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Departments of
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Immunology and
Surgery, and
Second Department of Anatomy, Kurume University School of Medicine, Fukuoka, Japan; and
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Cancer Vaccine Development Division, Kurume University Research Center for Innovative Cancer Therapy, Fukuoka, Japan
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A24-restricted and tumor-specific CTLs were established from PBMCs of an esophageal cancer patient (HLA-A*2402/2601) by a mixed lymphocyte tumor cell culture, as reported previously (15, 16). Both this CTL line and that used for cloning of the SART-3 gene (15) were originated from the parental HLA-A24-restricted CTL line reported previously (16), and these two sublines shared similar cytotoxic profiles. CTL clones and sublines from the CTL subline used in this study were established by the limiting dilution culture method, as reported previously (15). In brief, T cells at a concentration of 1, 10, or 100 cells/well of a round-bottom 96-well microculture plate were incubated with the cloning medium (25% RPMI 1640 medium (Life Technologies, Grand Island, NY), 55% AIM-V medium (Life Technologies), 20% FCS (Equitech Bio, Ingram, TX), 100 U/ml IL-2 (Shionogi Pharmaceutical, Osaka, Japan), 10 µg/ml PHA (Difco, Detroit, MI), and 0.1 mM MEM nonessential amino acids solution (Life Technologies)) in the presence of irradiated (50 Gy) allogenic HLA-A24+ PBMCs (2 x 105 cells/well) donated by three healthy volunteers as feeder cells. For inhibition of CTL activity of 10 µg/ml of anti-HLA class I (W6/32, IgG2a), anti-HLA-A23/24 (0041HA, IgG2a) as anti-HLA-A24, anti-CD8 (Nu-Ts/c, IgG2a), anti-CD4 (Nu-Th/i, IgG1), and anti-HLA-class II (H-DR1, IgG2a) were used. Anti-CD14 (H14, IgG2a) mAb or anti-CD13 mAb (MACS-2, IgG1) served as an isotype-matched control mAb.
Identification of a clone 12 gene
An expression gene-cloning method described previously
(14, 15) was used to identify a gene coding for a tumor Ag
recognized by the HLA-A24-restricted CTLs. Briefly, mRNAs of the KE-4
tumor cells were converted to cDNAs, ligated to the SalI
adapter, and inserted into the expression vector pSV-SPORT-1
(Life Technologies, Rockville, MD). Both 200 ng of plasmid DNA pools or
clones of the KE-4 cDNA library and 200 ng of the HLA-A*2402
or HLA-A*2601 cDNA as a negative control were mixed with 1
µl of Lipofectin (Life Technologies) in 70 µl of OPTI-MEM (Life
Technologies) for 15 min. A total of 30 µl of the mixture was then
added to the VA-13 (2 x 104) cells and
incubated for 5 h. Then 200 µl of the RPMI 1640 medium
containing 10% FCS was added and cultured for 2 days, followed by the
addition of cells of the HLA-A24-restricted CTL line
(104 cells/well). After an 18-h incubation, 100
µl of supernatant was collected to measure IFN-
by an ELISA in a
duplicate assay (limit of sensitivity: 10 pg/ml). DNA sequencing was
performed with the dideoxynucleotide sequencing method using a DNA
sequencing kit (Perkin-Elmer, Foster, CA) and analyzed by an ABI PRISM
377 DNA Sequencer (Perkin-Elmer).
Northern blot analysis
Nylon membranes (Hybond-N+; Amersham, Buckinghamshire, U.K.) with UV-fixed poly(A)+ RNA (2 µg/lane) from various tissues were prehybridized in a solution (7% SDS, 1 mM EDTA, 0.5 M NaH2PO4, pH 7.2) for 10 min and hybridized overnight at 65°C in the same solution containing the 32P-labeled clone 12 as a probe. The membranes were washed three times at 65°C in a washing buffer (1% SDS, 40 mM NaH2PO4, pH 7.2), and then autoradiographed. The relative expression level of the SART-2 mRNA was calculated by the following formula: index = (SART-2 density of a sample/ß-actin density of a sample) x (ß-actin density of the KE-4/SART-2 density of the KE-4).
Cloning of the SART-2 gene
We tentatively designated this gene, which encodes a tumor Ag recognized by the HLA-A24-restricted CTLs, a SART-2 gene (squamous cell carcinoma Ag recognized by T cells-2). The SART-2 clone was independently obtained from the cDNA libraries of both the KE-4 tumor cells and PBMCs of healthy donors by the standard colony hybridization method using the 32P-labeled clone 12 as a probe, as reported previously (14). The sequence of the SART-2 gene is available from EMBL/GenBank/DDBJ under accession number AF098066.
