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*
Division of Clinical Onco-Immunology, Ludwig Institute for Cancer Research, Lausanne, Switzerland;
Unité 463, Institut National de la Santé et de la Recherche Médicale, Nantes, France;
Instituto de Parasitología y Biomedicina, Consejo Superior de Investigaciones Científicas, Granada, Spain;
§
Centre Pluridisciplinaire d’Oncologie, CHUV, Lausanne, Switzerland; and
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Ludwig Institute for Cancer Research, Lausanne Branch, University of Lausanne, Epalinges, Switzerland
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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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Melan-A/MART-1 (Melan-A)3 is expressed by most fresh melanoma samples and by about 60% of melanoma cell lines (9, 10). Melan-A-specific CTL have been identified in both PBMC and tumor-infiltrating lymphocytes (TIL) from HLA-A*0201 melanoma patients (10, 11). Nine of 10 independent HLA-A*0201-restricted Melan-A-specific TIL lines were found to recognize the nonapeptide Melan-A27-35 (AAGIGILTV) (11). Melan-A-specific CTL could be induced by stimulation of PBMC from HLA-A*0201 normal donors and melanoma patients with peptide Melan-A27-35 (Refs. 12–15; and S. D’Souza, F. Lejeune, D. Rimoldi, D. Liénard, J.-C. Cerottini, and P. Romero, manuscript in preparation). Since this antigenic peptide appeared to be an immunodominant Melan-A epitope (11, 12), it has been proposed as a target for the development of vaccines in HLA-A*0201 patients with melanoma. More recently, we found that the decapeptide Melan-A26-35 (EAAGIGILTV) was better recognized than the nonapeptide Melan-A27-35 by 4 of 4 tumor-infiltrated lymph nodes (TILN) as well as by 10 of 13 Melan-A-specific CTL clones derived from melanoma patients (16). Whether the Melan-A decapeptide is more immunogenic than the nonapeptide when used in peptide-based vaccines has not yet been determined.
The majority of natural peptides bound to HLA-A*0201 have a restricted size of 9 to 10 amino acids and contain two dominant anchor residues within their sequence: leucine (L) or methionine (M) at position 2, and valine (V) at position 9 (17, 18). Amino acids present at other positions within the peptide may play a role in HLA-A*0201-peptide interactions (19). In particular, the presence of negatively charged residues at position 1 such as glutamic acid (E) or aspartic acid (D) was reported to be associated with poor binding to HLA-A*0201 (19). In this respect, it is noteworthy that both Melan-A antigenic peptides lack the major anchor residue at position 2. Moreover, the Melan-A26-35 peptide has glutamic acid at position 1.
In the current study, we introduced single amino acid substitutions in the Melan-A nonapeptide or decapeptide sequences with the aim of improving peptide binding to HLA-A*0201 and/or recognition by tumor-reactive CTL. This approach led to the identification of a Melan-A26-35 peptide analogue, which exhibited more stable binding to HLA-A*0201 and improved recognition by Melan-A-specific TILN and CTL clones from HLA-A*0201 melanoma patients. In addition, this particular peptide analogue was more efficient than the natural peptides in inducing Melan-A-specific, melanoma-reactive CTL responses in vitro in PBMC from HLA-A*0201 melanoma patients. These results suggest that the efficacy of Melan-A peptide-based immunization of melanoma patients may be enhanced by using such a selected peptide analogue.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Tumor cell lines and EBV-transformed B cell lines were maintained in DMEM (Life Technologies, Gaithersburg, MD) supplemented with 10% FCS, 0.55 mM Arg, 0.24 mM Asn, and 1.5 mM Gln. Melanoma cell lines Me 256, Me 260, Me 275, and Me 290 were established at the Ludwig Institute for Cancer Research, Lausanne Branch, from surgically excised melanoma metastases from patients LAU84, LAU149, LAU50, and LAU203, respectively. CTL line 198NS, which is specific for peptide MAGE-3271-279 presented by HLA-A2, was derived as previously described (20). Melan-A-specific CTL clones M77.80 (Vß3) and M77.86 (Vß14) were derived from TIL of melanoma patient M77 as described (21). Briefly, CTL clones were obtained from limiting dilution cultures in the presence of irradiated autologous tumor cells, EBV-transformed B lymphocytes, PHA, and rIL-2. Clones were derived from wells having a probability of clonality higher than 90% according to single hit Poisson distribution. They were subsequently expanded by plating 5 x 103 cells, every 3 to 4 weeks, into microtiter plates together with irradiated feeder cells (5 x 104 allogenic PBMC and 2 x 104 EBV-transformed-B-LAZ cells). CTL clones 1.13 (Vß14), 7.10 (Vß17), and Mel 1.33 (Vß9) were derived from PBMC of normal HLA-A2 donors (N. Gervois, S. Le Guiner, N. Labarriere, J.-F. Fonteneau, A. S. Beignon, E. Diez, and F. Jotereau, manuscript in preparation). All the clones used in this study efficiently recognized HLA-A*0201-positive Melan-A-expressing tumor cell lines.
Synthetic peptides
Peptides were synthesized by standard solid phase chemistry on a multiple peptide synthesizer (Applied Biosystems, Foster City, CA) by using standard F-moc for transient NH2-terminal protection, and were analyzed by mass spectrometry. All peptides were >90% pure as indicated by analytical HPLC. Lyophilized peptides were diluted in DMSO and stored at -20°C.
HLA-A*0201 binding assay
The peptide binding capacity to HLA-A*0201 was assessed in a functional competition assay based on inhibition of recognition of the antigenic peptide MAGE-3271-279 by the HLA-A*0201-restricted CTL line 198NS (20). Briefly, various concentrations of competitor peptides (50 µl) were incubated with 51Cr-labeled T2 cells (50 µl) (1000 cells/well) for 15 min at room temperature. A suboptimal dose (1 nM) of the antigenic peptide MAGE-3271-279 (50 µl) was then added together with specific CTL (5000 cells/well) (50 µl). Chromium release was measured after a 4-h incubation at 37°C. The concentration of each competitor peptide required to achieve 50% inhibition of target cell lysis was then determined and indicated as [nM] 50%. To facilitate comparison, the relative competitor activity of each peptide was calculated as the [nM] 50% of the unmodified Melan-A nonapeptide AAGIGILTV divided by the [nM] 50% of the competitor peptide.
Assessment of the stability of Melan-A-derived peptides/HLA-A*0201 complexes
The stability of peptide/HLA-A*0201 complexes was assayed using
the TAP-deficient T2 cells. These cells were loaded with peptide by
overnight incubation at room temperature with saturating concentrations
(10 µM) of the different peptides and human ß2 microglobulin (3
µg/ml) (Sigma, Buchs, Switzerland) in serum-free medium (X-VIVO 10;
BioWhittaker, Walkersville, MD). After peptide removal and addition of
emetine (10-4 M; Sigma) to block protein synthesis,
cells were incubated at 37°C for the indicated time periods. For each
time point, an aliquot of cells was stained with mAb BB7.2 (HLA-A2
specific) to measure HLA-A2 Ag expression. Influenza
matrix58-66 peptide, which has been shown to bind to
HLA-A*0201 with high affinity and form stable peptide/HLA-A*0201
complexes (22), was used as an internal standard. Results are expressed
as:
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Cytokines
Human rIL-2 (Glaxo, Geneva, Switzerland) was kindly provided by Dr. M. Nabholz (ISREC, Epalinges, Switzerland) and human rIL-7 was donated by Dr. N. Vita of Sanofi Recherche (Labège, France). One unit of IL-2 is defined as the concentration that gives 50% maximal proliferation of CTLL-2.
