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Experimental Therapeutics, Preclinical Pharmacology |
Department of Otolaryngology [M. E., K. C., F. K., A. O.] and Division of Viral Immunology [ Y. Sob., M. T.], Center for AIDS Research, Kumamoto University School of Medicine, Kumamoto 860-8556, Japan; National Cancer Institute, NIH, Bethesda, Maryland 20892 [Y. Son., E. A.]; and Division of Basic Research, University of Pittsburgh Cancer Institute [T. L. W., A. B. D.] and Departments of Otolaryngology [T. L. W.] and Pathology [T. L. W.], School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania 15213
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ABSTRACT |
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pretreatment of
HLA-A24+ SCCHN cell lines was necessary for lysis,
suggesting that a ligand density higher than that normally expressed by
tumor cells is required for these CTLs to mediate lysis. Moreover, a
cloned CTL, designated TH#99, isolated from the bulk population by
limiting dilution, lysed HLA-A24+ SCCHN targets more
efficiently than the bulk CTL population. Lysis was inhibited by
anti-HLA class I monoclonal antibody but not by anti-HLA-DR monoclonal
antibody. These results indicate that HLA-A24-restricted CTLs
recognizing the wild-type sequence p53125–134 can be
generated using autologous dendritic cells from precursors present in
peripheral blood lymphocytes obtained from normal HLA-A24+
donors. This finding suggests that vaccine strategies targeting
wild-type sequence p53 epitopes can be extended to a wider range of
cancer patients. |
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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p53 Mutation Analysis.
p53 mutation analysis of SCC cell lines used in this study was
performed using the yeast functional assay, as described previously
(27)
. Briefly, first-strand cDNA was synthesized from mRNA
with Moloney murine leukemia virus reverse transcriptase (Life
Technologies, Inc.) and random primers (Life Technologies, Inc.). cDNA
was amplified by PCR with recombinant Pfu polymerase
(Stratagene, La Jolla, CA) and the P3 and P4 primers (P3,
5'-ATTTGATGCTGTCCCCGGACGATATTGAAC-3'; P4,
5'-ACCCTTTTTGGACTTCAGGTGGCTGGAGTG-3'). The PCRproduct was confirmed
by a 1% agarose gel electrophoresis. Crude PCR product and
linearized p53 expression vector were cotransfected into the
yeast reporter strain yIG397, which was then plated and grown. For DNA
sequencing, p53 fragments (codon 53–364) were amplified from plasmids
by means of the PCR procedure using rTth DNA polymerase
(Perkin-Elmer, Norwalk, CT). The PCR products were sequenced by a Dye
Terminator kit (Perkin-Elmer) on an ABI 373S automated sequencer
(Perkin-Elmer). The conditions used for the DNA sequencing were as
described in the manufacturer’s protocol, and the same primers (P3 and
P4) were used.
Immunohistochemistry.
Immunohistochemical staining was performed to detect p53 protein with
the streptavidin-biotin method using the Histofine SAB-PO kit
(Nichirei, Tokyo, Japan). Tumor cell suspensions were placed on
silane-coated slides, and then the slides were fixed with 95% ethanol.
Endogenous peroxidase activity was blocked in methanol with 3%
hydrogen peroxidase for 30 min. Nonspecific conjugation was blocked
with 10% normal goat serum. p53 immunohistochemical staining was
performed with murine mAb DO-7 (Novocastra) and rabbit antibody RSP53
(Nichirei), which binds to both wild-type and mutant p53 protein.
Peptides.
Peptides were synthesized using standard
N-(9-fluorenyl)methoxycarbonyl methodology and
purified, and their sequences were confirmed by mass spectral analysis.
Peptides were dissolved in DMSO at a concentration of 2 mg/ml and
stored at -80°C.
Cytokines.
Cytokines used in this study were obtained from the following sources:
(a) GM-CSF, R&D Systems (Minneapolis, MN); (b)
IL-1
, Genzyme (Cambridge, MA); (c) IL-7, Sigma (St.
Louis, MO); and (d) IFN-, Endogen (Woburn, MA). Human
recombinant IL-2 and IL-4 were provided by Takeda Chemical Industries,
Ltd. (Osaka, Japan) and Ono Pharmaceutical Co., Ltd. (Osaka, Japan),
respectively.
Peptide Binding Assay by Flow Cytometry.
