of May 18, 2018.This information is current as

Tumor-Associated Inhibition In VitroPathway and Resistance of Dendritic Cells to

B CanonicalκPromotes Activation of NF-Chromosome 1 Open Reading Frame 190

Zhang, Zhujun Zhang and Rongcun YangZhang, Jingyi Liu, Miaomiao Zhang, Jiangbo Oyang, Yuan Zhizi Jing, Xin Yuan, Jing Zhang, Xin Huang, Zhiqian

http://www.jimmunol.org/content/185/11/6719doi: 10.4049/jimmunol.0903869November 2010;

2010; 185:6719-6727; Prepublished online 3J Immunol 

MaterialSupplementary

9.DC1http://www.jimmunol.org/content/suppl/2010/11/03/jimmunol.090386

Referenceshttp://www.jimmunol.org/content/185/11/6719.full#ref-list-1

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The Journal of Immunology

Chromosome 1 Open Reading Frame 190 PromotesActivation of NF-kB Canonical Pathway and Resistance ofDendritic Cells to Tumor-Associated Inhibition In Vitro

Zhizi Jing, Xin Yuan, Jing Zhang, Xin Huang, Zhiqian Zhang, Jingyi Liu,

Miaomiao Zhang, Jiangbo Oyang, Yuan Zhang, Zhujun Zhang, and Rongcun Yang

Tumor-associated dendritic cells (DCs) often induce T cell anergy or deletion and regulatory T cells instead of antitumor immunity.

Although many tumor-associated Ags have been found, there is still no effective vaccine for cancer. Thus, novel rational strategies to

enhance the immunogenicity of cancer-specific Ags are needed. Chromosome 1 open reading frame 190 (c1orf190), a gene that

encodes a 239-aa hypothetical protein and contains multiple kinase phosphorylation sites, has a wide relationship with multiple

signaling pathway molecules and can be regulated by multiple factors, such as TLR ligands. In this study, we demonstrate that

c1orf190 can activate NF-kB, drive the production of cytokines, and promote the Ag-presenting function and the priming ability

of DCs. Furthermore, c1orf190 can promote resistance of DCs to tumor-associated inhibition not only in the Ag-presenting

function but also in the priming ability to induce Ag-specific T lymphocytes. Thus, c1orf190, an NF-kB activator, may be a

candidate gene for regulating the function of DCs to resist tumor-associated factor-mediated dysfunction. We also found that

c1orf190-mediated cytokine release is achieved by activating the canonical but not the noncanonical NF-kB pathway. The

Journal of Immunology, 2010, 185: 6719–6727.

Dendritic cells (DCs) are key regulators of innate andadaptive immunity by selectively promoting or sup-pressing T cell responses (1). In addition to orchestrating

essential aspects of the adaptive immune response, DCs produceproinflammatory cytokines, such as IL-1b, IL-6, and IL-12 toregulate the functions of innate immune cells. DCs can be inducedfrom monocytes under certain conditions, including in the pres-ence of GM-CSF and various TLR ligands. Central to the im-munologic function of DCs is that their maturation is induced byTNF family members and TLR ligands. Activation of the canon-ical and the noncanonical NF-kB pathways is a common signalingresponse to these stimuli. A number of observations have dem-onstrated that the transcription factor NF-kB is essential for op-timal DC maturation. Inhibition of NF-kB or its upstream acti-vator IkB kinase (IKK)2 may block DC Ag presentation both in

vitro and in vivo (2–5). DC development and survival also requiresdistinct NF-kB subunits. For example, DC development is inhib-ited in RelB-deficient mice (6) and in bone marrow cells infectedwith adenoviruses harboring an IkB repressor (7).NF-kB, composed of NF-kB1 (p50), NF-kB2 (p52), RelA,

RelB, and c-Rel, plays a central role in regulating diverse biolog-ical processes (8–10), especially in inducing immune and inflam-matory responses and in regulating the expression of immuneresponse genes associated with innate and adaptive immunity (11).NF-kB can be activated in response to TNF-a, IL-1, TLR li-gands, and various growth factors. Various external stimuli lead tothe phosphorylation of IkB by IKKs, which results in its ubiq-uitination and proteolytic degradation at the proteasome. NF-kB isthen released from IkB, translocates into the nucleus, binds to itscognate DNA elements, and induces the transcription of variouscytokines, receptors, and other proinflammatory genes (12, 13).NF-kB/IkB may directly respond to many independent signalingmolecules, as reviewed by Verma and Stevenson (14). The mech-anisms by which extracellular signals are transmitted to NF-kBare still unclear despite intensive investigative efforts. Findinga novel functional molecule that activates NF-kB transcriptionfactor will cause an important consequence not only in under-standing the NF-kB–activating pathways, but also in developingnew adjuvants that enhance vaccine immunogenicity.Many kinases, such as protein kinase A, protein kinase C (PKC),

casein kinase II (CKII), glycogen synthase kinase-3b, T2K (TBK,NAK), and PI3K, have been shown to induce the phosphorylationof NF-kB directly or indirectly (8–10, 15, 16), which may benecessary for the transcriptional competence of NF-kB dimers byfacilitating their DNA binding, transcriptional coactivator recruit-ment, and acetylation. Chromosome 1 open reading frame 190(c1orf190), a hypothetical protein, contains multiple CKII, PKC,and cAMP- and cGMP-dependent protein kinase phosphorylationsites. In this study, we demonstrate that c1orf190 participates inthe activation of NF-kB, the induction of proinflammatory cyto-kines, and in the Ag-presenting and priming function of DCs.

Department of Immunology, Nankai University School of Medicine and Key Labo-ratory of Bioactive Materials, Nankai University, Ministry of Education, People’sRepublic of China

Received for publication December 10, 2009. Accepted for publication September22, 2010.

This work was supported in part by National Natural Science Foundation of ChinaGrants 30771967 and 30872315, the Ministry of Science and Technology (China)(863 Program, 2008AA02Z129), the National Key Basic Research and DevelopmentProgram of China (973 Program, 2007CB914803), and by the National Key ScientificProgram of China (2011CB964902).

