal Flag-tagged ULBP1, 2 and 3 were constructed by inserting the cDNA fragment into the mammalian expression vector pFLAG-CMV-1 (Sigma-Aldrich). IFN-ß was purchased from PeproTech (Rocky Hill, NJ, USA). LPS from Escherichia coli serotype 0111:B4 and polymyxin B were obtained from Sigma-Aldrich. Poly I:C was purchased from GE Healthcare (Buckinghamshire, UK). Synthetic Pam3CSK4 (Pam3) was obtained from Roche Diagnostics (Basel, Switzerland). A synthetic lipopeptide based upon the full-length mycoplasmal macrophage-activating lipopeptide-2 (Malp2) was obtained from Biosynthesis, Inc. (Lewisville, TX, USA). Lipopeptide was frozen at –20°C as a 200-µM stock solution in 25 mM octylglucoside.

Production of pAbs against ULBP1, 2 and 3
pAbs against ULBP1, 2 and 3 were raised in a rabbit. The protocol for antibody production was reported previously (21). Briefly, RK13 cells (1 x 107) were transiently transfected with ULBP1, 2 and 3/pFLAG-CMV-1 using Lipofectamine 2000 (Invitrogen). After 2 days, transfected RK13 cells were collected with 10 mM EDTA–PBS, washed with PBS three times and suspended in 0.5 ml of PBS. Then, RK13 cell suspensions were mixed with 0.6 ml of Freund's complete adjuvant (Invitrogen) and extensively agitated. The mixture was used to immunize rabbits four times at 7-day intervals with a boost injection before drawing blood. The antisera were harvested by centrifugation. IgG was purified from the sera according to the standard method. IgG (0.5 µg) was absorbed with RK13 cells (1 x 107) in FACS buffer (PBS containing 0.1% BSA and 0.1% sodium azide).

Virus propagation
MV Edmonston strain was passaged and titrated in Vero cells. Human RSV field-isolate strain (RSV2177) in subgroup B was isolated and propagated with Hep-2 cells (RSV2177 was kindly provided by K. Imai, Wakayama Prefecture Center). The accession numbers of NS1, NS2, N, G, F and SH genes are AB245473–AB245478. The titer of RSV2177 was determined by 50% tissue culture infective dose (TCID50) with Hep-2 cells. Flu A/PR8/34 (FluA; H1N1) was kindly provided by H. Kida (Hokkaido University, Sapporo). FluA was prepared and titered by TCID50 with MDCK cells as described previously (22). The method to generate infectious HCV particles in an in vitro system using the plasmid pJFH-1 was described previously (23, 24). The viral titer was expressed as focus-forming units per milliliter of supernatant, determined by the average number of core-positive foci detected at the highest dilutions. MV, FluA and RSV were inactivated by UV irradiation at 1.8 J cm–2.

Preparation of immature mDCs and NK cells
CD14+ monocytes were isolated from human PBMCs using a MACS system (Miltenyi Biotec, Bergisch Gladbach, Germany). Immature mDCs were generated from monocytes (1 x 106 cells ml–1) by culture for 6 days in RPMI 1640 medium supplemented with 10% heat-inactivated FCS in the presence of 500 IU ml–1 recombinant human granulocyte macrophage colony-stimulating factor (PeproTech) and 100 IU ml–1 recombinant human IL-4 (PeproTech). Preparations of immature mDCs were checked for the surface markers CD14–, CD40+, CD83–, CD80low, CD86low and CD1a+ before use. Autologous NK cells were negatively isolated from PBMCs by MACS. Cells purified by this technique had an average purity of 95%, as assessed by flow cytometry with anti-CD56 mAb. Autologous NK cells were stocked by Cell Banker (Diaton, Tokyo, Japan) at –80°C during induction of mDCs.