Generation of TILN and Melan-A-specific CTL
TILN were generated from tumor-infiltrated lymph nodes of HLA-A*0201+ melanoma patients obtained by surgery at the Centre Pluridisciplinaire d’Oncologie, CHUV, Lausanne, Switzerland. Tumoral lymph node fragments were minced to single cell suspensions and cultured in 24-well tissue culture plates (Costar corporation, Cambridge, MA) in 2 ml of Iscove’s Dulbecco medium supplemented with Asn, Arg, and Gln and 10% pooled human A+ serum in the presence of IL-2 and IL-7 (100 U/ml and 10 ng/ml, respectively). After 2 to 3 wk of cell culture, the TILNs were tested for cytolytic activity and their cell surface phenotype was determined by flow cytometry. The TILN chosen for this study were >90% CD3+CD8+ cells that had cytolytic activity against autologous or HLA-A*0201+ tumor cell lines and recognized the Melan-A(26)27-32 peptides presented by HLA-A*0201. Peptide-specific CTL were generated as previously described (20) with minor modifications. Briefly, PBMC from HLA-A*0201+ melanoma patients were isolated by centrifugation in Ficoll-Paque (Pharmacia, Uppsala, Sweden). After enrichment for CD3+ lymphocytes by treatment with Lympho-Kwik T (One Lambda, Canoga Park, CA), CD8+ T lymphocytes were isolated using a miniMACS device (Miltenyi Biotec GmBH, Sunnyvale, CA). The resulting populations routinely contained >75% CD8+ T cells and were used as responder cell populations. Purified CD8+ T cells were plated at 1 to 2 x 106 cells/well together with 2 x 106 stimulator cells/well in 24-well plates in a total volume of 2 ml of Iscove’s medium supplemented with 10% human serum, Asn, Arg, and Gln (complete medium) in the presence of IL-7 (10 ng/ml) and IL-2 (10 U/ml). Stimulator cells were prepared as follows: 2 x 106 autologous PBMC were incubated for 2 h at 37°C in serum-free medium (X-VIVO 10; BioWhittaker) with a Melan-A-derived peptide (20 µg/ml) and human ß2 microglobulin (3 µg/ml). Peptide-pulsed PBMC were then washed, irradiated (3000 rad), and adjusted to the appropriate volume before addition to the CD8+-enriched responder cell populations. On day 7, cells were restimulated with peptide-pulsed autologous PBMC in complete medium supplemented with IL-2 (10 U/ml). Subsequent restimulations were performed weekly with peptide-pulsed and irradiated autologous PBMC. CTL activity was first tested at the end of the second in vitro restimulation.
Assessment of Ag recognition by TILN, Melan-A-specific CTL clones, and peptide-induced CTL
Ag recognition was assessed using chromium-release assays.
Target cells were labeled with 51Cr for 1 h at
37°C and washed two times. Labeled target cells (1,000 cells in 50
µl) were then added to varying numbers of effector cells (50 µl) in
V-bottom microwells in the presence or absence of 1 µg/ml of the
antigenic peptide (50 µl). In the peptide titration experiments,
target cells (1,000 cells in 50 µl) were incubated in the presence of
various concentrations of peptide (50 µl) for 15 min at room
temperature before the addition of effector cells (50 µl). In each
case the effector cells were preincubated for at least 20 min at 37°C
with unlabeled K562 cells (50,000/well) to eliminate nonspecific lysis
due to NK-like effectors present in stimulated T cell populations.
Chromium release was measured in supernatant (100 µl) harvested after
4 h of incubation at 37°C. The percent specific lysis was
calculated as:
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Analysis of mRNA expression
The analysis of mRNA expression was performed as described (24). Briefly, total cellular RNA was extracted by the guanidine-isothiocyanate/cesium chloride procedure. cDNA synthesis from 2 µg of RNA was accomplished by priming with oligo(dT) and aliquots corresponding to 100 ng of RNA were amplified by 30 cycles of PCR using oligonucleotide primers specific for the gene Melan-A (24). A 10-µl aliquot from each reaction was run on a 2% agarose gel and visualized by ethidium bromide fluorescence. To verify RNA integrity, a 21-cycle PCR assay with primers specific for ß-actin was conducted in each case.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Several overlapping antigenic peptide sequences have been mapped
in the Melan-A protein region containing the immunodominant
HLA-A2-restricted CTL epitope (11). To determine which of these
peptides were recognized by TILN populations derived from two
HLA-A*0201 melanoma patients, we tested the peptides listed in Table I
. In an attempt to minimize Ag-specific
selection in vitro, we used TILN that were cultured for a short time
period in the presence of IL-2 and IL-7 alone. Two melanoma lines (Me
290 and Me 260) were used as a source of Melan-A-expressing target
cells. As shown in Figure 1, both TILN
populations were able to lyse the HLA-A*0201-positive melanoma line Me
290 equally well in the absence or presence of exogenously added
peptide Melan-A26-35. In contrast, the
HLA-A*0201-negative melanoma line Me 260 was not recognized by the two
TILN populations, either in the absence or in the presence of
exogenously added peptide. T2 target cells, which express HLA-A*0201
but not Melan-A, were lysed only in the presence of exogenously added
peptide. These results show that the two TILN populations used in this
study recognized Melan-A-derived epitopes expressed in
HLA-A*0201-positive melanoma cells.
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). In
contrast, peptide Melan-A27-36 (AAGIGITLVI) was poorly
recognized while the partially overlapping peptide
Melan-A32-40 (ILTVILGVL) was not recognized at all. Similar
results have been reported with TILN populations from three other
HLA-A*0201 melanoma patients (16). It thus appears that peptide
Melan-A26-35 is recognized most efficiently by TILN
populations from HLA-A*0201 melanoma patients compared with other
overlapping peptides. Based on these results, we selected peptides
Melan-A26-35 and Melan-A27-35 for further
studies. Binding of Melan-A peptide analogues to HLA-A*0201
In an attempt to identify Melan-A peptides with enhanced binding
activity to HLA-A*0201, we synthesized the peptide analogues listed in
Table II. Peptide analogues
Melan-A27-35 A28L and A28 M contain single amino acid
substitutions corresponding to the HLA-A*0201 dominant anchor residues
at position 2 of the nonapeptide. The same substitutions were
introduced at position 2 of the decapeptide (peptide analogues
Melan-A26-35 A27L and Melan-A26-35 A27M). For
comparison, we also synthesized the analogues Melan-A27-35
A27L and A27 M as well as Melan-A26-35 A28L and A28M. Two
additional peptide analogues were synthesized (peptides
Melan-A26-35 E26F and E26Y) in which E at position 1 of the
decapeptide (described as being associated with less efficient
peptide-HLA-A2 binding) was replaced by an aromatic amino acid (Y or F)
known to favor decapeptide binding to HLA-A2 (19).
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, binding of
the various peptides to HLA-A*0201 was evaluated in a functional
competition assay based on inhibition of specific recognition of
peptide MAGE-3271-279 by the
HLA-A*0201-restricted CTL line 198NS (20). The nonsubstituted
nonapeptide was a relatively weak competitor (50% inhibition at 60
µM) compared with the internal positive control peptide
(Influenza A matrix 58-66 peptide, 50% inhibition at 1
µM) (Fig. 2, Table II). As expected, the HLA-A1-restricted
MAGE-3168-176 peptide did not show competitor activity even
at the highest concentration tested. Substitution of A at position 2 of
the nonapeptide with either L or M (Melan-A27-35 A28L,
A28M) resulted in a 30- to 40-fold increase in competitor activity. In
contrast, substitution of A at position 1 of the nonapeptide with L or
M (Melan-A27-35 A27L, A27M) had no apparent effect. Peptide
Melan-A26-35 was a fourfold better competitor than the
peptide Melan-A27-35. Substitution of A at position 2 of
the decapeptide with L (Melan-A26-35 A27L) resulted in an
additional twofold increase in competitor activity, while substitution
with M (Melan-A26-35 A27M) had no further effect.
