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Immunology |
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
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ABSTRACT |
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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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60% of the
Japanese 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 adenocarcinoma and have reported a new CTL-directed tumor antigen
residing in the
ER.3 |
MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Identification of the ART-4 Gene.
The expression-gene cloning method was used to identify a
gene-encoding tumor rejection antigens recognized by the CTL line,
GK-CTL, as reported elsewhere (15)
. Briefly, cDNA derived
from the HT1376 bladder carcinoma cells was ligated to
SalI/NotI site of the expression vector
pSV-SPORT-1 (Life Technologies, Inc., Gaithersburg, MD). A
total of 1 x 105 clones from the
cDNA library were screened, and two positive pools (6A1 and 4E6, each
containing 100 clones) obtained from the first screening were
subdivided for further screening. 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
the 4E6 were analyzed for their bioactivity to stimulate
IFN-
production by the GK-CTLs at the second and third screening,
and one positive (4E6-2B9) and one negative (4E6-6F2) clone were
provided for this study. DNA sequencing was performed by the
dideoxynucleotide sequencing method using an AutoRead Sequencing kit
(Pharmacia Biotech, Uppsala, Sweden) and analyzed using an ALF express
DNA Sequencer (Pharmacia Biotech). Full-length cDNA clones were
obtained from an HT1376 and a PBMC cDNA library (SuperScript Human
Leukocyte cDNA library in pCMV-SPORT; Life Technologies) by
the standard colony hybridization method with
32P-labeled cDNA probe (clone 4E6-2B9) as
reported previously (13)
. We tentatively designated this
gene an ART-4 gene. The difference of the nt sequence at
position 758 of ART-4 cDNA between PBMCs and HT1376 cells
was confirmed by both repeated nt sequencing and susceptibility of PCR
products against AciI restriction enzyme digestion.
Amplification was performed for 30 cycles using the primers
5'-ATCCAAGCAGATCCAG CAGG-3' (sense) and 5'-AGTGTGAGCAGAACACTCGG-3'
(antisense).
Northern Blot Analysis.
Preparation of RNA, transfer to nylon membranes, and Northern
hybridization have been described elsewhere (13)
. A
32P-labeled 600-bp fragment of NcoI
cut ART-4 cDNA was used as a probe. The membranes were
washed and then autoradiographed. The relative expression of the
ART-4 mRNA was calculated with the following formula:
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Chromosome Mapping.
A genomic DNA panel of hybrids with 1000-kb resolution made from hybrid
cells of irradiated human HFL cells and hamster A23 cells (Gene bridge
4 Radiation Hybrid Panel; Research Genetics, Huntsville, AL; Ref.
21
) was used as a template for PCR. A 768-bp fragment of
the ART-4 gene was amplified by PCR from the panel. PCR
amplifications were performed with a sense primer, F2 (111–130;
5'-ATCCAAGTGCTTGCACTCACA-3'), and an antisense primer, 4R878 (859–878;
5'-AGTGTGAGCAGAACACTCGG-3'), under the following conditions: denaturing
at 95°C for 1 min, annealing at 58°C for 1 min, and extension at
72°C for 1 min for 35 cycles. The PCR products were subsequently
dot-blotted on a nitrocellulose filter and hybridized with the
32P-end-labeled sequence-specific oligonucleotide
probe R3 (452–471; 5'-AGGTTCTCAGGCTCACAAGC-3'), which specifically
hybridized with the human ART-4 gene but not with any
hamster-derived genes. After washing, the filter was subjected to
autoradiography as described previously (22)
. The results
obtained were analyzed using the radiation hybrid map software of the
Whitehead Institute/Massachusetts Institute of Technology Center for
Genome Research.
Preparation of Fusion Protein and Rabbit Antisera.