Stimulation and infection of immature mDCs
Immature mDCs (1 x 106 cells) were incubated for indicated time intervals with LPS (100 ng ml–1), poly I:C (50 µg ml–1), Pam3 (100 ng ml–1), Malp2 (100 nM) and IFN-ß (1000 U ml–1). These reagents were treated with polymyxin B (final concentration, 5 µg ml–1) for 1 h at 37°C before stimulation of the mDCs. The immature mDCs (1 x 106 cells) were infected with MV, FluA and RSV at a multiplicity of infection (moi) of 1, and 2 x 105 cells of immature mDCs were infected with HCV at a moi of 1. The cells were collected at 4°C by gentle pipetting with PBS containing 2 mM EDTA.

Western blotting
RK13 cells in 12-well plates were transiently transfected with ULBP1, 2, 3/pFLAG-CMV-1 and control vector (pFLAG-CMV-1) using Lipofectamine 2000 according to the manufacturer's protocol. After 24 h, the cells were solubilized in PBS containing 10 mM EDTA, 1 mM phenylmethylsulphonylfluoride (PMSF), and 1% Triton X-100. After standing at 4°C for 30 min, supernatants were separated by centrifugation at 10 000 x g for 3 min. Pellets of the Triton X-100 lysates were further solubilized in PBS containing 10 mM EDTA, 1 mM PMSF and 60 mM octylglucoside at 4°C for 30 min with constant agitation, and an octylglucoside-soluble fraction was obtained by centrifugation at 15 000 x g for 1 h at 4°C. The supernatants were subjected to SDS–PAGE under non-reducing conditions and transblotted onto nylon membranes. The membranes were incubated with pAbs against ULBP1, 2 or 3 for 2 h, washed three times with Tris-buffered saline (TBS) containing 0.5% Tween 20 and incubated with HRP-conjugated goat anti-rabbit Igs for 1 h at 37°C. Following the second incubation, the membranes were washed three times with TBS-Tween 20 and proteins were detected with an ECL chemiluminescence kit (GE Healthcare).

Immunoprecipitation
Octylglucoside-soluble fractions were obtained from 293FT cells (5 x 106 cells) and immature mDCs (5 x 107 cells) as described above. The cell lysates were incubated with indicated pAbs or mAbs for 2 h at 4°C. The immune complexes were precipitated with protein G-Sepharose and washed thoroughly. Immunoprecipitated proteins were eluted by adding SDS–PAGE sample buffer and boiling and were subjected to SDS–PAGE (12.5%) under non-reducing conditions followed by immunoblotting with indicated antibodies.

Flow cytometry
Cells were incubated with 0.5 µg of pAbs or mAbs together with human IgG (10 µg) for 30 min at 4°C in a FACS buffer. After the cells had been washed twice with the above buffer, FITC-labeled secondary antibody (American Qualex) was added, and the cells were incubated for an additional 30 min at 4°C. The cells were then washed and treated with FITC-labeled secondary antibodies for 30 min at room temperature. Ten percent goat serum was added to each reaction mixture to prevent non-specific binding. Cells were analyzed on a FACSCalibur (BD Biosciences PharMingen, San Diego, CA, USA).

Quantitative RT–PCR
Total RNA was extracted from immature mDCs infected with HCV by Trisol (GE Healthcare). After other virus infection or stimulation of immature mDCs, total RNA was extracted with an RNeasy mini kit (Qiagen, Bothell, WA, USA). A total of 1 µg of total RNA was incubated at 70°C for 5 min and then kept on ice for 2 min, and RT was performed with Moloney murine leukemia virus reverse transcriptase (Promega, Madison, WI, USA) at 37°C for 90 min followed by quantitative PCR. Primers for PCR were designed using Primer Express software (Perkin Elmer Applied Biosystems, Foster City, CA, USA). The following primers were used for PCR: ß-actin forward, 5'-CCTGGCACCCAGCACAAT-3' and reverse, 5'-GCCGATCCACACGGAGTACT-3'; ULBP1 forward, 5'-CAAGTGGAGAATTTAATACCCATTGAG-3' and reverse, 5'-TGTTGTTTGAGTCAAAGAGGA-3'; ULBP2 forward, 5'-TTACTTCTCAATGGGAGACTGT-3' and reverse, 5'-TGTGCCTGAGGACATGGCGA-3'; ULBP3 forward, 5'-CCTGATGCACAGGAAGAAGAG-3' and reverse, 5'-TATGGCTTTGGGTTGAGCTAAG-3', MICA forward, 5'-CCTTGGCCATGAACGTCAGG-3' and reverse, 5'-CCTCTGAGGCCTCGCTGCG-3'; MICB forward, 5'-ACCTTGGCTATGAACGTCACA-3' and reverse, 5'-CCCTCTGAGACCTCGCTGCA-3'; ULBP4 forward, 5'-CCTCAGGATGCTCCTTTGTGA-3' and reverse, 5'-CGACTTGCAGAGTGGAAGGATC-3' and RAET1G forward, 5'-TGGCCGACCCTCACTCTCT-3' and reverse, 5'-CCGTGGTCCAGGTCTGAACT-3'. The PCR reaction mixture contained 15 µl of Platinum SYBR Green qPCR SuperMix-UDG (Invitrogen), 200 nM of paired primers and 1 µl of cDNA in a total volume of 25 µl. ß-Actin was used as an internal control to normalize reactions. The PCR conditions were 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. Triplicate results of quantitative real-time PCR were analyzed by using the 2{delta}{delta}CT method as described previously (25).

