Tumor-Associated Inhibition in Vitro Pathway and Resistance of Dendritic Cells to B Canonical Κ Promotes Activation of NF

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Chromosome 1 Open Reading Frame 190 Promotes Activation of NF- κB Canonical Pathway and Resistance of Dendritic Cells to Tumor-Associated Inhibition In Vitro This information is current as of September 25, 2021. Zhizi Jing, Xin Yuan, Jing Zhang, Xin Huang, Zhiqian Zhang, Jingyi Liu, Miaomiao Zhang, Jiangbo Oyang, Yuan Zhang, Zhujun Zhang and Rongcun Yang J Immunol 2010; 185:6719-6727; Prepublished online 3 November 2010; Downloaded from doi: 10.4049/jimmunol.0903869 http://www.jimmunol.org/content/185/11/6719 http://www.jimmunol.org/ Supplementary http://www.jimmunol.org/content/suppl/2010/11/03/jimmunol.090386 Material 9.DC1 References This article cites 41 articles, 17 of which you can access for free at: http://www.jimmunol.org/content/185/11/6719.full#ref-list-1

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Chromosome 1 Open Reading Frame 190 Promotes Activation of NF-kB Canonical Pathway and Resistance of Dendritic 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 Downloaded from 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. http://www.jimmunol.org/

endritic cells (DCs) are key regulators of innate and vitro and in vivo (2–5). DC development and survival also requires adaptive immunity by selectively promoting or sup- distinct NF-kB subunits. For example, DC development is inhib- D pressing T cell responses (1). In addition to orchestrating ited in RelB-deficient mice (6) and in bone marrow cells infected essential aspects of the adaptive immune response, DCs produce with adenoviruses harboring an IkB repressor (7). proinflammatory cytokines, such as IL-1b, IL-6, and IL-12 to NF-kB, composed of NF-kB1 (p50), NF-kB2 (p52), RelA, regulate the functions of innate immune cells. DCs can be induced RelB, and c-Rel, plays a central role in regulating diverse biolog- from monocytes under certain conditions, including in the pres- ical processes (8–10), especially in inducing immune and inflam- ence of GM-CSF and various TLR ligands. Central to the im- matory responses and in regulating the expression of immune by guest on September 25, 2021 munologic function of DCs is that their maturation is induced by response genes associated with innate and adaptive immunity (11). TNF family members and TLR ligands. Activation of the canon- NF-kB can be activated in response to TNF-a, IL-1, TLR li- ical and the noncanonical NF-kB pathways is a common signaling gands, and various growth factors. Various external stimuli lead to response to these stimuli. A number of observations have dem- the phosphorylation of IkB by IKKs, which results in its ubiq- onstrated that the transcription factor NF-kB is essential for op- uitination and proteolytic degradation at the proteasome. NF-kBis timal DC maturation. Inhibition of NF-kB or its upstream acti- then released from IkB, translocates into the nucleus, binds to its vator IkB kinase (IKK)2 may block DC Ag presentation both in cognate DNA elements, and induces the transcription of various cytokines, receptors, and other proinflammatory genes (12, 13). NF-kB/IkB may directly respond to many independent signaling Department of Immunology, Nankai University School of Medicine and Key Labo- ratory of Bioactive Materials, Nankai University, Ministry of Education, People’s molecules, as reviewed by Verma and Stevenson (14). The mech- Republic of China anisms by which extracellular signals are transmitted to NF-kB Received for publication December 10, 2009. Accepted for publication September are still unclear despite intensive investigative efforts. Finding 22, 2010. a novel functional molecule that activates NF-kB transcription This work was supported in part by National Natural Science Foundation of China factor will cause an important consequence not only in under- Grants 30771967 and 30872315, the Ministry of Science and Technology (China) (863 Program, 2008AA02Z129), the National Key Basic Research and Development standing the NF-kB–activating pathways, but also in developing Program of China (973 Program, 2007CB914803), and by the National Key Scientific new adjuvants that enhance vaccine immunogenicity. Program of China (2011CB964902). Many kinases, such as protein kinase A, protein kinase C (PKC), Address correspondence and reprint requests to Dr. Rongcun Yang, Department of casein kinase II (CKII), glycogen synthase kinase-3b, T2K (TBK, Immunology, Nankai University School of Medicine, Nankai University, Tianjin 300071, People’s Republic of China. E-mail address: [email protected] NAK), and PI3K, have been shown to induce the phosphorylation The online version of this article contains supplemental material. of NF-kB directly or indirectly (8–10, 15, 16), which may be necessary for the transcriptional competence of NF-kB dimers by Abbreviations used in this paper: c1orf190, chromosome 1 open reading frame 190; CKII, casein kinase II; CRE, cAMP response element; DC, dendritic cell; FIV, feline facilitating their DNA binding, transcriptional coactivator recruit- immunodeficiency virus; GRE, glucocorticoid response element; HSE, heat shock ment, and acetylation. Chromosome 1 open reading frame 190 response element; IKK, IkB kinase; NBD, NF-kB essential modulator-binding domain; NEMO, NF-kB essential modulator; NIK, NF-kB–induding kinase; PKC, pro- (c1orf190), a hypothetical protein, contains multiple CKII, PKC, tein kinase C; RLU, relative light unit; SEAP, secreted alkaline phosphatase; siIKKa, and cAMP- and cGMP-dependent protein kinase phosphorylation small interfering RNA for IkB kinase-a; siNIK, small interfering RNA for NF-kB– sites. In this study, we demonstrate that c1orf190 participates in inducing kinase; siRNA, small interfering RNA; SRE, serum response element. the activation of NF-kB, the induction of proinflammatory cyto- Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00 kines, and in the Ag-presenting and priming function of DCs. www.jimmunol.org/cgi/doi/10.4049/jimmunol.0903869 6720 C1orf190 PROMOTES ACTIVATION OF NF-kB

