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,
*
University of Pittsburgh Cancer Institute and Departments of
Pathology and
Otolaryngology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213;
National Cancer Institute, Bethesda, MD 20892; and
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Department of Medical Microbiology, Johannes Gutenberg University, Mainz, Germany
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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analysis of two T cell lines isolated from bulk
populations of effectors reactive against the wt
p53264–272 peptide, using either the parental or the 7W
variant peptide, indicated that these T cells were expressing identical
TCR V13.6/complementarity-determining region 3/J region sequences.
This finding confirms the heteroclitic nature of at least one of the
variant peptides identified in this study. The use of variant peptides
of the wt p53264–272 epitope represents a promising
approach to overcoming the nonresponsiveness of certain cancer patients
to this self epitope, thereby enhancing its potential use in tumor
vaccines for appropriately selected cancer
patients. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To date, five MHC class I-restricted, naturally presented human wild-type (wt) sequence p53 epitopes have been identified. They have been shown to be able to induce epitope-specific CTL from PBMC obtained from healthy individuals (1, 6, 7, 8, 9, 10, 11). The p53125–134 epitope is HLA-A24 restricted (11), while the other four, p5365–73, p53149–157, p53217–225, and p53264–272, are HLA-A2.1 restricted. Among these, the wt p53264–272 peptide has been the most intensively investigated (1, 2, 6, 7, 8, 12).
The potential of wt p53 epitopes as targets for immunotherapy, however, remains uncertain due to the several critical concerns related to immunological recognition of this truly self tumor Ag. Using HLA-2.1-transgenic wt (p53+/+) and p53null (p53-/-) mice, Sherman and colleagues (13, 14, 15) have demonstrated that the CTL repertoire available for wt p53 self epitopes in p53+/+ mice is limited to intermediate affinity T cells, because the higher affinity CTL are either deleted or tolerized. Apparently, this situation occurs in humans as well, as only CTL with intermediate affinity for the wt p53264–272 epitope have been generated to date from PBMC obtained from normal donors as well as cancer patients (7, 12). This observation raises the question of whether such CTL are potent enough to be effective in tumor eradication.
Another concern relates to our experience that PBMC obtained from only some HLA-A2.1+ healthy donors and patients with OSCC were responsive to in vitro stimulation (IVS) with the wt p53264–272 peptide pulsed onto autologous dendritic cells (DC) (7, 12). Furthermore, CTL reactive against this epitope could only be generated from T cell precursors in PBMC of patients whose tumors were not likely to present this epitope. The analysis of these tumors indicated no accumulation of p53 or accumulation of mutant p53 with a missense mutation at codon 273, a site known to block processing of the wt p53264–272 epitope (16). In contrast, PBMC obtained from patients with tumors considered capable of presenting the wt p53264–272 epitope (i.e., tumors that accumulate mutant p53) were nonresponsive to IVS with wt p53264–272-pulsed autologous DC. These findings have led us to conclude that CTL specific for the wt p53264–272 epitope might play a role in the outgrowth of epitope-loss tumor cells, which are able to escape from the host immune system. This conclusion was further strengthened by the results of a recently completed study in our laboratories that used tetrameric peptide/MHC class I complexes to determine frequencies and characteristics of the p53264–272-specific CTL in unstimulated PBMC obtained from 30 OSCC patients and 31 normal donors (data not shown).4