mDC–NK co-culture and cytokine assay
Immature mDCs were infected with FluA, MV and RSV at a moi of 1 and harvested for mDC–NK co-culture at 24 h after infection. Immature mDCs (1 x 106 cells) were transfected with 5 µg of ULBP1, 2, 3/pFLAG-CMV-1 plasmid DNA by electroporation using a Human Dendritic Cell Nucleofector Kit (Amaxa, Gaithersburg, MD, USA). At 36 h after transfection, the mDCs were treated with mitomycin C. Autologous NK cells (1 x 105) were co-cultured with the mDCs (5 x 104) in 96-well round-bottom plates in the presence or absence of 100 IU ml–1 of IL-2 for 24 h and 3 days. Twelve hours before harvesting, [3H]thymidine (1 mCi/well) was added to the wells. Then the cells were harvested with a cell harvester, and radioactivity was measured by a liquid scintillation counter (Aloca, Tokyo, Japan). The supernatants of the mDC–NK co-culture were collected and assayed in commercial ELISA kit for IFN-{gamma} (GE Healthcare).


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 Funding
 References
 
Production of pAbs against ULBP1, 2 and 3
We produced pAbs against human ULBP1, 2 and 3 by immunizing rabbits with RK13 cells independently expressing ULBP1, 2 or 3. Successful production of pAbs was tested by immunoblotting and FACS analysis. To minimize non-specific reactions, these antibodies were further absorbed with intact RK13 cells. Immunoblotting and flow cytometric analysis using the anti-ULBP1, 2 and 3 pAbs showed that the antibodies did not cross-react with each other (Fig. 1A and B). HEK293FT cells were transfected with vectors for the expression of ULBP1, 2 and 3, and their levels of expression were determined by flow cytometry using the pAbs against anti-ULBP1, 2 or 3. The results were compared with those obtained with commercially available mAbs. The FACS profiles of ULBP1, 2 and 3 against pAbs were essentially identical to those against mAbs (Fig. 1C). The anti-ULBP3 pAb also made immunoprecipitation feasible (Fig. 1D); however, the anti-ULBP1 and 2 pAbs did not because of the low affinity toward antigens (data not shown). Our anti-ULBP3 pAb did not immunoprecipitate either ULBP1 or ULBP2 (Fig. 1D). Thus, we established tools to detect human ULBPs in a variety of human cells.