C1orf190 may be a candidate gene for regulating the function negative control p-TATA-like-SEAP reporter constructs were purchased of DCs. from BD Clontech (Palo Alto, CA) and used to cotransfect the cells with the plasmids. As described before (19), 293T cells were seeded in 96-well plates and cotransfected with c1orf190 construct combined with reporter Materials and Methods plasmids as indicated. Twenty-four hours after transfection with Lip- Preparation of monocyte-derived DCs ofectamine 2000, according to the manufacturer’s instructions, cells were treated with the indicated reagents or left untreated for 12 h. The samples Monocyte-derived DCs were prepared from monocytes according to our were harvested and processed as described above for determination of previously described protocol (17, 18). Briefly, after Ficoll-Hypaque sep- SEAP activity and normalized for the b-galactosidase activity (BD aration, peripheral blood cells were resuspended in RPMI 1640 medium Clontech). The amount of transfected DNA was equalized with empty supplemented with 10% FBS, 2 mM L-glutamine, and 1% penicillin- expression vectors, which were also used in the control, along with either streptomycin. The cells were incubated overnight at 37˚C, and the non- NF-kB or other SEAP reporter constructs. adherent cells were removed by gentle pipetting. The adherent cells were cultured in RPMI 1640 and supplemented with 10% FBS containing 1000 Small interfering RNA experiments U/ml GM-CSF (R&D Systems, Minneapolis, MN) and 1000 U/ml IL-4 (R&D Systems). After 6 d, floating DCs were harvested in PBS To knock down c1orf190, the small interfering RNA (siRNA) templates and cell morphology was determined by standard microscopic techniques. used to knock down the c1orf190 were selected and designed using the Phenotypes of DCs were analyzed by PE- or FITC-labeled anti-CD11c siRNA target finder (http://www.ambion.com/techlib/misc/siRNA_finder. (3.9), anti-human MHC class II-DR (L243), anti-human CD86 (IT2.2), html) (Supplemental Table II). To maximize knockdown and minimize off- anti-human CD80 (2D10.4), anti-human CD40 (HB14), anti-human B7H1 target effects, each gene was given two couples of the siRNA templates. (M1H1), and anti-human CD11b (ICRF44) that were purchased from BD Searches of the Homo sapiens genome database (BLAST) were carried out Pharmingen (San Diego, CA). to ensure that the sequences would not target other genes. Then the siRNA template was cloned into the p-feline immunodeficiency virus (FIV) Downloaded from Gene cloning double-promoter siRNA-expressing vector p-FIV-H1-U6-copGFP, which contains human U6 and mouse H1 double promoters according to the C1orf190 cDNAwas obtained by the PCR from monocyte-derived DCs with protocol described by the manufacturer (System Biosciences, Mountain TaqDNA polymerase. The PCR product was cloned into the eukaryotic View, CA). Irrelevant siRNA was used as a control. siRNA structures were expression vector pcDNA3.1/V5-His-TOPO as c1orf190 pcDNA (Invi- directly used to transfect the 293T cells using Lipofectine 2000 and used to trogen, Carlsbad, CA). C1orf190 pcDNA was used to transfect HEK transfect DCs using a Nucleofection approach. Silencing of the target 293T cells using Lipofectamine 2000 (Invitrogen) and used to transfect molecule was demonstrated using RT-PCR.