Because these observations suggest that it may be possible to accurately predict ex vivo the responsiveness of cancer patients to immunotherapy targeting this epitope, we felt that a means of circumventing the nonresponsiveness of individuals needed to be investigated to proceed with the development of wt p53-based vaccines. One solution is to identify a heteroclitic peptide or, in more precise terms, an altered peptide ligand (APL) with enhanced functional activity relative to the parental wt p53264–272peptide. By substituting amino acids at various positions of an epitope that contact MHC class I and/or TCR, an array of APL with biological potencies higher than those of the parental epitopes has been identified for various antigenic determinants (17, 18, 19, 20, 21, 22, 23, 24, 25, 26). In applying this strategy to the wt p53264–272 epitope, we anticipated that an APL might induce CTL-mediated responses that cross-react with the parental epitope and that these CTLs also might demonstrate enhanced avidities relative to CTLs induced by the parental peptide. Most importantly, we sought to determine whether an APL would be able to induce anti-wt p53264–272 CTL from PBMC that were nonresponsive to the parental peptide, particularly the PBMC obtained from patients whose tumors accumulate mutant p53 and are considered to have the potential to present this epitope.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The following HLA-A2+ OSCC cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA): SCC-4 and SCC-9. The SCC-4 cell line expresses and accumulates p53 expressing a missense mutation at codon 151 but does not present the wt p53264–272 epitope (6). The SCC-9 cell line expresses an altered p53 molecule with a deletion of codons 274–285. It does not accumulate p53 molecules, yet presents the wt p53264–272 epitope. In addition, the p53null osteosarcoma cell line, SaOS-2, was obtained from ATCC. The cloned p53+ cell line, SaOS-2Cl3, was derived by transduction of SaOS-2 cells with a p53 cDNA expressing a missense mutation in codon 143 (7). The HLA-A2+ OSCC cell line PCI-13 has been described previously (27). It expresses a p53 missense mutation in codon 286 (Glu to Lys) and presents the wt p53264–272 epitope. Tumor cells were cultured in plastic culture flasks (Costar, Cambridge, CA) under standard conditions (37°C, 5% CO2 in a fully humidified atmosphere) in complete medium consisting of DMEM (Life Technologies, Grand Island, NY) supplemented with 10% (v/v) heat-inactivated FBS, 2 mM L-glutamine, 50 µg/ml streptomycin, and 50 IU/ml penicillin (all from Life Technologies). The T2 cell line was also obtained from ATCC and maintained in RPMI 1640 (Life Technologies) containing 10% heat-inactivated FBS, 2 mM L-glutamine, and antibiotics. The cultures were routinely tested and found to be free of mycoplasma contamination (Gen-Probe, San Diego, CA).
Peptides
The CTL-defined, HLA-A2.1-binding peptide, LLGRNSFEV (1), corresponding to wt p53264–272, as well as single amino acid exchange variants of this peptide were synthesized by standard N-(9-fluorenyl)methoxycarbonyl methology. Peptides were purified by reversed-phase HPLC, and their amino acid sequence was confirmed by mass spectrometry analysis. All peptides were dissolved in DMSO (Fisher Scientific, Pittsburgh, PA) at 1 mg/ml and diluted with PBS just before use. The 19 variant peptides contain single amino acid exchanges with a bias toward retention of a high degree of similarity to the central region of the parental peptide. The variant peptides are designated 1E, 1F, 1V, 3L, 3F, 3W, 4K, 4L, 5K, 5L, 6G, 6T, 6Y, 7L, 7P, 7Y, 7W, 8A, and 8Y, in which numbers denote the position within the parental sequence and letters refer to exchanged amino acids.
MHC stabilization assay
T2 cells were incubated overnight at room temperature before use in this assay. Cells were washed and incubated at a cell density of 2 x 105/0.2 ml of complete medium with various peptides at final concentrations of 1 x 10-5–1 x 10-10 M for 3 h at room temperature, followed by a 3-h incubation period at 37°C. After washing with PBS, cells were incubated at 4°C for 30 min with anti-HLA class I mAb, W6/32 (HB95; ATCC), and then with FITC-conjugated goat anti-mouse Ig (Caltag Laboratories, Burlingame, CA) as a secondary Ab. Fluorescence of viable T2 cells was measured at 488 nm in a FACScan flow cytometer (BD Biosciences, San Jose, CA), and the level of MHC class I expression was determined by evaluating the mean fluorescence intensity of stained T2 cells. Cells incubated either at room temperature or 37°C in the absence of peptide served as controls.