Figure 1
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  		Fig. 1. Characterization of pAbs against ULBP1, 2 and 3. (A) Western blot analysis of RK13 cells transfected with ULBP1/pFLAG-CMV-1 (lane 1), ULBP2/pFLAG-CMV-1 (lane 2), ULBP3/pFLAG-CMV-1 (lane 3) and an empty vector (lane 4). The blots were probed with our anti-ULBP1, 2 and 3 pAbs and anti-Flag mAb as indicated. (B) Flow cytometric analysis of RK13 cells transfected with ULBP1, 2, 3/pFLAG-CMV-1. Cells were stained with our anti-ULBP1, 2 and 3 pAbs and anti-Flag mAb and FITC-labeled secondary antibody. (C) ULBP1, 2 and 3 expressions in 293FT cells determined by flow cytometry using anti-ULBP1, 2, 3 pAbs and mAb. Shaded histograms represent cells labeled with isotype-matched control antibody. (D) ULBP3 in 293FT cells was immunoprecipitated by our anti-ULPB3 pAb. Octylglucoside-soluble fractions obtained from 293FT cells were incubated with our anti-ULPB1, 2 and 3 pAbs. Commercial anti-ULBP1 pAb (AF1380), anti-ULBP2 pAb (AF1298) and anti-ULBP3 mAb (166514) were used to detect each ULBP for western blot analysis as indicated. Lysates of RK13 cells transfected with ULBP1, 2 and 3 were loaded in the right side of lane as positive control (PC).

 
ULBP1, 2 and 3 expression on mDCs
We then examined the expression profiles of ULBPs on mDCs by flow cytometry using the pAbs and mAbs against ULBPs. The most prominent finding was that the expression of ULBP3 was detected on immature mDCs only by anti-ULBP3 pAb (Fig. 2). The pAbs as well as mAbs conferred similar expression profiles of ULBP1 and 2 on immature mDCs (Fig. 2). Thus, the expression levels of ULBP3 were undetermined until this anti-ULBP3 pAb was developed: the commercially available mAb (166510) we have tested failed to detect ULBP3. We then performed an immunoprecipitation assay to confirm the expression of ULBP3 in mDCs. Another anti-ULBP3 mAb (166514) and our anti-ULBP3 pAb were used for immunoprecipitation of ULBP3 in immature mDCs and for immunoblotting analysis, respectively. A band specific to ULBP3 was detected from the extract of immature mDCs (Fig. 3). On mDCs, the fluorescence shifts due to the antibodies were usually slight but specific on flow cytometry. Thus, we concluded that human mDCs possess ULBP3, that we have first described herein.


Figure 2
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  		Fig. 2. Expression profiles of ULBP1, 2 and 3 on mDCs. Expression of ULBP1, 2 and 3 on mDCs was examined by flow cytometry using anti-ULBP1, 2 and 3 pAbs and mAbs. The results for four representative donors of six are shown. The values of mean fluorescence shift are shown in the insets.

 

Figure 3
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  		Fig. 3. Expression of ULBP3 in octylglucoside-soluble fractions of mDCs (lanes 1, 2) and non-transfected 293FT cells (lanes 3, 4). Cells were solubilized with 60 mM octylglucoside and ULBP expressions were examined by immunoprecipitation. The extracts were incubated with anti-ULBP3 mAb (166514) (lanes 1, 3) or isotype-matched control antibody (lanes 2, 4). Anti-ULBP3 pAb was used for probing the respective proteins by western blot analysis.

 
Another notable point is that the expression patterns of ULBP1, 2 and 3 were individually different on immature mDCs. Four of six representative profiles obtained by pAbs are shown in Fig. 2. mDCs from donor 1 expressed ULBP1, 2 and 3, while only ULBP2 and 3 were expressed on the mDCs from donor 2. On the mDCs from donor 3, only ULBP3 was detected among the ULBPs. Almost no ULBP expression, apart from a very slight expression of ULBP2, was observed on the mDCs from some donors, such as donor 4. Thus, ULBP1 was expressed at a lesser or undetectable level compared with the levels of ULBP2 and 3 on immature mDCs; the conclusion was confirmed with the mAbs (Fig. 2).

We next examined the levels of surface-expressed ULBPs in freshly isolated monocytes and cells during monocyte-to-mDC differentiation (Fig. 4). Like the case with mDCs, the anti-ULBP3 pAb, but not the other pAbs, detected ULBP3 on the surface of monocytes. ULBP1 was expressed on freshly isolated monocytes and its expression level diminished concomitant with the maturation state of the mDC. In contrast to ULBP1, the expression level of ULBP2 on monocytes increased in parallel with mDC differentiation. The results were confirmed by using anti-ULBP2 pAbs and mAbs. The level of surface expression of ULBP3 was also shown to be up-regulated during mDC maturation by using the anti-ULBP3 pAb.