Address correspondence and reprint requests to Dr. Rongcun Yang, Department ofImmunology, Nankai University School of Medicine, Nankai University, Tianjin300071, People’s Republic of China. E-mail address: ryang@nankai.edu.cn

The online version of this article contains supplemental material.

Abbreviations used in this paper: c1orf190, chromosome 1 open reading frame 190;CKII, casein kinase II; CRE, cAMP response element; DC, dendritic cell; FIV, felineimmunodeficiency virus; GRE, glucocorticoid response element; HSE, heat shockresponse element; IKK, IkB kinase; NBD, NF-kB essential modulator-binding do-main; NEMO, NF-kB essential modulator; NIK, NF-kB–induding kinase; PKC, pro-tein kinase C; RLU, relative light unit; SEAP, secreted alkaline phosphatase; siIKKa,small interfering RNA for IkB kinase-a; siNIK, small interfering RNA for NF-kB–inducing kinase; siRNA, small interfering RNA; SRE, serum response element.

Copyright� 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00

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C1orf190 may be a candidate gene for regulating the functionof DCs.

Materials and MethodsPreparation of monocyte-derived DCs

Monocyte-derived DCs were prepared from monocytes according to ourpreviously described protocol (17, 18). Briefly, after Ficoll-Hypaque sep-aration, peripheral blood cells were resuspended in RPMI 1640 mediumsupplemented with 10% FBS, 2 mM L-glutamine, and 1% penicillin-streptomycin. The cells were incubated overnight at 37˚C, and the non-adherent cells were removed by gentle pipetting. The adherent cells werecultured in RPMI 1640 and supplemented with 10% FBS containing 1000U/ml GM-CSF (R&D Systems, Minneapolis, MN) and 1000 U/mlIL-4 (R&D Systems). After 6 d, floating DCs were harvested in PBSand cell morphology was determined by standard microscopic techniques.Phenotypes of DCs were analyzed by PE- or FITC-labeled anti-CD11c(3.9), anti-human MHC class II-DR (L243), anti-human CD86 (IT2.2),anti-human CD80 (2D10.4), anti-human CD40 (HB14), anti-human B7H1(M1H1), and anti-human CD11b (ICRF44) that were purchased from BDPharmingen (San Diego, CA).

Gene cloning

C1orf190 cDNAwas obtained by the PCR frommonocyte-derived DCswithTaqDNA polymerase. The PCR product was cloned into the eukaryoticexpression vector pcDNA3.1/V5-His-TOPO as c1orf190 pcDNA (Invi-trogen, Carlsbad, CA). C1orf190 pcDNA was used to transfect HEK293T cells using Lipofectamine 2000 (Invitrogen) and used to transfectDCs using a Nucleofection approach according to the manufacturer’sprotocol (Amaxa Biosystems, Cologne, Germany) after sequencing. The for-ward primer 59-ATGGAGGGGACCGTGGAGTCCCAGACG-39 and reverseprimer 59-CAAGAAGGTCACATCATCCTGGCACTG-39 were used toclone human c1orf190 gene.

RT-PCR analysis

Total cellular RNA was prepared using TRIzol reagent (Invitrogen) asrecommended by the manufacturer. RT-PCR was performed by SuperScriptone-step RT-PCR with Platinum Taq according to the protocol provided(Invitrogen). The amplification conditions included the following steps: onecycle of cDNA synthesis and predenaturation (50˚C for 15 min, 94˚C for 2min), 40 cycles of PCR amplification (denaturation at 94˚C for 15 s,annealing at 55˚C for 30 s, and extension at 72˚C for 1 min/kb), and onecycle of final extension (72˚C for 10 min). The primers used in this studyare listed in Supplemental Table I.

Reporter assay

For the reporter assay, p-NF-kB–secreted alkaline phosphatase (SEAP),p-AP-1-SEAP, p-NF-AT-SEAP, p-MyC-SEAP, p-serum response element(SRE)-SEAP, p-cAMP response element (CRE)-SEAP, p-heat shock re-sponse element (HSE)-SEAP, and positive control p-SEAP2 as well as

negative control p-TATA-like-SEAP reporter constructs were purchasedfrom BD Clontech (Palo Alto, CA) and used to cotransfect the cells withthe plasmids. As described before (19), 293T cells were seeded in 96-wellplates and cotransfected with c1orf190 construct combined with reporterplasmids as indicated. Twenty-four hours after transfection with Lip-ofectamine 2000, according to the manufacturer’s instructions, cells weretreated with the indicated reagents or left untreated for 12 h. The sampleswere harvested and processed as described above for determination ofSEAP activity and normalized for the b-galactosidase activity (BDClontech). The amount of transfected DNA was equalized with emptyexpression vectors, which were also used in the control, along with eitherNF-kB or other SEAP reporter constructs.

Small interfering RNA experiments

To knock down c1orf190, the small interfering RNA (siRNA) templatesused to knock down the c1orf190 were selected and designed using thesiRNA target finder (http://www.ambion.com/techlib/misc/siRNA_finder.html) (Supplemental Table II). To maximize knockdown and minimize off-target effects, each gene was given two couples of the siRNA templates.Searches of the Homo sapiens genome database (BLAST) were carried outto ensure that the sequences would not target other genes. Then the siRNAtemplate was cloned into the p-feline immunodeficiency virus (FIV)double-promoter siRNA-expressing vector p-FIV-H1-U6-copGFP, whichcontains human U6 and mouse H1 double promoters according to theprotocol described by the manufacturer (System Biosciences, MountainView, CA). Irrelevant siRNAwas used as a control. siRNA structures weredirectly used to transfect the 293T cells using Lipofectine 2000 and used totransfect DCs using a Nucleofection approach. Silencing of the targetmolecule was demonstrated using RT-PCR.