For preparation of the ART-4/GST protein, the BamHI site was
introduced to the 5'-terminal of the ART-4 gene by a linker
PCR. The primer pair used for the PCR was as follows: sense
BamHI primer 5'-CTCACGGATCCAACATGGCTCC-3' and antisense
primer 5'-AATCGATGAGCTCACCTTAACCTT-3'. The amplified fragment (nt
3–364) was digested with BamHI and SacI and
ligated to a SacI/NotI fragment of
pSVSPORT/ART-4. Subsequently, the ligated fragment of the
ART-4 gene containing nt positions 3–1733 was cloned into
the BamHI/NotI site of the pGEX-4T-2
vector (Pharmacia Biotech). In this construct, the GST gene
flanked the 5' terminal of the ART-4 gene. The nt sequence
of the construct was confirmed by sequencing. Purification of the
ART-4/GST fusion protein from the bacterial lysate was performed
according to the manufacturer’s procedure. Polyclonal anti-ART-4/GST
Ab was prepared by immunization of rabbits with purified ART-4/GST
protein by the methods reported previously (13
, 23)
.
ART-4/myc and ART-4/GFP Constructs.
For preparation of the ART-4/myc, the XbaI site
was introduced to the 5' flanking region of the stop codon by PCR. The
primer pair used for the PCR was as follows: sense primer
5'-CGCTGCCATGGCTGTTT-3' and antisense XbaI primer
5'-TGCGGGAACTCGCTCTAGAC-3'. The amplified fragment (nt 821-1256) was
digested with XbaI and NcoI and ligated to a
KpnI/NcoI fragment of pSVSPORT-ART-4.
Subsequently, the ligated fragment of the ART-4 gene
containing nt positions 1–1251 was cloned into the
KpnI/XbaI site of the pcDNA3.1/Myc-His
vector (Invitrogen, Carlsbad, CA). A myc gene flanked the 3'
terminal of the ART-4 gene as a tag (ART-4/myc).
To prepare the ART-4/GFP protein, the SalI site was
introduced to the 5' flanking region of the stop codon by PCR. The
primer pair used for the PCR was as follows: sense primer
5'-TCGAATTCCACGCAGCCAA-3' and antisense SalI primer
5'-AACTCGGTCGACACCTTTTCTTCAC-3'. The amplified product (nt 1–1267) was
digested with EcoRI and SalI and cloned into an
EcoRI/SalI site of pEGFP-N2 vector
(Clontech, Palo Alto, CA). A GFP gene flanked the 3'
terminal of ART-4 gene as a tag (ART-4/GFP).
Western Blot Analysis.
Preparation of cytosol and nuclear fractions of tissues or cells have
been described (23)
. Obtained fractions were separated by
SDS-PAGE, blotted to a polyvinylidene difluoride membrane, and
subjected for the Western blot. Precise methods of the Western blot
analysis have been described previously (23)
.
Assays for Cellular Localization.
Intracellular localization of the ART-4/GFP fusion protein in the
transfectants was analyzed as follows. COS7 cells were transfected with
the ART-4/GFP, followed by serial observation under a Zeiss
confocal Ar-Kr laser scanning microscope with both fluorescence and
visible rays or fluorescence only. Localization of the ART-4/GFP
protein was recorded under an FITC filter (520 nm). The exposure
sequences and imaging were controlled by LSM version 3.70 imaging
software. The visible-ray image was studied by differential
interference microscopy. COS7 cells transfected with Living Colors
subcellular localization vectors pEYFP-ER,
pEYFP-Golgi, or pEYFP-Mito (Clontech) were also
analyzed to obtain representative fluorescence patterns of ER, Golgi,
or mitochondria-resident proteins, respectively. The
pEYFP-ER vector encodes a fusion protein consisting of EYFP,
the ER targeting sequence of calreticulin, and the ER retrieval
sequence, KDEL. pEYFP-Golgi encodes a fusion protein
consisting of EYFP and a sequence of the
NH2-terminal 81 amino acids of human
ß1,4-galactosyltransferase to sort the fusion protein to the Golgi
apparatus. pEYFP-Mito encodes a fusion protein consisting of
EYFP and the mitochondrial targeting sequence from subunit VIII of
human cytochrome c oxidase.