Figure 4
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  		Fig. 4. Variable expressions of ULBP1, 2 and 3 during induction of mDCs. Freshly isolated monocytes and monocytes cultured for 3 or 6 days in the presence of IL-4 and granulocyte macrophage colony-stimulating factor were analyzed for expression of ULBP1, 2 and 3 by flow cytometry using anti-ULBP1, 2 and 3 pAbs and mAbs. The values of mean fluorescence shift are shown in the insets.

 
Induction of NKG2DLs on mDCs by TLR ligands
Previous reports showed that ULBP1 and MICA/B are expressed on LPS- and IFN-{alpha}-stimulated mDCs, respectively (11, 20). We next examined the expression of ULBP1, 2 and 3 and MICA/B on mDCs stimulated with various TLR ligands (LPS, poly I:C, Pam3 and Malp2) as well as type I IFN with the use of flow cytometry (Table 1). The mDCs from four donors were assayed 48 h after stimulation. The cell surface expression of ULBP2, as well as that of ULBP1, was induced by LPS. Stimulation of mDCs with poly I:C resulted in an increased expression of ULBP2 on the surface of mDCs. The incremental expression of surface ULBP started 36 h after stimulation (data not shown). In our experimental setting, the cell surface expression of MICA and B was not induced by IFN-ß at any time beginning 48 h after stimulation, although mDC maturation stimulated by IFN-ß was confirmed by flow cytometry using anti-CD83 antibody.


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  		Table 1. LPS, poly I:C and RNA virus infection induce NKG2DLs

 
We analyzed the transcriptional regulation of NKG2DL mRNAs by stimulation with TLRs. NKG2DLs, ULBP1 and 2 and RAET1G were induced by TLR ligands (Fig. 5A). Results were obtained by transcriptional analysis with mDCs stimulated with LPS and poly I:C and confirmed the results obtained by flow cytometry (Fig. 5A). Poly I:C was also a strong inducer of RAET1G mRNA, which showed the highest level of all NKG2DLs on mDCs. MICA mRNA was not induced by any of the TLR ligands or IFN-ß. Transcription of MICB was slightly induced in mDCs stimulated with poly I:C and IFN-ß 24 h after stimulation (Fig. 5A), and declined thereafter (data not shown). Poly I:C and IFN-ß did not appear to induce sufficient MICB mRNA to influence cell surface expression of MICB on mDCs in our experimental setting (Fig. 3A).


Figure 5
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  		Fig. 5. Expression of various NKG2DLs in immature mDCs was induced by TLR ligands and virus infections. (A, B) mDCs were harvested at indicated time points after treatment with TLR ligands (Pam3, poly I:C, Malp2 and LPS), IFN-ß or viruses (MV, FluA, RSV and HCV). The mRNA levels of NKG2DLs in mDCs were measured by quantitative PCR. (C) UV inactivation of viruses abolished the induction of ULBP2 and RAET1G mRNA. Up-regulation of ULBP1 induced by RSV was resistant to UV inactivation.

 
Induction of NKG2DLs on mDCs by RNA virus infections
We showed that poly I:C induced ULBP2 and RAET1G on mDCs, which prompted us to examine the effects of RNA virus infections on the expression of NKG2DLs on mDCs. We chose four RNA viruses (MV, FluA, RSV and HCV) for testing NKG2DL induction. Immature mDCs have been reported to be infected with MV, FluA and RSV in vitro (26–28). Flow cytometric analysis using anti-ULBP1, 2 and 3 pAbs and anti-MICA and B mAbs showed that MV and FluA induced cell surface expression of ULBP2, while RSV induced ULBP1 (Table 1). Transcriptions of ULBP2 and RAET1G were induced at relatively higher levels than those of other TLR ligands in mDCs infected with MV, FluA and RSV. Above all, RSV was a strong inducer of ULBP1 mRNA. We next inactivated these RNA viruses by UV to examine whether induction of NKG2DLs was due to some virus-associated proteins or virus replication. UV-inactivated viruses precluded the cells from up-regulation of ULBP2 and RAET1G, although the up-regulation of ULBP1 by RSV was not affected by UV inactivation (Fig. 5C).