Generation of anti-p53 CTL with peptide-pulsed autologous DC
Peripheral blood or leukapheresis products were obtained from previously studied HLA-A2.1+ individuals: seven normal donors and six OSCC patients (12). PBMC were isolated by sedimentation over Ficoll-Hypaque (Amersham Pharmacia Biotech, Piscataway, NJ). The study was approved by the Institutional Review Board at the University of Pittsburgh, and written informed consent was obtained from each individual donating peripheral blood. PBMC were phenotyped for HLA-A2 expression by flow cytometry, using anti-HLA-A2 mAb, BB7.2 (HB82; ATCC), and a mouse IgG isotype as a control. The verification of the A0201 subtype was performed using PCR with sequence-specific primers, as previously described (12).
Human DC were generated from PBMC according to a modification of the
method of Sallusto and Lanzavecchia (28), as
described by us earlier (7). DC were harvested on day 6,
phenotyped by flow cytometry, and then resuspended in AIM-V medium
(2 x 106 cells/ml) containing 10 µg/ml
peptide and incubated at 37°C for 4 h. The peptide-pulsed DC
were then cocultured with autologous PBMC in 24-well tissue culture
plates (Costar) in a final volume of 2 ml/well AIM-V medium
supplemented with 10% (v/v) human AB serum (Pel-Freez Biologicals,
Brown Deer, WI) and 25 ng/ml IL-7 (Genzyme, Cambridge, MA) for the
first 72 h and, additionally, with 20 IU/ml IL-2 (Chiron-Cetus,
Emeryville, CA) for the remaining time in culture. The lymphocytes were
restimulated 1 wk later with peptide-pulsed autologous DC. Irradiated
(3000 rad) autologous PBMC were used as APC after the third round of
restimulations. Microcultures of CTL lines recognizing the wt
p53264–272 or 7W peptide were isolated from bulk
populations of effectors by limiting dilution (1 cell/well/96-well
plates), and the lines were maintained in cytokine-supplemented media
plus peptide-pulsed APC, as previously described (7).
Specificities of generated T cells were determined using one or more of
a panel of assays detailed below. The TCR V expression on T cells in
bulk CTL populations and cell lines derived from them was done using
the IOTest Mark TCR V Repertoire kit (Beckman Coulter, San
Diego, CA).
ELISPOT assay for IFN-
The ELISPOT assay was performed in 96-well plates
with nitrocellulose membrane inserts (Millipore, Bedford, MA), as
previously described by us (29). The capture and detection
anti-IFN- mAb were purchased from Mabtech (Nacka, Sweden). The
spots were counted by computer-assisted image analysis (ELISPOT 4.14.3;
Zeiss, Jena, Germany). For Ab-blocking experiments, target cells were
preincubated with anti-HLA class I mAb for 30 min. Cryopreserved
aliquots of PBMC obtained from a normal donor were thawed and, after
stimulation with PMA (1 ng/ml) and ionomycin (1 µM; both from
Sigma-Aldrich, St. Louis, MO), were used as a positive control for each
assay. The interassay reproducibility of the assay was acceptable with
a coefficient of variation = 15% (n =
30).
Cytotoxicity assay
The 4-h 51Cr release assay was performed
at various E:T ratios, as previously described (7).
Briefly, sensitized targets were labeled with
51Cr for 45 min at 37°C, washed, and added to
wells of 96-well plates (1 x 104
cells/well). Effector T cells were then added to give various E:T
ratios. When Ab-blocking experiments were performed, target cells were
incubated with anti-HLA class I mAb or the anti-HLA-DR mAb,
L243 (HB55; ATCC),for 30 min before adding effector cells. The
percentage of specific lysis was calculated according to the formula:
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Flow cytometry analysis using HLA-A2.1/peptide tetrameric complexes (tetramer)
The streptavidin-PE-labeled tetramers used in this study were obtained from the tetramer core facility of the National Institute of Allergy and Infectious Disease (Atlanta, GA). Three-color flow cytometry assays (FACScan; BD Biosciences) were performed with PerCP anti-CD3, FITC anti-CD8, and PE-tetramer. The specificity of the HLA-A2.1/p53264–272 tetramer was confirmed by its staining of a CTL line specific for this p53 epitope and by the lack of staining of irrelevant CTL or HLA-A2- PBMC of healthy donors, as previously described (30). The additional PE-conjugated HLA-A2.1/tetramer used in this study contained the 7W variant peptide. Generally, 75,000 events per sample were collected progressively after live gating on lymphocytes by forward and side scatter.