Substitution of L or M for A at position 3 of the decapeptide
(Melan-A26-35 A28L, A28M) reduced its competitor activity.
In contrast, substitution of Y or F for E at position 1 (Melan-A E26Y,
E26F) significantly enhanced the competitor activity of the decapeptide
(Table II).
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The stability of complexes formed between HLA-A*0201 and the
different Melan-A peptides was assayed on T2 cells, which lack TAP
function and consequently are defective in properly loading class I
molecules with antigenic peptides generated in the cytosol. The
association of exogenously added peptides with thermolabile, empty
HLA-A2 molecules stabilizes them and results in an increase in the
level of surface HLA-A2 recognizable by conformation-dependent
1/2-specific mAb such as W6/32 or BB7.2 (25). Indeed, overnight
incubation of T2 cells with saturating amounts of HLA-A*0201 binding
peptides and human ß2 microglobulin resulted in increased surface
expression of HLA-A*0201 molecules. After peptide removal and addition
of emetine to inhibit protein synthesis, T2 cells were incubated at
37°C and the amount of HLA-A*0201 molecules remaining at the cell
surface was determined after various incubation times (as illustrated
in Figure 3A for some
peptides). The stability of each peptide/HLA-A*0201 complex was then
normalized relative to that observed for the Influenza A matrix
58-66 peptide/HLA-A*0201 complex (Fig. 3, B–D). HLA-A*0201 complexes formed with
peptides Melan-A27-35 and Melan-A26-35 were
unstable, reaching background levels in less than 1 h of
incubation at 37°C (Fig. 3, A–C).
Similarly, complexes formed with peptides Melan-A27-35 A27L
and A27M dissociated rapidly (Fig. 3B). In contrast,
peptides Melan-A26-35 A27L, A27M (Fig. 3C), E26Y, and E26F (Fig. 3D)
formed complexes that were relatively stable over a 6-h period.
Complexes of intermediate stability were observed with the remaining
peptide analogues tested (Fig. 3, B and
C).
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The relative antigenic activity of the peptide analogues was
quantitated in a standard CTL assay using Melan-A-specific TILN and CTL
clones. A dose-response curve was generated for each peptide analogue
(Fig. 4). The antigenic activity of each
peptide analogue was then calculated relative to that of the
Melan-A27-35 peptide (which was 60 and 30 nM for 50%
maximal lysis by TILN LAU 203 and LAU 132, respectively, Table III). The two Melan-A-specific TILN
populations recognized the reference nonapeptide with a similar
efficiency (Table III). Substitution of A at position 1 of the
nonapeptide with L or M resulted in enhanced peptide recognition of
about 10-fold. Surprisingly, substitution of A at position 2 of the
nonapeptide with either L or M, which strongly increased peptide
binding to HLA-A*0201, resulted in a >50-fold reduction of their
antigenic activity.
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).
To further document these findings, we tested the different
peptides and their analogues for recognition by five independent,
HLA-A*0201-restricted Melan-A-specific CTL clones known to lyse
appropriate melanoma target cells. As shown in Table IV, the CTL clones recognized the peptide
Melan-A27-35 with variable efficiency, as reflected by the
concentration of peptide required to achieve 50% of maximal target
cell lysis (between 15 and 4000 nM). Substitution of A at position 1 of
the nonapeptide with L enhanced peptide recognition by four of the five
clones, whereas a similar substitution at position 2 resulted in a
general loss of antigenic activity. Three of the five CTL clones
recognized peptide Melan-A26-35 more efficiently than
peptide Melan-A27-35. Nevertheless, all five CTL clones
recognized very efficiently the decapeptide analogue containing L at
position 2. In contrast, substitution of A at this position with M
decreased efficiency of peptide recognition to variable degrees.
Moreover, substitution of L for A at position 3 of the decapeptide
resulted in greatly reduced efficiency of recognition by four of the
five clones. Surprisingly, substitution of A at position 3 with
M, in contrast with the findings obtained with TILN, improved peptide
recognition by four of the five clones. Finally, substitutions at
position 1 of Melan-A26-35 resulted in reduced recognition
by several of the clones tested.
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Induction of Melan-A-specific, tumor-reactive CTL by in vitro stimulation with Melan-A peptide analogues
Based on the above-mentioned data, the peptide analogue
Melan-A26-35 A27L was selected for in vitro CTL
induction studies. The ability of this peptide to induce
melanoma-reactive CTL specific for Melan-A was evaluated by stimulating
CD8+-enriched T lymphocytes derived from PBMC of melanoma
patients with autologous irradiated PBMC pulsed with peptide.
Stimulation with Melan-A27-35 and Melan-A26-35
peptides was also performed in each experiment to allow an appropriate
comparison between the CTL responses generated with the natural and the
modified peptides. The results obtained on day 7 after the second
restimulation of CD8+-enriched T cells from melanoma
patient LAU 203 are shown in Figure 5.
Peptide-specific CTL activity was barely detectable in the cultures
stimulated with the natural peptides, whereas the culture stimulated
with the Melan-A26-35 A27L peptide analogue exhibited
strong CTL activity. This activity was directed not only against the
peptide analogue used for stimulation but was also cross-reactive with
the nonsubstituted Melan-A decapeptide. More importantly, the CTLs
induced by stimulation with the Melan-A26-35 A27L peptide
analogue were able to lyse the Melan-A-expressing autologous melanoma
cell line Me 290 (Fig. 5). After three additional rounds of
stimulation, peptide-specific activity as well as tumor reactivity was
also clearly detectable in those cultures stimulated with the natural
peptides (data not shown). Similar results were obtained after
stimulation of PBMC CD8+ T cells from patient LAU 132 (data
not shown).
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HLA-A*0201-restricted Melan-A-specific tumor-reactive CTL were detected
in PBMC cultures from five of eight patients after stimulation with the
Melan-A26-35 A27L peptide analogue. In contrast, none of
the PBMC cultures stimulated with the nonsubstituted Melan-A nona- or
decapeptides exhibited specific cytolytic activity. Together, these
data indicate that the Melan-A26-35 A27L peptide analogue
displays greater immunogenicity than the two parental peptides with
regard to induction in vitro of Melan-A-specific and
tumor-reactive CTL.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the present study, we used short-term-cultured Melan-A-specific HLA*0201-restricted TILN from two melanoma patients. The TILN had been incubated in the presence of cytokines without addition of exogenous antigenic peptides to further define Melan-A peptide sequence(s) recognized by specific, tumor-reactive CTL. Quantitative assessment of peptide recognition indicated that the most efficiently recognized natural peptide was peptide Melan-A26-35, followed by peptide Melan-A27-35, whereas peptides Melan-A27-36 and Melan-A32-40 were not recognized. These results confirm and extend our previous study in which four TILN from HLA-A*0201 melanoma patients were tested for recognition of various Melan-A peptides (16). Similar analyses performed with monoclonal populations of Melan-A-specific, tumor-reactive CTL revealed that the majority (10 of 13 CTL clones tested) recognized more efficiently peptide Melan-A26-35 than peptide Melan-A27-35 (Ref. 16 and this study).