We next infected immature mDCs with HCV at a moi of 1 to evaluate the mDC response in terms of NKG2DLs against HCV. Flow cytometry and transcriptional analysis showed no detectable changes in NKG2DLs in mDCs infected with HCV (Table 1 and Fig. 5B).

Up-regulation of ULBPs on mDCs promote NK cell proliferation and IFN-{gamma} production
We examined whether NKG2DLs induced on mDCs were involved in mDC-mediated NK cell activation. NK cell proliferation and IFN-{gamma} production were determined by co-culturing NK cells with mDCs over-expressing ULBP1 or 2. Immature mDCs were transfected with plasmid containing cDNA of ULBP1 or 2 by electroporation. The surface expression of ULBP1 and 2 on these mDCs was confirmed 36 h after transfection (Fig. 6A). The transfected mDCs were treated with mitomycin C and co-cultured with autologous NK cells in the presence or absence of IL-2. IL-2 was essential for mDCs to induce NK cell proliferation and IFN-{gamma} production by the function of ULBPs (data not shown). In the presence of IL-2, NK cells efficiently proliferated when co-cultured with ULBP1- and 2-transfected mDCs (Fig. 6B). NK cells produced more IFN-{gamma} than those co-cultured with control mDCs (transfected with the vector only) (Fig. 6C). We next blocked the interaction between NKG2D and NKG2DLs by anti-NKG2D mAbs in the virus-infected mDCs–NK co-culture system. Then, the blockade of signals via NKG2D resulted in a decrease of NK cell IFN-{gamma} production (Fig. 6D). Taken together, the ULBPs expressed on mDCs may play a role in effecting NK cell proliferation and IFN-{gamma} production.


Figure 6
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  		Fig. 6. NK cell proliferation and IFN-{gamma} production via interaction between NKG2D and NKG2DLs. (A) mDCs were transfected with plasmid containing cDNA of ULBP1 or 2 and cultured for 36 h. Mock-transfected mDCs were used as a control. Cells were analyzed by flow cytometry using anti-Flag mAb. Shaded histograms represent cells transfected with control vecter. (B) mDCs either mock- or ULBP transfected were treated with mitomycin C and co-cultured with autologous NK cells for 3 days. NK cell proliferation was measured by [3H]thymidine assay. (C) mDCs were transfected with ULBP1 and 2. After 36 h culture, transfected mDCs were treated with mitomycin C and co-cultured with autologous NK cells in the presence of IL-2 for 24 h. (D) mDCs were infected with MV, FluA and RSV for 24 h and co-cultured with autologous NK cells for 24 h in the presence of anti-NKG2D mAb or isotype control. The supernatants of mDC–NK co-culture were assayed for IFN-{gamma}. Values are expressed as the means ± SD of triplicate determinations. Similar experiments were repeated twice, and representative results are shown. *Significantly different (P < 0.05) by Student's t-test.

 

   Discussion
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 Abstract
 Introduction
 Methods
 Results
 Discussion
Funding
 References
 
In the present study, we produced pAbs against ULBP1, 2 and 3. These antibodies were specific and suitable for detection of ULBP proteins by western blotting and flow cytometry. These pAbs allowed us to reveal that ULBP2 and 3, as well as ULBP1 and MICA/B, were expressed on the surfaces of mDCs. TLR stimulation with Pamps and RNA viruses caused a variable induction of NKG2DLs on mDCs, which augmented NK cell proliferation and IFN-{gamma} production.

There appears to be a discrepancy in ULBP3 protein expression judged by using the pAbs and commercially available mAbs. After many comparative studies using pAbs and mAbs, we concluded that mDCs harbor ULBP3 protein on their membranes. Thus, our pAbs were useful for the detection of ULBP1, 2 and 3. By using the pAbs, ULBP3, as well as ULBP1 and 2, can be analyzed by blotting and flow cytometry. Because pAbs recognize multiple epitopes in general, the results may predict that steric hinderance of membrane ULBP3 disturbs the access of mAbs to the epitope. On mDCs but not on 293FT cells, there may be some molecules which have a role in hiding the epitope of the anti-ULBP3 mAb (166514). If this is the case, a molecular assembly involving ULBP3 appears complex in mDCs beyond the probing abilities of anti-ULBP3 mAbs.

