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Departments of
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Immunology and
Surgery, and
First Department of Internal Medicine, Kurume University School of Medicine, Kurume, Japan;
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Division of Hematology, St. Mary Hospital, Kurume, Japan; and
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Division of Viral Immunology, Center for AIDS Research, Kumamoto University School of Medicine, Kumamoto, 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 (in a majority, of 95%, the genotype is
A*2402), 20% of Caucasians, and 12% of Africans (21).
Therefore, we have studied Ags recognized by HLA-A24-restricted and
tumor-specific CTLs to better understand the molecular basis of tumor
immunology, reporting that a cyclophilin B gene encoded
antigenic epitopes recognized by the CTLs. |
Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The HLA-A24-restricted and tumor-specific CTL (GK-CTL) line was
established from tumor-infiltrating lymphocytes
(TILs)3 of a patient
with lung adenocarcinoma (HLA-A*0206/A*2402, B39/B52, Cw7/) by
incubation of TILs with IL-2 alone for more than 60 days using
previously reported methods (15). Briefly, the patient’s
TILs were incubated with the culture medium (45% RPMI 1640 medium,
45% AIM-V medium; Life Technologies, Walkersville, MA), 10% FCS
(Equitech Bio, Ingram, TX) with 100 U/ml of IL-2 (Shiongi
Pharmaceutical, Osaka, Japan). These IL-2-activated TILs were tested
for CTL activity every 7 days to various cancer and normal cells by a
6-h 51Cr-release assay and an IFN-
assay with
an ELISA (limit of sensitivity: 10 pg/ml) as reported elsewhere
(15, 22). The HLA class I genotypes of these tumor cells
were described previously (15, 22, 23). GK-CTL sublines
were established from the parental CTL line by incubation of cells at
1, 2, or 4 cells/well of 96-well U-bottom microculture plates with the
culture medium and IL-2 (100 U/ml) in the presence of irradiated (30
Gy) HLA-A24+ allogenic PBMCs as feeder cells, as
reported (23). The surface phenotype of the CTL line and
sublines was investigated by an immunofluorescence assay with
FITC-conjugated anti-CD3, -CD4, or -CD8 mAb (22). For
inhibition of CTL activity, 100 µg/ml of anti-class I (W6/32,
IgG2a) or anti-CD8 (IgG2a), anti-class II (H-DR-1, IgG2a),
anti-CD4 (IgG1) mAb were used as reported previously
(22). Anti-CD14 mAb (IgG2a) or -CD 13 (IgG1) was served as
an isotype-matched control mAb. A two-tailed Student t test
was employed for the statistical analysis in this study.
Identification of the cyclophilin B gene
Methods for identification of a gene coding for tumor Ag
recognized by CTLs were reported previously (15). In
brief, poly(A)+ RNA of the HT1376 bladder
carcinoma cells was converted to cDNA, ligated to the SalI
adapter, and inserted into the expression vector pSV-SPORT-1 (Life
Technologies, Gaithersburg, MD). cDNA of HLA-A*2402 or
HLA-A*2601 was obtained by RT-PCR and was cloned into the
eukaryotic expression vector pCR3 (Invitrogen, San Diego, CA). Both 200
ng of plasmid DNA pools or clones of the HT1376 cDNA library and 200 ng
of the HLA-A*2402 cDNA were mixed with 1 µl of lipofectin
in 70 µl of OPTI-MEM (Life Technologies) for 15 min. Thirty
microliters of the mixture was then added to the COS-7 (2 x
104) cells and incubated for 5 h, and 200
µl of the RPMI 1640 medium containing 10% FCS was added and cultured
for 2 days, followed by incubation with CTLs (104
cells/well). After an 18-h incubation, 100 µl of supernatant was
collected to measure IFN- by an ELISA in a duplicate assay as
reported (15). This concentration (100 ng/well) of
HLA-A*2402 cDNA was chosen based on the fact that the
expression level of HLA-A24 Ags on the surface of COS-7 cells
transfected with 50, 100, 200, or 400 ng/well of HLA-A*2402
cDNA was 35, 42, 40, or 20% by FACScan analysis with anti-HLA-A24
mAb, respectively. A total of 1 x 105