The binding of peptides to HLA-A*2402 molecules was tested as described
previously (28
, 29)
. Briefly, RMA-S-A*2402 cells were
incubated at 26°C for 18–24 h. Cells (2 x
105) in 50 µl of PBS supplemented with 20% FBS
(PBS-FBS) were incubated at 26°C for 3 h with a 50-µl solution
of peptides at 10-3 to
10-8 M and then at 37°C for 3 h. After washing with PBS-FBS, cells (2 x
105) were incubated for 30 min on ice with an
appropriate dilution of mAb TP 25.99 (HLA-class I-specific mAb; a
generous gift from Dr. S. Ferrone, New York Medical College,
Valhalla, NY). After two washes with PBS-FBS, the cells were incubated
for 30 min on ice with an appropriate dilution of FITC-conjugated IgG
of sheep antibody specific to mouse immunogloblin (Silenus
Laboratories, Howthorn, Australia). Cells were then washed three times
with PBS-FBS, and the fluorescence intensity of the cells was measured
using a FACScan. RMA-S-A*2402 cells cultured at 26°C or at 37°C and
stained with mAb TP25.99 under the same experimental conditions served
as controls.
Peptides at a concentration of 10-4 M with over 25% of the MFI of RMA-S-A*2402 cells cultured at 26°C were evaluated as binding peptides. The 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 mAb TP25.99 from the MFI value of peptide-loaded RMA-S-A*2402 cells stained with mAb TP25.99. The BL50, which is the peptide concentration yielding the half-maximal MFI, was calculated. Binding peptides were classified according to their BL50 into three categories: (a) high binder (BL50 <10-5 M); (b) intermediate binder (10-5 M < BL50 <10-4 M); and (c) low binder (BL50 > 10-4 M).
In Vitro Anti-p53 CTL Induction.
Peptide-specific CTLs were generated as follows: PBMCs were isolated
using Lymphoprep (Nycomed Pharma, Oslo, Norway) density gradient
centrifugation from HLA-A24+ healthy donors. DCs
were generated from PBMCs using a modification of the method reported
by Sallusto and Lanzavecchia (30)
. In brief, PBMCs
were resuspended at 10 x 106 cells/ml in
AIM-V medium (Life Technologies, Inc.) and distributed in T-75 flasks
(Falcon, Becton Dickinson, NJ). After 2 h of incubation,
nonadherent cells were removed by gentle washing, and AIM-V medium
containing GM-CSF (1000 units/ml) and IL-4 (10 ng/ml) was added.
After 6 days of incubation, IL-1 (50 units/ml) was added to
the medium. One day later, nonadherent DCs were harvested and used as
antigen-presenting cells. The DCs were resuspended in AIM-V medium
containing 40 µg/ml peptide and incubated at 37°C for 4 h.
Subsequently, the peptide-pulsed DCs were irradiated (3000 rad) and
washed. At day 0, 3 x 106 PBMCs and 3 x 105 peptide-pulsed DCs were cocultured in
24-well tissue culture plates in a final volume of 2 ml of AIM-V medium
supplemented with 5% human AB serum and IL-7 (5 ng/ml). On day
7, and weekly thereafter, responder cells were restimulated with
peptide-pulsed autologous adherent PBMCs. These cells were prepared as
follows: irradiated PBMCs (4 x 106) were
incubated for 2 h in a final volume of 0.5 ml/well AIM-V medium
containing 10 µg/ml peptide in a 24-well plate; and then nonadherent
cells and excess peptide-containing medium were removed. The responder
lymphocytes in culture medium supplemented with IL-2 (10 IU/ml) and
IL-7 (5 ng/ml) were added to the 24-well plate containing
peptide-pulsed autologous adherent PBMCs. After five rounds of
stimulation, responder cells were analyzed for their phenotype and
tested for their specificity.
The CTLs were cloned from the bulk cell line at a density of 1 cell/well in 96-well plates. Each well contained 5 x 104 irradiated PBMCs from two different healthy donors in 0.2 ml/well AIM-V medium containing 5% AB serum, IL-2 (100 IU/ml), IL-4 (1 ng/ml), and 1 x 104 peptide-pulsed allogeneic HLA-A24+ EBV-B cells. The cloned CTLs were stimulated with irradiated allogeneic HLA-A24+ EBV-B cells that had been incubated with peptide (10 µg/ml) for 2 h and washed. Peptide-specific CTL clones were then restimulated every week and expanded using irradiated allogeneic PBMC mixtures and A24+ EBV-B cells pulsed with peptide as feeder and stimulator cells, respectively. After transferring to 24-well plates, irradiated allogeneic EBV-B cells were added as feeder cells to the clones instead of PBMC mixtures.
Flow Cytometric Analysis.