The second point is that the cell surface expression of NKG2DLs (ULBP1, 2 and 3 and MICA and B) is variable. The molecular mechanisms by which the expression of NKG2DLs is regulated remain largely undetermined. Polymorphism in the promoters of NKG2DLs may be one of the reasons for the variable expression. Indeed, the putative promoter regions of the NKG2DL genes have a number of potential binding sites for transcription factors, such as heat shock transcription factor 1, activation transcription factor 6, myeloid zinc finger 1, basonuclin, IFN regulatory factor 7 and NF-{kappa}B, which are involved in shock, danger signals and foreign material responses (26). However, none of these transcriptional factors has been proved to actually bind to the promoter regions and the precise role of these putative promoter regions has not yet been determined.

ULBP2 was induced by LPS and poly I:C but not by other TLR ligands on the mDCs (Table 1). Other ULBP proteins tend to be up-regulated by poly I:C and/or LPS. On mDCs, cytoplasmic and membrane receptors recognize poly I:C, whereas the TLR4 complex signals the presence of LPS (4). Hence, a possible interpretation of Fig. 5(A) is that the membrane receptors, TLR3 and TLR4, were involved in ULPB up-regulation. Since both TLR3 and TLR4 can activate the TIR-containing adapter molecule-1 (TICAM-1) (TIR domain-containing adaptor-inducing IFN) pathway, ULBP may be under the control of TICAM-1 signaling followed by activation of virus-activated kinases. When viruses replicate in mDCs, dsRNA is generated in the cytoplasm secondary to a viral infection, which mainly stimulates melanoma differentiation-associated gene 5 (MDA5) and retinoic acid-inducible gene-I (RIG-I) (27), namely the cytoplasmic RNA recognition pathway. Figure 5(B) also reveals that the cytoplasmic RNA recognition pathway led to up-regulation of ULBPs. It is rational to conclude that the extra- and intra-cytoplasmic RNA recognition results in a ULBP up-regulation profile in mDCs. RIG-I and MDA5 tightly link interferon-beta promoter stimulator 1 (IPS-1, MAVS, Cardif and VISA) to converge with the TICAM-1 pathway on the NAP1 adapter (28). Thus, our interpretation is consistent with the reported molecular cascades of the TICAM-1 pathway and the IPS-1 pathway. Total elevation of ULBP families on mDCs would be indispensable to the mDC–NK reciprocal activation.

Figure 5(B and C) suggests that each viral species differentially up-regulated ULBPs during infection. Viral replication inside the mDCs is essential for ULBP up-regulation because UV-irradiated viruses, except for RSV, lose the ULBP-inducing ability. UV-inactivated RSV still induced ULBP1, which was evident in this report. This finding may support a previous notion (29) that the F protein of RSV stimulates TLR4 to activate NF-{kappa}B. Other viruses, including MV, need RNA entry into the target cells for ULBP induction. In this context, no NKG2DLs were induced in HCV-infected mDCs, where HCV did not replicate productively (data not shown).

Although we have avoided mentioning each detail about viral infection versus ULBP regulation, ULBP2 and RAET1G were the main responders to viral replication. They are regulated by MDA5–RIG-I signaling secondary to viral replication (27). Human cytomegalovirus glycoprotein, UL16, binds NKG2DLs intracellularly and prevents cell surface expression (30). What happens in DNA virus infections in terms of ULBPs is an interesting issue.