clones from the cDNA library of the HT1376 cells were tested for their
ability to stimulate IFN- production by the GK-CTL after
cotransfection with the HLA-A*2402 cDNA into COS-7 cells in
the first screening. Namely, these pooled cDNA clones were subdivided
into 1000 different wells (expected number of clones per well: 100
clones/well) in the duplicate assays. Significant levels of IFN-
production were obtained in the 24 different wells. Before the second
screening, the plasmids were reobtained from each of these 24 positive
wells followed by distribution into 10 different wells. These cDNA
pools were tested for their activity to stimulate IFN- production by
the CTLs in the duplicate assays. One (6A1) of the plasmid pools
induced the significant IFN- production in 4 of 10 wells. Then, a
total of 400 independent plasmids were obtained from the positive well
(6A1) followed by testing their activity to stimulate IFN-
production in the duplicate assay as the second screening. Among 400
independent plasmids, two positive clones (6A1–4F2 and
6A1–3D9) and two negative clones (6A1–2F7 and
6A1–4A9)were provided for further analyses. Furthermore,
the other positive clone (4E6–2B5) was identified in the
first and second screening. This manuscript describes the results of
the 6A1–4F2 gene. A 6A1–2F7 gene was used as a
negative control. Investigation of the other two positive genes is
currently in progress. DNA sequencing was performed with
dideoxynucleotide sequencing method using a DNA Sequence kit
(Perkin-Elmer, Foster, CA), and the sequence was analyzed by the ABI
PRISM 377 DNA Sequencer (Perkin-Elimer).
Northern blot analysis
mRNA expression of a cyclophilin B (Cyp-B) gene on various tumor cells and tissues was investigated by Northern blot analysis with a 32P-labeled Cyp-B probe according to previously reported methods (15). ß-actin probe was used as a control. The relative expression level of the mRNA was calculated by the following formula: index = (Cyp-B density of a sample/ß-actin density of a sample) x (ß-actin density of unstimulated PBMCs/Cyp-B density of unstimulated PBMCs) (15). Expression of HLA class I or HLA-A24 Ags on these cells was studied by staining of the cells with anti-class I (w6/32) mAb recognizing a monomorphic region of class I molecule or anti-HLA-A24 mAb recognizing a polymorphic region of HLA-A24 molecule (One Lamda, Canoga Park, CA), and the expression was measured by FACScan (Becton Dickinson, San Jose, CA) as reported previously (23).
Detection of TCR repertoire
TCR Vß chain mRNA expression was investigated with the RT-PCR method described previously (24). Briefly, total RNA of the GK-CTL line and its sublines was converted to cDNA. cDNA was individually mixed with each TCR Vß primer pair, and then PCR was performed with 200 nM dNTP and 0.5 U Taq DNA polymerase (Promega, Madison, WI) for 35 cycles (94°C for 1.5 min, 60°C for 2 min, and 72°C for 3 min). Amplified DNA was analyzed with 1.3% agarose gel electrophoresis as reported (24, 25).
Peptides and evaluation of HLA-A*2402 binding
Eleven different peptide sequences capable of binding to HLA-A24 among the 208 amino acids of Cyp-B (Cyp-B77–85, Cyp-B79–89, Cyp-B84–92, Cyp-B91–99, Cyp-B110–120, Cyp-B114–122, Cyp-B119–129, Cyp-B124–132, Cyp-B144–153, Cyp-B152–161, and Cyp-B160–168) were synthesized based on a previous report regarding the HLA-A24-binding motif (26). HIV-derived peptide (RYPLTFGWCF) capable of binding to HLA-A*2402 Ags was used as a negative control (27). Cyp-B-1-9 peptide was also provided for a case in which the methionine of the upstream was used as the start codon (28, 29). The modified peptides of Cyp-B84–92 and Cyp-B91–99 at position 2 from phenylalanine to tyrosine (Cyp-B84–922F-Y and Cyp-B91–992F-Y, respectively) were synthesized because these modified peptides are expected to have a stronger binding motif to HLA-A2402 molecules (26). These peptides were kindly provided by Dr. Kanaoka (Sumitomo Pharmaceutical, Osaka), and their purity was 85–95%.