Subcellular organelles were fractionated by velocity-controlled sucrose gradient fractionation (24 , 25) . In brief, 5 x 107 COS7 cells transfected with the ART-4/myc gene were washed with ice-cold STE buffer [0.25 M sucrose, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 20 mM Tris-HCl (pH 8.0), and 1 mM EDTA]. Cells were suspended in a hypotonic buffer (0.025 M sucrose-STE), incubated for 30 min on ice, and homogenized with a 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 (284,000 x 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 1 and 2, 0.6 M; number 3, interface between 0.6 and 1 M; numbers 4 and 5, 1 M; number 6, interface between 1 and 1.3 M; numbers 7 an 8, 1.3 M; number 9, interface between 1.3 and 2 M; and number 10, 2 M. Each fraction was mixed with SDS-sample buffer and subjected to Western blot analysis. For detection of ER and Golgi marker proteins, rabbit anti-NADPH cytochrome P450 reductase (StressGen, Victoria, British Columbia, Canada) and anti-protein kinase C µ (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 from the deduced amino
acid sequence of ART-4 (purity was >70%) with binding motifs for
HLA-A2402 molecules in the literature (28)
, including
motifs of tyrosine or phenylalanine at position 2, and of isoleucine,
leucine, phenylalanine, or tryptophan at position 9, were obtained from
Biologicala (Nagoya, Japan). An HIV-derived peptide (RYPLTFGWCF)
capable of binding to HLA-A24 molecules was used as a negative control
(15)
. Peptides of >95% purity were used for experiments
of dose dependency and CTL induction. The estimated score of half time
of dissociation of each ART-4 peptide for HLA-A24 molecules was
calculated based on a computer search for HLA peptide 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 the analysis
because it is an 8-mer peptide. For detection of antigenic peptides
recognized by the GK-CTLs, COS7 cells (2 x 104) were transfected with HLA-A2402
or control HLA-A2601 cDNA. Two days after the transfection,
peptides at a concentration of 10 µM, unless
stated otherwise, were added to the culture. Two h later, the
supernatant was removed, and the GK-CTLs (1 x 105) were added to the culture and incubated for
an additional 18 h, and the concentration of IFN- in the
culture supernatants was measured by ELISA (limit of sensitivity, 10
pg/ml) in a duplicate assay. The surface phenotype of the CTL line and
sublines was investigated by an immunofluorescence assay with
FITC-conjugated anti-CD3, anti-CD4, or anti-CD8 mAb (15)
.
For inhibition of CTL activity, 10 µg/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 as an isotype-matched
control mAb. Two-tailed Student’s t test was used for the
statistical analysis in this study.
CTL Induction by Peptides.
PBMCs (1 x 106 per well) from
HLA-A2402+ healthy donors or cancer patients were
incubated with 10 µM of the peptide in a 24-well plate in
the presence of 100 units/ml interleukin 2 as reported
previously (15)
. At days 7 and 14 of culture, the cells
were restimulated 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 (10 µM) for 2 h. Responder
cells were harvested at day 21 of the culture and were further cultured
in a 96-well U-bottomed microculture plate in the presence of
irradiated autologous PBMCs (2 x 106 cells/well) as APCs that had been pulsed with
a corresponding peptide. Seven to 10 days later, the expanded cells
were transferred to a 24-well plate and cultured for additional 14–25
days with interleukin 2 alone. The cytotoxic activity was measured by a
standard 6-h 51Cr-release assay at different E:T
ratios, as reported previously (15)
.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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production by the GK-CTLs after
cotransfection with HLA-A2402 into the COS-7 cells. After
repeated cycles of the screening, one clone (4E6-2B9) was
confirmed to encode a tumor antigen recognized by the GK-CTLs when
cotransfected with HLA-A2402 but not when cotransfected with
control HLA-A2601 (Fig. 1)
. The 4E6-6F2 clone was served as a negative control in this
experiment.