GST-fusion proteins and rabbit antisera, and SART-2-tag fusion protein
For preparation of the clone 12/GST-fusion protein, the clone 12 was digested with SalI and NotI at the multiple cloning site of pSV-SPORT-1, then ligated into the pGEX-5X-3 vector (Pharmacia LKB Biotechnology, Uppsala, Sweden). Polyclonal anti-clone 12/GST Ab (designated anti-SART-2 Ab in this work) was prepared by immunizing rabbits with a purified recombinant clone 12/GST-fusion protein, as reported previously (14). The SART-2/green fluorescence protein (GFP) fusion protein was prepared for identification of cellular location and determining the size of the SART-2 protein. To prepare the SART-21–958/GFP (corresponding to aa positions 1–958 of the SART-2 protein), the SART-2 of positions 150-3023 was amplified by PCR using the forward primer 5'-AGATCAAGCTTCACGATGGGGACTCACACA-3' and the reverse primer 5'-GTCGACTACACTGTGATTGGGAACAAG-3'. The amplified product was digested with HindIII and SalI, and ligated to the pEGFP-N2 vector (Clontech, Palo Alto, CA). This gene was named SART-21–958/GFP. The GFP was ligated to the position 3023 before the stop codon of the second frame of SART-21–958. To prepare the three deletion mutants (SART-21–932/GFP, SART-21–901/GFP, and SART-232–958/GFP), the SART-2 of positions 150-2945, positions 150-2852, and positions 242-3023 were amplified by PCR using the forward primer 5'-AGATCAAGCTTCACGATGGGGACTCACACA-3' and the reverse primer 5'-ATAAAGACGGTACCGGCCATGTAGGCTCT-3'; the forward primer 5'-AGATCAAGCTTCACGATGGGGACTCACACA-3' and the reverse primer 5'-GAACAGGTACCTATAGGAAGCAGACAGTGAT-3'; and the forward primer 5'-GAGAAAAGCTTAGTTATGGTTCCCTTCACCA-3' and the reverse primer 5'-GTCGACTACACTGTGATTGGGAACAAG-3', respectively. The amplified products were digested with HindIII and KpnI (or, in the case of SART-232–958, with HindIII and SalI), and ligated to the pEGFP-N2 vector (Clontech).
Western blot analysis
The samples were lysed with a buffer consisting of 10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.5% Triton X-100, 0.2 mM PMSF (Sigma, St. Louis, MO), and 0.03 trypsin-inhibitory units (TIU)/ml aprotinin (Sigma), sonicated, and centrifuged at 14,000 rpm for 20 min, and the supernatant was used as the cytoplasm fraction. The pellet was lysed with a buffer consisting of 7.2 M urea, 1.6% Triton X-100, 0.8% DTT, and 2% lithium dodecyl sulfate, and then centrifuged. The supernatant was used as the nuclear fraction, and the lysate was separated by SDS-PAGE. The proteins in acrylamide gel were blotted to a Hybond-polyvinylidene difluoride membrane (Amersham) and were incubated with the appropriate Abs for 4 h at room temperature. Other methods used for the Western blot analysis were as described previously (14). The histologies and HLA class I genotypes of a majority of the various cancer cell lines (n = 66) used in this study have been described elsewhere (14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24), and thus were not shown in this manuscript.
