Identification of a New Endoplasmic Reticulum-resident Protein Recognized byHLA-A24-restricted Tumor-infiltrating Lymphocytes of Lung Cancer 1
Kouichiro Kawano, Shinya Gomi, Koji Tanaka, Naotake Tsuda, Toshiharu Kamura, Kyogo Itoh, and Akira Yamada2
Cancer Vaccine Development Division, Kurume University Research Center for Innovative Cancer Therapy [K. K., N. T., K. I., A. Y.], Departments of Immunology [S. G., K. T.,K. I., A. Y.] and Gynecology [T. K.], Kurume University School of Medicine, Kurume 830-0011, Japan
ABSTRACT
To help clarify the molecular basis of tumor immunology in lungcancer, we have investigated antigens recognized by HLA-A24-restrictedCTLs established from T cells infiltrating into lung adenocarcinoma andreport a new gene encoding tumor epitopes recognized by the CTLs. Thisgene was located on chromosome 4q31.22 and encoded an unreportedendoplasmic reticulum-resident protein with 412 deduced amino acids.This protein had a molecular mass of 46 kDa and was expressed in themajority of malignant cells and tissues tested, with the exception of T-cellleukemia cells, but was not expressed in a panel of normal cells andtissues, except in those of the testis, placenta, and fetal liver. Two peptidesat positions 13–20 and 75–84 were recognized by the CTLs and had anability to induce HLA-A24-restricted and tumor-specific CTLs in periph-eral blood mononuclear cells of lung cancer patients. Thus, these peptidesmight be appropriate molecules for use in the specific immunotherapy ofHLA-A24 1 patients with lung and other cancers.
INTRODUCTION
Many genes encoding tumor antigens recognized by CTLs havebeen identified from melanoma cDNA (1–8). CTL-directed tumorantigens have also been identified from tumors other than melanomas,including HER2/neu (9, 10), prostate-specific antigen (11, 12),SART-1 (13), SART-3 (14), and cyclophilin B (15). Immunotherapywith some of the peptides capable of inducing HLA-class I-restrictedCTLs has been shown to result in tumor regression in HLA-A02011
melanoma patients (16, 17). These results indicate that identificationof the peptides capable of inducing CTLs may provide a new modalityof cancer therapy. However, little information is available on peptide-based immunotherapy for cancer patients other than melanoma pa-tients. This may be attributable in part to the relative dearth of studieson target molecules recognized by CTLs infiltrating into cancers otherthan melanomas. Lung cancer is among the most commonly occurringmalignancies in the world and is one of the few that continues to showan increasing incidence. The HLA-A24 allele is found in;60% of theJapanese population, 20% of Caucasians, and 12% of Africans (18).Therefore, we have studied antigens recognized by HLA-A24-restricted CTLs established from T cells infiltrating into lung adeno-carcinoma and have reported a new CTL-directed tumor antigenresiding in the ER.3
MATERIALS AND METHODS
Cell Lines. A bladder carcinoma cell line, HT1376, was used for prepara-tion of the cDNA library. COS7 and C1R-A2402 (an HLA-A2402 transfectant)cells were used for the transfection and peptide-pulse experiments, respec-tively. The other cell lines used in this study were as follows: lung adenocar-cinomas (1-87, PC-9, and A549), lung squamous cell carcinomas (Sq-1 andQG56), small cell lung carcinomas (LK79 and LC-65A), large cell lungcarcinoma (86-2), esophageal squamous cell carcinomas (KE3, KE4, TE8, andTE9), head and neck cancers (HSC-4 and Kuma-1), hepatocellular carcinomas(HAK-3 and KYN-1), colon adenocarcinomas (KM12LM, colo201, andcolo205), gastric adenocarcinoma (MKN28), and Epstein-Barr virus-trans-formed B-cell line (Bec-1). The origins and HLA genotypes of these cell lineshave been described previously (13, 19, 20).
Identification of the ART-4 Gene. The expression-gene cloning methodwas used to identify a gene-encoding tumor rejection antigens recognizedby the CTL line, GK-CTL, as reported elsewhere (15). Briefly, cDNAderived from the HT1376 bladder carcinoma cells was ligated toSalI/NotIsite of the expression vectorpSV-SPORT-1(Life Technologies, Inc., Gaith-ersburg, MD). A total of 13 105 clones from the cDNA library werescreened, and two positive pools (6A1 and 4E6, each containing 100clones) obtained from the first screening were subdivided for furtherscreening. This study described the results for the 4E6-derived genes,whereas those for the 6A1-derived genes have been reported elsewhere(15). A total of 400 clones from the4E6were analyzed for their bioactivityto stimulate IFN-g production by the GK-CTLs at the second and thirdscreening, and one positive (4E6-2B9) and one negative (4E6-6F2) clonewere provided for this study. DNA sequencing was performed by thedideoxynucleotide sequencing method using an AutoRead Sequencing kit(Pharmacia Biotech, Uppsala, Sweden) and analyzed using an ALF expressDNA Sequencer (Pharmacia Biotech). Full-length cDNA clones were ob-tained from an HT1376 and a PBMC cDNA library (SuperScript HumanLeukocyte cDNA library in pCMV-SPORT; Life Technologies) by thestandard colony hybridization method with32P-labeled cDNA probe (clone4E6-2B9) as reported previously (13). We tentatively designated this geneanART-4gene. The difference of the nt sequence at position 758 ofART-4cDNA between PBMCs and HT1376 cells was confirmed by both repeatednt sequencing and susceptibility of PCR products againstAciI restrictionenzyme digestion. Amplification was performed for 30 cycles using theprimers 59-ATCCAAGCAGATCCAG CAGG-39 (sense) and 59-AGTGT-GAGCAGAACACTCGG-39(antisense).