Interestingly, a few Melan-A-specific CTL clones have been shown to recognize peptides Melan-A26-35 and Melan-A27-35 equally well (Refs. 11 and 16, and this study), or peptide Melan-A27-35 better than peptide Melan-A26-35 (11). Altogether, these results suggest a diversity in the fine specificity of recognition by CTL directed against the Melan-A immunodominant epitope. Although surprising, this finding is not without precedent. Indeed, an extensive analysis of Ag recognition by a large panel of H-2Kd-restricted CTL clones directed against a parasite nonapeptide, PbCS 252-260, showed not only cross-reactivity with the octapeptide PbCS 253-260, but also clonal diversity in the efficiency of recognition of the two peptides (28). Additional studies using single Ala-substituted peptide analogues of the Melan-A26-35 decapeptide are in progress to determine the extent of clonal diversity in Melan-A Ag recognition by CTL.
In agreement with a recent report (15), the current study indicates
that peptides Melan-A26-35 and 27-35 have
relatively low binding affinities for HLA-A2. Moreover, complexes
between these peptides and cell-associated HLA-A2 molecules were found
to be unstable at 37°C, with a half-life less than 1 h (Fig. 3).
In the same study mentioned above (15), it was reported that complexes
between peptide Melan-A26-35 and HLA-A2 molecules were less
stable than Melan-A27-35 peptide/HLA-A2 complexes, in
contrast to our own data.
Although the reason for this discrepancy may be due to technical differences in the procedures used to measure the stability of complexes, it is clear that both peptides lack one of the major anchor residues found in the majority of natural peptides associated to HLA-A*0201.
In an attempt to overcome the relatively poor binding of natural Melan-A peptides to HLA-A*0201, we synthesized a series of single amino acid substituted peptide analogues and tested them for 1) binding to HLA-A*0201, 2) ability to form stable peptide/HLA-A*0201 complexes, and 3) recognition by a panel of Melan-A-specific, HLA-A*0201-restricted TILN and CTL clones.
Among the peptide analogues tested, the decapeptide Melan-A26-35 that was substituted with L for A at position 2 (Melan-A26-35 A27L) was found to form stable complexes with cell-associated HLA*0201 molecules and, more importantly, to be recognized more efficiently than the natural decapeptide by TILN (6- to 60-fold) as well as by the Melan-A-specific CTL clones tested (4- to 1000-fold) (Tables III and IV).
Surprisingly, the same amino acid substitution at position 2 of the nonapeptide Melan-A27-35 resulted in a strong reduction in the efficiency of recognition by Melan-A-specific T cells in spite of the enhanced binding affinity to HLA-A2. Although it has been observed in another class I-restricted T cell recognition system that changes in peptide residues buried in the peptide binding cleft can negatively influence recognition by specific CTL (29), it is striking that introduction of L at position 2 in the nonapeptide is deleterious for Ag recognition whereas it has the opposite effect at position 2 in the decapeptide. It is conceivable that when the nonapeptide is engineered to bind with high affinity to the HLA-A2 molecule, it displays a qualitatively different epitope from that displayed by the corresponding high-affinity bound decapeptide analogue. In support of this interpretation is the crystallographic observation that the orientation of the side chains from the central peptide residues relative to the HLA-A2 binding site is similar for four nonapeptide co-crystals, but is nearly the opposite for one decapeptide co-crystal (30). The change in peptide amino acid side chain orientations would be induced by a zig-zag movement in the decapeptide main chain, allowing the longer peptide to fit within the limited size of the HLA-A2 peptide binding cleft.
Based on these results, the immunogenicity of Melan-A26-35 A27L analogue was assessed by its ability to induce CTL from PBMC of HLA-A2+ melanoma patients. Clearly, the Melan-A26-35 A27L peptide analogue was more efficient than the two natural peptides in inducing melanoma-reactive CTL. The enhanced CTL stimulatory capacity of the Melan-A26-35 A27L peptide analogue may have several applications. First, and most important, this peptide analogue could be used as a vaccine able to elicit potent antitumor CTL responses. It has been shown for a viral peptide that analogues with the ability to form long-lived complexes with HLA-A2 molecules were more immunogenic in vitro than their natural counterparts (23). Moreover, a high correlation has been found between overall peptide affinity for MHC class I molecules and in vivo peptide immunogenicity in HLA-A2Kb transgenic mice (31). An even better correlation with the peptide’s ability to form stable HLA-A2 complexes has been reported (22). Improved immunogenicity in HLA-A2Kb transgenic mice has also been reported for analogues of a self-peptide, namely the gp100154–162, displaying both higher affinity and more prolonged complex stability than the natural peptide (32). Of note, one of these analogues incorporated an amino acid substitution at a nonanchor peptide residue (32). Second, the Melan-A peptide analogues may be advantageous in the monitoring of CTL responses in melanoma patients against the natural tumor peptide Ags. In this regard, use of analogues of the peptides gp100 209-217 and gp100 280-288 has been shown to reduce the number of stimulations required to reveal a CTL response in patients immunized with the corresponding natural peptides (33). Finally, analogues with greater immunogenicity than their natural counterparts may be useful in shortening the stimulation time required to obtain the large numbers of peptide-specific effector CTL populations required for adoptive transfer therapy. As a first step toward possible applications of our findings to cancer patients, we are currently assessing the immunogenicity of the Melan-A peptide analogues described here in HLA-A2/Kb transgenic mice (34).
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Danila Valmori, Division d’Onco-Immunologie Clinique, Institut Ludwig, CHUV–BH 19-602, 1011 Lausanne, Switzerland. E-mail address:
3 Abbreviations used in this paper: Melan-A, Melan-A/MART-1; TIL, tumor-infiltrating lymphocyte; TILN, tumor-infiltrated lymph node.