TCR and CDR3 spectratyping
RNA was extracted from
p53264–272-specific CTL lines generated using
parental or the 7W variant peptide, followed by reverse transcription
into cDNA, as previously described (31). Screening for
expression of TCR V chains was performed using the primers described by
Puisieux et al. (31) for TCR V amplification,
followed by a runoff reaction with fluorophore-labeled primers specific
for the C region of the TCR (5'-TGTGCACCTCCTTCCCATTCACC) chain.
Labeled runoff products were subjected to DNA fragment analysis, as
described (32). Finally, amplified products were directly
subjected to DNA sequence analysis using ABI 310 sequencer
(PerkinElmer, Weiterstadt, Germany).
Statistical analysis
A two-tailed Wilcoxon rank sum test was performed to analyze ELISPOT data. An unpaired two-tailed Student’s t test was used to interpret differences in CTL reactivities against different target cells and in the presence of blocking Ab in cytotoxic assays, and differences between the number of spots obtained from T cells incubated with T2 cells pulsed with relevant p53 peptides vs that obtained using T2 cells pulsed with the irrelevant gp100 peptide in ELISPOT assays. Differences were considered significant when p < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Because the parental peptide binds efficiently to HLA-A2.1 molecules, all of the APL considered in this study represent single amino acid exchanges at nonanchor residues for the purpose of enhancing the interactions of the variant peptides with the TCR rather than MHC class I molecules. Nineteen variants of the wt p53264–272 peptide were screened for their recognition by a bulk population of anti-wt p53264–274-specific CTL that was maintained in our laboratory (7). T2 cells pulsed with the individual peptides at a fixed concentration of 1 x 10-6 M peptide served as targets for these CTL in a 51Cr release cytotoxic assay. Significant cytotoxic reactivity against T2 cells pulsed with three of the 19 variant peptides, namely, 6T, 7W, and 7P, was detected (data not shown). Therefore, these three variant peptides were selected for further characterization.
Variant peptide binding to HLA-A2.1 molecules
Binding of the 6T, 7W, and 7P variant peptides to HLA-A2.1
molecules was compared with that of the parental peptide in an MHC
stabilization assay. The relative mean fluorescence intensity of
parental and variant peptide-stabilized HLA-A2 molecules on T2 cells is
shown in Fig. 1
. All the peptides showed
stabilization of HLA-A2 molecules in a dose-dependent manner within the
concentration range of 1 x 10-5–1 x
10-9 M. However, in general, the binding
affinities of the variant peptides to HLA-A2.1 molecules on T2 cells
were slightly lower than that of the parental wt peptide (wt >
6T 
7W > 7P).
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The affinity of the bulk population of
anti-p53264–272-specific CTL for the variant
peptides was determined in a 4-h 51Cr release
assay using T2 cells pulsed with these peptides at concentrations
ranging from 1 x 10-5 to 1 x
10-12 M as target cells. As shown in Fig. 2, at concentrations <1 x
10-8 M, the dose-response curves of the three
variant peptides were shifted to the left relative to that of the
parental wt peptide. Because the increased responsiveness of
the CTL for these variant peptides cannot be attributed to enhanced
binding to HLA-A2.1 molecules, these results are consistent with an
increased affinity of TCR for the variant peptides.
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Previously, we reported that CTL reactive against the wt
p53264–272 epitope could be generated from
PBMC obtained from only two of the seven
HLA-A2.1+ normal donors tested (12).
Analyses involving multiple cryopreserved samples derived from
leukopaks obtained from two of the normal donors (a responder and a
nonresponder) confirmed the consistency of responses of these donors’
PBMC to the parental peptide. In the same experiments in which the
seven donors’ PBMC were tested for induction using the parental
peptide, CTL reactive against this peptide could be generated from five
of these seven PBMC using either the 6T or 7W variant peptide. Included
in this group were three nonresponsive PBMC (Table I); PBMC obtained from donors 6 and 7
responded to the 7W variant, while PBMC obtained from donor 4 responded
to the 6T peptide. None of the seven PBMC tested responded to the 7P
variant peptide.