DCs using a Nucleofection approach according to the manufacturer’s siRNA for NF-kB–inducing kinase (siNIK), IKKa (siIKKa), or control http://www.jimmunol.org/ protocol (Amaxa Biosystems, Cologne, Germany) after sequencing. The for- siRNA (50 nmol/ml) was used to transfect the cells using Lipofectamine ward primer 59-ATGGAGGGGACCGTGGAGTCCCAGACG-39 and reverse 2000 according to the manufacturer’s instruction. The siRNA sequences primer 59-CAAGAAGGTCACATCATCCTGGCACTG-39 were used to used to target human NIK and IKKa mRNA were as follows: siNIK, 59- clone human c1orf190 gene. GCUCCGUCUACAAGCUUGATT-39 (sense) and siIKKa,59-GGCCU- RT-PCR analysis GUGAUGUUCCUGAATT-39 (sense). Nonsilencing control siRNA is an irrelevant siRNA with random nucleotides (59-ACUATCUAAGUUACTA- Total cellular RNA was prepared using TRIzol reagent (Invitrogen) as CCCCTT-39). Sequences were synthesized and annealed by Shanghai San- recommended by the manufacturer. RT-PCR was performed by SuperScript gon Biological Engineering Technology and Services (Shanghai, China). one-step RT-PCR with Platinum Taq according to the protocol provided NF-kB essential modulator (NEMO) siRNA (M-003767-02) and non- (Invitrogen). The amplification conditions included the following steps: one targeting control (D001201-03) were obtained from Dharmacon (Lafay- by guest on September 25, 2021 cycle of cDNA synthesis and predenaturation (50˚C for 15 min, 94˚C for 2 ette, CO) as a pool of four annealed dsRNA oliogonucleotides. min), 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 one NEMO-binding domain peptide-mediated NF-kB inhibition cycle of final extension (72˚C for 10 min). The primers used in this study For NEMO-binding domain (NBD) peptide-mediated inhibition, both the are listed in Supplemental Table I. functional wild-type TAT-NBD (YGRKKRRQRRR–G–TTLDWSWLQME) Reporter assay and the negative control mutant TAT-NBD (YGRKKRRQRRR–G–TTL- DASALQME) described by others (20, 21) were synthesized by Shanghai For the reporter assay, p-NF-kB–secreted alkaline phosphatase (SEAP), Sangon Biological Engineering Technology and Services. NBD-mediated p-AP-1-SEAP, p-NF-AT-SEAP, p-MyC-SEAP, p-serum response element NF-kB inhibition was done according to the protocol described by Tas (SRE)-SEAP, p-cAMP response element (CRE)-SEAP, p-heat shock re- et al. (20, 21). The c1orf190-transfected DCs were incubated for 2 h with sponse element (HSE)-SEAP, and positive control p-SEAP2 as well as the 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 chromosome 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 microscopy (original magnification 3400) (C2). An image collected using bright light was used as a control (C1). D, The transcriptional levels of c1orf190 in DCs after exposure to different TLR ligands. DCs were cultured with 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. The Journal of Immunology 6721 and cytokine release were analyzed after 24 h. NBD and control mutant Samples were examined by fluorescence microscopy (Fluoview FV300; peptides were used at a concentration of 50 mM. Olympus Optical, Tokyo, Japan). Functional analysis Western blotting analysis To determine the function of DCs transfected by c1orf190, we first observe For Western blot analyses, the cells were harvested and subjected to SDS- the effect of c1orf190 on the ability of DCs to stimulate the Ag-specific PAGE. Following the transfer to a Hybond-P membrane (Amersham T cells. Biosciences, Piscataway, NJ), the samples were analyzed by Western We prepared influenza peptide-specific HLA-A0201–restricted CD8+ blotting with anti-p-IkBa or anti-inactive IkBa Ab (Cell Signaling Tech- CTLs using our previous protocol (18). In brief, isolated CD8+ T lym- nology, Beverly, MA). The protein–Ab complexes were detected using the phocytes with a purity of .95% were seeded into 48-well plates (Poly- peroxidase-conjugated secondary Ab (Boehringer Mannheim, Mannheim, Sorb; Nunc, Roskilde, Denmark) at a concentration of 5 3 105 cells/well Germany) and ECL (Amersham Biosciences). in 10% human AB serum-RPMI 1640 medium. As APCs, autologous DCs were incubated with 20 mg/ml peptides in serum-free RPMI 1640 medium Immunofluorescence and fluorescence microscopy for 2 h at 37˚C in 5% CO2. After washing, DCs were added to the plates at a concentration of 1 3 106 cells/well. IL-7 (10 ng/ml) was added at the The cells were washed with PBS, fixed with 3.7% formaldehyde solution for initiation of the cultures. After incubation for 48 h, IL-2 (10 U/ml) was 10 min, permeabilized with 0.1% (v/v) Triton X-100 in BPS for 5 min, and added to the cultures. As the controls, CD8 T cells were cultured with blocked with PBS containing 1% BSA for 30 min. For the transfected cells, cytokines alone at the same time. In some cases, at day 14 and day 21, plasmid expressing V5 FITC-labeled mAb (Invitrogen) was added at 1 CD8 T cells were stimulated with peptide-loaded, irradiated autologous mg/ml to detect V5. For the nuclear localization of RelA/p65, the DCs. Influenza virus-specific CTLs were then cocultured with HLA-A2– transfected cells were incubated for 1 h with a 1/1000 dilution of the restricted peptide-pulsed DCs transfected with c1orf190 or c1orf190 specific polyclonal antiserum against RelA/p65 (sc-372; Santa Cruz Bio- siRNA. After incubation for 48 h, supernatants were collected and IFN-g technology, Santa Cruz, CA). The cells were then washed with PBS and was measured by ELISA. To load DCs with peptides, DCs were pulsed Downloaded from labeled with a Cy3-conjugated secondary Ab (Millipore, Bedford, MA). with influenza-restricted peptide (20 mg/ml) in RPMI 1640 medium for 3 h http://www.jimmunol.org/ by guest on September 25, 2021