Assays for cellular location
For immunoprecipitation analysis, the 293T human embryonic kidney cells and those transfected with the SART-21–958/GFP (1 x 107) were washed twice with PBS and labeled with 37 M Bq of Na125I (IMS-30; Amersham, Arlington Heights, IL) by lactoperoxidase-catalyzed iodination, as previously described (25). After iodination, cells were washed three times with PBS and then lysed in 1 ml of lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, 1 mM PMSF, 0.05 TIU/ml aprotinin, 0.02% NaN3) on ice for 30 min. The cell lysate was centrifuged at 12,000 rpm for 20 min at 4°C and further precleared overnight at 4°C with Formalin-fixed Staphylococcus aureus Cowan I (Mercian, Tokyo, Japan). The precleared lysate was precipitated with anti-GFP mAb (Clontech) or control normal mouse IgG-coupled protein A-AffiGel beads (Bio-Rad, Richmond, CA) at 4°C overnight. Precipitates were washed twice with the lysis buffer, once with the lysis buffer containing 0.5 M NaCl, once with 0.5% deoxycholate, and once with the lysis buffer alone. Immunoprecipitates were solubilized with SDS-PAGE sample buffer and analyzed by 6% SDS-PAGE under reducing conditions. Gels were then fixed, dried, and subjected to autoradiography. For cellular location analysis, subcellular organelles were fractionated velocity-controlled sucrose gradient fractionation, as described previously (26, 27). In brief, 5 x 107 COS7 cells transfected with SART-21–958/GFP gene were washed with ice-cold STE buffer (0.25 M sucrose, 1 mM PMSF, 0.05 TIU/ml aprotinin, 20 mM Tris-HCl, pH 8, and 1 mM EDTA). Cells were suspended in a hypotonic buffer (0.025 M sucrose-STE) and incubated for 30 min, and homogenized with tight fitting Dounce homogenizer. The homogenate was centrifuged at 500 x g for 5 min to remove the nuclei and undisrupted cells. The supernatant was layered on a discontinuous sucrose gradient consisting of 1 ml of 2 M sucrose, 3 ml of 1.3 M sucrose, 3 ml of 1 M sucrose, and 2.5 ml of 0.6 M sucrose, and then centrifuged at 40,000 rpm 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: 0.6 M in the fraction number 1 and 2; between 0.6 M and 1 M in number 3; 1 M in number 4 and 5; between 1 M and 1.3 M in number 6; 1.3 M in number 7 and 8; between 1.3 M and 2 M in number 9; 2 M in number 10. Each fraction was mixed with SDS-sample buffer and subjected to Western blot analysis. For detection of endoplasmic reticulum (ER) and Golgi marker proteins, rabbit anti-NADPH cytochrome P450 reductase (StressGen, Victoria, BC, Canada) and sheep anti-protein kinase C µ (The Binding Site, Birmingham, U.K.) Abs were used, respectively. Normal rabbit sera and sheep sera were used as negative controls.
The SART-21–958/GFP gene or the pEGFP-N2 vector alone as a control was transfected in COS7 cells, as previously described (15), followed by serial observation under a Zeiss confocal Ar-Kr laser-scanning microscope. Control vectors used in this study were pEYFP-ER, which encodes a fusion protein consisting of enhanced yellow fluorescent protein (EYFP), the ER targeting sequence of calreticulin, and the ER retrieval sequence, KDEL, pEYFP-Golgi, which encodes EYFP and a sequence encoding the N-terminal 81 aa of human ß-1,4-galactosyltransferase, and pEYFP-Mito, which encodes a fusion of EYFP and mitochondrial targeting sequence from subunit VIII of human cytochrome c oxidase (Clontech). The SART-21–958/GFP gene was also transfected in MCF-7 breast cancer cells. Localization of the SART-2-GFP protein was recorded under an FITC filter (520 nm). The exposure sequences and imaging were controlled by LSM imaging software, version 3.7.
Constructions of deletion mutants
The SART-2/pCMV-SPORT plasmid was digested with Eco47III for the preparation of deletion mutants. The linearized DNA was subjected to second restriction enzyme SphI digestion to generate sensitivity to ExoIII at one end. ExoIII nuclease/Mung bean nuclease treatment was performed according to the manufacturer’s instructions (TaKaRa, Ootsu, Japan) to obtain five deletion mutants of SART-2 (SART-21–2817 corresponding to nucleotide positions 1–2817 of the SART-2 gene, SART-21–2000, SART-21–1525, SART-21–1209, and SART-21–540).
Peptides and assay
Among the many different peptide sequences with motifs for
binding to the HLA-A24 molecules in the deduced amino acid sequences of
the SART-2 Ag, 10 different peptides reported to have strong binding
motifs for HLA-A24 (tyrosine at position 2, and isoleucine,
phenylalanine, or leucine at position 9) (28) were used.
These peptides were obtained from Biologica (Nagoya, Japan) and had a
purity of >95%. For detection of antigenic peptides recognized by the
CTLs, the HLA-A*2402 or HLA-A*2601 cDNA (a
negative control) was transfected to the VA-13 (2 x
104) cells and incubated for 5 h. Then 200
µl of the RPMI 1640 medium containing 20% FCS was added and cultured
for 2 days, followed by the addition of a peptide at a concentration of
10 µM in most experiments or of 10 nM to 50 µM in other
experiments. In certain experiments, C1R-A*2402 cells (kindly provided
by Dr. Takiguchi, Kumamoto University, Kurume, Japan) (15)
were used as peptide-loading cells. Two hours later, the supernatant
was removed, and the HLA-A24-restricted CTLs (104
cells/well) were added and incubated for 18 h. A total of 100 µl
of the supernatant was collected to measure IFN- using an ELISA kit
in a triplicate assay, as reported previously (14).