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and
cytotoxicity assays. Fig. 3, A
and B, shows the results obtained with the effectors
generated from PBMC obtained from donor 7. Unpulsed T2 target cells or
T2 cells pulsed with an irrelevant HLA-A2.1-binding peptide, the
melanoma-associated gp100 peptide (33), were not
recognized by these CTL in either assay to any noticeable extent. More
importantly, these effector cells were also capable of recognizing the
naturally presented epitope, as evidenced by their ability to lyse
PCI-13 and SCC-9 tumor cells as well as mutant p53-transfected SaOS-2
Cl3 cell lines in a MHC class I-restricted manner (Fig. 3C).
No significant cytotoxicity was noted against
HLA-A2+ tumor cell lines, SCC-4 and SaOS-2, which
do not present the epitope.
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assay (Fig. 4A), and
cytolytic against the OSCC lines, SCC-9 and PCI-13, as well as
SaOS-2Cl3 (Fig. 4B). This response was blocked by
anti-HLA class I mAb but not anti-HLA-DR mAb. No significant
reactivity was obtained against the tumor cell line SCC-4. The
reactivity of these effectors against SaOS-2 cells in the analysis
shown in Fig. 4B was higher than normally detected against
this p53null cell line, using bulk populations of
anti-p53 effectors (7, 12). However, the reactivity of
the 6T-induced effectors against SaOS-2 targets was not significantly
blocked by anti-HLA class I mAb and thus could be attributed to
nonspecific effectors present in the bulk population. In
summary, variant-induced effector T cells had similar reactivities
against the parental epitope as those reported previously for the
parental peptide-induced effectors from responsive normal donors as
well as OSCC patients (7, 12).
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The critical test of the variant peptides was whether their use
could induce CTL capable of recognizing the anti-wt
p53264–272 epitope from nonresponsive patients
whose tumors were considered capable of presenting this epitope
(12). The nonresponsiveness of PBMC obtained from at least
one of these donors, patient 3, has been repeatedly confirmed during
the past 2 years using blood samples obtained at different times, as
well as multiple cryopreserved leukapheresis samples obtained from this
patient. As shown in Table II, none of
the PBMC from three of these patients responded to the 6T or 7P variant
peptides. However, the 7W variant peptide did induce the ex vivo
generation of anti-wt p53264–272 CTL from
PBMC of patient 3, whose autologous tumor, PCI-13, presents this
epitope (7, 12). The affinity of these effectors for the
parental epitope was comparable with that of a bulk population of
CTL induced using the parental epitope, and was in the range of
1 x 10-9 M (Figs. 2
and 5A). Furthermore, the
7W-induced CTL were cytotoxic against a panel of tumor cell lines
naturally presenting the wt p53264–272 epitope,
including the autologous PCI-13 cell line, and this reactivity was MHC
class I restricted (Fig. 5B). This result clearly
illustrates the potential value of the 7W variant peptide in
immunotherapy targeting the wt p53264–272
epitope in individuals like OSCC patient 3.
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usage by T cell microcultures
reactive against parental and/or variant peptides
The ability of the variant peptides to induce the generation of
CTL specific for wt p53264–272 from nonresponder
PBMC raised the question of the relationship between these CTL and
those induced by the parental peptide in responder PBMC. The need to
investigate this relationship became evident when the cross-reactive
bulk population of CD8+ T cells induced with the
7W variant peptide from normal donor 7 was stained with the parental or
variant tetramer. Whereas only 
2% tetramerdim
cells were detected with the parental tetramer, a cluster of 40%
tetramerbright cells was detected with the 7W
tetramer (Fig. 6, A and
B). One possible explanation for this observed difference
was that the variant peptide induced a single
CD8+ T cell population that bound the variant
tetramer with higher avidity/stability than did the parental tetramer.
