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Immunology |
Department of Immunology [S. N., K. H., K. I., S. S.], Cancer Vaccine Development Division, Kurume University Research Center for Innovative Cancer Therapy [K. I.], and the First Department of Internal Medicine [S. N., K. O.], Kurume University School of Medicine, Kurume 830-0011, Japan, and the First Department of Surgery, Okayama University School of Medicine [S. G.], Okayama 700-9559, 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 (22)
. We reported
previously that HLA-A24-restricted CTLs (GK-CTLs) established from T
cells infiltrating into lung adenocarcinoma recognized the cyclophilin
B peptides (23)
. In this study, we also investigated other
antigens recognized by the GK-CTLs and report a new gene coding for
CTL-directed antigenic epitopes. |
MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cloning of a Gene.
An HLA-A24-restricted and tumor-specific CTL (GK-CTL) line was used in
this study as a source of effector cells to detect tumor antigens. This
line was established from TILs of a patient with lung adenocarcinoma
(HLA-A*0206/A*2402, B39/B52, Cw7/) by incubation of TILs with
interleukin 2 alone for >60 days. Its characteristics have been
reported elsewhere (23)
. Methods for identification of a
gene coding for the tumor antigen recognized by CTLs were also reported
previously (23)
. In brief, poly(A)+
RNA of the HT1376 bladder carcinoma cells was converted to cDNA,
ligated to SalI adapter, and inserted into the expression
vector pSV-SPORT-1 (Life Technologies, Inc., Rockville, MD). Both 200
ng of plasmid DNA pools or clones of the HT1376 cDNA library and 200 ng
of the HLA-A*2402 or HLA-A*2601 (as a negative
control) cDNA were mixed with 1 µl of LipofectAMINE reagent (Life
Technologies, Inc.) in 100 µl of Opti-MEM (Life Technologies, Inc.)
for 15 min. Fifty µl of the mixture were then added to the COS-7
cells and incubated for 5 h, and then 200 µl of RPMI 1640
containing 10% FCS were added and cultured for 2 days, followed by
incubation with the CTLs. After an 18-h incubation, 100 µl of
supernatant were collected to measure IFN-
by an ELISA (Ref.
3
; limiting sensitivity, 10 pg/ml). 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 (23)
. Anti-CD14 (JML-H14, IgG2a) or anti-CD13
(MCS-2, IgG1) mAb was used as an isotype-matched control mAb.
Two-tailed Student’s t test was used for the statistical
analysis in this study.
A cDNA library derived from HT1376 cells and containing a total of 1 x 105 clones was screened, and two positive clones (6A1-3D9 and 6A1-4F2) were selected for additional analysis. This report describes the results for the 6A1-3D9 gene. A 6A1-4A9 gene was used as a negative control. The result for the 6A1-4F2 gene has been reported elsewhere (23) . The mRNA expression of a 6A1-3D9 gene on various tumor cells and tissues was investigated by Northern blot analysis with a 32P-labeled 6A1-3D9 as a probe, with a ß-actin probe as control by the methods reported previously (23) . The relative expression of the ART-1 mRNA was calculated with the following formula: index = (6A1-3D9 density of a sample/ß-actin density of a sample) x (ß-actin density of the unstimulated PBMCs/6A1-3D9 density of the unstimulated PBMCs). Mann-Whitney U test was used for the statistical analysis.
The full-length of the 6A1-3D9 clone was obtained from both a PBMC cDNA library (SuperScript Human Leukocyte cDNA library in pCMV-SPORT; Life Technologies, Inc.) and an HT1376 cDNA library by the colony hybridization method with 32P-labeled 6A1-3D9 cDNA. DNA sequencing was performed by the dideoxynucleotide sequencing method using a DNA Sequencing kit (Perkin-Elmer Corp., Foster, CA), and the sequence was analyzed by the ABI PRISM 377 DNA Sequencer (Perkin-Elmer). Then the full-length 6A1-3D9 gene was tentatively designated ART-1 (adenocarcinoma antigen recognized by T cell-1).
Subcellular Localization Analysis.