Received for publication October 10, 1997. Accepted for publication October 30, 1997.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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M. A. DeBenedette, D. M. Calderhead, H. Ketteringham, A. H. Gamble, J. M. Horvatinovich, I. Y. Tcherepanova, C. A. Nicolette, and D. G. Healey Priming of a Novel Subset of CD28+ Rapidly Expanding High-Avidity Effector Memory CTL by Post Maturation Electroporation-CD40L Dendritic Cells Is IL-12 Dependent J. Immunol., October 15, 2008; 181(8): 5296 - 5305. [Abstract] [Full Text] [PDF]
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Y. Hou, B. Kavanagh, and L. Fong Distinct CD8+ T Cell Repertoires Primed with Agonist and Native Peptides Derived from a Tumor-Associated Antigen J. Immunol., February 1, 2008; 180(3): 1526 - 1534. [Abstract] [Full Text] [PDF]
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L. Derre, M. Ferber, C. Touvrey, E. Devevre, V. Zoete, A. Leimgruber, P. Romero, O. Michielin, F. Levy, and D. E. Speiser A Novel Population of Human Melanoma-Specific CD8 T Cells Recognizes Melan-AMART-1 Immunodominant Nonapeptide but Not the Corresponding Decapeptide J. Immunol., December 1, 2007; 179(11): 7635 - 7645. [Abstract] [Full Text] [PDF]
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C. Barbey, P. Baumgaertner, E. Devevre, V. Rubio-Godoy, L. Derre, G. Bricard, P. Guillaume, I. F. Luescher, D. Lienard, J.-C. Cerottini, et al. IL-12 Controls Cytotoxicity of a Novel Subset of Self-Antigen-Specific Human CD28+ Cytolytic T Cells J. Immunol., March 15, 2007; 178(6): 3566 - 3574. [Abstract] [Full Text] [PDF]
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K. M. Zirlik, D. Zahrieh, D. Neuberg, and J. G. Gribben Cytotoxic T cells generated against heteroclitic peptides kill primary tumor cells independent of the binding affinity of the native tumor antigen peptide Blood, December 1, 2006; 108(12): 3865 - 3870. [Abstract] [Full Text] [PDF]
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G. Bioley, C. Jandus, S. Tuyaerts, D. Rimoldi, W. W. Kwok, D. E. Speiser, J.-M. Tiercy, K. Thielemans, J.-C. Cerottini, and P. Romero Melan-A/MART-1-Specific CD4 T Cells in Melanoma Patients: Identification of New Epitopes and Ex Vivo Visualization of Specific T Cells by MHC Class II Tetramers J. Immunol., November 15, 2006; 177(10): 6769 - 6779. [Abstract] [Full Text] [PDF]
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D. E. Speiser, P. Baumgaertner, C. Barbey, V. Rubio-Godoy, A. Moulin, P. Corthesy, E. Devevre, P.-Y. Dietrich, D. Rimoldi, D. Lienard, et al. A Novel Approach to Characterize Clonality and Differentiation of Human Melanoma-Specific T Cell Responses: Spontaneous Priming and Efficient Boosting by Vaccination J. Immunol., July 15, 2006; 177(2): 1338 - 1348. [Abstract] [Full Text] [PDF]
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S. Colombetti, T. Fagerberg, P. Baumgartner, L. Chapatte, D. E. Speiser, N. Rufer, O. Michielin, and F. Levy Impact of Orthologous Melan-A Peptide Immunizations on the Anti-Self Melan-A/HLA-A2 T Cell Cross-Reactivity. J. Immunol., June 1, 2006; 176(11): 6560 - 6567. [Abstract] [Full Text] [PDF]
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L. Chapatte, M. Ayyoub, S. Morel, A.-L. Peitrequin, N. Levy, C. Servis, B. J. Van den Eynde, D. Valmori, and F. Levy Processing of Tumor-Associated Antigen by the Proteasomes of Dendritic Cells Controls In vivo T-Cell Responses. Cancer Res., May 15, 2006; 66(10): 5461 - 5468. [Abstract] [Full Text] [PDF]
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P. Baumgaertner, N. Rufer, E. Devevre, L. Derre, D. Rimoldi, C. Geldhof, V. Voelter, D. Lienard, P. Romero, and D. E. Speiser Ex vivo Detectable Human CD8 T-Cell Responses to Cancer-Testis Antigens Cancer Res., February 15, 2006; 66(4): 1912 - 1916. [Abstract] [Full Text] [PDF]
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L. Chapatte, S. Colombetti, J.-C. Cerottini, and F. Levy Efficient Induction of Tumor Antigen-Specific CD8+ Memory T Cells by Recombinant Lentivectors Cancer Res., January 15, 2006; 66(2): 1155 - 1160. [Abstract] [Full Text] [PDF]
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A. Margalit, H. M. Sheikhet, Y. Carmi, D. Berko, E. Tzehoval, L. Eisenbach, and G. Gross Induction of Antitumor Immunity by CTL Epitopes Genetically Linked to Membrane-Anchored {beta}2-Microglobulin J. Immunol., January 1, 2006; 176(1): 217 - 224. [Abstract] [Full Text] [PDF]
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C. L. Smith, F. Mirza, V. Pasquetto, D. C. Tscharke, M. J. Palmowski, P. R. Dunbar, A. Sette, A. L. Harris, and V. Cerundolo Immunodominance of Poxviral-Specific CTL in a Human Trial of Recombinant-Modified Vaccinia Ankara J. Immunol., December 15, 2005; 175(12): 8431 - 8437. [Abstract] [Full Text] [PDF]
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V. Vignard, B. Lemercier, A. Lim, M.-C. Pandolfino, Y. Guilloux, A. Khammari, C. Rabu, K. Echasserieau, F. Lang, M.-L. Gougeon, et al. Adoptive Transfer of Tumor-Reactive Melan-A-Specific CTL Clones in Melanoma Patients Is Followed by Increased Frequencies of Additional Melan-A-Specific T Cells J. Immunol., October 1, 2005; 175(7): 4797 - 4805. [Abstract] [Full Text] [PDF]
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A. Bredenbeck, F. O. Losch, T. Sharav, M. Eichler-Mertens, M. Filter, A. Givehchi, W. Sterry, P. Wrede, and P. Walden Identification of Noncanonical Melanoma-Associated T Cell Epitopes for Cancer Immunotherapy J. Immunol., June 1, 2005; 174(11): 6716 - 6724. [Abstract] [Full Text] [PDF]
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D. Dieckmann, E. S. Schultz, B. Ring, P. Chames, G. Held, H. R. Hoogenboom, and G. Schuler Optimizing the exogenous antigen loading of monocyte-derived dendritic cells Int. Immunol., May 1, 2005; 17(5): 621 - 635. [Abstract] [Full Text] [PDF]
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R. Mortarini, A. Scarito, D. Nonaka, M. Zanon, I. Bersani, E. Montaldi, E. Pennacchioli, R. Patuzzo, M. Santinami, and A. Anichini Constitutive Expression and Costimulatory Function of LIGHT/TNFSF14 on Human Melanoma Cells and Melanoma-Derived Microvesicles Cancer Res., April 15, 2005; 65(8): 3428 - 3436. [Abstract] [Full Text] [PDF]
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N. Schaft, J. Dorrie, P. Thumann, V. E. Beck, I. Muller, E. S. Schultz, E. Kampgen, D. Dieckmann, and G. Schuler Generation of an Optimized Polyvalent Monocyte-Derived Dendritic Cell Vaccine by Transfecting Defined RNAs after Rather Than before Maturation J. Immunol., March 1, 2005; 174(5): 3087 - 3097. [Abstract] [Full Text] [PDF]
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L. Chapatte, C. Servis, D. Valmori, O. Burlet-Schiltz, J. Dayer, B. Monsarrat, P. Romero, and F. Levy Final Antigenic Melan-A Peptides Produced Directly by the Proteasomes Are Preferentially Selected for Presentation by HLA-A*0201 in Melanoma Cells J. Immunol., November 15, 2004; 173(10): 6033 - 6040. [Abstract] [Full Text] [PDF]