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1.8 Kbp in size was observed in each of the 21 different
tumor cell lines tested, including 3 lung adenocarcinomas and 2 lung
squamous cell carcinomas, and in all of the normal tissues tested
(heart, brain, placenta, lung, liver, skeletal muscle, kidney,
pancreas, spleen, thymus, prostate, testis, ovary, small intestine, and
colon). Representative results are shown in Fig. 2A
. The relative level of mRNA expression ranged from 0.90 to
1.24 in tumor cell lines and from 0.78 to 4.40 in normal tissues, when
the levels of the expression in the KE4 esophageal cancer cells were
taken as 1.0 (Fig. 2A)
. In the normal tissues, relatively
high levels of expression were observed in the pancreas (4.4), kidney
(2.8), and ovary (2.4), and low level expression was seen in skeletal
muscle (0.6) and PBMCs (0.7). These results suggest that this gene is
expressed ubiquitously at the mRNA level, and that the clone 4E6-2B9
with a length of 1584-bp might be a truncated form.
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. This gene
was tentatively designated ART-4. The sequence of
ART-4 cloned from HT1376 cDNA is available from
EMBL/GenBank/DDBJ (accession number AB026125). The nt sequences of
these clones were identical with the exception of the position 708
(guanine in HT1376 versus adenine in the PBMCs). This
difference is attributable to a genetic polymorphism rather than to a
point mutation, because the position 708 was a guanine in the HT1376,
RMG I, HSC-4, and LC-1 cells but an adenine in the PBMCs, MKN28, and
86-2 cells. PBMCs from another healthy donor and a tissue sample of the
testis contained both guanine and adenine at position 708. The amino
acids translated from these codons were different (arginine or
glutamine). This gene showed 99% similarity at the nucleotide level
with the previously reported gene HP-10 (30)
isolated from placental cDNA and having an unknown function
(30)
. Despite the high homology between the
ART-4 and HP-10 gene at the nt levels, the
homology between the two genes at the deduced amino acids level was
only 50%. This discrepancy was mainly attributable to the
difference of start positions for the translation and the subsequent
frame shift of the codons. We next attempted to clone the
HP-10 from a human placental cDNA library using PCR methods;
however, the nt sequences of all 20 clones obtained from PCR products
were identical with that of ART-4. Furthermore, the
susceptibility of these clones to several restriction enzymes that
distinguish the differences between ART-4 and
HP-10 verified the sequencing results (data not shown).
Therefore, we could not confirm the presence of HP-10 in
human samples under the used conditions.
The deduced amino acid sequence of the ART-4 gene is shown
in Fig. 2C
. Hydrophobicity analysis showed that a protein
encoded by the 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 and 401–407 and a
di-lysine (KKXX) motif-like ER membrane retention signal (FVKK) at the
COOH terminus positions 408–411 (Fig. 2C)
. The ability of
the ART-4 gene to stimulate IFN- production by the
GK-CTLs was confirmed (Fig. 2E)
, i.e., the
ART-4 gene stimulated IFN- production by the CTLs in a
dose-dependent manner when COS7 cells transfected with both the
ART-4 and HLA-A2402 gene were used as stimulator
cells.
Chromosome mapping of the ART-4 gene was performed by the radiation hybrid mapping method as reported elsewhere (21) . A genomic DNA panel of hybrids with 1000-kb resolution made from hybrid cells of irradiated human HFL cells and hamster A23 cells was used as a template for PCR. The PCR with the primer pair F2 and 4R878 amplified the 768-bp fragment from genomic DNA isolated from PBMCs (data not shown). The PCR product did not contain an intron. A faint band corresponding to a PCR product of the same size was also amplified from hamster A23-derived genomic DNA under the same conditions. Therefore, the PCR products from the radiation hybrid panel were dot-blotted and subsequently hybridized with the human ART-4 sequence-specific oligonucleotide probe R3. This probe specifically hybridized with the PCR product from the human HFL cell-derived genomic DNA but not with the hamster A23-derived genomic DNA (data not shown). Database analysis for the radiation hybrid mapping indicated that the ART-4 gene was located on the chromosome 4, 2.12 centirays distal from the AFM350VH9 marker gene, compatible with the classical 4q31.22.