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. 6722 C1orf190 PROMOTES ACTIVATION OF NF-kB at 37˚C, according to our previously reported method (18), and then ELISA washed three times before use. To investigate whether c1orf190-transfected DCs could resist the effect Commercial sandwich ELISA kits were used to quantify IFN-g, IL-1b, of tumor-associated factors on Ag-presenting function of DCs, DCs were IL-6, and IL-12p70 (Pierce/Endogen, Rockford, IL). The OD value of each first transfected with c1orf190, c1orf190 siRNA, or control vectors and then of the samples was measured at 450 nm using a SpectraMax 190 ELISA cocultured with ovarian carcinoma SK-OV3, cervical carcinoma HeLa cells plate reader. Cytokine levels were quantified by two to three titrations and SiHa cells in 24-Transwell plates, TGF-b1 (10 ng/ml), or IL-10 (10 ng/ using standard curves and expressed in picograms per milliliter. ml) for 18 h. These treated DCs, after having been loaded with HLA-A2– restricted peptides, were cocultured with influenza virus-specific CTLs. Statistical analysis After 48 h, supernatants were collected and IFN-g was measured by For statistical analysis, we used the Student t test, and a 95% confidence ELISA. , To investigate whether c1orf190-transfected DCs resist the effect of limit was taken to be significant (defined as p 0.05) tumor-associated factors on the priming ability of DCs, we initially generated synthetic Melan-A/MART-1 mRNA, which can induce MHC class Results I-restricted cytotoxic T cells in vivo and in vitro (22, 23). A full-length k cDNA fragment from the plasmid (American Type Culture Collection, C1orf190 activates NF- B Manassas, VA) was subcloned into the plasmid pSP-64 (poly(A)) at Hinc1 C1orf190, a hypothetical protein, which can be detected in human and XmaI (Promega, Madison, WI) in front of a synthetic poly(A) tail, which allows in vitro transcription under the control of an SP6 promoter. DCs (GEO DataSets [http://www.ncbi.nlm.nih.gov/sites/entrez]: The plasmids were linearized behind the poly(A) tail by restriction enzyme GDS2750/235214_at/C1orf190/Homo sapiens, GDS2453/235214_at/ digestion at FSP1 site and in vitro transcribed with the SP6 mMESSAGE C1orf190/Homo sapiens, and GDS1249/235214_at/C1orf190/Homo mMACHINE kit (Ambion, Austin, TX) according to the protocol provided sapiens), includes a 720-bp open reading frame that encodes a by the manufacturer. Purification of in vitro transcripts was performed 239-aa protein (LOC541468) (Fig. 1). A ScanProsite analysis (http:// Downloaded from with RNeasy Mini anion-exchange spin columns (Qiagen, Valencia, CA) according to the RNA cleanup protocol provided by the manufacturer. us.expasy.org/tools/scanprosite/) showed that c1orf190 contained N- These synthetic Melan-A/MART-1 mRNAs were used to cotransfect DCs myristoylation sites (MYRISTYL), an amidation site (AMIDATION), with c1orf190 vectors, c1orf190 siRNA, or control vectors using Nucle- CKII phosphorylation sites (CK2_PHOPHO_SITES), a leucine zip- ofector technology (Amaxa Biosystems, Gaithersburg, MD). The trans- per pattern (LEUCINE_ZIPPER), a cAMP- and cGMP-dependent fected DCs, after having been cocultured with ovarian carcinoma SK-OV3, cervical carcinoma HeLa cells, and SiHa cells in a 24-Transwell protein kinase phosphorylation site (CAMP_PHOSPHO_SITE) plate, TGF-b1 (10 ng/ml), or IL-10 (10 ng/ml) for 18 h, were then used to (Fig. 1), and a protein kinase C phosphorylation site (PKC_ http://www.jimmunol.org/ induce Melan-A/MART-1–specific T lymphocytes according to the above- PHOSPHO_SITE). A fusion protein of c1orf190 tagged with V5 + described method. The sp. act. of induced CD8 T cells was analyzed in could be detected in the cytoplasm of transfected 293T cells (Fig. response to autologous DC-loaded HLA-A0201–restricted Melan-A/MART-1 1C). Importantly, c1orf190 expression in DCs could be regulated peptides or HLA-A0201–restricted unrelated peptides, according to our previous method (18), on day 5 after the last restimulation in IFN-g by multiple factors, including TLR ligands (GEO DataSets: GDS2750/ ELISA assay. 235214_at/C1orf190/Homo sapiens;Fig.1D), hypoxia (GEO Data- The mAb BB7.2 (HB82; American Type Culture Collection) was used Sets: GDS2750/235214_at/C1orf190/Homo sapiens), and peroxi- to detect HLA-A0201. HLA-A0201–binding influenza peptide MP58–66 some proliferator-activated receptor-g ligand or the retinoic acid (GILGFVFTL), HLA-A0201–binding Melan-A/MART-1 (EAAGI- 26–35 receptor-a antagonist (GEO DataSets: GDS2453/235214_at/ GILTV), and HLA-A0201–restricted unrelated binding peptide (QLFF- by guest on September 25, 2021 DNYAL) were synthesized by a solid phase method, using a multiple pep- C1orf190/Homo sapiens). CKII (24), PKC (25, 26), cAMP, and tide synthesizer, and purified by HPLC, as previously described (18). cGMP-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. The Journal of Immunology 6723 kinases, may phosphorylate many different proteins. Furthermore, C1orf190 promotes resistance of DCs to tumor-associated because TLRs and hypoxia-associated genes play a critical role in inhibition controlling differentiation of DCs, c1orf190 might have an im- Tumor-associated factors, especially IL-10 and TGF-b, can inhibit portant role in regulating the differentiation and function of DCs. the production of cytokines and decrease the functional capacity To explore the biological function of c1orf190, we investigated of DCs (28–31). Because c1orf190 could activate NF-kB and pro- whether c1orf190 could regulate the activity of transcription factors mote the Ag-presenting and priming function of DCs, we next NF-kB, AP-1, NF-AT, glucocorticoid response element (GRE), investigated whether c1orf190-transfected DCs could resist the HSE, CRE, MyC, and SRE. Because HEK 293T cells and DCs effect of tumor-associated factors on DCs. To test this idea, could express endogenous c1orf190 (Fig. 2A), we first knocked c1orf190-transfected DCs were exposed to TGF-b, IL-10, or dif- down the c1orf190 to observe the effect of c1orf190 degradation ferent kinds of tumor supernatants. Morphological analysis on the activity of transcription factors. siRNA templates of showed that while c1orf190-transfected DCs were exposed to tu- c1orf190 were designed, synthesized, and cloned into p-FIV-H1- mor supernatants, DCs still maintained their morphological struc- U6-copGFP. Whereas NF-kB-SEAP, NFAT-SEAP, GRE-SEAP, ture with higher levels of expression of MHC class II-DR, CD40, CRE-SEAP, MYC-SEAP, and HSE-SEAP were cotransfected in- to 293T cells or DCs with c1orf190-targeted siRNA with demonstrated transfection (Fig. 2A), NF-kB and NF-AT activity was significantly reduced, whereas other transcription factors such as AP-1, CRE, SRE, GRE, HSE, and MyC were not affected or were only slightly affected (Fig. 2B and Supplemental Fig. 1). More- Downloaded from over, downregulation of NF-kB activity mediated by c1orf190- targeted siRNAs was dose-dependent (Fig. 2C1). When the dose of c1orf190-targeted siRNA structures was .500 ng, suppression of 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, ectopic http://www.jimmunol.org/ c1orf190 was used to cotransfect 293T cells or DCs with each of the reporter plasmids. The overexpression of ectopic c1orf190 in 293T cells and DCs was demonstrated with RT-PCR and Western blotting (Fig. 2A). The results show that ectopic c1orf190 remarkably upregulated the activity of NF-kB and NF-AT, whereas the activity of other transcription factors GRE, AP-1, HSE, SRE, CRE, MyC, and SRE was only slightly upregulated or stayed constant (Fig. 2B and Supplemental Fig. 1). Ectopic c1orf190- mediated NF-kB activity was also dose-dependent (Fig. 2C2). by guest on September 25, 2021 C1orf190 not only affected NF-kB activity but also impacted nuclear localization of p65 proteins. As shown in Fig. 2D, the ectopic c1orf190 increased the nuclear localization of p65 in both 293T cells and DCs. Thus, these data suggest that c1orf190 may regulate the activity of transcription factors, especially NF-kB.