siRNA for NF-kB–inducing kinase (siNIK), IKKa (siIKKa), or controlsiRNA (50 nmol/ml) was used to transfect the cells using Lipofectamine2000 according to the manufacturer’s instruction. The siRNA sequencesused to target human NIK and IKKa mRNA were as follows: siNIK, 59-GCUCCGUCUACAAGCUUGATT-39 (sense) and siIKKa, 59-GGCCU-GUGAUGUUCCUGAATT-39 (sense). Nonsilencing control siRNA is anirrelevant siRNA with random nucleotides (59-ACUATCUAAGUUACTA-CCCCTT-39). Sequences were synthesized and annealed by Shanghai San-gon Biological Engineering Technology and Services (Shanghai, China).NF-kB essential modulator (NEMO) siRNA (M-003767-02) and non-targeting control (D001201-03) were obtained from Dharmacon (Lafay-ette, CO) as a pool of four annealed dsRNA oliogonucleotides.

NEMO-binding domain peptide-mediated NF-kB inhibition

For NEMO-binding domain (NBD) peptide-mediated inhibition, both thefunctional wild-type TAT-NBD (YGRKKRRQRRR–G–TTLDWSWLQME)and the negative control mutant TAT-NBD (YGRKKRRQRRR–G–TTL-DASALQME) described by others (20, 21) were synthesized by ShanghaiSangon Biological Engineering Technology and Services. NBD-mediatedNF-kB inhibition was done according to the protocol described by Taset al. (20, 21). The c1orf190-transfected DCs were incubated for 2 h withthe NBD peptide or control mutant NBD. The effect on the NF-kB activity

FIGURE 1. Characterization of c1orf190 gene. A,

The ideogram of human chromosome 1 with c1orf190.

C1orf190 is located in 46669006–46686928pb on chro-

mosome 1p34. B, The schematic diagram of c1orf190

protein showing 26 hits with a high probability of

occurrence by ScanProsite (http://us.expasy.org/tools/

scanprosite/). C, The expression of c1orf190 as a cyto-

plasmic protein. 293T cells were transfected with V5-

tagged c1orf190 vectors and stained using FITC-labeled

anti-V5 Abs. An image was collected by confocal mi-

croscopy (original magnification3400) (C2). An image

collected using bright lightwas used as a control (C1).D,

The transcriptional levels of c1orf190 in DCs after ex-

posure to different TLR ligands. DCswere culturedwith

5 mg/ml bacterium DNA, 1 mg/ml LPS, and 2.5 mg/ml

polyinosinic-polycytidylic acid. After 24 h, c1orf190

transcriptional levels were detected using RT-PCR.

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and cytokine release were analyzed after 24 h. NBD and control mutantpeptides were used at a concentration of 50 mM.

Western blotting analysis

For Western blot analyses, the cells were harvested and subjected to SDS-PAGE. Following the transfer to a Hybond-P membrane (AmershamBiosciences, Piscataway, NJ), the samples were analyzed by Westernblotting with anti-p-IkBa or anti-inactive IkBa Ab (Cell Signaling Tech-nology, Beverly, MA). The protein–Ab complexes were detected using theperoxidase-conjugated secondary Ab (Boehringer Mannheim, Mannheim,Germany) and ECL (Amersham Biosciences).

Immunofluorescence and fluorescence microscopy

The cells were washed with PBS, fixed with 3.7% formaldehyde solution for10 min, permeabilized with 0.1% (v/v) Triton X-100 in BPS for 5 min, andblocked with PBS containing 1% BSA for 30 min. For the transfected cells,plasmid expressing V5 FITC-labeled mAb (Invitrogen) was added at 1mg/ml to detect V5. For the nuclear localization of RelA/p65, thetransfected cells were incubated for 1 h with a 1/1000 dilution of thespecific polyclonal antiserum against RelA/p65 (sc-372; Santa Cruz Bio-technology, Santa Cruz, CA). The cells were then washed with PBS andlabeled with a Cy3-conjugated secondary Ab (Millipore, Bedford, MA).

Samples were examined by fluorescence microscopy (Fluoview FV300;Olympus Optical, Tokyo, Japan).

Functional analysis

To determine the function of DCs transfected by c1orf190, we first observethe effect of c1orf190 on the ability of DCs to stimulate the Ag-specificT cells.

We prepared influenza peptide-specific HLA-A0201–restricted CD8+

CTLs using our previous protocol (18). In brief, isolated CD8+ T lym-phocytes with a purity of .95% were seeded into 48-well plates (Poly-Sorb; Nunc, Roskilde, Denmark) at a concentration of 5 3 105 cells/wellin 10% human AB serum-RPMI 1640 medium. As APCs, autologous DCswere incubated with 20 mg/ml peptides in serum-free RPMI 1640 mediumfor 2 h at 37˚C in 5% CO2. After washing, DCs were added to the plates ata concentration of 1 3 106 cells/well. IL-7 (10 ng/ml) was added at theinitiation of the cultures. After incubation for 48 h, IL-2 (10 U/ml) wasadded to the cultures. As the controls, CD8 T cells were cultured withcytokines alone at the same time. In some cases, at day 14 and day 21,CD8 T cells were stimulated with peptide-loaded, irradiated autologousDCs. Influenza virus-specific CTLs were then cocultured with HLA-A2–restricted peptide-pulsed DCs transfected with c1orf190 or c1orf190siRNA. After incubation for 48 h, supernatants were collected and IFN-gwas measured by ELISA. To load DCs with peptides, DCs were pulsedwith influenza-restricted peptide (20 mg/ml) in RPMI 1640 medium for 3 h