Another possible explanation was that the variant peptide induced two
distinct populations of CD8+ T cells; one was
cross-reactive and bound both tetramers (most likely with different
avidities), while the other was specific for the 7W variant and bound
the 7W tetramer with high avidity. The two possibilities could be
distinguished based on TCR usage of the T cells involved in recognition
of these peptides. To accomplish this, T cell microcultures were
established by limiting dilution from bulk CTL populations induced with
either parental or variant peptide. Several T cell clones from each
type of microculture were expanded for further analysis. Based in part
on their rates of proliferation as well as peptide specificities (Fig. 7), four oligoclonal T cell lines,
designated 2, 4, 53, and 68, were selected for TCR analysis by
complementarity-determining region (CDR)3 spectratyping.
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). Although tetramer analysis of the bulk population suggested that
the vast majority of CTL were 7W specific, one of the several T
cell clones isolated was cross-reactive. The 53 cell line, which was
specific for the 7W variant, was found to express V9. The 68 cell
line, which recognized the variant as well as the parental peptide, was
found to express V13.6 with completely different CDR3 and J regions
from those expressed by line 53 (Table III). Consistent with these findings was
the result of a combined tetramer/V expression flow analysis
of the bulk population from which the 53 and 68 cell lines were
derived. We observed that the 40% cells that stained as a distinct
cluster with the 7W tetramer were V13.6-,
whereas the 2% cells that stained weakly with this tetramer were
V13.6+ (Fig. 6C).
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), respectively. The 2 cell line was
found to express V13.6, with identical motifs for the CDR3 and J
regions as the 68 cell line, which was induced from a different
individual using the 7W peptide (Table III). The observation that
cross-reactive T cells induced by the variant peptide from PBMC
obtained from one donor express the identical TCR/CDR3/J region as that
expressed by T cells induced with the parental peptide from another
donor illustrates the heteroclitic nature of the 7W variant
peptide.
The 4 cell line was shown to express V1 with a CDR3 and J region
sequence distinct from those of any of the other cell lines analyzed
(Table III). Interestingly, V analysis of the bulk population of
effectors from which the 4 cell line was isolated detected mainly V1
and V13 CD8+ cells. Another bulk population of
parental peptide-induced CTL, which was obtained from a different
normal donor (7) and used to identify 7W and 6T peptides
as potential APLs (Table II), was found to consist of >90%
V1+ cells (data not shown). These results,
summarized in Table IV, are strongly
suggestive of a relatively limited TCR V usage being involved in
recognition by CTL of the HLA-A2.1-restricted, wt
p53264–272 epitope, regardless of whether these
cells are induced by the parental or variant peptide.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Since the parental wt p53264–272 peptide has a
reasonable affinity for HLA-A2.1 molecules (>1 x
10-9 M), the 19
p53264–272 variants designed for this study had
unmodified anchor positions. Among the amino acid exchanges tested,
those at position 6 (6T) and position 7 (7W) appeared to be promising.
Since both variants have lower affinities than the parental peptide for
HLA-A2.1 molecules, their ability to increase the frequency of
anti-p53264–272 CTL responses
generated from nonresponsive PBMC does not appear to be due to their
enhanced binding to HLA-A2.1 molecules. Instead, their increased
immunogenicity might be due to the replacement or counterbalancing of
residues causing adverse TCR-peptide interactions. Such a replacement
could result in an improved interaction of the peptide/MHC complex with
TCR and a subsequent expansion of T cells capable of recognizing the
parental epitope (23, 24). Two lines of evidence support
this conclusion. First, using the parental tetramer to determine the
frequency of tetramer+ precursor T cells in
unstimulated PBMC obtained from normal donors and patients with cancer,
we found that most of the nonresponsive individuals had markedly lower
frequencies of these cells in their peripheral circulation than did the
responders (data not shown).4 Second, the
parental and variant peptides were found to engage and expand T cells
expressing the same TCR in PBMC obtained from responsive and
nonresponsive donors (see Tables III and IV). These findings support
the concept that increased stability of interaction with the TCR is the
basis for the enhanced functional activity of the 7W variant
peptide.
Although the use of variant peptides did reverse the nonresponsiveness
in IVS of PBMC obtained from some donors, their use did not yield
high-affinity CTL. The persistence of low-affinity CTL against self
tumor peptides, such as wt p53 epitopes, which is considered a true
consequence of tolerance (15), might be due to a limited
TCR repertoire being available for recognition of these epitopes. Our
analyses detected the predominant use of only two TCR V families,
V1 and V13.6, being involved in CTL recognition of the wt
p53264–272 epitope in four different donors.