C1orf190 promotes the production of proinflammatory FIGURE 4. C1orf190 promotes resistance of DCs to the tumor-associ- cytokines and the Ag-presenting function of DCs ated inhibition in presenting Ag(s). A, Ag-presenting function of DCs NF-kB plays a critical role in DC activation and the expression of transfected by c1orf190 is not affected by TGF-b1 and IL-10. Influenza proinflammatory cytokines and chemokines (21). Thus, we ex- virus-specific CTLs were cocultured with HLA-A2–restricted peptide- amined the effect of c1orf190 on the production of proin- pulsed DCs (CTLs/DCs, 10:1), which were transfected with c1orf190 flammatory cytokines and on the Ag-presenting function of DCs. (C1orf190) or Ctr. and then exposed to TGF-b1 or IL-10 for 3 d. Ctr. in A1 C1orf190, but not the control vectors, resulted in the upregulation and A2, DCs transfected by control vectors without TGF-b1 or IL-10 treatment; TGFbCtr. in A1, DCs transfected by control vectors with TGF- of IL-1b, IL-6, and IL-12. As illustrated in Fig. 3A and 3B, the b1 (10 ng/ml) treatment; IL10Ctr. in A2, DCs transfected by control levels of IL-1b, IL-6, and IL-12 in the c1orf190-transfected DCs vectors with IL-10 (10 ng/ml) treatment; C1orf190 in A1 and A2, DCs were significantly higher than those in control vector-transfected transfected by c1orf190 vectors without TGF-b1 or IL-10 treatment; DCs (p , 0.05). Conversely, c1orf190 siRNA but not control TGFbc1orf190 in A1, DCs transfected by c1orf190 vectors with TGF-b1 siRNAs downregulated the mRNA and protein expression of IL- (10 ng/ml) treatment; IL10c1orf190 in A2, DCs transfected by c1orf190 1b, IL-6, and IL-12 (p , 0.05; Fig. 3). Importantly, c1orf190 vectors with IL-10 (10 ng/ml) treatment. B, The Ag-presenting function of promoted the Ag-presenting function of DCs. As shown in Fig. DCs transfected by c1orf190 is not affected by tumor-associated factors. 3C, peptide-pulsed DCs transfected with c1orf190 could stimulate Vector.ctr, c1orf190, MocksiRNA, or siRNA are, respectively, the relative influenza peptide-specific CD8 T cells to produce higher levels of secretion of peptide-specific CTLs after coculture with DCs transfected by IFN-g than could peptide-pulsed control vector-transfected DCs control vectors, c1orf190 vectors, control siRNA, or c1orf190 siRNA upon exposure to different treatments, including control medium (Med.), ovarian (p , 0.05), whereas peptide-specific CD8 T cells cocultured with carcinoma SK-OV3 (Tu1; American Type Culture Collection), cervical peptide-pulsed DCs transfected by c1orf190 siRNA only produced carcinoma HeLa cells (Tu2; American Type Culture Collection), SiHa cell , lower levels of IFN-g as compared with control siRNA (p (Tu3; American Type Culture Collection), TGF-b1 (TGFbeta; 10 ng/ml) or 0.05). Thus, c1orf190 as an NF-kB activator is capable of regu- IL-10 (10 ng/ml). Relative secretion is the cytokine secretion (pg/ml) in lating inflammatory cytokine production and Ag-presenting func- CTLs stimulated with the treated DCs divided by the cytokine secretion tion of DCs. (pg/ml) in CTLs stimulated with the control-treated DCs. 6724 C1orf190 PROMOTES ACTIVATION OF NF-kB