FIGURE 2. C1orf190 activates NF-kB. A, The expression of c1orf190 in the transfected 293T cells and DCs. A1, The transcriptional levels of c1orf190

in 293T cells (A1.1) and DCs (A1.2) transfected with c1orf190-targeted siRNA (siRNA) or control siRNA (mocksiRNA); A2, The protein levels of V5-fused

c1orf190 in 293T cells (A2.1) and DCs (A2.2) transfected by V5-tagged c1orf190 vectors (C1orf190/V5) or control vectors (Vector.ctr). 293T cells and DCs

were transfected using the protocol described in Materials and Methods. The transcriptional levels of c1orf190 were detected using RT-PCR. C1orf190/V5

fusion protein was detected by anti-V5 Abs (Invitrogen) using Western blotting. B, Effect of c1orf190 siRNAs and ectopic c1orf190 on the activity of

transcriptional factors NF-kB, AP-1, and NF-AT. C1orf190-specific siRNAs downregulated the activity of NF-kB and NF-AT, whereas ectopic c1orf190

upregulated the activity of NF-kB and NF-AT in both 293T cells (B1) and DCs (B2). MocksiRNA, siRNA, Vector.ctr, and c1orf190 are 293T cells (B1) or

DCs (B2) transfected by control siRNA, c1orf190 siRNA, control vectors, and c1orf190 vectors, respectively. C, The effect of c1orf190 siRNA and ectopic

c1orf190 on NF-kB activity is dose-dependent. Increasing amounts of c1orf190 siRNA or c1orf190 vectors were cotransfected with p-SEAP reporter

constructs (250 ng) into 293T cells. The supernatants were harvested and tested using a SEAP reporter assay after 24 h. Data are presented as the percentage

inhibition of SEAP activity in cells transfected with c1orf190 siRNA as compared with cells transfected with control siRNA (C1) or presented as the fold

change of SEAP activity in 293T cells transfected with c1orf190 as compared with the cells transfected with control vectors (C2). D, Ectopic c1orf190

promotes the nuclear translocation of p65. 293T cells (D1) or DCs (D2) transfected by c1orf190 vector (C1orf190) or control vector (Vector.ctr) were plated

on gelatin-coated coverslips and the subcellular localization of NF-kB p65 was analyzed by immunofluoresence with a specific polyclonal antiserum

against p65. The nuclei were stained with DAPI (blue). Original magnification 340. RLU, relative light unit.

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at 37˚C, according to our previously reported method (18), and thenwashed three times before use.

To investigate whether c1orf190-transfected DCs could resist the effectof tumor-associated factors on Ag-presenting function of DCs, DCs werefirst transfected with c1orf190, c1orf190 siRNA, or control vectors and thencocultured with ovarian carcinoma SK-OV3, cervical carcinoma HeLa cellsand SiHa cells in 24-Transwell plates, TGF-b1 (10 ng/ml), or IL-10 (10 ng/ml) for 18 h. These treated DCs, after having been loaded with HLA-A2–restricted peptides, were cocultured with influenza virus-specific CTLs.After 48 h, supernatants were collected and IFN-g was measured byELISA.

To investigate whether c1orf190-transfected DCs resist the effect oftumor-associated factors on the priming ability of DCs, we initially gen-erated synthetic Melan-A/MART-1 mRNA, which can induce MHC classI-restricted cytotoxic T cells in vivo and in vitro (22, 23). A full-lengthcDNA fragment from the plasmid (American Type Culture Collection,Manassas, VA) was subcloned into the plasmid pSP-64 (poly(A)) at Hinc1and XmaI (Promega, Madison, WI) in front of a synthetic poly(A) tail,which allows in vitro transcription under the control of an SP6 promoter.The plasmids were linearized behind the poly(A) tail by restriction enzymedigestion at FSP1 site and in vitro transcribed with the SP6 mMESSAGEmMACHINE kit (Ambion, Austin, TX) according to the protocol providedby the manufacturer. Purification of in vitro transcripts was performedwith RNeasy Mini anion-exchange spin columns (Qiagen, Valencia, CA)according to the RNA cleanup protocol provided by the manufacturer.These synthetic Melan-A/MART-1 mRNAs were used to cotransfect DCswith c1orf190 vectors, c1orf190 siRNA, or control vectors using Nucle-ofector technology (Amaxa Biosystems, Gaithersburg, MD). The trans-fected DCs, after having been cocultured with ovarian carcinomaSK-OV3, cervical carcinoma HeLa cells, and SiHa cells in a 24-Transwellplate, TGF-b1 (10 ng/ml), or IL-10 (10 ng/ml) for 18 h, were then used toinduce Melan-A/MART-1–specific T lymphocytes according to the above-described method. The sp. act. of induced CD8+ T cells was analyzed inresponse to autologous DC-loaded HLA-A0201–restricted Melan-A/MART-1peptides or HLA-A0201–restricted unrelated peptides, according to ourprevious method (18), on day 5 after the last restimulation in IFN-gELISA assay.

The mAb BB7.2 (HB82; American Type Culture Collection) was usedto detect HLA-A0201. HLA-A0201–binding influenza peptide MP58–66(GILGFVFTL), HLA-A0201–binding Melan-A/MART-126–35 (EAAGI-GILTV), and HLA-A0201–restricted unrelated binding peptide (QLFF-DNYAL) were synthesized by a solid phase method, using a multiple pep-tide synthesizer, and purified by HPLC, as previously described (18).

ELISA

Commercial sandwich ELISA kits were used to quantify IFN-g, IL-1b,IL-6, and IL-12p70 (Pierce/Endogen, Rockford, IL). The OD value of eachof the samples was measured at 450 nm using a SpectraMax 190 ELISAplate reader. Cytokine levels were quantified by two to three titrationsusing standard curves and expressed in picograms per milliliter.