Northern Blot Analysis. Preparation of RNA, transfer to nylon mem-branes, and Northern hybridization have been described elsewhere (13). A32P-labeled 600-bp fragment ofNcoI cutART-4cDNA was used as a probe.The membranes were washed and then autoradiographed. The relative expres-sion of theART-4mRNA was calculated with the following formula:
Index5ART-4 density of a sample
b-actin density of a sample3
b-actin density of the KE4 cells
ART-4 density of the KE4 cells
Chromosome Mapping. A genomic DNA panel of hybrids with 1000-kbresolution made from hybrid cells of irradiated human HFL cells and hamsterA23 cells (Gene bridge 4 Radiation Hybrid Panel; Research Genetics, Hunts-ville, AL; Ref. 21) was used as a template for PCR. A 768-bp fragment of theART-4gene was amplified by PCR from the panel. PCR amplifications wereperformed with a sense primer, F2 (111–130; 59-ATCCAAGTGCTTGCACT-CACA-39), and an antisense primer, 4R878 (859–878; 59-AGTGTGAGCA-GAACACTCGG-39), under the following conditions: denaturing at 95°C for 1min, annealing at 58°C for 1 min, and extension at 72°C for 1 min for 35cycles. The PCR products were subsequently dot-blotted on a nitrocellulose
Received 12/28/99; accepted 5/3/00.The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby markedadvertisementin accordance with18 U.S.C. Section 1734 solely to indicate this fact.
1 Supported in part by Grants-in-Aid 08266266, 09470271, 10153265, 09770985, and09671401 from the Ministry of Education, Science, Sports and Culture of Japan and byGrant H10-Genome-003 from the Ministry of Health and Welfare, Japan.
2 To whom requests for reprints should be addressed, at Department of Immunology,Kurume University School of Medicine, 67 Asahi-machi, Kurume 830-0011, Japan.Phone: 81-942-31-7744; Fax: 81-942-31-7745; E-mail: [email protected].
3 The abbreviations used are: ER, endoplasmic reticulum; ART-4, adenocarcinomaantigen recognized by T cells 4; GST, glutathioneS-transferase; Ab, antibody; mAb,monoclonal Ab; EYFP, enhanced yellow fluorescent protein; GFP, green fluorescentprotein; GST, glutathioneS-transferase; nt, nucleotide; PBMC, peripheral blood mono-nuclear cell; APC, antigen-presenting cell.
filter and hybridized with the32P-end-labeled sequence-specific oligonucleo-tide probe R3 (452–471; 59-AGGTTCTCAGGCTCACAAGC-39), which spe-cifically hybridized with the humanART-4gene but not with any hamster-derived genes. After washing, the filter was subjected to autoradiography asdescribed previously (22). The results obtained were analyzed using theradiation hybrid map software of the Whitehead Institute/Massachusetts Insti-tute of Technology Center for Genome Research.
Preparation of Fusion Protein and Rabbit Antisera. For preparation ofthe ART-4/GST protein, theBamHI site was introduced to the 59-terminal ofthe ART-4gene by a linker PCR. The primer pair used for the PCR was asfollows: senseBamHI primer 59-CTCACGGATCCAACATGGCTCC-39andantisense primer 59-AATCGATGAGCTCACCTTAACCTT-39. The amplifiedfragment (nt 3–364) was digested withBamHI andSacI and ligated to aSacI/NotI fragment ofpSVSPORT/ART-4. Subsequently, the ligated fragmentof theART-4gene containing nt positions 3–1733 was cloned into theBamHI/NotI site of thepGEX-4T-2vector (Pharmacia Biotech). In this construct, theGSTgene flanked the 59terminal of theART-4gene. The nt sequence of theconstruct was confirmed by sequencing. Purification of the ART-4/GST fusionprotein from the bacterial lysate was performed according to the manufactur-er’s procedure. Polyclonal anti-ART-4/GST Ab was prepared by immunizationof rabbits with purified ART-4/GST protein by the methods reported previ-ously (13, 23).
ART-4/myc and ART-4/GFP Constructs. For preparation of theART-4/myc, theXbaI site was introduced to the 59flanking region of the stop codonby PCR. The primer pair used for the PCR was as follows: sense primer59-CGCTGCCATGGCTGTTT-39and antisenseXbaI primer 59-TGCGG-GAACTCGCTCTAGAC-39. The amplified fragment (nt 821-1256) was di-gested withXbaI andNcoI and ligated to aKpnI/NcoI fragment ofpSVSPORT-ART-4. Subsequently, the ligated fragment of theART-4gene containing ntpositions 1–1251 was cloned into theKpnI/XbaI site of thepcDNA3.1/Myc-Hisvector (Invitrogen, Carlsbad, CA). Amycgene flanked the 39terminal of theART-4gene as a tag (ART-4/myc). To prepare the ART-4/GFP protein, theSalIsite was introduced to the 59flanking region of the stop codon by PCR. Theprimer pair used for the PCR was as follows: sense primer 59-TCGAATTC-CACGCAGCCAA-39 and antisenseSalI primer 59-AACTCGGTCGACAC-CTTTTCTTCAC-39. The amplified product (nt 1–1267) was digested withEcoRI andSalI and cloned into anEcoRI/SalI site ofpEGFP-N2 vector(Clontech, Palo Alto, CA). AGFP gene flanked the 39terminal ofART-4geneas a tag (ART-4/GFP).