CD80, CD86, and B7-H1 (Supplemental Fig. 2), whereas DCs We also investigated the effect of c1orf190 on the priming ability transfected with control vectors lost typical structures and at- of DCs in the presence of tumor-associated factors. Toward this tachment ability with a lower expression of MHC class II-DR, end, we employed the tumor-associated Ag Melan-A/MART-1, CD40, CD80, CD86m and B7-H1 (Supplemental Fig. 2), imply- which has been demonstrated to induce Ag-specific HLA-A0201– ing that the c1orf190-tranfected DCs have an ability to resist the restricted cytotoxic CD8+ lymphocytes (22, 23). We first investi- effect of tumor-associated factors on DCs. Importantly, unlike gated whether c1orf190 could promote DCs to induce Ag-specific control vector-transfected DCs that had the reduced Ag-presenting T lymphocytes. As shown in Fig. 5B, indeed, Ag-specific T lym- functions upon exposure to tumor-associated factor TGF-b or phocytes induced by DCs transfected by c1orf190 released higher IL-10, c1orf190-transfected DCs still had a strong Ag-presenting levels of IFN-g than did those induced by DCs transfected by the function even in the presence of tumor-associated factor TGF-b control plasmids in response to Melan-A/MART-1 HLA-A0201– or IL-10 as compared with the control (p , 0.05; Fig. 4A). To fur- restricted peptide-loaded DCs, whereas c1orf190-specific siRNA ther determine the resistance of c1orf190-transfected DCs against reduced the priming ability of DCs. Next, we sought to determine tumor-associated factors, c1orf190-transfected DCs were exposed whether DCs transfected by c1orf190 could resist the effect of to different kinds of tumors, including ovarian carcinomas and tumor-associated factors on the priming ability of DCs. As shown cervical carcinomas, in a Transwell plate. C1orf190-transfected in Fig. 5C, the ability of DCs untransfected or transfected by DCs indeed exhibited strong resistance to the tumor-associated control empty plasmid or control siRNA in inducing Melan-A/ factors and produced higher levels of IFN-g as compared with MART-1–specific T lymphocytes could be significantly reduced controls (p , 0.05), whereas c1orf190 siRNA-transfected DCs by IL-10, TGF-b, or tumor supernatants (p , 0.05). Especially, had reduced Ag-presenting function and produced lower levels DCs transfected with c1orf190 siRNA had the more remarkably Downloaded from of IFN-g compared with controls (p , 0.05), especially after decrease in inducing Melan-A/MART-1–specific T lymphocytes exposure to tumor supernatants (Fig. 4B). in the presence of IL-10, TGF-b, or tumor supernatants (p , 0.001). http://www.jimmunol.org/ by guest on September 25, 2021