Estimated scores of half time of dissociation of each Cyp-B peptide for HLA-A24 molecule were calculated based on HLA peptide motif search results with computer analysis (30) as follows; Cyp-B77–85, 11.0; Cyp-B79–89, 6.0; Cyp-B84–92, 28.0; Cyp-B84–922F-Y, 280.0; Cyp-B91–99, 15.0; Cyp-B91–992F-Y, 150.0; Cyp-B110–120, 6.6; Cyp-B114–122, 79.2; Cyp-B124–132, 30.0; Cyp-B152–161, 3.6;, and Cyp-B160–168, 3.5. Among them, the four peptides (Cyp-B84–92, Cyp-B84–922F-Y, Cyp-B91–99, and Cyp-B91–992F-Y) capable of inducing CTL activity in PBMCs were further evaluated for their binding activity to HLA-A24 molecules using RMA-S-A*2402 cells, as reported previously (31). In brief, peptides at a concentration of 10-4 M, with more than 25% of the mean fluorescence intensity (MFI) of RMA-S-A*2402 cells cultured at 26°C, were evaluated as binding peptides. Relative MFI of RMA-S-A*2402 cells was obtained by subtracting the MFI value of peptide-unloaded RMA-S-A*2402 cells stained with TP25.99 mAb from that of peptide-loaded cells. The half-maximal binding level (BL50), which is the peptide concentration yielding the half-maximal MFI, was calculated. Binding peptides were classified according to the BL50 into three categories: high binder (BL50 < 10-5 M), medium binder (10-5 M < BL50 < 10-4 M), and low binder (BL50 > 10-4 M).
CTL induction by peptide
PBMCs were incubated with 10 µM of a peptide in one well of a
24-well plate. At days 7 and 14 of culture, the cells were restimulated
with the irradiated (30 Gy) autologous PBMCs as APCs that had been
preincubated with the same peptide (10 µM) for 2 h at an
effector cell to APC ratio of 4 to 1. Effector cells were harvested at
day 21 of the culture and tested for both their surface phenotypes and
activity to produce IFN- in response to various target cells by an
ELISA at different E:T cell ratios. For a
51Cr-release assay, the cryopreserved PBMCs from
day 21 of culture were thawed and cultured in wells of 96-well U-bottom
microculture plates in the absence of any peptide but in the presence
of 100 U/ml of IL-2 and irradiated autologous PBMCs (2 x
105 cells/well) as feeder cells.
HLA-A24+ allogenic PBMCs from healthy donors were
used as feeder cells in some leukemia patients. Proliferating cells
were transferred to wells of the 24-well plate at days 10–14 after
reculture in the absence of feeder cells, and were further expanded in
culture with IL-2 alone. These CTLs were retested for their surface
phenotypes and CTL activity by an IFN- assay at around day 25 of
reculture, and then these cells were tested for their cytotoxicity by a
6-h 51Cr-release assay at different E:T ratios at
around day 28–35. CTL sublines were established from the parental CTL
line by incubation of cells at 1, 10, or 100 cells/well of 96-well
U-bottom microculture plates with the culture medium and IL-2 (100
U/ml) in the presence of autologous PBMCs as feeder cells as reported
(15, 23).