Western Blot Analysis. Preparation of cytosol and nuclear fractions oftissues or cells have been described (23). Obtained fractions were separated bySDS-PAGE, blotted to a polyvinylidene difluoride membrane, and subjectedfor the Western blot. Precise methods of the Western blot analysis have beendescribed previously (23).
Assays for Cellular Localization. Intracellular localization of the ART-4/GFP fusion protein in the transfectants was analyzed as follows. COS7 cellswere transfected with theART-4/GFP,followed by serial observation under aZeiss confocal Ar-Kr laser scanning microscope with both fluorescence andvisible rays or fluorescence only. Localization of the ART-4/GFP protein wasrecorded under an FITC filter (520 nm). The exposure sequences and imagingwere controlled by LSM version 3.70 imaging software. The visible-ray imagewas studied by differential interference microscopy. COS7 cells transfectedwith Living Colors subcellular localization vectorspEYFP-ER, pEYFP-Golgi,or pEYFP-Mito(Clontech) were also analyzed to obtain representative fluo-rescence patterns of ER, Golgi, or mitochondria-resident proteins, respectively.The pEYFP-ERvector encodes a fusion protein consisting of EYFP, the ERtargeting sequence of calreticulin, and the ER retrieval sequence, KDEL.pEYFP-Golgiencodes a fusion protein consisting of EYFP and a sequence ofthe NH2-terminal 81 amino acids of humanb1,4-galactosyltransferase to sortthe fusion protein to the Golgi apparatus.pEYFP-Mitoencodes a fusion proteinconsisting of EYFP and the mitochondrial targeting sequence from subunitVIII of human cytochromec oxidase.
Subcellular organelles were fractionated by velocity-controlled sucrosegradient fractionation (24, 25). In brief, 53 107 COS7 cells transfected withtheART-4/mycgene were washed with ice-cold STE buffer [0.25M sucrose, 1mM phenylmethylsulfonyl fluoride, 10mg/ml aprotinin, 20 mM Tris-HCl (pH8.0), and 1 mM EDTA]. Cells were suspended in a hypotonic buffer (0.025M
sucrose-STE), incubated for 30 min on ice, and homogenized with a tightfitting Dounce homogenizer. The homogenate was centrifuged at 5003 g for
5 min to remove the nuclei and undisrupted cells. The supernatant was layeredon a discontinuous sucrose gradient consisting of 1 ml of 2M sucrose, 3 ml of1.3 M sucrose, 3 ml of 1M sucrose, and 2.5 ml of 0.6M sucrose, and thencentrifuged at 40,000 rpm (284,0003 g) for 4 h at 4°C in a P40ST rotor(Hitachi, Tokyo, Japan). Fractions were collected from the top of the tube.Sucrose concentration in each fraction was as follows: the fraction numbers 1and 2, 0.6M; number 3, interface between 0.6 and 1M; numbers 4 and 5, 1M;number 6, interface between 1 and 1.3M; numbers 7 an 8, 1.3M; number 9,interface between 1.3 and 2M; and number 10, 2M. Each fraction was mixedwith SDS-sample buffer and subjected to Western blot analysis. For detectionof ER and Golgi marker proteins, rabbit anti-NADPH cytochrome P450reductase (StressGen, Victoria, British Columbia, Canada) and anti-proteinkinase Cm (Santa Cruz Biotechnology, Santa Cruz, CA) Abs were used,respectively (26, 27). Normal rabbit serum was used as a negative control.
Peptides and Assays.Eighteen different synthetic peptides derived fromthe deduced amino acid sequence of ART-4 (purity was.70%) with bindingmotifs for HLA-A2402 molecules in the literature (28), including motifs oftyrosine or phenylalanine at position 2, and of isoleucine, leucine, phenylala-nine, or tryptophan at position 9, were obtained from Biologicala (Nagoya,Japan). An HIV-derived peptide (RYPLTFGWCF) capable of binding toHLA-A24 molecules was used as a negative control (15). Peptides of.95%purity were used for experiments of dose dependency and CTL induction. Theestimated score of half time of dissociation of each ART-4 peptide forHLA-A24 molecules was calculated based on a computer search for HLApeptide motifs (29) as follows: ART-450–59, 30; ART-461–69, 198; ART-475–84,75; ART-4156–164, 36; ART-4238–246,20; ART-4265–274, 150; ART-4309–317,20; or ART-4365–373, 150. The value of ART-413–20 was not obtained by theanalysis because it is an 8-mer peptide. For detection of antigenic peptidesrecognized by the GK-CTLs, COS7 cells (23 104) were transfected withHLA-A2402or control HLA-A2601cDNA. Two days after the transfection,peptides at a concentration of 10mM, unless stated otherwise, were added tothe culture. Two h later, the supernatant was removed, and the GK-CTLs(1 3 105) were added to the culture and incubated for an additional 18 h, andthe concentration of IFN-g in the culture supernatants was measured by ELISA(limit of sensitivity, 10 pg/ml) in a duplicate assay. The surface phenotype ofthe CTL line and sublines was investigated by an immunofluorescence assaywith FITC-conjugated anti-CD3, anti-CD4, or anti-CD8 mAb (15). For inhi-bition of CTL activity, 10mg/ml of anti-class I (W6/32, IgG2a), anti-HLA-A24(A11.1 M, IgG3), anti-CD8 (Nu-Ts/c, IgG2a), anti-class II (H-DR-1, IgG2a),and anti-CD4 (Nu-Th/i, IgG1) mAbs were used as reported previously (15).Anti-CD14 (JML-H14, IgG2a) or anti-CD13 mAb (MCS-2, IgG1) was used asan isotype-matched control mAb. Two-tailed Student’st test was used for thestatistical analysis in this study.