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 in Materials 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. The Journal of Immunology 6725

However, the priming ability of DCs transfected with c1orf190 noncanonical pathway did not remarkably affect the production to induce Ag-specific T lymphocytes was not remarkably affected of IL-1b, IL-6, or IL-12 in the c1orf190-transfected DCs as by IL-10, TGF-b, or tumor supernatants. Thus, our results clearly compared with controls (Supplemental Fig. 3). The degraded reveal that c1orf190 may promote the resistance of DCs to tumor- IKKa and NIK by siRNA for the noncanonical NF-kB pathway- associated inhibition. associated kinases IKKa (siIKKa) and NIK (siNIK) could be observed (Supplemental Fig. 3). Thus, our results suggest that C1orf190-driven proinflammatory cytokine introduction occurs c1orf190-driven production of proinflammatory cytokines and im- via the canonical NF-kB pathway proved Ag-presenting function are mediated by activating the ca- NF-kB is an inducible transcription factor that is controlled by nonical NF-kB pathway, not the noncanonical pathway. two principal signaling cascades, the classical/canonical NF-kB activation pathway and the alternative/noncanonical pathway (32). Discussion Next, we examined whether c1orf190 drives proinflammatory In the studies presented, we have demonstrated that c1orf190 can cytokine introduction via the canonical NF-kB pathway or the activate the activity of NF-kB, drive the production of proin- noncanonical pathway using the NBD peptide, which is a highly flammatory cytokines, and promote the Ag-presenting and prim- selective inhibitor of the canonical NF-kB pathway and IKKa- ing function of DCs via the canonical NF-kB pathway. Thus, targetted and/or NIK-targetted siRNA, which acts to silence the c1orf190, as an NF-kB activator, may be involved in the reg- noncanonical pathway as described by others (20, 21, 33). In ulation of DC function and play an important role in the induc- MDCs cotransfected with NBD peptide and c1orf190, NBD tion of innate and adaptive immune responses. Meanwhile, we peptide not only blocked c1orf190-mediated IKBa phosphoryla- have also examined the ability of c1orf190-transfected DCs to re- Downloaded from tion but also decreased IL-1b, IL-6, and IL-12 production medi- sist the effect of tumor-associated factors on the Ag-presenting ated by c1orf190 (p , 0.05; Fig. 6B). As a control, mutant NBD and priming function of DCs. This might suggest an approach for peptide had no effect (Fig. 6B). We also detected the effect of enhancing vaccine immunogenicity. NEMO siRNA on the canonical NF-kB pathway. NEMO siRNA Examination of circulating and tumor-infiltrating DCs in tumor- also inhibited c1orf190-mediated IKBa phosphorylation and re- bearing animals and in cancer patients has revealed that DCs are

duced IL-1b, IL-6, and IL-12 production mediated by c1orf190 functionally impaired in their ability to induce T cell responses. http://www.jimmunol.org/ (p , 0.05; Fig. 6). However, siRNA-mediated knockdown of the Tumor-associated factors, such as IL-10 (28) and TGF-b (29, 34), by guest on September 25, 2021