The effector cells were incubated with FITC-conjugated mAbs to CD3,
CD4, CD8, or CD16 (all purchased from Becton Dickinson, Mountain View,
CA) for 30 min on ice and washed twice. All incubations and washes were
performed in PBS containing 0.1% NaN3 and 2%
FBS. Cells were analyzed on a FACScan (Becton Dickinson).
Chromium Release Assay.
Chromium release assays were performed as described previously
(31)
. Briefly, 1 x 106 target
cells were incubated with 51Cr for 1 h.
Effector cells were added to 2 x 103 target
cells in triplicate wells of V-bottomed microtiter plates (Limbro; Flow
Laboratories, McLean, VA) in a final volume of 0.2 ml. After a 4-h
incubation, the supernatant was harvested, and chromium release was
measured. Tumor cell lines were pretreated for 48 h with IFN-
(1000 units/ml) before the lysis assay.
Antigen specificity of tumor cell lysis was further determined in a cold target inhibition assay by analysis of the ability of the peptide-pulsed unlabeled (cold) HLA-A24+ EBV-B cell line (MO24) to inhibit lysis of 51Cr-labeled tumor cell lines.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, all of the peptides
synthesized bound to HLA-A24 molecules, but with different affinities.
Two peptides, p53125–134 and
p53204–212, bound to HLA-A24 with high affinity,
and four peptides were intermediate to low binders (Table 1)
.
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. A flow cytometric analysis of the
responding cells was performed. On day 40, most responding cells were
CD3+ and CD8+
(CD3+, 99.7%; CD4+, 3.7%;
CD8+, 96.4%; CD16+,
0.2%). The other peptide with high affinity to HLA-A24
molecules, p53204–212, did not elicit
peptide-specific CTLs from these donors using this culture system.
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. As
shown in Fig. 3
, none of these
HLA-A24+ tumor target cells were lysed by the
bulk anti-p53125–134 CTL cell line. In parallel
to these assays, the tumor cells were pulsed with relevant peptide and
shown to be effectively lysed, indicating that the
anti-p53125–134 CTLs can mediate cytolysis of
targets presenting artificially high levels of the epitope (Fig. 3)
.
These results suggested that the expression of epitope on tumor cells
was too low to allow efficient recognition of tumor cell lines by CTLs.
Therefore, to enhance HLA-class I and antigen presentation, target
cells were pretreated with IFN- as described by Theobald et
al. (18)
. Of five A24+ SCCHN
cell lines tested, three (HSC-4, Kuma-3, and Ca9-22) were lysed, and
two (HSQ-89 and FS-1) were not (Fig. 4A
). The lysis of the three
SCCHN cell lines was blocked by anti-HLA-class I mAb, but not by
anti-HLA-DR mAb (Fig. 4B
).
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, this CTL showed strong reactivity
against peptide-pulsed target cells at E:T cell ratios similar to the
bulk population. Moreover, a comparative analysis of the CTL clone and
the bulk population showed that the affinities of both effector cells
are in the intermediate range and are comparable. They mediated
half-maximal lysis of peptide-pulsed target cells at approximate
concentrations of 10-7 to
10-8 M, respectively (Fig. 6)
. Three HLA-A24+
SCCHNs that were sensitive to the bulk anti-p53 CTL cell line were
pretreated with IFN- and tested as targets. As expected, the cloned
CTL TH#99 efficiently lysed these SCCHN targets (Fig. 7A
), and the lysis was blocked
by anti-HLA-class I mAb, but not by HLA-DR mAb (Fig. 7B
).
The antigen specificity of the killing of HSC-4 tumor cells by this CTL
clone was confirmed by showing that cold (unlabeled) targets that were
pulsed with peptide p53125–134, but not targets
pulsed with an irrelevant peptide p53204–212,
specifically inhibited the lysis of HSC-4 cells (Fig. 7C
).