CTL Induction by Peptides. PBMCs (1 3 106 per well) from HLA-A24021 healthy donors or cancer patients were incubated with 10mM of thepeptide in a 24-well plate in the presence of 100 units/ml interleukin 2 asreported previously (15). At days 7 and 14 of culture, the cells were restim-ulated at a responder to stimulator ratio of 4:1 with the irradiated (30 Gy)autologous PBMCs as APCs that had been incubated with the same peptide (10mM) for 2 h. Responder cells were harvested at day 21 of the culture and werefurther cultured in a 96-well U-bottomed microculture plate in the presence ofirradiated autologous PBMCs (23 106 cells/well) as APCs that had beenpulsed with a corresponding peptide. Seven to 10 days later, the expanded cellswere transferred to a 24-well plate and cultured for additional 14–25 days withinterleukin 2 alone. The cytotoxic activity was measured by a standard 6-h51Cr-release assay at different E:T ratios, as reported previously (15).
RESULTS
Identification and Characterization of the ART-4 Gene. TheCTL line used for identification of the gene was the HLA-A24-restricted and tumor-specific effector CTL (GK-CTL) line withCD31CD42CD81 phenotype that was established from T cells infil-trating into the lung adenocarcinoma, and its characteristics have beenreported elsewhere (15). A total of 105 cDNA clones from the cDNAlibrary of the HT1376 tumor cells were tested for their ability tostimulate IFN-g production by the GK-CTLs after cotransfection withHLA-A2402into the COS-7 cells. After repeated cycles of the screen-
ing, one clone (4E6-2B9) was confirmed to encode a tumor antigenrecognized by the GK-CTLs when cotransfected withHLA-A2402butnot when cotransfected with controlHLA-A2601(Fig. 1). The 4E6-6F2 clone was served as a negative control in this experiment.
The clone 4E6-2B9 contained a 1584-bp-long DNA insert. Expres-sion of this gene at the mRNA level was investigated by Northern blotanalysis. A band of;1.8 Kbp in size was observed in each of the 21different tumor cell lines tested, including 3 lung adenocarcinomasand 2 lung squamous cell carcinomas, and in all of the normal tissuestested (heart, brain, placenta, lung, liver, skeletal muscle, kidney,pancreas, spleen, thymus, prostate, testis, ovary, small intestine, andcolon). Representative results are shown in Fig. 2A. The relative levelof mRNA expression ranged from 0.90 to 1.24 in tumor cell lines andfrom 0.78 to 4.40 in normal tissues, when the levels of the expressionin the KE4 esophageal cancer cells were taken as 1.0 (Fig. 2A). In thenormal tissues, relatively high levels of expression were observed inthe pancreas (4.4), kidney (2.8), and ovary (2.4), and low levelexpression was seen in skeletal muscle (0.6) and PBMCs (0.7). Theseresults suggest that this gene is expressed ubiquitously at the mRNAlevel, and that the clone 4E6-2B9 with a length of 1584-bp might bea truncated form.
A cDNA library of HT1376 cells was further screened to obtain thefull-length of cDNA clones containing the sequence of 4E6-2B9 by astandard colony hybridization technique, and a 1733-bp cDNA clonewas obtained. A cDNA clone of the same length was also cloned fromthe cDNA library of PBMCs of a healthy donor using the 4E6-2B9 asa probe. The nt sequences of these clones are shown in Fig. 2B. Thisgene was tentatively designatedART-4. The sequence ofART-4cloned from HT1376 cDNA is available from EMBL/GenBank/DDBJ(accession number AB026125). The nt sequences of these clones wereidentical with the exception of the position 708 (guanine in HT1376versusadenine in the PBMCs). This difference is attributable to a
genetic polymorphism rather than to a point mutation, because theposition 708 was a guanine in the HT1376, RMG I, HSC-4, and LC-1cells but an adenine in the PBMCs, MKN28, and 86-2 cells. PBMCsfrom another healthy donor and a tissue sample of the testis containedboth guanine and adenine at position 708. The amino acids translatedfrom these codons were different (arginine or glutamine). This geneshowed 99% similarity at the nucleotide level with the previouslyreported geneHP-10 (30) isolated from placental cDNA and havingan unknown function (30). Despite the high homology between theART-4andHP-10gene at the nt levels, the homology between the twogenes at the deduced amino acids level was only;50%. This dis-crepancy was mainly attributable to the difference of start positionsfor the translation and the subsequent frame shift of the codons. Wenext attempted to clone theHP-10 from a human placental cDNAlibrary using PCR methods; however, the nt sequences of all 20 clonesobtained from PCR products were identical with that ofART-4.Furthermore, the susceptibility of these clones to several restrictionenzymes that distinguish the differences betweenART-4andHP-10verified the sequencing results (data not shown). Therefore, we couldnot confirm the presence ofHP-10 in human samples under the usedconditions.