CTL precursor frequency analysis
The detailed methods of CTL precursor frequency analysis were
described elsewhere (23). In this study, the
Cyp-B84–92 peptide-stimulated PBMC from a
leukemia patient were plated at 1, 2, 5, 10, 20, 40, and 80 cells per
well of 96-well microculture plates, and were cultured with the cloning
medium (25% RPMI 1640 medium, 55% AIM-V medium, 20% FCS, 100 U/ml of
IL-2, 10 µg/ml of PHA, and 0.1 mM MEM nonessential amino acids
solution) in the presence of irradiated allogenic PBMCs of three
healthy volunteers as feeder cells. PBMCs cultured without peptide were
served as a negative control. Cells from each well were tested at 9 to
15 days of the culture for IFN- production in response to target
cells. Wells were considered positive if they contained effector cells
producing much higher levels (>100 pg/ml) of IFN- in response to
the HLA-A*2402+ MT-2 and Bec-2 cells as compared
with the IFN- levels in response to the
HLA-A*2402- RPMI8402 and Ban-B1 cells,
respectively. Data were analyzed by the minimum
X2 method with the 95% confidence intervals, and
the CTL precursor frequency was calculated by Taswell’s method
(32).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The cells of the GK-CTL
(CD3+CD4-CD8+)
line, derived from TILs of lung adenocarcinoma, produced significant
levels of IFN- by recognition of the majority of
HLA-A*2402+ epithelial cancer cells and the other
cancer cells, leukemia cells, and virus-transformed cells, but did not
react to any of the HLA-A*2402- target cells.
This CTL line failed to respond to several
HLA-A*2402+ epithelial cancer cells.
Representative results are shown in Table I
. This CTL line showed significant
levels of cytotoxicity against HLA-A*2402+ 11-18
lung adenocarcinoma cells, HT1376 bladder tumor cells, KOPT-K1 T cell
lymphoma cells, and virus-transformed B cells (SSB and Bec-2), but not
against any of the HLA-A*2402- target cells or
COS-7 cells (Fig. 1A). This
CTL line, however, did not lyse HLA-A24+
PHA-activated normal T cells (PHA-blast cells) from the PBMCs of
healthy donors, whereas lymphokine-activated killer (LAK) cells showed
HLA class I-nonrestricted cytotoxicity to all the target cells tested
(Fig. 1B). CTL-mediated IFN- production by recognition of
tumor cells was inhibited by 100 µg/ml of anti-class I (W6/32) or
anti-CD8, but not by anti-class II (H-DR-1), anti-CD4, or
isotype-matched irrelevant anti-CD13 or anti-CD14 mAb (Fig. 1C). These results indicate that the GK-CTL line showed
HLA-A24-restricted cytotoxicity against tumor cells and
virus-transformed cells, but not against normal cells. Because of its
wide spectrum of reactivity, this CTL line may recognize widely shared
Ags on HLA-A*2402 molecules.
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A total of 105 clones from the cDNA library
of the HT1376 tumor cells, which induced the highest IFN- production
by the GK-CTLs, were tested for their ability to stimulate IFN-
production by the CTLs after cotransfection with the
HLA-A*2402 cDNA into COS-7 cells. This method allows
identification of genes encoding tumor-rejection Ags (15).
After the first and second screening, one clone (6A1-4F2) was found to
confer recognition by the GK-CTLs by the third screening (Fig. 2). Namely, the CTLs produced significant
amounts of IFN- by recognition of the COS-7 cells transfected with
HLA-A*2402 cDNA and 6A1-4F2 gene, but
neither with HLA-A*2601 cDNA (a negative control) and
6A1-4F2 gene nor with HLA-A*2402 cDNA
and 6A1-2F7 gene (a negative control). The
nucleotide sequence of this cDNA clone was proved to be 893 bp long and
to show 100% homology with that of Cyp-B gene (28, 29, 33). Cyp-B, a family of cyclophilins involved in T cell
activation (34, 35), is reported to be ubiquitously
expressed in normal cells and tissues, with a higher expression in
lymphoblastoid cells (28, 29, 33, 34, 35). We have
investigated mRNA expression of Cyp-B by Northern blot
analysis, and a part of the results is shown in Fig. 3. Cyp-B mRNA was ubiquitously
expressed in tumor cells, PHA-blast cells, and unstimulated PBMCs, with
the highest expression in HT1376 tumor cells from which
Cyp-B gene was cloned.