These results confirm that the cloned CTLs can recognize the naturally
occurring epitope but that a ligand density higher than that normally
expressed by tumor cells is required for these CTLs to mediate
cytolysis.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In the present study, we used peptide-pulsed autologous DCs as
antigen-presenting cells and succeeded in generating an
HLA-A24-restricted CTL cell line from PBMCs of a healthy donor in
vitro that recognizes the wild-type sequence
p53125–134 epitope. The peptide-specific CTL
cell line could lyse some HLA-A24+ SCCHN cell
lines. Moreover, a cloned CTL, TH#99, isolated from the bulk population
of effectors by limiting dilution, lysed HSC-4, Kuma-3, and Ca9-22
cells more efficiently than the bulk CTL cell line. The killing
activity was blocked by anti-HLA class I mAb. These results indicate
that the p53125–134 peptide is endogenously
processed and presented on the surface of those tumor cells in
association with HLA-A24 molecules. Interestingly, HSC-4 and Ca9-22
have point mutations at the same p53 codon (Table 2)
. However, HSC-4
was more sensitive to the CTLs than was Ca9-22. Treatment of tumor
target cells with IFN- has been reported to enhance the expression
of MHC class I antigens and proteasomes (33, 34, 35, 36)
. In this
study, three tumor cell lines that were sensitive to the CTLs expressed
the HLA-A24 molecule, and their expression was enhanced by treatment
with IFN- (data not shown). The HLA-A24 antigen expression of HSC-4
was much higher level than that of Ca9-22 (data not shown). These
results may explain the difference in sensitivity to the CTLs. It is
worthwhile to note that HSC-4, Kuma-3, and Ca9-22 were sensitive to the
CTLs, whereas FS-1 and HSQ-89, which also express and accumulate mutant
p53 molecules, were not sensitive to the CTLs. The relation between p53
mutations and presentation of CTL-defined p53 epitopes still remains to
be elucidated. Ropke et al. (19)
reported that
accumulation of p53 protein was not an absolute requirement for killing
because SCC-9 carcinoma cells, in which p53 protein was not detectable,
could induce both tumor necrosis factor secretion and killing by
p53-specific CTLs. The proteasome system represents a major source for
the generation of MHC class I ligands (33
, 34)
, and
degradation of p53 protein is dependent on proteasomal processing
(35)
. CTL epitopes are produced via proteasomal processing
and can be affected by mutations outside of epitopes in addition to
mutational alterations within epitopes. For example, the p53 mutation
at residue 273 affected p53264–272-specific CTL
recognition of tumor cells carrying this mutation (36)
.
Namely, because the R to H mutation at residue 273 inhibits proteolytic
cleavage between residues 272 and 273, the flanking peptide epitope
p53264–272 is not produced sufficiently in tumor
cells with this type of p53 mutation to be recognized by the
epitope-specific and HLA-A*0201-restricted CTLs. In contrast, the same
cells transfected with p53 genes harboring mutations
at residues 143 or 175 were sensitive to lysis by the same effector
cells. In our studies, two SCCHN cell lines (FS-1 and HSQ-89), which
accumulate mutant p53 expressing missense mutation outside of the
codons encoding the epitope, were not recognized by
anti-p53125–134 CTLs. The alterations in these
tumor cells that might interfere with the processing and presentation
of this epitope are presently unknown. As additional MHC class I and
II-restricted p53 T-cell-defined epitopes are identified
(37)
, and as tumor cell lines are characterized for their
antigen processing and presentation, a more definitive pattern
concerning p53 alterations/mutations that influence the presentation of
CTL-defined epitopes will become evident. This relatively novel aspect
of antigen processing might be a crucial component in immunoselection
of p53 antigen-loss variants and the applicability of p53-based
immunotherapy.
In conclusion, our studies indicate that HLA-A24-restricted CTLs that recognize the wild-type p53125–134 epitope can be generated using autologous DCs from precursors present in peripheral blood lymphocytes obtained from normal HLA-A24+ donors. We are currently evaluating the presence of p53125–134-specific CTL precursors in HLA-A24+ patients with head and neck cancer.
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FOOTNOTES |
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1 Supported in part by Grants-in-Aid for
Scientific Research and Priority Areas (Cancer) from the Ministry of
Education, Science, Sports and Culture, Japan. 
2 To whom requests for reprints should be
addressed, at Department of Otolaryngology, Kumamoto University School
of Medicine, 1-1-1 Honjo, Kumamoto 860-8556, Japan. Fax:
81-96-373-5256; E-mail: eura{at}gpo.kumamoto-u.ac.jp
3 The abbreviations used are: TAA,
tumor-associated antigen; PBMC, peripheral blood mononuclear cell; DC,
dendritic cell; SCCHN, squamous cell carcinoma of the head and neck;
SCC, squamous cell carcinoma; IL, interleukin; mAb, monoclonal
antibody; FBS, fetal bovine serum; MFI, mean fluorescence intensity;
BL50, half-maximal binding level; GM-CSF, granulocyte
macrophage colony-stimulating factor; EBV-B, EBV transformed B.
Received 8/ 4/99; revised 12/ 6/99; accepted 12/ 6/99.
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REFERENCES |
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. J. Exp. Med., 179: 1109-1118, 1994.This article has been cited by other articles:
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