FIGURE 6. C1orf190 drives proinflammatory cytokine production via the canonical but not the noncanonical NF-kB pathway. A, NBD peptide selectively 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. 6726 C1orf190 PROMOTES ACTIVATION OF NF-kB potentially inhibit the function of Ag-presenting cells, such as responses, it will be interesting to study whether c1orf190 induces DCs, through a repression of inflammatory cytokine production Th1 immune responses in vivo. and MHC class II and costimulatory molecule expression. Tumor- associated DCs often induce T cell anergy or deletion and regu- Disclosures latory T cells instead of antitumor immunity. As a result, although The authors have no financial conflicts of interest. there are many tumor-associated Ags found, there is still no effective vaccine for cancer. Thus, novel rational strategies to enhance the immunogenicity of pathogen-specific Ags and cancer- References specific Ags are needed. Some immunological adjuvants have 1. Steinman, R. M., D. Hawiger, K. Liu, L. Bonifaz, D. Bonnyay, K. Mahnke, T. Iyoda, J. Ravetch, M. Dhodapkar, K. Inaba, and M. Nussenzweig. 2003. been shown to activate NF-kB among their multiple actions, but Dendritic cell function in vivo during the steady state: a role in peripheral tol- they are, however, limited in use by their lack of specific, local- erance. Ann. N. Y. Acad. Sci. 987: 15–25. 2. Ardeshna, K. M., A. R. Pizzey, S. Devereux, and A. Khwaja. 2000. The PI3 ized, and coordinated effects and consequently by toxicity (35). kinase, p38 SAP kinase, and NF-kB signal transduction pathways are involved in C1orf190, as an NF-kB activator, results in the upregulation of the survival and maturation of lipopolysaccharide-stimulated human monocyte- cytokines that contain NF-kB sites on the promoters of their genes derived dendritic cells. Blood 96: 1039–1046. 3. Rescigno, M., M. Martino, C. L. Sutherland, M. R. Gold, and P. Ricciardi- and promote the Ag-presenting function and priming ability of Castagnoli. 1998. Dendritic cell survival and maturation are regulated by dif- DCs. Importantly, c1orf190 could resist the effect of tumor- ferent signaling pathways. J. Exp. Med. 188: 2175–2180. associated factors. Thus, c1orf190 might be a candidate gene for 4. Andreakos, E., C. Smith, C. Monaco, F. M. Brennan, B. M. Foxwell, and M. Feldmann. 2003. IkB kinase 2 but not NF-kB-inducing kinase is essential for improving the immunogenicity of tumor vaccine. effective DC antigen presentation in the allogeneic mixed lymphocyte reaction.

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FIGURE S1. The effect of the degradation or overexpression of c1orf190 on the transcriptional factors. A, knockdown of c1orf190 down-regulated the activity of

NF-κB and NF-AT, whereas the activities of other transcription factors such as AP-1,

GRE, HSE, CRE, MyC and SRE were not affected or were only slightly affected. B, ectopic c1orf190 upregulated the activity of NF-κB and NF-AT, whereas the activity of AP-1, GRE, HSE, CRE, MyC and SRE transcription factors were not remarkably affected. MocksiRNA, siRNA, ctr. vector and clorf190 were 293 T cells transfected by vectors containing control siRNA, c1orf190 siRNA, control empty vectors and vectors containing c1orf190, respectively. A and B were respectively from different experiments. RLU, relative light unit.

FIGURE S2. A, the tumor associated DCs under light microscope. DC+Med, DCs cultured in 1640 medium; Ctr. DC+Tu, control vectors transfecetd DCs cocultured with human ovarian carcinoma SK-OV3 cells (ATCC) in a transwell plate;

C1orf190DC+Tu, c1orf190 transfected DCs cocultured with SK-OV3 tumor cells in a transwell plate. B, the expression of MHCII-DR (MHCIIDR), CD40, CD80, CD86 and B7-H1 in the DCs transfected with c1orf190 or c1orf190 siRNA in the presence of tumor associated factors. The transfected DCs were cocultured with human ovarian carcinoma SK-OV3 cells (ATCC) in a transwell plate for 24 hours. siRNA, mocksiRNA, clorf190 and vector.ctr. were respectively DCs transfected by c1orf190

1 siRNA, control siRNA, c1orf190 and control vectors. Ctr, antibody isotypic control;

G..M., geometric mean.

FIGURE S3. siRNA-mediated knockdown of the noncanonical pathway does not remarkably affect the production of IL-1β, IL-6 or IL-12 by c1orf190-transfected DCs.

A, the expression of IKK˞and NIK in the DCs transfected with IKK˞VL51$or NIK siRNA. Degraded IKKα and NIK by IKKα-targeted siRNA (A1) or NIK-targeted siRNA (A2) for the noncanonical NF-κB pathway-associated kinases IKKα and NIK were demonstrated by RT-PCR. B, siRNA-mediated knockdown of the noncanonical pathway does not remarkably affect the production of IL-1β, IL-6 or IL-12. DCs were cotransfected by c1orf190 vectors or control vectors with siIKKα, siNIK or control irrelevant siRNA (MocksiRNA), and then supernatants were harvested after 24 hours and secreted IL-1β, IL-6 and IL-12p70 (pg/ml) were measured by ELISA. Results are expressed as means ± SD from one representative experiment of three performed in triplicate (P<0.05). Ctr. control vectors; c1orf190, c1or190 vectors; siIKKα,

IKKα-targeted siRNA; siNIK, NIK-targeted siRNA.

TABLE S1. Primers used in this study.

TABLE S2. siRNA templates used in this study.