Peptide-binding assay
For peptide-binding assay, RMA-S-A*2402/Kb
were kindly provided by H. Takasu (Research Institution of Sumitomo
Pharmaceutical, Osaka, Japan). To provide these cells, a chimera gene
A*2402/Kb encoding with the 
1 and 2 domains
of HLA-A*2402 molecule and the 3, transmembrane, and intracellular
domains of H-2Kb molecule were established. The
exon 1 to 3 of HLA-A*2402 cDNA and the exon 4 to 8 of
H-2Kb cDNA were ligated into pcDNA3.1(+)
(Invitrogen, Groningen, The Netherlands). Thereafter, this gene was
transfected into RMA-S cells (mouse lymphoma cell line)
(29). These cells were named
RMA-S-A*2402/Kb. These cells were incubated at
26°C for 18 h. After washing with PBS, the cells (2 x
105) were suspended with OPTI-MEM (Life
Technologies) containing 3 µg/ml of human
ß2-microglobulin (Cortex Biochem, Sanleandro,
CA) and 100 µg/ml of peptide, followed by incubation at 26°C for
3 h and 37°C for 3 h. After washing with PBS, the cells
were incubated with anti-HLA-A24 mAb at 4°C for 30 min, followed
by the cells incubated with PE-conjugated rabbit anti-mouse IgG Ab
(Cappel, Aurora, OH) at 4°C for 30 min. The cells were then suspended
with 1 ml of PBS containing 1% formaldehyde, and analyzed with FACScan
(Becton Dickinson, Mountain View, CA). Binding activity was evaluated
by the mean fluorescence intensity of the HLA-A*2402 molecule of the
RMA-S-A*2402/Kb cells that were pulsed with a
peptide (100 µM). Estimated score of half time of dissociation of
each SART-2 peptide for HLA-A24-molecule was also calculated based on
HLA peptide motif search results with computer analysis
(30), as follows: SART-293–101,
66.0; SART-2161–169, 288.0;
SART-2229–305, 360.0;
SART-2297–305, 140.0;
SART-2472–480, 66.0;
SART-2476–484, 120.0;
SART-2645–653, 420.0;
SART-2661–669, 105.0; or
SART-2899–907, 288.0; and
SART-2921–927, 240.0.
Induction of CTLs by peptides
PBMCs (1 x 106) from
HLA-A24+ healthy donors or cancer patients were
preincubated with a peptide (10 µM). These cells were washed with
PBS, irradiated (50 Gy), and used as APCs. PBMCs (1 x
106) from the same donor or patient were cultured
with the autologous APCs in the well of a 24-well plate containing 1 ml
of the culture medium (45% RPMI 1640, 45% AIM-V medium, and 10% FCS
with 100 U/ml IL-2) at a responder to stimulator ratio of 1:1. At days
7 and 14 of the culture, cells were collected, washed, and stimulated
with the autologous irradiated APCs that were loaded with the same
peptide, as described above. Cells were harvested at day 21 of the
culture, and some of them were cryopreserved at -196°C. Most of them
were further incubated in wells of a round-bottom 96-well microculture
plate with the culture medium in the presence of irradiated autologous
PBMCs used as feeder cells. These cells from the microculture were
further expanded in wells of a 24-well plate in the presence of IL-2
(100 U/ml) alone. Cells were then tested for both their surface
phenotypes by immunofluorescent techniques with anti-CD3,
anti-CD4, and anti-CD8 mAb, and FACScan, as reported previously
(15), and their cytotoxicity to the various target cells
at different E:T ratios in a 6-h 51Cr release
assay at days 22 to 28 of reculture, as reported previously (15, 16). The frequency of CTL precursors reacting to a corresponding
peptide used for stimulation was analyzed in the PBMCs after the third
stimulation, as described previously (16). In brief, these
PBMCs were plated at 1, 2, 5, 10, 20, 40, or 80 cells/ well of 96-well
microculture plates and cultured with the cloning medium in the
presence of feeder cells. The PBMCs cultured without any peptide as a
negative control were plated at 25, 50, 100, 200, or 500 cells/well.