Statistical analysis

For statistical analysis, we used the Student t test, and a 95% confidencelimit was taken to be significant (defined as p , 0.05)

ResultsC1orf190 activates NF-kB

C1orf190, a hypothetical protein, which can be detected in humanDCs (GEO DataSets [http://www.ncbi.nlm.nih.gov/sites/entrez]:GDS2750/235214_at/C1orf190/Homo sapiens, GDS2453/235214_at/C1orf190/Homo sapiens, and GDS1249/235214_at/C1orf190/Homosapiens), includes a 720-bp open reading frame that encodes a239-aa protein (LOC541468) (Fig. 1). A ScanProsite analysis (http://us.expasy.org/tools/scanprosite/) showed that c1orf190 contained N-myristoylation sites (MYRISTYL), an amidation site (AMIDATION),CKII phosphorylation sites (CK2_PHOPHO_SITES), a leucine zip-per pattern (LEUCINE_ZIPPER), a cAMP- and cGMP-dependentprotein kinase phosphorylation site (CAMP_PHOSPHO_SITE)(Fig. 1), and a protein kinase C phosphorylation site (PKC_PHOSPHO_SITE). A fusion protein of c1orf190 tagged with V5could be detected in the cytoplasm of transfected 293T cells (Fig.1C). Importantly, c1orf190 expression in DCs could be regulatedby multiple factors, including TLR ligands (GEO DataSets: GDS2750/235214_at/C1orf190/Homo sapiens; Fig. 1D), hypoxia (GEO Data-Sets: GDS2750/235214_at/C1orf190/Homo sapiens), and peroxi-some proliferator-activated receptor-g ligand or the retinoic acidreceptor-a antagonist (GEO DataSets: GDS2453/235214_at/C1orf190/Homo sapiens). CKII (24), PKC (25, 26), cAMP, andcGMP-dependent protein kinase (27), as protein serine/threonine

FIGURE 3. C1orf190 drives the production of proinflammatory cytokines and promotes the Ag-presenting function of DCs. A, The transcriptional levels

of IL-1b, IL-6, and IL-12 in DCs transfected by c1orf190 siRNA or ectopic c1orf190. Ctr., C1orf190, MocksiRNA, and siRNA are DCs transfected by

control vectors, c1orf190, control siRNA, and c1orf190 siRNA, respectively. B, Cytokine secretion by DCs transfected by c1orf190 or c1orf190 siRNA.

Supernatants from culture medium were collected and subjected to an ELISA assay using ELISA kits for the cytokines IL-1b, IL-6, and IL-12 as described

in Materials and Methods. Ctr., C1orf190, mocksiRNA, and siRNA are, respectively, DCs transfected by control vectors, c1orf190 vectors, control siRNA,

and c1orf190 siRNA. C, C1orf190 promotes the Ag-presenting function of DCs. Influenza virus-specific CTLs were cocultured with different numbers of

HLA-A2–restricted peptide-pulsed DCs, which were transfected with control vectors, c1orf190 vectors (C1orf190), control siRNA (MocksiRNA), or

c1orf190 siRNA (siRNA). The supernatants were collected and IFN-g was measured by ELISA after incubation for 48 h.

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kinases, may phosphorylate many different proteins. Furthermore,because TLRs and hypoxia-associated genes play a critical role incontrolling differentiation of DCs, c1orf190 might have an im-portant role in regulating the differentiation and function of DCs.To explore the biological function of c1orf190, we investigated

whether c1orf190 could regulate the activity of transcription factorsNF-kB, AP-1, NF-AT, glucocorticoid response element (GRE),HSE, CRE, MyC, and SRE. Because HEK 293T cells and DCscould express endogenous c1orf190 (Fig. 2A), we first knockeddown the c1orf190 to observe the effect of c1orf190 degradationon the activity of transcription factors. siRNA templates ofc1orf190 were designed, synthesized, and cloned into p-FIV-H1-U6-copGFP. Whereas NF-kB-SEAP, NFAT-SEAP, GRE-SEAP,CRE-SEAP, MYC-SEAP, and HSE-SEAP were cotransfected in-to 293T cells or DCs with c1orf190-targeted siRNA with dem-onstrated transfection (Fig. 2A), NF-kB and NF-AT activity wassignificantly reduced, whereas other transcription factors such asAP-1, CRE, SRE, GRE, HSE, and MyC were not affected or wereonly slightly affected (Fig. 2B and Supplemental Fig. 1). More-over, downregulation of NF-kB activity mediated by c1orf190-targeted siRNAs was dose-dependent (Fig. 2C1). When the doseof c1orf190-targeted siRNA structures was .500 ng, suppressionof the NF-kB activity was .50%.To further confirm the effect of c1orf190 on transcription factors

NF-kB, AP-1, NF-AT, GRE, HSE, CRE, MyC, and SRE, ectopicc1orf190 was used to cotransfect 293T cells or DCs with each ofthe reporter plasmids. The overexpression of ectopic c1orf190 in293T cells and DCs was demonstrated with RT-PCR and Westernblotting (Fig. 2A). The results show that ectopic c1orf190 re-markably upregulated the activity of NF-kB and NF-AT, whereasthe activity of other transcription factors GRE, AP-1, HSE, SRE,CRE, MyC, and SRE was only slightly upregulated or stayedconstant (Fig. 2B and Supplemental Fig. 1). Ectopic c1orf190-mediated NF-kB activity was also dose-dependent (Fig. 2C2).C1orf190 not only affected NF-kB activity but also impactednuclear localization of p65 proteins. As shown in Fig. 2D, theectopic c1orf190 increased the nuclear localization of p65 in both293T cells and DCs. Thus, these data suggest that c1orf190 mayregulate the activity of transcription factors, especially NF-kB.