For preparation of the ART-1/GFP fusion gene, a fragment of
positions 1–1324 of ART-1 was obtained by digestion of
ART-1/pCMV-SPORT with EcoRI and
HindIII. The fragment of positions 1219–1622 of
ART-1 was amplified by PCR using a forward primer
5'-TGTCAGTGAGGAGCTGGAGG-3' and a reverse primer
5'-CTCCCTCTGTCGACTTTGTATTTTCC-3', followed by digestion with
SalI and HindIII. These two fragments were
ligated into a pEGFP-N2 vector (Clontech, Palo Alto, CA), which was
digested by EcoRI and SalI. The sequence of the
PCR-amplified region was checked. The ART-1/GFP gene or the
pEGFP-N2 vector alone as a control was transfected to 293T cells
(SV-40-transformed human embryonic kidney cells). The
paraformaldehyde-fixed samples were observed under a confocal Ar-Kr LSM
(Carl Zeiss, Oberkochen, Germany). Localization of the ART-1/GFP
protein was recorded under an FITC filter (520 nm). The exposure
sequences and imaging were controlled by LSM imaging software (version
3.7; Carl Zeiss). Western blot analysis was used to detect estimated
molecules of the ART-1 protein.
The ART-1-transfected 293T cells were lysed with RIPA buffer (20 mM Tris-HCl, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS, 2 mM EDTA, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 1 µM phenylmethylsulfonyl fluoride), rotated for 30 min at 4°C, and centrifuged at 15,000 rpm for 30 min. The supernatant was separated by SDS-PAGE. The proteins in acrylamide gel were blotted to a Hybond-poly(vinylidene difluoride) membrane (Amersham Pharmacia, Buckinghamshire, United Kingdom) and were incubated with anti-GFP polyclonal antibody (Clontech) for 3 h at room temperature. The other methods used for the Western blot analysis were described previously (19) .
Peptide and CTL Assay.
The literature was searched for ART-1-derived peptides capable of
binding to the HLA-A*2402 molecules (24)
, resulting in the
16 different ART-1 peptides used in this study. These peptides were
kindly provided by Dr. Kanaoka (Sumitomo Pharmaceutical, Osaka, Japan),
and their purity was >95%. Among them, the three peptides at
positions 158–165, 170–179, and 188–196
(ART-1158–165,
ART-1170–179, and
ART-1188–196, respectively), which were
recognized by the CTLs, were also evaluated for their binding activity
to HLA-A24 molecules. For the peptide-binding assay, a chimera gene
A*2402/Kb encoding for the 
1 and 2
domains of the HLA-A*2402 molecule and for the 3 transmembrane and
intracellular domains of the H-2Kb molecule was
established. Exons 1–3 of HLA-A*2402 cDNA and exons 4–8 of
H-2Kb cDNA were ligated into
pcDNA3.1 (Invitrogen, CH Groningen, the Netherlands). This gene was
then transfected into RMA-S cells (mouse lymphoma cell line). These
cells expressed HLA-A*2402 molecules that were detectable with
anti-HLA-A23/A24 mAb (One Lambda, Canoga Park, CA). 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, Inc.) containing 3 µg/ml human
ß2-microglobulin (Cortex Biochem, San Leandro,
CA) and the appropriate concentration of peptide, followed by
incubation at 26°C for 3 h and at 37°C for 3 h. The cells
were then incubated with anti-HLA-A23/A24 mAb at 4°C for 30 min,
washed with PBS, and incubated with phycoerythrin-conjugated rabbit
antimouse IgG antibody (Cappel, Aurora, OH) at 4°C for 30 min. After
washing with PBS, the cells were suspended with 1 ml of PBS containing
1% formaldehyde and analyzed by FACScan (Becton Dickinson, San Jose,
CA). An HIV-derived peptide (RYLRDQQLGI) capable of binding to
HLA-A*2402 (25)
and SART-1736–745
(KGSGKMKTER) capable of binding to HLA-A*2601 (19)
were
used as positive and negative controls, respectively.
For detection of antigenic peptides recognized by the GK-CTL line,
C1R-A*2402 cells (2 x 104) were
pulsed with a peptide at a final concentration of 10 µM
for 2 h. The GK-CTLs (1 x 104) were then added and incubated for 18 h,
and 100 µl of supernatant were collected to measure IFN- by ELISA
in triplicate assays as reported previously (23)
. To test
peptide specificity, GK-CTL sublines were established from the parental
CTL line by the limiting dilution culture method at 1 or 10 cells/well
as described previously (23)
.
CTL Induction by Peptides.