Cells from each well were tested between 8 and 14 days of culture for
their activity. A well was considered positive if it contained effector
cells producing a much higher level (>100 pg/ml) of IFN- in
response to a corresponding SART-2 peptide loaded on C1R-A*2402 cells
as compared with that in response to C1R-A*2402 cells without any
peptide and that in response to those loaded with an HIV-envelope
peptide with an HLA-A*2402 molecule-binding motif (RYLRDQQLLGI) (as a
negative control). The data were analyzed by the minimum
2 method with 95% confidence intervals. The
CTL precursor frequency was calculated by determining the cell
concentration at which 37% of the wells were negative, using a graph
of the percentage of negative wells on the ordinate and the responder
cell concentration on the abscissa (31).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The HLA-A24-restricted and tumor-specific CTL line
(CD3+CD4-CD8+)
was established from PBMCs of an esophageal cancer patient (KE-4,
HLA-A*2402/2601) after stimulation with the autologous KE-4 tumor cells
by the standard mixed lymphocyte-tumor cell culture, and its
characteristics have been described elsewhere (16). The
CTL subline used in this study was established from that parental CTL
line, and its CTL activity is shown in Fig. 1
. This CTL subline showed the
cytotoxicity against HLA-A*2402+ esophageal SCCs
(KE-4 autologous tumor cells and KE-3) and lung adenocarcinoma cells
(PC-9). In contrast, the CTL subline failed to lyse any of the
HLA-A*2402- tumor cells or
HLA-A*2402+ PHA-blastoid cells from PBMCs of
healthy donors, K562, or VA-13 fibroblast cells (Fig. 1). The
cytotoxicity of this CTL subline with a
CD3+4-8+
phenotype was inhibited by anti-CD8 or anti-HLA-class I (W6/32)
mAb, but not by anti-CD4 or anti-HLA class II mAb (data not
shown).
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A total of 105 cDNA clones from the cDNA
library of the KE-4 tumor cells was tested for their ability to
stimulate IFN- production by the HLA-A24-restricted CTLs shown above
after cotransfection with HLA-A*2402 into the VA-13
fibroblast cells. After repeated experiments for several candidate
clones, one clone (clone 12) was confirmed to encode a tumor
Ag recognized by the HLA-A24-restricted CTLs when cotransfected with
HLA-A*2402, but not with HLA-A*2601 as a negative
control (Fig. 2A). Expression
of this gene was investigated by Northern blot analysis, and a band of
about 4 kb was observed in all eight SCC cell lines and in two of six
adenocarcinoma cell lines tested. A portion of the data is shown in
Fig. 3A. This gene was also
expressed in all of the normal tissues tested (Fig. 3B), and
the relative levels of mRNA expression were within the range of 0.633
± 0.420 in all of the tissue samples, with higher expression in the
kidney (relative expression level, 1.5) or ovary (1.1), and lower
expression in the colon (0.16), thymus (0.15), or brain (0.1). These
results suggest that this gene was expressed in all of the SCCs and
normal tissues, but was expressed in only a part of adenocarcinomas at
the mRNA level, and that the 2311-bp-long cDNA was incomplete. Then, a
3958-bp-long gene was independently cloned from the cDNA libraries of
both KE-4 and PBMCs of healthy donors, using clone 12 as a
probe and the standard methods for colony hybridization, and its
characteristics are shown in Fig. 4. The
sequences of these clones were identical, and all contained the
clone 12 at positions 776-3086. The entire nucleotide
sequence of this gene was found within the genomic DNA of chromosome
6q22 (EMBL accession number, Z84488) by a search of GenBank
(32). These results indicate that this cDNA clone is
transcribed from the chromosome 6q22, and consists of six exons (Fig. 4). The deduced 958-aa sequence encoded by an open reading frame of
this gene at positions 150- 3023 showed no similarity to any known
amino acid sequences. A hydrophobicity analysis showed that a protein
encoded by this gene had one highly hydrophobic region at the N
terminus, potentially allowing it to act as a signal peptide (Fig. 4).
In addition, it had two highly hydrophobic regions at the C terminus,
creating potential transmembrane regions, as well as five putative
N-glycosylation sites (Fig. 4). There was a di-arginine
(XXRR) motif-like ER membrane retention signal at position 2–5 (RTHT).