The deduced amino acid sequence of theART-4gene is shown inFig. 2C. Hydrophobicity analysis showed that a protein encoded bythe ART-4 gene had a hydrophobic region at the NH2 terminus(positions 1–21), potentially allowing it to act as a signal peptide (Fig.2D). There are nuclear localization signals at positions 396–412 and401–407 and a di-lysine (KKXX) motif-like ER membrane retentionsignal (FVKK) at the COOH terminus positions 408–411 (Fig. 2C).The ability of theART-4gene to stimulate IFN-g production by theGK-CTLs was confirmed (Fig. 2E), i.e., the ART-4gene stimulatedIFN-g production by the CTLs in a dose-dependent manner whenCOS7 cells transfected with both theART-4 and HLA-A2402genewere used as stimulator cells.
Chromosome mapping of theART-4gene was performed by theradiation hybrid mapping method as reported elsewhere (21). Agenomic DNA panel of hybrids with 1000-kb resolution made fromhybrid cells of irradiated human HFL cells and hamster A23 cells wasused as a template for PCR. The PCR with the primer pair F2 and4R878 amplified the 768-bp fragment from genomic DNA isolatedfrom PBMCs (data not shown). The PCR product did not contain anintron. A faint band corresponding to a PCR product of the same sizewas also amplified from hamster A23-derived genomic DNA underthe same conditions. Therefore, the PCR products from the radiationhybrid panel were dot-blotted and subsequently hybridized with thehumanART-4sequence-specific oligonucleotide probe R3. This probespecifically hybridized with the PCR product from the human HFLcell-derived genomic DNA but not with the hamster A23-derivedgenomic DNA (data not shown). Database analysis for the radiationhybrid mapping indicated that theART-4 gene was located on thechromosome 4, 2.12 centirays distal from theAFM350VH9markergene, compatible with the classical 4q31.22.
Expression of the ART-4 Protein. Expression of ART-4 at theprotein level in various cells and tissues was examined by Westernblot analysis using rabbit anti-ART-4/GST polyclonal Ab. Thismethod visualized anMr 46,000 band of a recombinant ART-4 proteinafter cleavage with thrombin (data not shown). When theART-4genewas transfected to COS7 cells, an intensiveMr 46,000 band wasobserved in both the nuclear and cytosol fractions (Fig. 3A). Further-more, both the polyclonal anti-ART-4/GST Ab and anti-myc mAbrecognized anMr 51,000 band in the nuclear and cytosol fractions ofCOS7 cells transfected with theART-4/mycgene. The difference inmigration between these bands (Mr 46,000 and 51,000) was probablyattributable to the myc-tag peptide (Mr ;5,000). TheMr 46,000 band
Fig. 1. Recognition of a4E6-2B9gene product by the GK-CTLs. COS7 cells weretransfected with 100 ng of clone 4E6-2B9 or 4E6-6F2 (as a negative control) DNA alongwith 100 ng ofHLA-A2402or HLA-A2601cDNA (as a negative control) and were testedfor their ability to stimulate IFN-g production by the GK-CTLs at an effector:stimulatorratio of 10. Values represent the means of the triplicate assays.
was detected in both the cytosol and nucleus of all of the tested tumorcell lines established from various organs, including the lung(n 5 10), esophagus (n5 3), stomach (n5 3), head and neck (n5 4),uterus (n5 8), ovary (n 5 8), and breast (n5 6). It was also
detectable in leukemia cell lines, with the exception of the three celllines (Jurkat, MOLT-4, and RPMI8402) from T-cell leukemia. Thisband was also detectable in both the cytosol and nucleus of themajority of cancer tissues from various organs tested, including the
Fig. 2. Characterization of theART-4gene.A, expression of theART-4gene at the mRNA level was studied by Northern blot analysis. Twenty-two tumor cell lines described in“Materials and Methods,” Bec-1, COS7, and PBMCs, and 15 normal tissues were provided for Northern blot analysis with a probe of4E6-2B9or b-actin as a control. Representativeresults are shown. Relative expression levels are also given.B, nt sequence of theART-4gene, 1733 bp long cloned, from the HT1376 cDNA library. The sequence of the clone 4E6-2B9corresponds to the positions 1–1584 of theART-4gene. TheART-4had an open reading frame of 1236 bp in length and encoding 412 amino acids when the first ATG (17–19,underlinedin bold) and stop codon (1253–1255,double-underlined) were used for protein synthesis. There was only one nt difference, at position 708 (p), between theART-4of HT1376 andPBMCs (guanine in the HT1376versusadenine in the PBMCs). These sequence data are available from EMBL/GenBank/DDBJ under accession number AB026125.C, the deducedamino acid sequence of the ART-4 protein.Underlining indicates the peptide sequences capable of inducing HLA-A24-restricted CTLs, and abox indicates an ER-retention signal.D, hydrophobicity plot (K-D plot) of ART-4 protein. Hydrophobicity was analyzed with GeneWorks Version 2.4 software (IntelliGenetics, Mountain View, CA). Thetwo short linesabove the peaks indicate the positions of the CTL-directed epitopes.E, bioactivity of theART-4gene. COS7 cells were transfected with various doses of theART-4gene and 100 ngof HLA-A2402or controlHLA-A2601cDNA, and their stimulatory effects on IFN-g production by the GK-CTLs were tested. Values represent the means of triplicate assays. Thetwo-tailed Student’st test was used for the statistical analysis between the IFN-g production by the CTLs in response to COS7 cells transfected withART-4andHLA-A2402genesand that of transfected withART-4andHLA-A2601genes.p, P , at least 0.05.
lung (n 5 18), head and neck (n5 9), stomach (n5 8), uterus(n 5 16), and breast (n5 8). In contrast, anMr 46,000 protein ofART-4 was not detectable in either the cytosol or nuclear fraction ofany of the normal tissues tested, except for those of the testis,placenta, and fetal liver. Some of these results are shown in Fig. 3B,and a summary is shown in Table 1.