PBMCs (1 x 106 per well) from
HLA-A*2402+ healthy volunteers or cancer patients
were incubated with 10 µM of each peptide in a 24-well
plate in the presence of 100 IU/ml interleukin 2 as reported previously
(23)
. At days 7 and 14 of culture, the cells were
restimulated at a responder: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. These cells were cultured
additionally in a 96-well, U-bottomed microculture plate in the
presence of irradiated autologous PBMCs (2 x 106 cells/well) that had been pulsed with the
corresponding peptide and were used as APCs. Seven to 10 days later,
the expanded cells were transferred to a 24-well plate and additionally
cultured for 14–25 days without peptide and without APCs. The
cytotoxic activity was measured by a standard 6-h
51Cr release assay at different E:T ratios as
reported previously (23)
. The target cells used were from
the lung adenocarcinoma cell lines 11-18, PC-9, and RERF-LC-MS, and the
lung SCC cell lines LC-1/sq and QG56. PHA blasts from
HLA-A24+ healthy volunteers were also used as
target cells as described by Gomi et al. (23)
.
Expression of HLA-class I or HLA-A24 antigens on these cells was
studied by staining of the cells with anti-HLA-class I (W6/32) mAb
recognizing a monomorphic region of the HLA-class I molecule or
anti-HLA-A24 mAb recognizing a polymorphic region of the HLA-class I
molecule (One Lamda, Inc.), and the expression was measured by FACScan
(Becton Dickinson) as reported previously (23)
.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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production by the GK-CTLs, were tested for their
ability to stimulate IFN- production by the CTLs against the COS-7
cells after cotransfection with HLA-A*2402 cDNA. This method
allows identification of genes encoding tumor-rejection antigens as
reported previously (23)
. In the third screening, one
clone (6A1-3D9) was found to confer recognition by the GK-CTLs,
i.e., the CTLs produced significant amounts of IFN- by
recognition of the COS-7 cells transfected with the
HLA-A*2402 and 6A1-3D9 genes but not by
recognition of those transfected with the HLA-A*2601 cDNA (a
negative control) and the 6A1-3D9 genes, nor by those
transfected with the HLA-A*2402 and 6A1-4A9 genes
(a negative control; Fig. 1A
).
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. The nucleotide
sequence of this 6A1-3D9 gene was almost identical to the
corresponding position of the KIAA0764 gene, which is 4261
bp in length and is registered in GenBank (accession no. AB018307; Ref.
26
; Fig. 1C
), i.e., the sequence of
6A1-3D9 is identical to that of KIAA0764, except
at position 848 (at which C is replaced by T). The KIAA0764
gene was isolated from a human brain cDNA library in the human gene
cloning project. To our knowledge, little is known about
KIAA0764, other than its sequence, mRNA expression in some
tissues, and chromosomal location. The 6A1-3D9 gene encoding
414 amino acids in the first ATG at nucleotide positions 363–365 is
used as a start codon, which is identical to the translated
amino acids sequence of KIAA0764 if its ATG at positions 344–346 is
used as a start codon (Fig. 1C)
.
The expression of the 6A1-3D9 gene at the mRNA level was
investigated by Northern blot analysis, and one major band of 2 kb
in length along with several dim bands between 2 and 4 kb in length
were observed in all of the malignant cells and normal tissues tested,
with higher expression in the lung adenocarcinoma and gliomas and lower
expression in the esophageal SCCs and hematological malignant cells
(Fig. 1D)
. These results suggest that the 1784-bp-long
6A1-3D9 gene was a truncated gene. A 2021-bp-long gene was
independently cloned from the cDNA libraries of HT1376 tumor (four of
four clones tested) and PBMCs of healthy donors (five of seven clones
tested) using the 6A1-3D9 gene as a probe by the colony
hybridization method (Fig. 1C)
. This 2021-bp-long gene,
containing all of the sequences of 6A1-3D9 gene (Fig. 1C)
, also encoded antigens recognized by the GK-CTLs (data
not shown). We designated this gene an adenocarcinoma antigen
recognized by T cell-1 (ART-1) gene (GenBank accession no.
AF197954).