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production
Levels of IFN- production by CTLs largely differed among the
HLA-A*2402 tumor cells, as shown in Table I
. Therefore, we investigated
the correlation between levels of HLA-A24 expression on the surface and
the activity to stimulate IFN- production by the CTLs in the
HLA-A*2402 homozygous tumor cells (Fig. 3). PBMCs of an
HLA-A24+ homozygous donor were served as a
control. Both HLA-A24 expression and the ability to stimulate IFN-
production were highest or higher in HT1376 or SKG-I tumor cells,
respectively. In contrast, HLA-24 expression was very low in MKN-45 and
LC99A tumor cells, and was undetectable in LK79 cells. None of them
stimulated IFN- production by CTLs. The Cyp-B mRNA was
detectable in all these tumor cells. These results suggest that the
level of HLA-A24 expression, but not that of Cyp-B
expression, correlates with the ability to stimulate IFN-
production.
Identification of Cyp-B peptide recognized by GK-CTLs
Twelve different Cyp-B-derived were synthesized, loaded onto the
HLA-A*2402-transfected COS-7 cells at a concentration of 10 µM, and
were tested for their ability to induce IFN- production by GK-CTLs
(Fig. 4A). Among these
peptides, three peptides (Cyp-B77–85,
Cyp-B84–92, and
Cyp-B91–99) stimulated the significant levels of
IFN- production by CTLs. Among these peptides, two peptides
(Cyp-B84–92 and
Cyp-B91–99) stimulated IFN- production in a
dose-dependent fashion (Fig. 4B). The significant level of
IFN- production was induced by Cyp-B91–99
peptide as low as 0.01 µM or by Cyp-B84–92
peptide as low as 10 µM. In contrast, a dose dependency was not
obtained in the remaining peptide (data not shown). The CTLs failed to
respond to an HIV-derived peptide capable of binding to HLA-A*2402
molecules taken as a negative control (Fig. 4, A and
B). Subsequently, Cyp-B84–92 and
Cyp-B91–99 peptides along with their modified
peptides were mainly studied in the following experiments.
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GK-CTL sublines were established from the parental line by
incubation at 1, 2, or 4 cells/well, and their peptide specificity were
tested. Among 80 different sublines tested, four sublines recognized
CIR-A*2402 cells pulsed with Cyp-B84–92, but not
either Cyp-B91–99 or HIV-derived peptide as a
negative control, whereas the other three sublines recognized
CIR-A*2402 cells pulsed with Cyp-B91–99, but not
the others. Representative results of each subline are shown in Fig. 5. The remaining sublines failed to
respond to any peptides tested. Then, the TCR Vß repertoire of these
two sublines shown in Fig. 5 along with the parental GK-CTL lines were
analyzed by the RT-PCR. TCR Vß usage in the parental line, 2-40 and
2-3 sublines were polyclonal with the preferential uses of Vß-1 and
Vß-15 in the parental line, Vß-1 and Vß-16 in 2-40 subline, and
Vß-1 and Vß-16 in the 2-3 line.
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Cyp-B84–92,
Cyp-B84–922F-Y,
Cyp-B91–99 and
Cyp-B91–992F-Y peptides were tested for their
respective ability to stimulate IFN- production in PBMCs of
HLA-A24+ leukemia patients. HIV-derived peptide
was used as a negative control in certain experiments. PBMCs from all
five HLA-A24+ patients (two patients with acute
lymphoblastic leukemia [ALL], two with acute myeloid leukemia, and
one with Hodgkin’s lymphoma) produced significant levels of IFN- in
response to the HLA-A*2402+ MT-2 and Bec-2 cells
when stimulated three times in vitro by either
Cyp-B84–92 or Cyp-B91–99.
The modified peptides induced CTL activity with levels of activity
lower than induced by their wild-types. Representative results of one
ALL patient are shown in Table II. PBMCs
from these five patients were comprised of less than 10% leukemia
cells at the time of harvesting. IFN- production by the
peptide-induced CTLs in response to HLA-A*2402+
tumor cells was inhibited by anti-CD8, but not by anti-CD4 mAb
(data not shown).
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in response to HLA-A*2402+ target
cells when stimulated by Cyp-B84–922F-Y or
Cyp-B91–992F-Y, but not when stimulated by the
wild peptides. Representative results of a stomach cancer patient are
shown in Table II.