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J. Bae, J. A. Martinson, and H. G. Klingemann Heteroclitic CD33 Peptide With Enhanced Anti-Acute Myeloid Leukemic Immunogenicity Clin. Cancer Res., October 15, 2004; 10(20): 7043 - 7052. [Abstract] [Full Text] [PDF]
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Q. Lou, R. J. Kelleher Jr, A. Sette, J. Loyall, S. Southwood, R. B. Bankert, and S. H. Bernstein Germ line tumor-associated immunoglobulin VH region peptides provoke a tumor-specific immune response without altering the response potential of normal B cells Blood, August 1, 2004; 104(3): 752 - 759. [Abstract] [Full Text] [PDF]
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A. Zippelius, P. Batard, V. Rubio-Godoy, G. Bioley, D. Lienard, F. Lejeune, D. Rimoldi, P. Guillaume, N. Meidenbauer, A. Mackensen, et al. Effector Function of Human Tumor-Specific CD8 T Cells in Melanoma Lesions: A State of Local Functional Tolerance Cancer Res., April 15, 2004; 64(8): 2865 - 2873. [Abstract] [Full Text] [PDF]
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N. C. V. Verra, A. Jorritsma, K. Weijer, J. J. Ruizendaal, A. Voordouw, P. Weder, E. Hooijberg, T. N. M. Schumacher, J. B. A. G. Haanen, H. Spits, et al. Human Telomerase Reverse Transcriptase-Transduced Human Cytotoxic T Cells Suppress the Growth of Human Melanoma in Immunodeficient Mice Cancer Res., March 15, 2004; 64(6): 2153 - 2161. [Abstract] [Full Text] [PDF]
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C. M. Coughlin, B. A. Vance, S. A. Grupp, and R. H. Vonderheide RNA-transfected CD40-activated B cells induce functional T-cell responses against viral and tumor antigen targets: implications for pediatric immunotherapy Blood, March 15, 2004; 103(6): 2046 - 2054. [Abstract] [Full Text] [PDF]
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S. Rothenfusser, V. Hornung, M. Ayyoub, S. Britsch, A. Towarowski, A. Krug, A. Sarris, N. Lubenow, D. Speiser, S. Endres, et al. CpG-A and CpG-B oligonucleotides differentially enhance human peptide-specific primary and memory CD8+ T-cell responses in vitro Blood, March 15, 2004; 103(6): 2162 - 2169. [Abstract] [Full Text] [PDF]
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K. Fleischer, B. Schmidt, W. Kastenmuller, D. H. Busch, I. Drexler, G. Sutter, M. Heike, C. Peschel, and H. Bernhard Melanoma-Reactive Class I-Restricted Cytotoxic T Cell Clones Are Stimulated by Dendritic Cells Loaded with Synthetic Peptides, but Fail to Respond to Dendritic Cells Pulsed with Melanoma-Derived Heat Shock Proteins In Vitro J. Immunol., January 1, 2004; 172(1): 162 - 169. [Abstract] [Full Text] [PDF]
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H. Benlalam, B. Linard, Y. Guilloux, A. Moreau-Aubry, L. Derre, E. Diez, B. Dreno, F. Jotereau, and N. Labarriere Identification of Five New HLA-B*3501-Restricted Epitopes Derived from Common Melanoma-Associated Antigens, Spontaneously Recognized by Tumor-Infiltrating Lymphocytes J. Immunol., December 1, 2003; 171(11): 6283 - 6289. [Abstract] [Full Text] [PDF]
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A. Anichini, A. Scarito, A. Molla, G. Parmiani, and R. Mortarini Differentiation of CD8+ T Cells from Tumor-Invaded and Tumor-Free Lymph Nodes of Melanoma Patients: Role of Common {gamma}-Chain Cytokines J. Immunol., August 15, 2003; 171(4): 2134 - 2141. [Abstract] [Full Text] [PDF]
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R. Mortarini, A. Piris, A. Maurichi, A. Molla, I. Bersani, A. Bono, C. Bartoli, M. Santinami, C. Lombardo, F. Ravagnani, et al. Lack of Terminally Differentiated Tumor-specific CD8+ T Cells at Tumor Site in Spite of Antitumor Immunity to Self-Antigens in Human Metastatic Melanoma Cancer Res., May 15, 2003; 63(10): 2535 - 2545. [Abstract] [Full Text] [PDF]
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P.-Y. Dietrich, F.-A. Le Gal, V. Dutoit, M. J. Pittet, L. Trautman, A. Zippelius, I. Cognet, V. Widmer, P. R. Walker, O. Michielin, et al. Prevalent Role of TCR {alpha}-Chain in the Selection of the Preimmune Repertoire Specific for a Human Tumor-Associated Self-Antigen J. Immunol., May 15, 2003; 170(10): 5103 - 5109. [Abstract] [Full Text] [PDF]
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V. Dutoit, P. Guillaume, M. Ayyoub, C. S. Hesdorffer, I. F. Luescher, and D. Valmori Decreased Binding of Peptides-MHC Class I (pMHC) Multimeric Complexes to CD8 Affects Their Binding Avidity for the TCR But Does Not Significantly Impact on pMHC/TCR Dissociation Rate J. Immunol., May 15, 2003; 170(10): 5110 - 5117. [Abstract] [Full Text] [PDF]
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J. S. Gold, C. R. Ferrone, J. A. Guevara-Patino, W. G. Hawkins, R. Dyall, M. E. Engelhorn, J. D. Wolchok, J. J. Lewis, and A. N. Houghton A Single Heteroclitic Epitope Determines Cancer Immunity After Xenogeneic DNA Immunization Against a Tumor Differentiation Antigen J. Immunol., May 15, 2003; 170(10): 5188 - 5194. [Abstract] [Full Text] [PDF]
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M. G. Carrabba, C. Castelli, M. J. Maeurer, P. Squarcina, A. Cova, L. Pilla, N. Renkvist, G. Parmiani, and L. Rivoltini Suboptimal Activation of CD8+ T Cells by Melanoma-derived Altered Peptide Ligands: Role of Melan-A/MART-1 Optimized Analogues Cancer Res., April 1, 2003; 63(7): 1560 - 1567. [Abstract] [Full Text] [PDF]
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V. Pullarkat, P. P. Lee, R. Scotland, V. Rubio, S. Groshen, C. Gee, R. Lau, J. Snively, S. Sian, S. L. Woulfe, et al. A Phase I Trial of SD-9427 (Progenipoietin) with a Multipeptide Vaccine for Resected Metastatic Melanoma Clin. Cancer Res., April 1, 2003; 9(4): 1301 - 1312. [Abstract] [Full Text] [PDF]
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N. Meidenbauer, J. Marienhagen, M. Laumer, S. Vogl, J. Heymann, R. Andreesen, and A. Mackensen Survival and Tumor Localization of Adoptively Transferred Melan-A-Specific T Cells in Melanoma Patients J. Immunol., February 15, 2003; 170(4): 2161 - 2169. [Abstract] [Full Text] [PDF]
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M. Ayyoub, A. Zippelius, M. J. Pittet, D. Rimoldi, D. Valmori, J.-C. Cerottini, P. Romero, F. Lejeune, D. Lienard, and D. E. Speiser Activation of Human Melanoma Reactive CD8+ T Cells by Vaccination with an Immunogenic Peptide Analog Derived from Melan-A/Melanoma Antigen Recognized by T Cells-1 Clin. Cancer Res., February 1, 2003; 9(2): 669 - 677. [Abstract] [Full Text] [PDF]
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I. D. Davis, M. Jefford, P. Parente, and J. Cebon Rational approaches to human cancer immunotherapy J. Leukoc. Biol., January 1, 2003; 73(1): 3 - 29. [Abstract] [Full Text] [PDF]