The mean relative level of mRNA expression of the ART-1 of
2 kb in length in adenocarcinomas was significantly higher than that
of either of the SCCs (P = 0.018) and
malignant hematological disease (P = 0.0002;
Table 1
). Among SCCs, the expression of the mRNA in the lung cancers was three
times higher than that of esophageal SCCs. The mRNA was also expressed
in a panel of normal cells and tissues tested different levels of
expression (highest in the thymus; high in the testis, placenta, and
PHA blast; lower in the lung, kidney, and PBMCs; and lowest in the
stomach, small intestine, and skeletal muscle). Table 1
summarizes the
data on the relative levels of mRNA expression. Taken together, these
results suggest that the ART-1 gene with 2021-bp length was
dominantly expressed at the mRNA level in both malignant and normal
cells, although its expression levels varied largely among the samples
tested.
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) from the PBMC cDNA library was
different from those of ART-1 and KIAA0764 gene
and encode 412 amino acids if the ATG at nucleotide positions
1228–1230 is used as starting codon that is identical to the 414 amino
acids of ART-1 peptide sequence, except the second (N) and fourth (Q)
amino acids were deleted. Therefore, there might be an ART-1
gene family that includes such genes as the P17 gene and the
KIAA0764 gene, with much lower expression levels than
ART-1 itself.
Localization of the ART-1 Protein.
Localization of the ART-1 protein in cells was studied by transfection
of the ART-1/GFP fusion gene into 293T cells. The ART-1/GFP
protein was expressed in the nucleus of the 293T cells transfected with
the fusion gene (Fig. 1E)
. Anti-GFP polyclonal antibody
recognized a Mr 87,000 band in the
extraction of these transfected 293T cells, indicating that the
expected Mr of the ART-1 protein was
60,000 (Fig. 1F)
.
ART-1 Peptides Recognized by the CTLs.
Sixteen different ART-1-derived peptides with motifs of binding to
HLA-A*2402 molecules were loaded onto the
HLA-A*2402-transfected COS-7 cells at a concentration of 10
µM and then tested for their ability to induce
IFN- production by the GK-CTLs. Three of these 16 peptides,
ART-1158–165,
ART-1170–179, and
ART-1188–196, stimulated significant levels of
IFN- production in a dose-dependent manner (Fig. 2, A and B)
. The other 13 peptides failed to
stimulate IFN- production by the CTLs. The lowest dose of peptide
capable of inducing significant levels of IFN- production by the
GK-CTLs was 0.01 µM in
ART-1158–165, 1 µM in
ART-1170–179, and 0.01
µM in ART-1188–196.
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GK-CTL sublines were established from the parental GK-CTL line by
incubation at 1, 10, or 100 cells/well, and their peptide specificities
were tested. Two cyclophilin B peptides at positions 84–92 and 91–99
were used as negative control, because the same GK-CTL line was also
used for identification of these cyclophilin B peptides
(23)
. The HIV-derived peptide capable of binding to
HLA-A*2402 molecules was used as a negative control. Among the 120
different sublines tested, one, two, or three sublines recognized
C1R-A*2402 cells pulsed with ART-1158–165,
ART-1170–179, or
ART-1188–196, respectively. Representative
results for each subline in response to the corresponding peptides are
shown in Fig. 2C
. IFN- production by these sublines in
response to the corresponding peptides was inhibited by anti-HLA-class
I (W6/32) or anti-CD8 mAb but not by anti-HLA-class II (H-DR-1) or
anti-CD4 mAb (data not shown).
Induction of CTLs by Peptides.