Similarly, these peptides were tested with PBMCs of healthy donors.
PBMCs from five of six HLA-A24+ healthy donors
showed HLA-A24-restricted IFN- production when stimulated by the
modified peptides, but not when stimulated by any of the wild peptides.
Representative results are shown in Table II. In PBMCs from the
remaining healthy donor, the Cyp-B91–99 induced
higher levels of HLA-A24-restricted IFN- production. The
Cyp-B91–992F-Y induced lower but significant
levels of CTL activity (data not shown).
Twenty-five to 40% of
CD3+CD4-CD8+
cells were observed in the population of the PBMCs showing the
HLA-A24-restricted CTL activity at day 21 by analysis of their surface
phenotypes (data not shown). The remaining cells were mostly
CD3+CD4+CD8-.
To confirm CTL activity by a 6-h 51Cr-release
assay, these PBMCs at 21 days were further incubated for an additional
23–28 days in the absence of any peptides but in the presence of IL-2
and feeder cells (irradiated autologous PBMCs for most cases,
HLA-A24+ allogenic PBMCs for some leukemia
patients). CTL activity of these cells was rechecked by an IFN-
assay before their use in a 6-h 51Cr-release
assay. When stimulated with Cyp-B84–92 (Fig. 6a) or
Cyp-B84–922F-Y (Fig. 6b), PBMCs of a
HLA-A24+ leukemia patient lysed the
HLA-A*2402+ tumor cells (BALL-1 cells from B cell
type ALL, or 11-18 lung adenocarcinoma cells) and the virus-transformed
cells (MT-2 or Bec-2 cells), but did not lyse either
HLA-A24+ PHA-activated T cells from a healthy
donor or any HLA-A24- target cells (RPMI8402,
HL-60 cells of monocyte type from a patient with acute promyelocytic
leukemia, or COS-7 cells). When stimulated with the
Cyp-B84–922F-Y, PBMCs of a
HLA-A24+ stomach cancer patient also lysed the
HLA-A*2402+ KOPT-K1 and the MT-2 cells, but did
not lyse any HLA-A24- target cells (Fig. 6c). Similarly, when stimulated with the
Cyp-B84–922F-Y (Fig. 6d) or the
Cyp-B91–992F-Y (Fig. 6e), PBMCs of
HLA-A24+ healthy donors lysed the
HLA-A*2402+ tumor cells (KOPT-K1, MT-2, and 11-18
tumor cells), but did not lyse any HLA-A24+
PHA-activated T cells from healthy donors, including the autologous T
cells, or any HLA-A24- target cells. Forty to
50% of
CD3+CD4-CD8+
cells were observed in the population of PBMCs showing the
HLA-A24-restricted CTL activity at the time of
51Cr-release assay. The remaining cells were
mostly
CD3+CD4+CD8-.
In contrast, PBMCs that failed to show HLA-A24-restricted IFN-
production at day 21 did not proliferate well in most cases. LAK cell
activity was observed in the remaining few cases in which PBMCs
proliferated well (data not shown).
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Peptide-specificity and CTL precursor frequency
To confirm the peptide specificity, the sublines were established
from the PBMCs from a healthy donor that had been stimulated with
Cyp-B91–992F-Y peptide. Among the four sublines
tested, one subline 100-1 responded to CIR-A*2402 cells pulsed with
Cyp-B91–992F-Y peptide used for stimulation, but
not pulsed with Cyp-B84–922F-Y (Fig. 7). This CTL-mediated IFN- production
was inhibited by 100 µg/ml of anti-class I (W6/32) or
anti-CD8, but not by anti-class II (H-DR-1), anti-CD4, or
isotype-matched irrelevant mAb (Fig. 7).
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Binding affinities of the four peptides were analyzed. HBV-derived
peptide (WFHISCLTF) capable of binding to HLA-A2402 and HIV-derived
peptide (VPVKLKPGM) capable of binding to HLA-B35 were served as
positive and negative controls, respectively (31). All the
four peptides bound to HLA-A*2402 molecules with different affinities
(Fig. 9).