Expression of the ART-4 Protein.
Expression of ART-4 at the protein level in various cells and tissues
was examined by Western blot analysis using rabbit anti-ART-4/GST
polyclonal Ab. This method visualized an
Mr 46,000 band of a recombinant ART-4
protein after cleavage with thrombin (data not shown). When the
ART-4 gene was transfected to COS7 cells, an intensive
Mr 46,000 band was observed in both
the nuclear and cytosol fractions (Fig. 3A)
. Furthermore, both the polyclonal anti-ART-4/GST Ab and
anti-myc mAb recognized an Mr 51,000
band in the nuclear and cytosol fractions of COS7 cells transfected
with the ART-4/myc gene. The difference in migration between
these bands (Mr 46,000 and 51,000) was
probably attributable to the myc-tag peptide
(Mr 5,000). The
Mr 46,000 band was detected in both
the cytosol and nucleus of all of the tested tumor cell lines
established from various organs, including the lung
(n = 10), esophagus (n = 3), stomach (n = 3), head and neck
(n = 4), uterus (n = 8), ovary (n = 8), and breast
(n = 6). It was also detectable in leukemia
cell lines, with the exception of the three cell lines (Jurkat, MOLT-4,
and RPMI8402) from T-cell leukemia. This band was also detectable in
both the cytosol and nucleus of the majority of cancer tissues from
various organs tested, including the lung (n = 18), head and neck (n = 9), stomach
(n = 8), uterus (n = 16), and breast (n = 8). In contrast,
an Mr 46,000 protein of ART-4 was not
detectable in either the cytosol or nuclear fraction of any 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
.
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. On the basis of a comparison of the fluorescence
distribution pattern of ART-4/GFP to that of control EYFP-fusion
proteins (EYFP-ER, EYFP-Golgi, and EYFP-Mito), the ART-4/GFP protein in
the cytosol might have been expressed at the ERs, but it would not have
been expressed on the Golgi apparatuses or the mitochondrias.
Furthermore, this ART-4 protein was detected in the fractions 1, 2, 7,
and 9 when the subcellular organelles from the ART-4/myc
transfectants were fractionated by the velocity-controlled sucrose
gradient method (Fig. 3D)
. The distribution pattern of this
protein was very similar to that of NADPH cytochrome P450 reductase, a
marker protein for ER-resident protein, but differed largely from that
of protein kinase Cµ, a Golgi marker protein. These results
together with the presence of an ER membrane retention signal at the
COOH terminus at positions 408–411 strongly suggest that the ART-4
protein is an ER-resident protein.
Antigenic Peptides Recognized by the CTLs.
Each of the 18 different ART-4-derived synthetic peptides with motifs
of binding to HLA-A2402 molecules was loaded onto the
HLA-A2402-transfected COS7 cells at a concentration of 10
µM and then tested for its ability to induce
IFN- production by the GK-CTLs. Two of these peptides,
ART-413–20 and
ART-475–84, stimulated significant levels of
IFN- production in a dose-dependent manner (Fig. 4, A and B)
. Similar dose-dependent effects of the
two peptides were also observed when the peptides were loaded onto
irradiated autologous PBMCs or C1R-A2402 cells (data not shown). The
other 16 peptides failed to stimulate IFN- production by the CTLs.