Furthermore, in two different donors, identical usage by the parental
and variant peptide was detected. In contrast, an analysis of responses
in HLA-A2.1+ patients to repeated immunizations
with an anchor position-variant peptide of the melanoma-associated
gp100209–217 epitope demonstrated that the
appearance of higheraffinity T cells was associated with an expansion
of the TCR repertoire rather than an increased oligoclonal response
(33). In the future, additional data on TCR usage of
cross-reactive and variant-specific CTL cell could allow for extensive
molecular modeling of the interactions within the trimeric complexes
and, perhaps, the design of APL with more enhancing properties
than those of the 6T and 7W variants. These variants might engage more
diverse populations of T cells that are capable of cross-recognition of
the parental epitope with, perhaps, higher avidity. However, the
apparent outgrowth of epitope-loss tumors in OSCC patients responsive
to this epitope suggests that even intermediate-affinity CTL
recognizing wt p53264–272 might be effective in
tumor eradication (12).
To fully estimate the potential of p53-based vaccines in immunotherapy
of cancer, it is becoming increasingly apparent that an array of T
cell-defined wt p53 epitopes needs to be analyzed, and strategies for
optimal induction of T cells recognizing these epitopes need to be
further evaluated. In this regard, the use of genetically modified DC
expressing intact wt p53 appears to enhance the generation and increase
the frequency of antitumor effectors from PBMC of normal donors and
cancer patients (34). The p53-based immunotherapy also
might be critically dependent on targeting the right epitopes and
matching a patient’s ability to respond ex vivo to wt p53 epitopes
with the potential of his/her tumor to present these epitopes for
immune recognition. Again, of course, it is necessary to be aware that
a patient’s ex vivo responsiveness to these epitopes does not
guarantee a successful in vivo response to immunization with them. In
this study, HLA-A2.1+ patient 3 with OSCC, for
whom the tumor cell line and tumor-specific CTL are available in the
laboratory, has been of particular interest. The tumor cell line
established from thispatient and designated PCI-13 accumulates
p53 molecules expressing a missense mutation at codon 286 and
naturally presents the p53264–272 epitope,
albeit following pretreatment with IFN- (7, 12). The
ability to generate anti-p53264–272 CTL with
the 7W variant from this patient’s PBMC, which were nonresponsive to
the parental peptide, provides a basis for the potential use of
the 7W variant peptide in immunotherapy of this patient and,
perhaps, other nonresponsive OSCC patients with tumors expressing
similar characteristics. Concurrently, it needs to be determined
whether the trends observed in OSCC patients regarding their
responsiveness to wt p53 epitopes and the potential of their tumors to
present these epitopes are also apparent in patients with other types
of cancers.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Albert B. DeLeo, University of Pittsburgh Cancer Institute, University of Pittsburgh School of Medicine, Division of Basic Research, Biomedical Science Tower W956, 211 Lothrop Street, Pittsburgh, PA 15213. E-mail address: deleo{at}imap.pitt.edu
3 Abbreviations used in this paper: OSCC, oral squamous cell carcinoma; APL, altered peptide ligand; CDR, complementarity-determining region; DC, dendritic cell; IVS, in vitro stimulation; wt, wild type.
4 T. K. Hoffmann, A. Donnenberg, S. Finkelstein, K. Chikamatsu, V. Donnenberg, U. Friebe, E. Appella, A. B. DeLeo, and T. L. Whiteside. Frequencies of tetramer+ T cells specific for the wild type sequence p53264–272 peptide in the circulation of patients with head and neck cancer. Submitted for publication.