ART-1158–165,
ART-1170–179, and
ART-1188–196 peptides were tested for their
ability to induce HLA-A24-restricted and tumor-specific CTLs from the
PBMCs of HLA-A24+ patients with lung cancer
(n = 7; six with adenocarcinomas and one with
SCCs) and HLA-A24+ healthy volunteers
(n = 5). PBMCs from the three cancer patients
who received chemotherapy at the time of blood sampling did not
proliferate well under the condition used. Those from the remaining
four patients (three with adenocarcinoma and one with SCC) did
proliferate well; analysis of their surface phenotypes showed that
CD3+CD4-CD8+
cells made up 10–30% of the population of PBMCs at day 21 of the
culture period (data not shown). These PBMCs at day 21 were incubated
for an additional 23–28 days, followed by a 6-h
51Cr release assay to measure CTL activity
against various target cells (Fig. 3A)
. The PBMCs that were stimulated by the
ART-1188–196 peptide but not those stimulated by
either the ART-1158–165 or
ART-1170–179 peptide showed significant levels
of cytotoxicity to HLA-A24+ lung cancer cells
(11-18, LC-1/sq, and PC9; Fig. 3
). In contrast, these PBMCs did not
lyse either HLA-A24+ PHA blasts from healthy
volunteers or HLA-A24- lung cancer cells
(RERF-LC-MS and QG-56). These CTLs failed to lyse
HLA-A24+ PHA blasts in the presence of exogenous
ART-1188–196 peptide added in culture, although
they showed cytotoxicity to the C1R-A*2402 cells pulsed with the
peptide under the same condition (Fig. 4A)
. The percentages of
CD3+CD4-CD8+
cells in the population of PBMCs showing the HLA-A24-restricted and
tumor-specific CTL activity at the time of the 6-h
51Cr release assay were 42% in patient 1, 46%
in patient 2, 32% in patient 3, and 21% in patient 4. The CTL
sublines were established from patient 2 by incubation at 1 and 10
cells/well, and their peptide specificities were tested. Among the 40
different sublines tested, one subline (no. 10) recognized C1R-A*2402
cells pulsed with ART-1188–196 but did not
recognize C1R-A*2402 cells pulsed with the other peptides (Fig. 4B)
. The IFN- production by this subline in response to
HLA-A24 tumor cells (HT1376) was inhibited by anti-HLA-class I (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; Fig. 4C
).
These results suggest that the ART-1188–196
peptide has the ability to induce HLA-A24restricted and
tumor-specific CTLs in PBMCs of lung cancer patients.
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. None of the three
ART-1 peptides tested induced the HLA-A24-restricted CTL activity in
PBMCs of five healthy donors. These PBMCs showed either no cytotoxicity
or MHC nonrestricted cytotoxicity (data not shown). |
DISCUSSION |
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2 kb between these genes at the nucleotide level in
the noncoding region of the COOH terminus. Results from the Northern
blotting suggest that the ART-1 gene, with a 2021-bp length,
was dominantly expressed at the mRNA level in both malignant and normal
cells, although its expression levels varied widely among the samples
tested. Furthermore, there might be an ART-1 gene family
that includes such genes as the P17 gene and the
KIAA0764 gene, with much lower expression levels than
ART-1 itself.
The ART-1 mRNA was ubiquitously expressed in normal tissues
and malignant tumor cell lines at different levels. Among tumor cell
lines, ART-1 mRNA was highly expressed in adenocarcinomas
and particularly in lung adenocarcinomas. It was also highly expressed
in gliomas. The expression in lung SCCs (n = 3; mean, 0.39) was three times higher than that in esophageal SCCs
(n = 4; mean, 0.13), and ART-1
mRNA was scarcely expressed in malignant hematological cells.
Therefore, the ART-1 peptide might be applicable for use in
peptide-based immunotherapy of patients with non-small cell lung
cancers and gliomas. Among normal tissues, ART-1 mRNA
expression was highest in the thymus, high in the testis and placenta,
low in the lung and several other tissues, and lowest in the stomach,
small intestine, and skeletal muscle. ART-1 mRNA was highly
expressed in PHA blasts but only slightly expressed in unstimulated
PBMCs. It is of note that the expression in 293T cells, a 293 cell line
with SV-40 large T, was three times higher than that in 293 cells
(Table 1)
. The results of the present laser-confocal microscopic
analysis of the GFP-tagged ART-1 transfectants indicate that the ART-1
protein is localized at the nucleus. The other tumor-rejection
antigens, SART-3 and SART-1800, were
preferentially expressed in both normal and malignant proliferating
cells but were not detectable in normal tissues or unstimulated normal
cells as determined by the Western blot analysis. SART-3 and
SART-1 genes were ubiquitously expressed at the mRNA level
by Northern blot analysis (19
, 20)
. Taken together, these
present results and our previous results (19
, 20
, 23)
suggest that ART-1 is a nuclear protein with a probable biological role
in cellular proliferation. Further studies, including detection of
ART-1 at the protein level, will be needed to clarify the biological
function of ART-1 and its family. On the basis of the present results,
however, it can be conclusively stated that the ART-1,
P17, and KIAA0764 genes, respectively, encode the
GK-CTL-recognized peptides ART-1158–165,
ART-1170–179, and
ART-1188–196. Therefore, proteins encoded by
these three genes might be appropriate target molecules for use in
peptide-based specific immunotherapy of lung and gliomas.