To confirm the presence of peptide-specific CTLs, the sublines were
established from the parental GK-CTL line by a limiting dilution
culture at 1 or 10 cells/well, and their peptide specificities were
tested. Among the 68 different sublines tested, 7 sublines recognized
C1R-A2402 cells pulsed with ART-413–20 but not
with either ART-475–84 or HIV-derived peptide as
a negative control. The CTL activity of those 7 sublines was inhibited
by 10 µg/ml of anti-A24, anti-class I HLA (W6/32) or anti-CD8 mAb but
not by anti-class II (H-DR-1), anti-CD4, or isotype-matched irrelevant
control mAbs (anti-CD13 and anti-CD14). Representative results for one
subline, #1-2, are shown in Fig. 4C
. The other 6 sublines
recognized C1R-A2402 cells pulsed with
ART-475–84 but not those pulsed with the other
peptides, and their CTL activity was inhibited by anti-HLA-A24,
anti-class I HLA (W6/32), or anti-CD8 mAb but not by the other mAbs.
Representative results for one subline, #10-1, are shown in Fig. 4D
. The remaining 55 sublines failed to respond to any
peptides tested (data not shown).
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. In all three cases, the PBMCs failed to lyse
HLA-A24+ PHA-blasts, VA13 fibroblast cells, or
K562 tumor cells. These PBMCs possessed 20–60% of
CD3+CD4-CD8+
cells, and the remaining cells were mostly
CD3+CD4+CD8-
cells at the time of assays (data not shown). To confirm the peptide
specificity, sublines derived from the patient 1 shown in Fig. 5A
were further established from the peptide-induced PBMCs,
followed by a test of their reactivity to
ART-413–20, ART-475–84,
or HIV peptide. Three of 69 sublines tested from the PBMCs stimulated
with the ART-413–20 responded to the
C1R-HLA-A2402 cells pulsed with ART-413–20.
IFN- production by these sublines was inhibited by 10 µg/ml of
anti-HLA-A24, anti-class I HLA (W6/32), or anti-CD8 mAb but not by
anti-class II (H-DR-1), anti-CD4, or irrelevant control mAbs (anti-CD13
and anti-CD14). The results for one subline,
#1-10-9, are shown in Fig. 5B
. On the other hand, 2 of 17 sublines tested from the
PBMCs stimulated with the ART-475–84 responded
to C1R-HLA-A2402 cells pulsed with ART-475–84
peptide, and the CTL activity of these sublines was inhibited by 10
µg/ml of anti-HLA-A24, anti-class I HLA (W6/32), or anti-CD8 mAb but
not by the other mAbs. The results for one subline,
#6-10-7, are shown in Fig. 5C
. In the remaining two cancer patients, HLA-A24-restricted
CTL activity was not induced in the PBMCs stimulated with either
peptide. In these PBMCs, the percentages of
CD3+CD4-CD8+
cells were very low (<8%). HLA-A24-restricted CTL activity was also
undetectable in the PBMCs of all healthy donors (n = 5) after stimulation with either peptide (data not shown).
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DISCUSSION |
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There are several motifs in the sequence of the ART-4 protein: a nuclear 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 the COOH terminus. The KKXX motif has been found previously at the COOH terminus of several ER-resident types I and III membrane proteins and shown to be responsible for the localization to the ERs (31) . Membrane-topology prediction suggests that the ART-4 is a type Ib membrane protein. Results of the present laser-confocal microscopic analysis of the GFP-tagged ART-4 transfectants suggest that the ART-4 protein was localized at the ER and in the nucleus but not at the Golgi or on the plasma membrane. The localization of ART-4 at the ER was further confirmed by the subcellular organelle fractionation of a tagged ART-4 protein in the transfectants. All of the results suggest that the ART-4 is an ER-resident protein encoding tumor-rejection antigen recognized by the HLA-A24-restricted CTLs. We have identified recently cyclophilin B as the tumor-rejection antigen recognized by the GK-CTLs (15) , and this molecule is also known as an ER-resident protein involved in the signal transduction of cellular proliferation (32) . ER is well known as a major site of peptide loading to HLA molecules (33 , 34) . Therefore, it might be interesting to study the relationship between CTL-directed epitopes and ER-resident proteins. However, currently identified functional domains involved in the signal transduction of cellular proliferation, such as kinase domains or src homologous domains, are not found in the deduced ART-4 sequence. Therefore, we were unable to hypothesize any biological functions of this newly identified ER-resident protein, and further studies on the function of the ART-4 will be needed.