The SART-2 gene encoded the Ags recognized by the CTLs when
cotransfected with HLA-A*2402, but not HLA-A*2601
(Fig. 2B).
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The SART-21–958 (full-length)/GFP protein
was expressed in the cytoplasm of COS7 cells that were transfected with
the SART-21–958/GFP (Fig. 5A, left side).
This protein could be expressed in ERs, Golgi apparatuses, and in part
secretory vesicles, but not on cell surface or plasma membrane, based
on the pattern of expression of conventional ER marker (calrecticulin),
Golgi marker (ß-1,4-galactosyltransferase), or mitochondrial marker
(subunit VIII of human cytochrome c oxidase) (Fig. 5, B and C) (33, 34). The similar
expression pattern, except for that in the secretory vesicles, was
observed in the MCF-7 breast cancer cells that were transfected with
the SART-21–958/GFP (Fig. 5C).
A similar, but weak and diffused expression pattern was observed in the
COS7 cells that were transfected with the deletion mutant
SART-21–932 protein, in which one of the two
C-terminal hydrophobic regions at positions 933–958 was deleted (Fig. 5A, right side). In contrast, neither a mutant
lacking the N-terminal hydrophobic region at positions 1–31 nor a
mutant lacking the two C-terminal hydrophobic regions at positions
902–958 was expressed in the COS7 cells that were transfected with
SART-232–958/GFP or
SART-21–901/GFP, respectively (data not
shown). To confirm that the SART-2 protein is not expressed on the cell
surface, we immunoprecipitated the surface-labeled
SART-21–958/GFP transfectants or the
parental untransfected 293T cells with anti-GFP mAb. Neither the
130-kDa band of SART-21–958/GFP that was
detectable by Western blot analysis (see below) nor any other band was
immunoprecipitated from the transfectants (data not shown).
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130-kDa band was recognized by both the
anti-GFP mAb and anti-SART-2 polyclonal Ab (Fig. 5
D). The size of the tagged GFP was 27 kDa. Therefore,
the size of the translated SART-2 protein in COS7 cells was expected to
be 100 kDa. Most parts of this SART-2 protein were detected in the
fractions 1, 2, and 9 when the subcellular organelles from the
SART-21–958/GFP transfectants were
fractionated velocity-controlled sucrose gradient method (Fig. 5E). The SART-21–958/GFP protein was
mainly detected in fractions 1 and 2, similar to NADPH cytochrome P450
reductase, a marker protein for ER-resident protein. It was also
detected in part in fraction 9, similar to protein kinase C µ, a
Golgi maker protein. All of these results strongly suggest that the
SART-2 protein mainly exists in ERs and also in part in Golgi
apparatuses. Expression of the SART-2 protein in cancer cells and tissues
Expression of the SART-2 protein in various cells and tissues was
investigated by Western blot analysis using anti-SART-2 polyclonal
Ab. A portion of the results is shown in Fig. 5F, and the
summary is shown in Table I. Among the
normal cells and tissues tested, including testis, spleen, and heart,
none expressed the SART-2 protein under the employed condition. In
contrast, the 100-kDa band of SART-2 was expressed in the cytoplasm of
all the SCC cell lines tested that were established from various
tissues (head and neck, esophagus, lung, and uterus). It was also
expressed in all the glioma cell lines tested, in the majority of renal
cell carcinoma and uterine adenocarcinoma cell lines, and in half of
the lung adenocarcinomas tested, but was not expressed at all in breast
cancer cell lines. In cancer tissues, it was expressed in the majority
of SCCs from various tissues (in 15 of 16 SCCs of the head and neck,
5/9 of those from the esophagus, 6/10 of those from the lung, and 18/29
of those from the uterus). This band was also expressed in the majority
of renal cell carcinoma tissues (5/9) and brain tumors (10/14 of
gliomas and 11/14 of nongliomas), and in 30–40% of melanomas (3/8),
lung adenocarcinomas (4/14), and uterine adenocarcinomas (16/46). In
contrast, it was not expressed at all in breast cancer tissues (0/16).
These results suggest that the SART-2 is a cancer-specific protein
expressed in ERs of the vast majority of SCCs from various tissues, in
the majority of other histological types of cancers, and in about
one-third of adenocarcinomas from various tissues other than
breast.