Among the three peptides with HLA-A24 antigen-binding motifs tested, only the ART-1188–196 peptide was able to induce HLA-A24-restricted CTLs from PBMCs of HLA-A24+ lung cancer patients. This difference among the three peptides may have been related to be the different levels of CTL precursors in PBMCs. The frequency of CTL precursors reacting to the ART-1188–196 peptide might have been higher than that of the others. Indeed, the frequency of CTL sublines from the GK-CTL line reacting to this peptide was highest among the three peptides. The amount of ART-1188–196 expressed on the groove of HLA-A*2402 molecules of cancer cells might have been larger than that of the others expressed. The binding affinity of the ART-1188–196 to HLA-A*2402 molecules was also highest among the three peptides. Therefore, the binding affinity of T cells to the peptide-HLA complex might be higher than the binding of T cells to the other peptides.
Although ART-1 is highly expressed in activated T cells, neither the GK-CTLs nor the CTLs induced by the ART-1186–196 peptide lysed PHA-activated normal T cells, even in the presence of excess amounts of a corresponding peptide in culture. These peptide-induced CTLs showed the cytotoxicity to C1R-A*2402 cells in the presence of an excess amounts of the peptide. Therefore, certain molecules, including a family of serpin, on activated T cells that play a role in protection from lysis by self-CTLs (27) , might be responsible for this phenomenon. Alternatively, the different posttranslational modification of the epitopes between normal and cancer cells might be involved in this issue. Some of the CTL epitopes that were identified previously have posttranslational modifications, and these modifications have had a significant impact on the ability of the CTLs to recognize these peptides (28) . Further studies, including determination of molecules involved in the resistance to the lysis, will be needed to clarify this issue.
In conclusion, the results of this study suggest that the ART1188–196 peptide is one of the target epitopes that is recognized by HLA-A24-restricted CTLs at the sites of lung cancer and that this peptide has the ability to induce HLA-A24-restricted CTLs from PBMCs of non-small cell cancer patients. The HLA-A24 allele is found in 60% of Japanese (with 95% of these cases being genotypically A*2402), in 20% of Caucasians, and in 12% of Africans (17) . The one ART-1-derived peptide was able to induce HLA-A24-restricted and tumor-specific CTLs in PBMCs of lung cancer patients. These ART-1 peptides could be useful for specific immunotherapy of HLA-A24+ patients with non-small cell lung cancer as well as in the development of a cancer vaccine for glioma patients.
|
ACKNOWLEDGMENTS |
|---|
for ELISA; Dr.
Masafumi Takiguchi of Kumamoto University, Kumamoto, Japan, for kindly
providing C1R-A*2402 cells for the study; and Hideo Takasu of Sumitomo
Pharmaceutical Company, Osaka, Japan, for providing the
RMA-S-A*2402/Kb cells. |
FOOTNOTES |
|---|
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, Fukuoka 830-0011, Japan. Phone:
81-942-31-7551; Fax: 81-942-31-7699; E-mail: shichijo{at}med.kurume-u.ac.jp
3 The abbreviations used are: HLA, human leukocyte
antigen; LSM, laser scanning microscope; TIL, tumor-infiltrating
lymphocyte; PBMC, peripheral blood mononuclear cell; mAb, monoclonal
antibody; APC, antigen-presenting cell; PHA, phytohemaglutinin;
SCC, squamous cell carcinoma; GFP, green fluorescent protein.
Received 2/ 7/00. Accepted 6/29/00.
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REFERENCES |
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T. Mine, Y. Sato, M. Noguchi, T. Sasatomi, R. Gouhara, N. Tsuda, S. Tanaka, H. Shomura, K. Katagiri, T. Rikimaru, et al. Humoral Responses to Peptides Correlate with Overall Survival in Advanced Cancer Patients Vaccinated with Peptides Based on Pre-existing, Peptide-Specific Cellular Responses Clin. Cancer Res., February 1, 2004; 10(3): 929 - 937. [Abstract] [Full Text] [PDF]
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,
11-18 (A24+); 
, LC-1/sq (A24+); •, PC9 (A24+); +, QG56 (A24-);
, RERF-LC-MS (A24-); *, PHA blasts (A24+).