Cellular Localization of the ART-4 Protein. The ART-4/GFPprotein was found in both the cytosol and nucleus of COS7 cellstransfected with theART-4/GFPgene (Fig. 3C). On the basis of acomparison of the fluorescence distribution pattern of ART-4/GFP tothat of control EYFP-fusion proteins (EYFP-ER, EYFP-Golgi, andEYFP-Mito), the ART-4/GFP protein in the cytosol might have beenexpressed at the ERs, but it would not have been expressed on the
Golgi apparatuses or the mitochondrias. Furthermore, this ART-4protein was detected in the fractions 1, 2, 7, and 9 when the subcel-lular organelles from theART-4/myctransfectants were fractionatedby the velocity-controlled sucrose gradient method (Fig. 3D). Thedistribution pattern of this protein was very similar to that of NADPHcytochrome P450 reductase, a marker protein for ER-resident protein,but differed largely from that of protein kinase Cm, a Golgi markerprotein. These results together with the presence of an ER membraneretention signal at the COOH terminus at positions 408–411 stronglysuggest that the ART-4 protein is an ER-resident protein.
Antigenic Peptides Recognized by the CTLs.Each of the 18different ART-4-derived synthetic peptides with motifs of binding toHLA-A2402 molecules was loaded onto theHLA-A2402-transfected
Fig. 3. Characterization of the ART-4 protein.A, expression of the ART-4 protein in transfectants. Expression of the ART-4 protein in COS7 cells transfected with theART-4orART-4/mycgene or in the parental COS-7 cells was analyzed by Western blotting with anti-ART-4/GST polyclonal Ab or anti-myc mAb.B, expression of the ART-4 protein in variouscell lines and tissues was studied by Western blot analysis with anti-ART4/GST polyclonal Ab. A summary of the results is shown in Table 1.C, expression of the ART-4/GFP fusionprotein in the transfectants. COS7 cells were transfected with theART-4/GFPgene followed by serial observation under a Zeiss confocal Ar-Kr laser scanning microscope with bothfluorescence and visible rays (F1V) or fluorescence only (F). Localization of the ART-4/GFP protein was recorded under an FITC filter (520 nm). The exposure sequences and imagingwere controlled by LSM version 3.70 imaging software. The visible-ray image was studied by differential interference microscopy. COS7 cells transfected with Living Colorssubcellular localization vectors,pEYFP-ER,pEYFP-Golgi, or pEYFP-Mito(Clontech), show representative fluorescence patterns of ER, Golgi, or mitochondria-resident proteins,respectively.D, subcellular localization of ART-4 protein in theART-4/myctransfectants. Homogenate of the transfectants was fractionated by sucrose density gradient centrifugation,followed by Western blot analysis. The ART-4/myc fusion protein was detected by anti-myc mAb. For detection of ER and Golgi marker proteins, anti-NADPH cytochrome P450reductase and anti-protein kinase Cm antibodies, respectively, were used.
COS7 cells at a concentration of 10mM and then tested for its abilityto induce IFN-g production by the GK-CTLs. Two of these peptides,ART-413–20 and ART-475–84, stimulated significant levels of IFN-gproduction in a dose-dependent manner (Fig. 4,A and B). Similardose-dependent effects of the two peptides were also observed whenthe peptides were loaded onto irradiated autologous PBMCs or C1R-A2402 cells (data not shown). The other 16 peptides failed to stimu-late IFN-g production by the CTLs. To confirm the presence ofpeptide-specific CTLs, the sublines were established from the parentalGK-CTL line by a limiting dilution culture at 1 or 10 cells/well, andtheir peptide specificities were tested. Among the 68 different sublinestested, 7 sublines recognized C1R-A2402 cells pulsed with ART-413–20 but not with either ART-475–84 or HIV-derived peptide as anegative control. The CTL activity of those 7 sublines was inhibitedby 10mg/ml of anti-A24, anti-class I HLA (W6/32) or anti-CD8 mAbbut not by anti-class II (H-DR-1), anti-CD4, or isotype-matchedirrelevant control mAbs (anti-CD13 and anti-CD14). Representativeresults for one subline, #1-2, are shown in Fig. 4C. The other 6sublines recognized C1R-A2402 cells pulsed with ART-475–84but notthose pulsed with the other peptides, and their CTL activity wasinhibited by anti-HLA-A24, anti-class I HLA (W6/32), or anti-CD8mAb but not by the other mAbs. Representative results for onesubline, #10-1, are shown in Fig. 4D. The remaining 55 sublines failedto respond to any peptides tested (data not shown).