C1orf190 promotes the production of proinflammatorycytokines and the Ag-presenting function of DCs

NF-kB plays a critical role in DC activation and the expression ofproinflammatory cytokines and chemokines (21). Thus, we ex-amined the effect of c1orf190 on the production of proin-flammatory cytokines and on the Ag-presenting function of DCs.C1orf190, but not the control vectors, resulted in the upregulationof IL-1b, IL-6, and IL-12. As illustrated in Fig. 3A and 3B, thelevels of IL-1b, IL-6, and IL-12 in the c1orf190-transfected DCswere significantly higher than those in control vector-transfectedDCs (p , 0.05). Conversely, c1orf190 siRNA but not controlsiRNAs downregulated the mRNA and protein expression of IL-1b, IL-6, and IL-12 (p , 0.05; Fig. 3). Importantly, c1orf190promoted the Ag-presenting function of DCs. As shown in Fig.3C, peptide-pulsed DCs transfected with c1orf190 could stimulateinfluenza peptide-specific CD8 T cells to produce higher levels ofIFN-g than could peptide-pulsed control vector-transfected DCs(p , 0.05), whereas peptide-specific CD8 T cells cocultured withpeptide-pulsed DCs transfected by c1orf190 siRNA only producedlower levels of IFN-g as compared with control siRNA (p ,0.05). Thus, c1orf190 as an NF-kB activator is capable of regu-lating inflammatory cytokine production and Ag-presenting func-tion of DCs.

C1orf190 promotes resistance of DCs to tumor-associatedinhibition

Tumor-associated factors, especially IL-10 and TGF-b, can inhibitthe production of cytokines and decrease the functional capacityof DCs (28–31). Because c1orf190 could activate NF-kB and pro-mote the Ag-presenting and priming function of DCs, we nextinvestigated whether c1orf190-transfected DCs could resist theeffect of tumor-associated factors on DCs. To test this idea,c1orf190-transfected DCs were exposed to TGF-b, IL-10, or dif-ferent kinds of tumor supernatants. Morphological analysisshowed that while c1orf190-transfected DCs were exposed to tu-mor supernatants, DCs still maintained their morphological struc-ture with higher levels of expression of MHC class II-DR, CD40,

FIGURE 4. C1orf190 promotes resistance of DCs to the tumor-associ-

ated inhibition in presenting Ag(s). A, Ag-presenting function of DCs

transfected by c1orf190 is not affected by TGF-b1 and IL-10. Influenza

virus-specific CTLs were cocultured with HLA-A2–restricted peptide-

pulsed DCs (CTLs/DCs, 10:1), which were transfected with c1orf190

(C1orf190) or Ctr. and then exposed to TGF-b1 or IL-10 for 3 d. Ctr. in A1

and A2, DCs transfected by control vectors without TGF-b1 or IL-10

treatment; TGFbCtr. in A1, DCs transfected by control vectors with TGF-

b1 (10 ng/ml) treatment; IL10Ctr. in A2, DCs transfected by control

vectors with IL-10 (10 ng/ml) treatment; C1orf190 in A1 and A2, DCs

transfected by c1orf190 vectors without TGF-b1 or IL-10 treatment;

TGFbc1orf190 in A1, DCs transfected by c1orf190 vectors with TGF-b1

(10 ng/ml) treatment; IL10c1orf190 in A2, DCs transfected by c1orf190

vectors with IL-10 (10 ng/ml) treatment. B, The Ag-presenting function of

DCs transfected by c1orf190 is not affected by tumor-associated factors.

Vector.ctr, c1orf190, MocksiRNA, or siRNA are, respectively, the relative

secretion of peptide-specific CTLs after coculture with DCs transfected by

control vectors, c1orf190 vectors, control siRNA, or c1orf190 siRNA upon

exposure to different treatments, including control medium (Med.), ovarian

carcinoma SK-OV3 (Tu1; American Type Culture Collection), cervical

carcinoma HeLa cells (Tu2; American Type Culture Collection), SiHa cell

(Tu3; American Type Culture Collection), TGF-b1 (TGFbeta; 10 ng/ml) or

IL-10 (10 ng/ml). Relative secretion is the cytokine secretion (pg/ml) in

CTLs stimulated with the treated DCs divided by the cytokine secretion

(pg/ml) in CTLs stimulated with the control-treated DCs.

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CD80, CD86, and B7-H1 (Supplemental Fig. 2), whereas DCstransfected with control vectors lost typical structures and at-tachment ability with a lower expression of MHC class II-DR,CD40, CD80, CD86m and B7-H1 (Supplemental Fig. 2), imply-ing that the c1orf190-tranfected DCs have an ability to resist theeffect of tumor-associated factors on DCs. Importantly, unlikecontrol vector-transfected DCs that had the reduced Ag-presentingfunctions upon exposure to tumor-associated factor TGF-b orIL-10, c1orf190-transfected DCs still had a strong Ag-presentingfunction even in the presence of tumor-associated factor TGF-bor IL-10 as compared with the control (p , 0.05; Fig. 4A). To fur-ther determine the resistance of c1orf190-transfected DCs againsttumor-associated factors, c1orf190-transfected DCs were exposedto different kinds of tumors, including ovarian carcinomas andcervical carcinomas, in a Transwell plate. C1orf190-transfectedDCs indeed exhibited strong resistance to the tumor-associatedfactors and produced higher levels of IFN-g as compared withcontrols (p , 0.05), whereas c1orf190 siRNA-transfected DCshad reduced Ag-presenting function and produced lower levelsof IFN-g compared with controls (p , 0.05), especially afterexposure to tumor supernatants (Fig. 4B).

We also investigated the effect of c1orf190 on the priming abilityof DCs in the presence of tumor-associated factors. Toward thisend, we employed the tumor-associated Ag Melan-A/MART-1,which has been demonstrated to induce Ag-specific HLA-A0201–restricted cytotoxic CD8+ lymphocytes (22, 23). We first investi-gated whether c1orf190 could promote DCs to induce Ag-specificT lymphocytes. As shown in Fig. 5B, indeed, Ag-specific T lym-phocytes induced by DCs transfected by c1orf190 released higherlevels of IFN-g than did those induced by DCs transfected by thecontrol plasmids in response to Melan-A/MART-1 HLA-A0201–restricted peptide-loaded DCs, whereas c1orf190-specific siRNAreduced the priming ability of DCs. Next, we sought to determinewhether DCs transfected by c1orf190 could resist the effect oftumor-associated factors on the priming ability of DCs. As shownin Fig. 5C, the ability of DCs untransfected or transfected bycontrol empty plasmid or control siRNA in inducing Melan-A/MART-1–specific T lymphocytes could be significantly reducedby IL-10, TGF-b, or tumor supernatants (p , 0.05). Especially,DCs transfected with c1orf190 siRNA had the more remarkablydecrease in inducing Melan-A/MART-1–specific T lymphocytesin the presence of IL-10, TGF-b, or tumor supernatants (p, 0.001).