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S. Mantovani, B. Palermo, S. Garbelli, R. Campanelli, G. Robustelli della Cuna, R. Gennari, F. Benvenuto, E. Lantelme, and C. Giachino Dominant TCR-{alpha} Requirements for a Self Antigen Recognition in Humans J. Immunol., December 1, 2002; 169(11): 6253 - 6260. [Abstract] [Full Text] [PDF]
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V. Rubio-Godoy, V. Dutoit, Y. Zhao, R. Simon, P. Guillaume, R. Houghten, P. Romero, J.-C. Cerottini, C. Pinilla, and D. Valmori Positional Scanning-Synthetic Peptide Library-Based Analysis of Self- and Pathogen-Derived Peptide Cross-Reactivity with Tumor-Reactive Melan-A-Specific CTL J. Immunol., November 15, 2002; 169(10): 5696 - 5707. [Abstract] [Full Text] [PDF]
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S. Mandruzzato, E. Rossi, F. Bernardi, V. Tosello, B. Macino, G. Basso, V. Chiarion-Sileni, C. R. Rossi, C. Montesco, and P. Zanovello Large and Dissimilar Repertoire of Melan-A/MART-1-Specific CTL in Metastatic Lesions and Blood of a Melanoma Patient J. Immunol., October 1, 2002; 169(7): 4017 - 4024. [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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S. Graff-Dubois, O. Faure, D.-A. Gross, P. Alves, A. Scardino, S. Chouaib, F. A. Lemonnier, and K. Kosmatopoulos Generation of CTL Recognizing an HLA-A*0201-Restricted Epitope Shared by MAGE-A1, -A2, -A3, -A4, -A6, -A10, and -A12 Tumor Antigens: Implication in a Broad-Spectrum Tumor Immunotherapy J. Immunol., July 1, 2002; 169(1): 575 - 580. [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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M. S. von Bergwelt-Baildon, R. H. Vonderheide, B. Maecker, N. Hirano, K. S. Anderson, M. O. Butler, Z. Xia, W. Y. Zeng, K. W. Wucherpfennig, L. M. Nadler, et al. Human primary and memory cytotoxic T lymphocyte responses are efficiently induced by means of CD40-activated B cells as antigen-presenting cells: potential for clinical application Blood, May 1, 2002; 99(9): 3319 - 3325. [Abstract] [Full Text] [PDF]
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D. Valmori, V. Dutoit, V. Schnuriger, A.-L. Quiquerez, M. J. Pittet, P. Guillaume, V. Rubio-Godoy, P. R. Walker, D. Rimoldi, D. Lienard, et al. Vaccination with a Melan-A Peptide Selects an Oligoclonal T Cell Population with Increased Functional Avidity and Tumor Reactivity J. Immunol., April 15, 2002; 168(8): 4231 - 4240. [Abstract] [Full Text] [PDF]
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T. Luft, P. Luetjens, H. Hochrein, T. Toy, K.-A. Masterman, M. Rizkalla, C. Maliszewski, K. Shortman, J. Cebon, and E. Maraskovsky IFN-{alpha} enhances CD40 ligand-mediated activation of immature monocyte-derived dendritic cells Int. Immunol., April 1, 2002; 14(4): 367 - 380. [Abstract] [Full Text] [PDF]
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V. Rubio-Godoy, C. Pinilla, V. Dutoit, E. Borras, R. Simon, Y. Zhao, J.-C. Cerottini, P. Romero, R. Houghten, and D. Valmori Toward Synthetic Combinatorial Peptide Libraries in Positional Scanning Format (PS-SCL)-based Identification of CD8+ Tumor-reactive T-Cell Ligands: A Comparative Analysis of PS-SCL Recognition by a Single Tumor-reactive CD8+ Cytolytic T-Lymphocyte Clone Cancer Res., April 1, 2002; 62(7): 2058 - 2063. [Abstract] [Full Text] [PDF]
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 H. Pilch, H. Hohn, K. Freitag, C. Neukirch, A. Necker, P. Haddad, B. Tanner, P. G. Knapstein, and M. J. Maeurer Improved Assessment of T-Cell Receptor (TCR) VB Repertoire in Clinical Specimens: Combination of TCR-CDR3 Spectratyping with Flow Cytometry-Based TCR VB Frequency Analysis Clin. Vaccine Immunol., March 1, 2002; 9(2): 257 - 266. [Abstract] [Full Text] [PDF]
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D. Valmori, C. Scheibenbogen, V. Dutoit, D. Nagorsen, A. M. Asemissen, V. Rubio-Godoy, D. Rimoldi, P. Guillaume, P. Romero, D. Schadendorf, et al. Circulating Tumor-reactive CD8+ T Cells in Melanoma Patients Contain a CD45RA+CCR7- Effector Subset Exerting ex Vivo Tumor-specific Cytolytic Activity Cancer Res., March 1, 2002; 62(6): 1743 - 1750. [Abstract] [Full Text] [PDF]
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 A. Zippelius, M. J. Pittet, P. Batard, N. Rufer, M. de Smedt, P. Guillaume, K. Ellefsen, D. Valmori, D. Lienard, J. Plum, et al. Thymic Selection Generates a Large T Cell Pool Recognizing a Self-Peptide in Humans J. Exp. Med., February 19, 2002; 195(4): 485 - 494. [Abstract] [Full Text] [PDF]
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V. Dutoit, V. Rubio-Godoy, M.-A. Doucey, P. Batard, D. Lienard, D. Rimoldi, D. Speiser, P. Guillaume, J.-C. Cerottini, P. Romero, et al. Functional Avidity of Tumor Antigen-Specific CTL Recognition Directly Correlates with the Stability of MHC/Peptide Multimer Binding to TCR J. Immunol., February 1, 2002; 168(3): 1167 - 1171. [Abstract] [Full Text] [PDF]
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I. Miconnet, S. Koenig, D. Speiser, A. Krieg, P. Guillaume, J.-C. Cerottini, and P. Romero CpG Are Efficient Adjuvants for Specific CTL Induction Against Tumor Antigen-Derived Peptide J. Immunol., February 1, 2002; 168(3): 1212 - 1218. [Abstract] [Full Text] [PDF]
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R. Campanelli, B. Palermo, S. Garbelli, S. Mantovani, P. Lucchi, A. Necker, E. Lantelme, and C. Giachino Human CD8 co-receptor is strictly involved in MHC-peptide tetramer-TCR binding and T cell activation Int. Immunol., January 1, 2002; 14(1): 39 - 44. [Abstract] [Full Text] [PDF]
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H. Terasawa, K.-Y. Tsang, J. Gulley, P. Arlen, and J. Schlom Identification and Characterization of a Human Agonist Cytotoxic T-Lymphocyte Epitope of Human Prostate-specific Antigen Clin. Cancer Res., January 1, 2002; 8(1): 41 - 53. [Abstract] [Full Text] [PDF]
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J.-S. Blanchet, D. Valmori, I. Dufau, M. Ayyoub, C. Nguyen, P. Guillaume, B. Monsarrat, J.-C. Cerottini, P. Romero, and J. E. Gairin A New Generation of Melan-A/MART-1 Peptides That Fulfill Both Increased Immunogenicity and High Resistance to Biodegradation: Implication for Molecular Anti-Melanoma Immunotherapy J. Immunol., November 15, 2001; 167(10): 5852 - 5861. [Abstract] [Full Text] [PDF]
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 D. Rimoldi, K. Muehlethaler, S. Salvi, D. Valmori, P. Romero, J.-C. Cerottini, and F. Levy Subcellular Localization of the Melanoma-associated Protein Melan-AMART-1 Influences the Processing of Its HLA-A2-restricted Epitope J. Biol. Chem., November 9, 2001; 276(46): 43189 - 43196. [Abstract] [Full Text] [PDF]