Received for publication August 7, 2001. Accepted for publication November 27, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. J. Exp. Med. 179:1109.This article has been cited by other articles:
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  C. Visus, D. Ito, A. Amoscato, M. Maciejewska-Franczak, A. Abdelsalem, R. Dhir, D. M. Shin, V. S. Donnenberg, T. L. Whiteside, and A. B. DeLeo Identification of Human Aldehyde Dehydrogenase 1 Family Member A1 as a Novel CD8+ T-Cell Defined Tumor Antigen in Squamous Cell Carcinoma of the Head and Neck Cancer Res., November 1, 2007; 67(21): 10538 - 10545. [Abstract] [Full Text] [PDF]
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 D. Ito, A. Albers, Y. X. Zhao, C. Visus, E. Appella, T. L. Whiteside, and A. B. DeLeo The Wild-Type Sequence (wt) p5325-35 Peptide Induces HLA-DR7 and HLA-DR11-Restricted CD4+ Th Cells Capable of Enhancing the Ex Vivo Expansion and Function of Anti-wt p53264-272 Peptide CD8+ T Cells J. Immunol., November 15, 2006; 177(10): 6795 - 6803. [Abstract] [Full Text] [PDF]
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P. Riedl, A. Bertoletti, R. Lopes, F. Lemonnier, J. Reimann, and R. Schirmbeck Distinct, Cross-Reactive Epitope Specificities of CD8 T Cell Responses Are Induced by Natural Hepatitis B Surface Antigen Variants of Different Hepatitis B Virus Genotypes J. Immunol., April 1, 2006; 176(7): 4003 - 4011. [Abstract] [Full Text] [PDF]
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M. A. Lyman, C. T. Nugent, K. L. Marquardt, J. A. Biggs, E. G. Pamer, and L. A. Sherman The Fate of Low Affinity Tumor-Specific CD8+ T Cells in Tumor-Bearing Mice J. Immunol., March 1, 2005; 174(5): 2563 - 2572. [Abstract] [Full Text] [PDF]
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 N. Sirianni, P. K. Ha, M. Oelke, J. Califano, W. Gooding, W. Westra, T. L. Whiteside, W. M. Koch, J. P. Schneck, A. DeLeo, et al. Effect of Human Papillomavirus-16 Infection on CD8+ T-Cell Recognition of a Wild-Type Sequence p53264-272 Peptide in Patients with Squamous Cell Carcinoma of the Head and Neck Clin. Cancer Res., October 15, 2004; 10(20): 6929 - 6937. [Abstract] [Full Text] [PDF]
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A. M. Ercolini, J.-P. H. Machiels, Y. C. Chen, J. E. Slansky, M. Giedlen, R. T. Reilly, and E. M. Jaffee Identification and Characterization of the Immunodominant Rat HER-2/neu MHC Class I Epitope Presented by Spontaneous Mammary Tumors from HER-2/neu-Transgenic Mice J. Immunol., April 15, 2003; 170(8): 4273 - 4280. [Abstract] [Full Text] [PDF]
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 J. Hernandez, F. Garcia-Pons, Y. C. Lone, H. Firat, J. D. Schmidt, P. Langlade-Demoyen, and M. Zanetti Identification of a human telomerase reverse transcriptase peptide of low affinity for HLA A2.1 that induces cytotoxic T lymphocytes and mediates lysis of tumor cells PNAS, September 17, 2002; 99(19): 12275 - 12280. [Abstract] [Full Text] [PDF]
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E. Huarte, P. Sarobe, J. Lu, N. Casares, J. J. Lasarte, J. Dotor, M. Ruiz, J. Prieto, E. Celis, and F. Borras-Cuesta Enhancing Immunogenicity of a CTL Epitope from Carcinoembryonic Antigen by Selective Amino Acid Replacements Clin. Cancer Res., July 1, 2002; 8(7): 2336 - 2344. [Abstract] [Full Text] [PDF]
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T. K. Hoffmann, A. D. Donnenberg, S. D. Finkelstein, V. S. Donnenberg, U. Friebe-Hoffmann, E. N. Myers, E. Appella, A. B. DeLeo, and T. L. Whiteside Frequencies of Tetramer+ T Cells Specific for the Wild-Type Sequence p53264-272 Peptide in the Circulation of Patients with Head and Neck Cancer Cancer Res., June 1, 2002; 62(12): 3521 - 3529. [Abstract] [Full Text] [PDF]
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