Cyp-B84–922F-Y and
Cyp-B91–992F-Y were evaluated as high binders,
Cyp-B91–99 as a median binder, and
Cyp-B84–92 as a low binder.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Among 12 peptides tested, relative affinities of the Cyp-B84–92 and Cyp-B91–99 were modest and lower than the several other Cyp-B peptides by HLA peptide motif search results. These results are consistent with those from the others (40). Peptides with intermediate affinity, but not with high affinity, seem to be recognized by HLA-A2-restricted melanoma-specific CTLs (40).
Cyp-B mRNA was ubiquitously expressed in tumor cells with
the various levels of expression. Capability of tumor cells to
stimulate IFN- production by the CTLs correlated with the levels of
HLA-A24 molecules, but not with Cyp-B mRNA level. Therefore,
HLA-class I expression on tumor cells could be a major limiting factor
in CTL-mediated recognition of Cyp-B peptide.
TCR Vß usage of the GK-CTL line and its sublines tested were
polyclonal with the preferential use of several Vß-chains. These
results are consistent with our previous investigation determined by
the methods of DNA sequencing of TCR V
and Vß usage of CTL clones
established from TILs (25). These results along with the
other studies of sublines suggest that the GK-CTL line consists of a
polyclonal population of CTLs reactive to
HLA-A24+ tumor cells, and a few of them recognize
the Cyp-B-derived peptides.
The wild-type Cyp-B peptides failed to induce HLA-A24-restricted CTL activity against tumor cells in PBMCs in most cases tested except for those from leukemia patients. These results suggest that T cells in peripheral blood, except for those from the peripheral blood of leukemia patients, are immunologically tolerant to the Cyp-B. T cells in peripheral blood of healthy donors also seem to be usually immunologically tolerant to other self Ags (16, 17, 18, 19, 20, 41). Administration of the gp100 peptide failed to induce CTLs in most cases of melanoma patients (18), suggesting that T cells in the circulation of melanoma patients are also generally tolerant to self Ags, regardless of the fact that CTLs reacting to these Ags were detectable in vitro by repeated stimulation with a relevant peptide (16, 17, 18, 19, 20, 41). CTLs reacting to MAGE-3 are also not detectable in the PBMC of melanoma patients, even after in vivo administration (42). Cumulatively, these results suggest that T cells in the circulation are immunologically tolerant to self Ags recognized by CTLs that are located at the tumor site or at an immuno-privilege site.
There might be another type of self Ag to which CTLs are easily detectable in PBMCs of healthy donors by in vitro stimulation with a relevant peptide. These self Ags would include tyrosinase (43), prostate-specific Ag (PSA) (12), and SART1 (23). Although mechanisms involved in this reactivity are not clear at the present time, one explanation might be based on antigenic mimicry. In particular, the SART1 peptide at positions 690–698 is highly homologous to a peptide of the membrane protein of Saccharomyces cerevisiae, a well characterized nonpathogenic fungus (23).
The modified Cyp-B peptides induced HLA-A24-restricted CTL activity against tumor cells in PBMCs from leukemia patients, epithelial cancer patients, and healthy donors. The modified gp100 peptide, which is more efficient for induction of CTLs in vitro, was able to induce CTLs in vivo, and the subsequent administration of IL-2 resulted in tumor regression in some melanoma patients (18). Therefore, the Cyp-B84–922F-Y and Cyp-B91–992F-Y peptides would be applicable for use in specific immunotherapy of HLA-A24+ cancer patients. We are now studying whether Cyp-B-derived peptides capable of binding to HLA-A2 molecules can be recognized by HLA-A2-restricted CTLs established from TILs of an HLA-A*0207+ colon cancer patient.
Binding activity of Cyp-B91–99 to HLA-A24 molecules was >10 times higher than that of Cyp-B84–92, which is consistent with the results of the experiments of dose dependency. Cyp-B84–922F-Y and Cyp-B91–992F-Y peptides were >10,000 and >10 times higher than Cyp-B84–92 and Cyp-B91–99 peptides, respectively. Although the binding activity of Cyp-B91–99 2F-Y was >10 times higher than that of Cyp-B84–922F-Y, large differences between the two modified peptides with regard to ability to induce CTLs were not observed in this study.