Induction of CTLs by Peptides. ART-413–20 and ART-475–84
peptides were tested for their ability to induce CTLs from the PBMCsof six HLA-A241 patients with lung cancer (four with adenocarcino-mas and two with squamous cell carcinomas) and five HLA-A241
healthy donors. The PBMCs stimulated with either peptide in the fourpatients (three with adenocarcinoma and one with squamous cellcarcinoma) showed significant levels of cytotoxicity to HLA-A241
11-18 lung cancer cells but not to HLA-A242 QG-56 lung cancercells. Low but significant lytic activity of the CTL against 11-18 cellswas probably attributable to the low expression of HLA-A24 mole-cules but not to that of the ART-4 peptides on the 11-18 cells, becausea significant augmenting effect of exogenously loaded ART-4 peptideonto the 11-18 cells on the recognition by the CTLs was not observed(data not shown). Results of the three cases are shown in Fig. 5A. Inall three cases, the PBMCs failed to lyse HLA-A241 PHA-blasts,VA13 fibroblast cells, or K562 tumor cells. These PBMCs possessed20–60% of CD31CD42CD81 cells, and the remaining cells weremostly CD31CD41CD82 cells at the time of assays (data not shown).To confirm the peptide specificity, sublines derived from the patient1 shown in Fig. 5Awere further established from the peptide-inducedPBMCs, followed by a test of their reactivity to ART-413–20, ART-475–84, or HIV peptide. Three of 69 sublines tested from the PBMCsstimulated with the ART-413–20 responded to the C1R-HLA-A2402cells pulsed with ART-413–20. IFN-g production by these sublines wasinhibited by 10mg/ml of anti-HLA-A24, anti-class I HLA (W6/32), oranti-CD8 mAb but not by anti-class II (H-DR-1), anti-CD4, or irrel-evant control mAbs (anti-CD13 and anti-CD14). The results for onesubline, #1-1029, are shown in Fig. 5B. On the other hand, 2 of 17sublines tested from the PBMCs stimulated with the ART-475–84
responded to C1R-HLA-A2402 cells pulsed with ART-475–84peptide,and the CTL activity of these sublines was inhibited by 10mg/ml ofanti-HLA-A24, anti-class I HLA (W6/32), or anti-CD8 mAb but notby the other mAbs. The results for one subline, #6-1027, are shown inFig. 5C. In the remaining two cancer patients, HLA-A24-restrictedCTL activity was not induced in the PBMCs stimulated with eitherpeptide. In these PBMCs, the percentages of CD31CD42CD81 cellswere very low (,8%). HLA-A24-restricted CTL activity was alsoundetectable in the PBMCs of all healthy donors (n5 5) afterstimulation with either peptide (data not shown).
DISCUSSION
This study reports that theART-4gene encodes antigenic epitopesrecognized by the HLA-A24-restricted and tumor-specific CTLs. TheART-4had 99 and 50% homology with theHP-10 at the nt and thededuced amino acid levels, respectively. TheHP-10 was originallycloned from a human placenta cDNA library using human hexokinasemAb (30). However, theHP-10did not show such hexokinase activ-ity, and the function of the HP-10 protein has been reported asregulation of protein phosphorylation only inEscherichia coli, not ineukaryotes (30). When we screened the HT1376 and PBMC cDNAlibraries to obtain the full-lengthART-4gene by colony hybridization,none of the obtained clones had theHP-10sequence. To confirm theexistence of theHP-10gene in human samples, we then tried to clonethe HP-10 gene from a human placenta cDNA library by means ofPCR. We analyzed 20 clones isolated from the PCR products by ntsequencing and also by digestion with restriction enzymes, and theresults indicated that all of them were theART-4 gene (data notshown). With the exception ofHP-10, none of the genes reportedpreviously have significant homology to theART-4gene, based on asearch of the literature. Therefore, we were unable to confirm thepresence of theHP-10gene in human samples under the used condi-tions. TheHP-10 might be a family gene of theART-4 gene, themessage of which was rarely expressed in the placenta, HT-1376tumor cells, and PBMCs.
There are several motifs in the sequence of the ART-4 protein: anuclear localization signal at positions 396–412, a cAMP- or cGMP-dependent protein kinase phosphorylation site at positions 294–297,and a di-lysine (KKXX)-like ER membrane retention signal at theCOOH terminus. The KKXX motif has been found previously at the
Table 1 Summary of the expression of the ART-4 protein in normal and cancer cellsand tissuesa
COOH terminus of several ER-resident types I and III membraneproteins and shown to be responsible for the localization to the ERs(31). Membrane-topology prediction suggests that the ART-4 is a typeIb membrane protein. Results of the present laser-confocal micro-scopic analysis of the GFP-taggedART-4 transfectants suggest thatthe ART-4 protein was localized at the ER and in the nucleus but notat the Golgi or on the plasma membrane. The localization of ART-4at the ER was further confirmed by the subcellular organelle fraction-ation of a tagged ART-4 protein in the transfectants. All of the resultssuggest that the ART-4 is an ER-resident protein encoding tumor-rejection antigen recognized by the HLA-A24-restricted CTLs. Wehave identified recently cyclophilin B as the tumor-rejection antigenrecognized by the GK-CTLs (15), and this molecule is also known asan ER-resident protein involved in the signal transduction of cellularproliferation (32). ER is well known as a major site of peptide loading
to HLA molecules (33, 34). Therefore, it might be interesting to studythe relationship between CTL-directed epitopes and ER-resident pro-teins. However, currently identified functional domains involved inthe signal transduction of cellular proliferation, such as kinase do-mains or src homologous domains, are not found in the deducedART-4 sequence. Therefore, we were unable to hypothesize anybiological functions of this newly identified ER-resident protein, andfurther studies on the function of the ART-4 will be needed.