FIGURE 5. C1orf190 promotes resistance of DCs to the tumor-associated inhibition in inducing Ag-specific T cells. A, The expression of Melan-A/

MART-1 and c1orf190 in DCs cotransfected with Melan-A/MART-1 (Mart-1) and c1orf190 siRNA (C1orf190 siRNA, DC2), siRNA control (Ctr.siRNA,

DC3), control vector (Ctr. Vector, DC4), c1orf190 (C1orf190, DC5), or c1orf190 plus c1orf190 siRNA (C1orf190 siRNA, DC6). DC1, DCs cotransfected

with control vector (Ctr. Vector) and control siRNA (Ctr.siRNA). The transcriptional levels of Melan-A/MART-1 and c1orf190 in cotransfected DCs were

detected using RT-PCR. B, DCs transfected with Melan-A/MART-1 induce Melan-A/MART-1–specific CD8+ T lymphocytes. Melan-A/MART-1–specific

CD8+ T cells were induced according to the protocol described in Materials and Methods. DC1T, DC2T, DC3T, DC4T, DC5T, and DC6T are Melan-A/

MART-1–specific CD8+ T cells induced by DC1, DC2, DC3, DC4, DC5, and DC6, respectively. The release of IFN-g by DC1T, DC2T, DC3T, DC4T,

DC5T, and DC6T were detected in response to HLA-A0201–restricted MART-1 peptide-loaded autologous DCs (Mart-1 DC) or control peptide-loaded

autologous DCs (Ctr.DC). C, C1orf190 promotes resistance of DCs to tumor-associated factors in inducing Ag-specific CD8+ T lymphocytes. MocksiRNA,

siRNA, Vector.ctr, and c1orf190 are, respectively, the relative secretion of Melan-A/MART-1–specific CD8+ T lymphocytes, which were induced by the

treated DC2, DC3, DC4, and DC5 in response to HLA-A2010–restricted Melan-A/MART-1 peptide-loaded autologous DCs. The treated DC2, DC3, DC4,

and DC5 were generated after exposed to medium (Ctr.), ovarian carcinoma SK-OV3 (Tu1), cervical carcinoma HeLa cells (Tu2), and SiHa cells (Tu3),

TGF-b1 (TGFbeta; 10 ng/ml), or IL-10 (10 ng/ml) according to the protocol described inMaterials and Methods. As described above, DC2, DC3, DC4, and

DC5 are DCs cotransfected with Melan-A/MART-1 mRNA and c1orf190 siRNA, c1orf190 siRNA control, control vector, or c1orf190. Relative secretion is

the cytokine secretion (pg/ml) in Melan-A/MART-1–specific T cells induced by the treated DCs divided by the cytokine secretion (pg/ml) in Melan-A/

MART-1–specific T cells induced by the control-treated DCs.

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However, the priming ability of DCs transfected with c1orf190to induce Ag-specific T lymphocytes was not remarkably affectedby IL-10, TGF-b, or tumor supernatants. Thus, our results clearlyreveal that c1orf190 may promote the resistance of DCs to tumor-associated inhibition.

C1orf190-driven proinflammatory cytokine introduction occursvia the canonical NF-kB pathway

NF-kB is an inducible transcription factor that is controlled bytwo principal signaling cascades, the classical/canonical NF-kBactivation pathway and the alternative/noncanonical pathway (32).Next, we examined whether c1orf190 drives proinflammatorycytokine introduction via the canonical NF-kB pathway or thenoncanonical pathway using the NBD peptide, which is a highlyselective inhibitor of the canonical NF-kB pathway and IKKa-targetted and/or NIK-targetted siRNA, which acts to silence thenoncanonical pathway as described by others (20, 21, 33). InMDCs cotransfected with NBD peptide and c1orf190, NBDpeptide not only blocked c1orf190-mediated IKBa phosphoryla-tion but also decreased IL-1b, IL-6, and IL-12 production medi-ated by c1orf190 (p , 0.05; Fig. 6B). As a control, mutant NBDpeptide had no effect (Fig. 6B). We also detected the effect ofNEMO siRNA on the canonical NF-kB pathway. NEMO siRNAalso inhibited c1orf190-mediated IKBa phosphorylation and re-duced IL-1b, IL-6, and IL-12 production mediated by c1orf190(p , 0.05; Fig. 6). However, siRNA-mediated knockdown of the

noncanonical pathway did not remarkably affect the productionof IL-1b, IL-6, or IL-12 in the c1orf190-transfected DCs ascompared with controls (Supplemental Fig. 3). The degradedIKKa and NIK by siRNA for the noncanonical NF-kB pathway-associated kinases IKKa (siIKKa) and NIK (siNIK) could beobserved (Supplemental Fig. 3). Thus, our results suggest thatc1orf190-driven production of proinflammatory cytokines and im-proved Ag-presenting function are mediated by activating the ca-nonical NF-kB pathway, not the noncanonical pathway.