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To identify the antigenic peptides in the SART-2, we initially
investigated the ability of deletion mutants of the SART-2
gene to stimulate IFN- production by the HLA-A24-restricted and
tumor-specific CTL line. The highest level of IFN- production was
observed in the CTLs when the
SART-21–3958 (full length) and the
HLA-A*2402 were cotransfected into VA-13 cells (Fig. 6A). IFN- production was
reduced to approximately one-third to one-half of the full-length level
when the SART-21–2817 or one of the other
four mutants was used. These results suggest that the main regions
recognized by this CTL line are split into two regions, one located at
the N terminus at positions 1–130 and the other at the C terminus at
positions 891–958. We then tested the reactivity of this CTL line to
the 10 different SART-2-derived peptides with HLA-A24-binding motifs
(tyrosine at position 2, and isoleucine, phenylalanine, or leucine at
position 9), as reported previously (28). Among them, the
three peptides at positions 93–101, 161–169, and 899–907 had the
ability to stimulate the CTLs to produce IFN-. Namely, the
HLA-A24-restricted CTLs produced significant levels of IFN- through
the recognition of HLA-A*2402-transfected VA-13 cells that
had been pulsed with the SART-293–101,
SART-2161–169, or
SART-2899–907 in a dose-dependent manner (Fig. 6B). The lowest doses capable of stimulating IFN-
production were 50 nM for SART-293–101, 130 nM
for SART-2161–169, and 3.1 µM for
SART-2899–907.
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). That of HIV-envelope peptide with
HLA-A*2402 molecule-binding motif (RYLRDQQLLGI) (as a positive control)
or SART-1736–744 peptide with HLA-A*2601
molecule-binding motif (KGSGKMKTE) (as a negative control) was 27.4 or
11.2, respectively (Table II).
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C. The subline 1-8, 100-24, or
10-5 recognized the SART-293–101,
SART-2161–169, or
SART-2899–907, respectively. Induction of CTLs by the peptides
The three peptides described above were tested for their ability
to induce HLA-A24-restricted and tumor-specific CTLs from
HLA-A24+ PBMCs from the three SCC patients (two
with esophageal SCC, one with lung SCC). All of the three nonapeptides
were able to induce the CTLs in PBMCs from all of the SCC patients.
Namely, PBMCs stimulated with each of the three peptides (the
SART-293–101,
SART-2899–907, and
SART-2161–169) induced significant levels of
cytotoxicity against the HLA-A24+ KE-4 tumor
cells, but failed to lyse either HLA-A24- QG56
lung SCC cells or VA-13 fibroblast cells. Representative results are
shown in Fig. 7A. These CTL
activities were inhibited by 10 µg/ml of anti-CD8, anti-HLA
class I, or anti-HLA-A24, but not by anti-CD4, anti-HLA
class II, or isotype-matched irrelevant mAb, and representative results
are shown in Fig. 7B. PBMCs from all four
HLA-A24+ healthy donors failed to respond to any
of the three peptides (data not shown). To confirm the specificity of
these peptide-induced CTLs, the PBMCs of an
HLA-A24+ esophageal cancer patient that had been
stimulated three times by the SART-293–101,
SART-2161–169, or
SART-2899–907 were provided for a
peptide-specific CTL precursor frequency analysis (Fig. 8). The frequency of the CTL precursors
recognizing the SART-293–101,
SART-2161–169, or
SART-2899–907 peptide was 1/63, 1/631, or 1/90,
respectively. In contrast, the frequency was an undetectable level
(<1/30,000) in the PBMCs cultured without any peptide as a negative
control.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The SART-2 mRNA was expressed in all the SCC cell lines tested, but was not expressed in several adenocarcinoma cell lines tested. It was ubiquitously expressed in a panel of normal tissues with higher expression in the kidney and ovary, and lower expression in the brain, colon, and thymus. These results suggest that, in some adenocarcinoma cell lines, the expression of SART-2 mRNA was too weak for detection by Northern blotting. To clarify this issue, we attempted to detect the mRNA expression using RT-PCR with the SART-2-specific primers. SART-2 mRNA expression was undetectable in the two SART-2 mRNA-negative adenocarcinoma cell lines (COLO201 and MCF-7) when 20 cycles of the RT-PCR were employed, but it became detectable when 25 cycles of the RT-PCR were employed (data not shown). The expression was strongly positive in KE-4 tumor cells used as a positive control under the same conditions. These results suggest that, in some adenocarcinoma cell lines, the SART-2 is not deleted, but only weakly expressed. This tendency might be due in part to the lower expression of SART-2 mRNA in some normal tissues, including those of the normal colon, although the detailed mechanisms involved in this issue are not clear at present.