Under the present conditions, the ART-4 protein was not detectable byWestern blot analysis with polyclonal anti-ART-4/GST Ab in any of thenormal tissues, except for the testis, fetal liver, and placenta, despite theubiquitous expression of theART-4gene at the mRNA level. We havereported similar expression patterns for the SART-1 (13) and SART-3(14) proteins, but the mechanisms involved in the discrepancy betweenthe mRNA and protein expression are presently unclear.
Fig. 4. Identification of epitopes recognized by the CTLs.A,determination of the antigenic peptides derived from the ART-4protein. Each of the 18 different peptides was loaded onto COS7cells transfected withHLA-A2402. The GK-CTLs were added tothe peptide-loaded cells and incubated for 18 h, and the culturesupernatant was harvested to measure IFN-g production byELISA (limit of sensitivity, 10 pg/ml) in triplicate assays. Valuesrepresent the means of triplicate assays. The background of IFN-gproduction by the GK-CTLs in response to each peptide loadedonto COS7 cells transfected withHLA-A2601 was subtractedfrom the values.B, dose dependency of the ART-4 peptides.Indicated doses of the peptides were loaded onto COS7 cellstransfected withHLA-A2402or controlHLA-A2601cDNA, andthen the ability of the peptides to stimulate IFN-g production bythe GK-CTLs was tested. Values represent the means of triplicateassays. The background of IFN-g production (50 pg/ml) by theGK-CTLs in response to theHLA-A2601transfectants was sub-tracted from the values.C and D, peptide specificity of theGK-CTL sublines. Sublines were established from the parentalGK-CTL line by a limiting dilution culture, and the 68 differentsublines with CD31CD42CD81 phenotype were tested for theirreactivity to C1R-A2402 cells pulsed with ART-413–20, ART-475–84, or HIV-derived peptide as a negative control at a respond-er/stimulator ratio of five in triplicate assays. Seven and sixsublines showed the reactivity to ART-413–20 and ART-475–84
peptide, respectively. IFN-g production by the sublines in re-sponse to 11-18 tumor cells at a responder:stimulator ratio of 4was tested in the presence of 10mg/ml of anti-HLA-class I(IgG1), anti-HLA-A24 (IgG1), anti-HLA-class II (IgG2a), anti-CD4 (IgG1), or anti-CD8 (IgG2a) mAb. Anti-CD13 (IgG1) andanti-CD14 (IgG2a) mAbs were used as isotype matched controls.Values represent the mean IFN-g levels of triplicate assays.p,values differed significantly from the controls withP , 0.01 bythe two-tailed Student’st test. Representative results of each ofsublines #1-2 and #10-1 are shown inC andD, respectively. Theother sublines failed to respond to any peptides tested.
Among the 18 peptides with HLA-A24 antigen-binding motifstested, the two ART-4-derived peptides, ART-413–20and ART-475–84,were consistently recognized by the HLA-A24-restricted CTLs inrepeated experiments. Because of the presence of each of the peptide-specific CTL sublines, the parental GK-CTL line would consist of amixture of these peptide-specific CTL clones. A dose dependency wasobserved in the two peptides, and.0.01mM of each the peptides wererequired for the recognition of target cells by the CTLs. Both peptideswere able to induce HLA-A24-restricted CTLs from PBMCs of theHLA-A241 lung cancer patients but not from those of the HLA-A241
healthy donors. These results suggest that there is a preferentialpresence of the CTL precursors reacting to the ART-4 epitopes inPBMCs of these cancer patients. In contrast, the CTL precursors maynot exist in PBMCs of healthy donors.
Regardless of the numerous unsolved issues, including identifica-tion of its biological roles, the ART-4 antigen might be an ideal targetmolecule for use in specific immunotherapy of relatively large num-bers of cancer patients, because it could be a cancer-specific proteinexpressed in the cytosol (ER) of the vast majority of cancers fromvarious tissues with different histological types. The other tumor-rejection antigens, including MAGE family antigens (1, 7), SART-1(13), and SART-3 (14), are also preferentially expressed in cancercells with different histological types. Although the mutated antigens,including mutant CDK4 (35) and mutant CASP 8 (36), are recognizedby CTLs, these types of antigens are expressed only in certain cancercells, and the mutated products are usually detected in only a smallpart of tumor samples. Thus, these molecules would not be applicableas targets in specific immunotherapy of a large number of cancerpatients.
The HLA-A24 allele is found in 60% of Japanese (with 95% ofthese cases being genotypically A2402), in 20% of Caucasians, and in12% of Africans (18). The two ART-4-derived peptides were able toinduce HLA-A24-restricted and tumor-specific CTLs in PBMCs oflung cancer patients. These ART-4 peptides might be appropriatemolecules for use in the specific immunotherapy of HLA-A241
patients with lung and other cancers.
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2000;60:3550-3558. Cancer Res Kouichiro Kawano, Shinya Gomi, Koji Tanaka, et al. Lymphocytes of Lung CancerProtein Recognized by HLA-A24-restricted Tumor-infiltrating Identification of a New Endoplasmic Reticulum-resident
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