DiscussionIn the studies presented, we have demonstrated that c1orf190 canactivate the activity of NF-kB, drive the production of proin-flammatory cytokines, and promote the Ag-presenting and prim-ing function of DCs via the canonical NF-kB pathway. Thus,c1orf190, as an NF-kB activator, may be involved in the reg-ulation of DC function and play an important role in the induc-tion of innate and adaptive immune responses. Meanwhile, wehave also examined the ability of c1orf190-transfected DCs to re-sist the effect of tumor-associated factors on the Ag-presentingand priming function of DCs. This might suggest an approach forenhancing vaccine immunogenicity.Examination of circulating and tumor-infiltrating DCs in tumor-

bearing animals and in cancer patients has revealed that DCs arefunctionally impaired in their ability to induce T cell responses.Tumor-associated factors, such as IL-10 (28) and TGF-b (29, 34),

FIGURE 6. C1orf190 drives proinflammatory cytokine production via the canonical but not the noncanonical NF-kB pathway. A, NBD peptide se-

lectively inhibits c1orf190-mediated phosphorylation of IKBa. DCs were transfected with c1orf190 (C1orf190) or control vectors (Ctr. Vector) and then

preincubated with either NBD peptide (NBD) or control mutant NBD peptide (NBDMut) for 2 h. Cell lysates were analyzed by Western blotting. B, NBD

peptide decreases IL-1b, IL-6, and IL-12p70 production by c1orf190-transfected DCs. MDCs were transfected by c1orf190 (C1orf190) or control vectors

(Ctr. Vector) and then incubated with either NBD peptide (NBD) or control peptide (NBDMut) for 2 h. Supernatants were harvested after 24 h, and secreted

IL-1b, IL-6, and IL-12p70 (pg/ml) were measured by ELISA. Results are expressed as means 6 SD from one representative experiment of three performed

in triplicate. p , 0.05. C, NEMO siRNAs inhibit c1orf190-mediated IKBa phosphorylation and IL-1b, IL-6, and IL-12 production. DCs were transfected

with control vector (Ctr. Vector), c1orf190 plasmid (C1orf190), or cotransfected using c1orf190 plasmids (C1orf190) with NEMO-specific siRNA

(NEMOsiRNA; 100 nM) or control siRNA (Ctr. siRNA; 100 nM). The transcriptional levels of NEMO and GAPDH were analyzed using RT-PCR (C1).

Phosphorylated IKBa was analyzed by Western blotting (C2). Supernatants were harvested after 24 h, and secreted IL-1b, IL-6, and IL-12p70 (pg/ml) were

measured by ELISA (C3). Results are expressed as means 6 SD from one representative experiment of three performed in triplicate.

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potentially inhibit the function of Ag-presenting cells, such asDCs, through a repression of inflammatory cytokine productionand MHC class II and costimulatory molecule expression. Tumor-associated DCs often induce T cell anergy or deletion and regu-latory T cells instead of antitumor immunity. As a result, althoughthere are many tumor-associated Ags found, there is still no ef-fective vaccine for cancer. Thus, novel rational strategies to en-hance the immunogenicity of pathogen-specific Ags and cancer-specific Ags are needed. Some immunological adjuvants havebeen shown to activate NF-kB among their multiple actions, butthey are, however, limited in use by their lack of specific, local-ized, and coordinated effects and consequently by toxicity (35).C1orf190, as an NF-kB activator, results in the upregulation ofcytokines that contain NF-kB sites on the promoters of their genesand promote the Ag-presenting function and priming ability ofDCs. Importantly, c1orf190 could resist the effect of tumor-associated factors. Thus, c1orf190 might be a candidate gene forimproving the immunogenicity of tumor vaccine.C1orf190 contains multiple CKII phosphorylation sites, PKC

phosphorylation sites, and cAMP- and cGMP-dependent proteinkinase phosphorylation sites, and it has a wide relationship withmultiple signaling pathway molecules (Supplemental Fig. 5). Thesemultiple sites may play an important role in mediating NF-kBactivity and proinflammatory cytokine production. Indeed, a sub-stantial amount of evidence shows that multiple kinases, such asPKC, CKII, protein kinase A, glycogen synthase kinase-3b, T2K(TBK, NAK), PI3K, AKT, p38, NIK, and even IKK, can inducephosphorylation of NF-kB (9, 10, 16). The distribution of NF-kB1, NF-kB2, RelA, RelB, and c-Rel is widespread and is re-ceptive to many extracellular and intracellular signals. Knock-down of c1orf190 partners affects the activity of NF-kB and theproduction of proinflammatory cytokines, suggesting that c1orf190-mediated NF-kB activation and proinflammatory cytokine produc-tion may be dependent on multiple signaling pathways. Furtherstudies are required to elucidate the biological function of theseinteractions.C1orf190-driven proinflammatory cytokine production is me-

diated by activating the canonical NF-kB pathway but not thenoncanonical pathway. C1orf190-mediated IKBa phosphorylationand IL-1b, IL-6, and IL-12 production by DCs may be blocked byNBD peptides or NEMO siRNA. The NBD peptide can block theassociation of NEMO with the IKK complex and inhibit cytokine-induced NF-kB activation and NF-kB–dependent gene expression(21). NF-kB inhibition by NBD peptide results in blockade ofIKK-mediated IkBa phosphorylation and subsequent nucleartranslocation and DNA binding of NF-kB p65 in DCs (20). Spe-cific inhibition of the canonical pathway in DCs has been dem-onstrated to cause immunoregulation not only in vitro (36, 37) butalso in vivo (37). Others have also shown that the production ofproinflammatory cytokine IL-12p70 and IL-6 can be blocked byinhibiting the canonical pathway using NBD peptide (20, 33). Thenoncanonical pathway is strictly dependent on IKKa homodimersand requires neither IKKb nor NEMO/IKKg (15, 38). Knockdownof IKKa or NIK may result in the increased production ofproinflammatory IL-12p70 and IL-6 production in DCs (20, 33).Recent studies in macrophages have also suggested a role forIKKa in the negative regulation of inflammation (39, 40). How-ever, siRNA-mediated knockdown of the noncanonical pathwayIKKa and NIK did not remarkably affect the production ofc1orf190-mediated IL-1b, IL-6, and IL-12.Additionally, IL-12, a heterodimeric proinflammatory cytokine

that induces the production of IFN-g, favors differentiation ofTh1 cells and forms a link between innate resistance and adaptiveimmunity (41). Because IL-12 plays a critical role in inducing Th1

responses, it will be interesting to study whether c1orf190 inducesTh1 immune responses in vivo.

DisclosuresThe authors have no financial conflicts of interest.

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