The 100 kDa of SART-2 protein was detectable in the cytoplasm of all the SCC cell lines without exception, in the majority of SCC tissues and the other histological types of cancers, and also in about one-third of adenocarcinomas. In contrast, the SART-2 protein was not detectable at all under the employed conditions in any of the normal cells or normal tissues tested, including PHA-blastoid cells and testis and fetal liver in which MAGE proteins and the other cancer-testis Ags as well as SART-1 and SART-3 proteins were detectable (1, 2, 13, 14, 15). We have reported previously that the other SCC Ag, SART-1, is not detectable at the protein level in any normal tissues or cells, with the exception of the testis and fetal liver, regardless of ubiquity of expression at the mRNA level (14). Reasons for this discrepancy are not presently known. Posttranscriptional regulation might be responsible for this discrepancy, although its mechanisms in human mRNAs are not well understood at the present time. The SART-2 gene could be a novel tool for exploring this mechanism, in part because its genomic structure has already been reported (32).
Regardless of these unsolved issues, including identification of its biological roles, the SART-2 Ag might be an ideal target molecule for use in specific immunotherapy of relatively large numbers of cancer patients, because it may be a cancer-specific protein expressed in the ERs of the vast majority of SCCs from various tissues, in the majority of other histological types of cancers, and also in about one-third of adenocarcinomas from various organs other than the breast. In contrast, the other tumor-rejection Ags, including Ags of the MAGE family (1, 2), melanoma-related Ags (3, 4, 5, 6, 7, 8), prostate-specific Ag (39, 40), HER-2/neu (41, 42), SART-1 (14), SART-3 (15), and cyclophilin B (37), were expressed in both cancer cells and certain normal cells with preferential expression in cancer cells. Although the mutated Ags and peptides recognized by the CTLs, including mutant CDK4 (43) and mutant CASP-8 (6), were cancer-specific Ags, these mutated products are usually observed in only a small part of tumor samples, and therefore may not be applicable as target molecules for use in specific immunotherapy of a large number of cancer patients.
Three different peptides from the SART-2 with HLA-A24-binding motifs
were recognized in a dose-dependent manner by the HLA-A24-restricted
and tumor-specific CTLs used in this study. The relative affinities of
these peptides are more modest than the several other SART-2 peptides
with HLA-A24-binding motif. These results are consistent with those
from the others (44). Peptides with intermediate affinity,
but not with high affinity, seem to be recognized by HLA-A2-restricted
melanoma-specific CTLs (44). Three of these peptides,
SART-293–101,
SART-2161–169, and
SART-2899–907, possessed the ability to induce
CTLs in vitro in PBMCs from SCC patients. The
SART-293–101 and
SART-2161–169 peptides are located in
the N terminus, whereas the remaining
SART-2899–907 is located in the C terminus.
These results are mostly consistent with the data from experiments on
deletion mutants, suggesting that the main antigenic regions are split
into the N terminus at positions 1–130 and the C terminus at positions
891–958. The peptide-induced CTLs from PBMCs of cancer patients showed
the HLA-A24-restricted and peptide-specific CTL activity against cancer
cells, but not against normal cells. The HLA-A24 allele is found in
60% of Japanese (the majority (95%) are genotypically HLA-A*2402),
20% of Caucasians, and 12% of Africans (45, 46).
Therefore, these peptides could be useful as a cancer vaccine in
specific immunotherapy for relatively large numbers of
HLA-A24+ cancer patients with SCC and the other
histological types of cancers.
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Acknowledgments |
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for ELISA; Dr. Masafumi Takiguchi of Kumamoto University for kindly
providing C1R-A*2402 cells to the study; and Mr. Hideo Takasu of
Sumitomo Pharmaceutical Company for providing the
RMA-S-A*2402/Kb cells. |
Footnotes |
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2 Address correspondence and reprint requests to Dr. Masanobu Nakao, Department of Immunology, Kurume University School of Medicine, 67 Asahi-machi, Kurume, Fukuoka 830-0011, Japan. E-mail address:
3 Abbreviations used in this paper: SCC, squamous cell carcinoma; ER, endoplasmic reticulum; EYFP, enhanced yellow fluorescent protein; GFP, green fluorescence protein; SART, squamous cell carcinoma Ag recognized by T cells; TIU, trypsin-inhibitory unit.
Received for publication September 14, 1999. Accepted for publication December 13, 1999.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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