Although Cyp-B mRNA is ubiquitously expressed in normal cells, with a higher expression in lymphoblastic cell lines (28, 29, 33), neither the GK-CTL nor the CTLs induced by Cyp-B peptide lysed PHA-activated normal T cells. In contrast to PHA-activated T cells, the cells of melanocyte cell lines were susceptible to lysis by the MART-1 peptide-induced CTLs (17). This discrepancy might be partly due to the fact that the cells from melanocyte cell lines used in the study are well adapted for in vitro culture, whereas PHA-activated T cells are not. EBV-transformed B cells that are also adapted for in vitro culture were susceptible to lysis by both the GK-CTL line and the CTLs induced by the Cyp-B peptides. Several molecules in the activated T cells, including a family of serpin are involved in their resistance for the CTL-mediated lysis (44, 45). Another possibility can be attributed to the different posttranslational modification of the epitopes between normal and cancer cells. Some of the previously identified CTL epitopes have posttranslational modifications, and the modifications have had a significant impact on the ability of the CTLs to recognize those peptides (46, 47). Further studies, including determination of molecules involved in the resistance to lysis, are needed to clarify this issue.
This study used autologous PBMCs as peptide-pulsed APCs in all the cases to avoid allogenic stimulation. Autologous PBMCs were also used as feeder cells for expansion of the peptide-stimulated PBMCs in most cases. However, in few cases of leukemia patients, allogenic but HLA-A24+ PBMCs from healthy donors were used because of limiting number of available cells. Therefore, we investigated the possible negative influence of the allogenic stimulation. The Cyp-B91–992F-Y-stimulated PBMCs from a healthy donor were expanded in the presence of either autologous PBMCs or allogenic but HLA-A24+ PBMCs as feeder cells followed by testing their CTL activity. Both effector cells showed the HLA-A24+ restricted CTL activity to tumor cells tested, and the levels of cytotoxicity were significantly higher in the former CTLs, which also failed to lyse any of the three PHA-blast cells (two with HLA-24+ and one with HLA-A24-). In contrast, the CTLs expanded in the presence of allogenic feeder cells showed the cytotoxicity to one (HLA-A24-) of the three PHA-blast cells (data not shown). These results suggest that the autologous feeder cells are better if available, and the allogenic HLA-A24+ feeder cells could be alternatively useful with attention to the allogenic influence.
In summary, this study showed that the wild-type Cyp-B peptides could
induce HLA-A24-restricted CTL activity in PBMCs from leukemia patients,
while their modified peptides could induce in those from leukemia
patients and epithelial cancer patients. The HLA-A24 allele is found in
60% of the Japanese population (in a majority, of 95%, the
genotype is A2402), 20% of Caucasians, and 12% of Africans
(21). Therefore, the wild-type peptides could be
applicable for use in specific immunotherapy of a relatively large
number of leukemia patients, while the modified peptides could be
applicable for leukemia patients and epithelial cancer patients.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Kyogo Itoh, Department of Immunology, Kurume University School of Medicine, 67 Asahi-machi, Kurume, 830-0011, Japan. E-mail address:
3 Abbreviations used in this paper: TIL, tumor-infiltrating lymphocyte; MFI, mean fluorescence intensity; BL50, half-maximal binding level; LAK, lymphokine-activated killer; ALL, acute lymphocytic leukemia; Cyp-B, cyclophilin B.
Received for publication March 8, 1999. Accepted for publication August 16, 1999.
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and ß genes for cytotoxic T lymphocytes in
human metastatic melanoma: possible involvement of TCR in tumor-cell
recognition. Int. J. Cancer 58:497.
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), A24/A33 (
), and A2/A30
(
), and of HLA-A24+ EBV-transformed B cells (SSB) (
)
by the GK-CTL line (left side) and LAK cells
(right side) that were generated by 5-day incubation of
PBMC from a HLA-A24+ healthy donor with 100 U/ml of IL-2.
C, GK-CTL-mediated IFN-