Results of in vitro studies support the previous finding that cytokines and cell–cell contact play important roles in mDC-mediated NK cell activation (6, 8). Cytokines from mDCs as well as NK cells contribute to the reciprocal activation (6, 8). Except for the NKG2D–NKG2DLs interaction, various cell surface molecules are likely involved in the mDC–NK cell interaction. Other ligands for NK inhibitory or activating receptors on mDCs are possibly associated with mDC-mediated NK cell activity (6, 31, 32). We showed herein that mDCs allow autologous NK cells to produce IFN-{gamma} by expression of a single species of NKG2DL, ULBP1 or 2. mDCs transfected with ULBP1/pFLAG-CMV-1 expressed a high level of ULBP1. Other NKG2DLs, such as ULBP2 and 3, and MICA and B were not induced on mDCs by electroporation (data not shown). When the mDCs were treated with mitomycin C, secondary mDC responses stimulated by NK cells and cytokines from mDCs did not influence mDC-mediated NK cell activation. A notable point is the requirement for IL-2 in NK cell activation via up-regulated ULBPs on mDCs, which in part reflects previous studies showing that IL-2 is required for NK cell activation in mDC–NK co-cultures (7, 8). An important point of our human studies was that autologous NK cell IFN-{gamma} production was augmented by NKG2DL-expressing mDCs and IL-2.

Under physiologic conditions, we showed that the blockade of NKG2D resulted in a decrease of IFN-{gamma} production in the virus-infected mDCs–NK cell co-culture system (Fig. 6D). Then, we next examined the influence of a single NKG2DL by the blockade of a single NKG2DL, such as ULBP1, 2 and 3, by our pAbs. However, no significant difference was observed in IFN-{gamma} production (data not shown). Therefore, the entire repertoire of NKG2DLs was involved in the NKG2D–NKG2DLs interaction. In this context, expression of ULBP3 on mDCs contributed to the amount of NKG2DLs on mDCs although we did not find any stimulation which induced ULBP3 on mDCs in this study.

In murine macrophages, transcription of RAE-1 family proteins, the murine homologues of ULBPs, are induced by TLR ligands, LPS, Pam3 and poly I:C. Of these Pamps, LPS and poly I:C are the strongest inducers of RAE-1 family proteins. Among the RAE-1 family proteins, RAE-1{delta} and RAE-1{epsilon} are up-regulated in a myeloid differentiation factor 88-dependent manner (33). In another study, infection with Mycobacterium tuberculosis induced the up-regulation of ULBP1 expression in human monocytes via TLR2, although TLR4-mediated up-regulation of ULBP1 has not been examined (34). Our human studies suggested that the TLR4 pathway, rather than the TLR2 pathway, is crucial for ULBP1 up-regulation. RAE-1 and ULBPs are likely to be differentially regulated in mice and humans in bacterial and viral infections. In principle, mDC-mediated NK activation relies on the total level of expression of the RAE-1–ULBP family, which is regulated by TLR signaling in mDCs. This event, TLRs participating in determining the magnitude of NK cell activation by mDCs, is largely common across mice and humans.


   Funding
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Funding
References
 
CREST; Japan Science and Technology Corporation; Grants-in-Aid from the Ministry of Education, Science and Culture (Specified Project for Advanced Research); HCV project in National Institutes of Health of Japan; Northtic Foundation; Uehara Memorial Foundation; Mitsubishi Foundation; Takeda Foundation.


   Acknowledgements
 
We are grateful to K. Funami, A. Ishii and M. Sasai in our laboratory for their critical discussions.


   Abbreviations
 
DC, Dendritic cell
Flu, influenza virus
HCV, hepatitis C virus
IPS-1, interferon-beta promoter stimulator 1
Malp2, macrophage-activating lipopeptide-2
MDA5, melanoma differentiation-associated gene 5
mDC, monocyte-derived dendritic cell
MIC, MHC class I-related chains
MV, measles virus
NKG2DL, NKG2D ligand
pAb, polyclonal antibody
Pamps, pathogen-associated molecular patterns
PMSF, phenylmethylsulphonylfluoride
RAET1G, retinoic acid early transcript 1G
RIG-I, retinoic acid-inducible gene-I
RSV, respiratory syncytial virus
RT, reverse transcription
TBS, Tris-buffered saline
TCID50, 50% tissue culture infective dose
TICAM-1, TIR-containing adapter molecule-1
TLR, Toll-like receptor
ULBP, UL16-binding proteins

   Notes
 
Transmitting editor: K. Okumura

Received 27 November 2006, accepted 11 June 2007.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Funding
 References
 

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