Under the present conditions, the ART-4 protein was not detectable by Western blot analysis with polyclonal anti-ART-4/GST Ab in any of the normal tissues, except for the testis, fetal liver, and placenta, despite the ubiquitous expression of the ART-4 gene at the mRNA level. We have reported similar expression patterns for the SART-1 (13) and SART-3 (14) proteins, but the mechanisms involved in the discrepancy between the mRNA and protein expression are presently unclear.
Among the 18 peptides with HLA-A24 antigen-binding motifs tested, the two ART-4-derived peptides, ART-413–20 and ART-475–84, were consistently recognized by the HLA-A24-restricted CTLs in repeated experiments. Because of the presence of each of the peptide-specific CTL sublines, the parental GK-CTL line would consist of a mixture of these peptide-specific CTL clones. A dose dependency was observed in the two peptides, and >0.01 µM of each the peptides were required for the recognition of target cells by the CTLs. Both peptides were able to induce HLA-A24-restricted CTLs from PBMCs of the HLA-A24+ lung cancer patients but not from those of the HLA-A24+ healthy donors. These results suggest that there is a preferential presence of the CTL precursors reacting to the ART-4 epitopes in PBMCs of these cancer patients. In contrast, the CTL precursors may not exist in PBMCs of healthy donors.
Regardless of the numerous unsolved issues, including identification of its biological roles, the ART-4 antigen might be an ideal target molecule for use in specific immunotherapy of relatively large numbers of cancer patients, because it could be a cancer-specific protein expressed in the cytosol (ER) of the vast majority of cancers from various 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 cancer cells with different histological types. Although the mutated antigens, including mutant CDK4 (35) and mutant CASP 8 (36) , are recognized by CTLs, these types of antigens are expressed only in certain cancer cells, and the mutated products are usually detected in only a small part of tumor samples. Thus, these molecules would not be applicable as targets in specific immunotherapy of a large number of cancer patients.
The HLA-A24 allele is found in 60% of Japanese (with 95% of these cases being genotypically A2402), in 20% of Caucasians, and in 12% of Africans (18) . The two ART-4-derived peptides were able to induce HLA-A24-restricted and tumor-specific CTLs in PBMCs of lung cancer patients. These ART-4 peptides might be appropriate molecules for use in the specific immunotherapy of HLA-A24+ patients with lung and other cancers.
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FOOTNOTES |
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1 Supported in part by Grants-in-Aid 08266266,
09470271, 10153265, 09770985, and 09671401 from the Ministry of
Education, Science, Sports and Culture of Japan and by Grant
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: akiymd{at}med.kurume-u.ac.jp
3 The abbreviations used are: ER, endoplasmic
reticulum; ART-4, adenocarcinoma antigen recognized by T cells 4; GST,
glutathione S-transferase; Ab, antibody; mAb, monoclonal
Ab; EYFP, enhanced yellow fluorescent protein; GFP, green fluorescent
protein; GST, glutathione S-transferase; nt, nucleotide;
PBMC, peripheral blood mononuclear cell; APC, antigen-presenting
cell.
Received 12/28/99. Accepted 5/ 3/00.
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), HLA-A24+ PHA-blasts (
), HLA-A24- lung
cancer cells (QG56, 
), or VA13 fibroblast cells (
) was measured
by a 6-h 51Cr release assay at different E:T ratios. Values
represent the means of triplicate assays. B and
C, CTL sublines were established from the PBMCs
stimulated with the ART-413–20 or ART-475–84
peptide by a limiting dilution culture, and these sublines were tested
for their reactivity to C1R-A2402 cells pulsed with
ART-413–20 peptide, ART-475–84 peptide, or
HIV peptide (as a negative control) at a responder:stimulator ratio of
2. The CTL activities of these sublines in response to 11-18 tumor
cells were also measured at a responder:stimulator ratio of 4 in the
presence of 10 µg/ml of various mAbs. Values represent the mean
IFN-