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S. Tangri, G. Y. Ishioka, X. Huang, J. Sidney, S. Southwood, J. Fikes, and A. Sette Structural Features of Peptide Analogs of Human Histocompatibility Leukocyte Antigen Class I Epitopes that Are More Potent and Immunogenic than Wild-Type Peptide J. Exp. Med., September 17, 2001; 194(6): 833 - 846. [Abstract] [Full Text] [PDF]
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P. Sliz, O. Michielin, J.-C. Cerottini, I. Luescher, P. Romero, M. Karplus, and D. C. Wiley Crystal Structures of Two Closely Related but Antigenically Distinct HLA-A2/Melanocyte-Melanoma Tumor-Antigen Peptide Complexes J. Immunol., September 15, 2001; 167(6): 3276 - 3284. [Abstract] [Full Text] [PDF]
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T. Luft, M. Rizkalla, T. Y. Tai, Q. Chen, R. I. MacFarlan, I. D. Davis, E. Maraskovsky, and J. Cebon Exogenous Peptides Presented by Transporter Associated with Antigen Processing (TAP)-Deficient and TAP-Competent Cells: Intracellular Loading and Kinetics of Presentation J. Immunol., September 1, 2001; 167(5): 2529 - 2537. [Abstract] [Full Text] [PDF]
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V. Rubio-Godoy, V. Dutoit, D. Rimoldi, D. Lienard, F. Lejeune, D. Speiser, P. Guillaume, J.-C. Cerottini, P. Romero, and D. Valmori Discrepancy between ELISPOT IFN-gamma secretion and binding of A2/peptide multimers to TCR reveals interclonal dissociation of CTL effector function from TCR-peptide/MHC complexes half-life PNAS, August 17, 2001; (2001) 181348898. [Abstract] [Full Text] [PDF]
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C. Pinilla, V. Rubio-Godoy, V. Dutoit, P. Guillaume, R. Simon, Y. Zhao, R. A. Houghten, J.-C. Cerottini, P. Romero, and D. Valmori Combinatorial Peptide Libraries as an Alternative Approach to the Identification of Ligands for Tumor-reactive Cytolytic T Lymphocytes Cancer Res., July 1, 2001; 61(13): 5153 - 5160. [Abstract] [Full Text] [PDF]
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M. J. Pittet, A. Zippelius, D. E. Speiser, M. Assenmacher, P. Guillaume, D. Valmori, D. Lienard, F. Lejeune, J.-C. Cerottini, and P. Romero Ex Vivo IFN-{{gamma}} Secretion by Circulating CD8 T Lymphocytes: Implications of a Novel Approach for T Cell Monitoring in Infectious and Malignant Diseases J. Immunol., June 15, 2001; 166(12): 7634 - 7640. [Abstract] [Full Text] [PDF]
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I. Miconnet, I. Coste, F. Beermann, J.-F. Haeuw, J.-C. Cerottini, J.-Y. Bonnefoy, P. Romero, and T. Renno Cancer Vaccine Design: A Novel Bacterial Adjuvant for Peptide-Specific CTL Induction J. Immunol., April 1, 2001; 166(7): 4612 - 4619. [Abstract] [Full Text] [PDF]
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P.-Y. Dietrich, P. R. Walker, A.-L. Quiquerez, G. Perrin, V. Dutoit, D. Liénard, P. Guillaume, J.-C. Cerottini, P. Romero, and D. Valmori Melanoma Patients Respond to a Cytotoxic T Lymphocyte-defined Self-Peptide with Diverse and Nonoverlapping T-Cell Receptor Repertoires Cancer Res., March 1, 2001; 61(5): 2047 - 2054. [Abstract] [Full Text]
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M. V. Dhodapkar, J. W. Young, P. B. Chapman, W. I. Cox, J. F. Fonteneau, S. Amigorena, A. N. Houghton, R. M. Steinman, and N. Bhardwaj Paucity of Functional T-Cell Memory to Melanoma Antigens in Healthy Donors and Melanoma Patients Clin. Cancer Res., December 1, 2000; 6(12): 4831 - 4838. [Abstract] [Full Text]
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P. R. Dunbar, C. L. Smith, D. Chao, M. Salio, D. Shepherd, F. Mirza, M. Lipp, A. Lanzavecchia, F. Sallusto, A. Evans, et al. A Shift in the Phenotype of Melan-A-Specific CTL Identifies Melanoma Patients with an Active Tumor-Specific Immune Response J. Immunol., December 1, 2000; 165(11): 6644 - 6652. [Abstract] [Full Text] [PDF]
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L. Jenne, J.-F. Arrighi, H. Jonuleit, J.-H. Saurat, and C. Hauser Dendritic Cells Containing Apoptotic Melanoma Cells Prime Human CD8+ T Cells for Efficient Tumor Cell Lysis Cancer Res., August 1, 2000; 60(16): 4446 - 4452. [Abstract] [Full Text]
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D. Valmori, V. Dutoit, D. Liénard, D. Rimoldi, M. J. Pittet, P. Champagne, K. Ellefsen, U. Sahin, D. Speiser, F. Lejeune, et al. Naturally Occurring Human Lymphocyte Antigen-A2 Restricted CD8+ T-Cell Response to the Cancer Testis Antigen NY-ESO-1 in Melanoma Patients Cancer Res., August 1, 2000; 60(16): 4499 - 4506. [Abstract] [Full Text]
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J.-L. Chen, P. R. Dunbar, U. Gileadi, E. Jager, S. Gnjatic, Y. Nagata, E. Stockert, D. L. Panicali, Y.-T. Chen, A. Knuth, et al. Identification of NY-ESO-1 Peptide Analogues Capable of Improved Stimulation of Tumor-Reactive CTL J. Immunol., July 15, 2000; 165(2): 948 - 955. [Abstract] [Full Text] [PDF]
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R. Mortarini, A. Borri, G. Tragni, I. Bersani, C. Vegetti, E. Bajetta, S. Pilotti, V. Cerundolo, and A. Anichini Peripheral Burst of Tumor-specific Cytotoxic T Lymphocytes and Infiltration of Metastatic Lesions by Memory CD8+ T Cells in Melanoma Patients Receiving Interleukin 12 Cancer Res., July 1, 2000; 60(13): 3559 - 3568. [Abstract] [Full Text]
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M. Oelke, U. Moehrle, J.-L. Chen, D. Behringer, V. Cerundolo, A. Lindemann, and A. Mackensen Generation and Purification of CD8+ Melan-A-specific Cytotoxic T Lymphocytes for Adoptive Transfer in Tumor Immunotherapy Clin. Cancer Res., May 1, 2000; 6(5): 1997 - 2005. [Abstract] [Full Text]
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N. Gervois, N. Labarriere, S. Le Guiner, M.-C. Pandolfino, J.-F. Fonteneau, Y. Guilloux, E. Diez, B. Dreno, and F. Jotereau High Avidity Melanoma-reactive Cytotoxic T Lymphocytes Are Efficiently Induced from Peripheral Blood Lymphocytes on Stimulation by Peptide-pulsed Melanoma Cells Clin. Cancer Res., April 1, 2000; 6(4): 1459 - 1467. [Abstract] [Full Text]
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T. N. J. Bullock, T. A. Colella, and V. H. Engelhard The Density of Peptides Displayed by Dendritic Cells Affects Immune Responses to Human Tyrosinase and gp100 in HLA-A2 Transgenic Mice J. Immunol., March 1, 2000; 164(5): 2354 - 2361. [Abstract] [Full Text] [PDF]
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M. H. Andersen, L. O. Pedersen, J. C. Becker, and P. t. Straten Identification of a Cytotoxic T Lymphocyte Response to the Apoptosis Inhibitor Protein Survivin in Cancer Patients Cancer Res., February 1, 2000; 61(3): 869 - 872. [Abstract] [Full Text]
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D. Valmori, F. Levy, I. Miconnet, P. Zajac, G. C. Spagnoli, D. Rimoldi, D. Lienard, V. Cerundolo, J.-C. Cerottini, and P. Romero Induction of Potent Antitumor CTL Responses by Recombinant Vaccinia Encoding a Melan-A Peptide Analogue J. Immunol., January 15, 2000; 164(2): 1125 - 1131. [Abstract] [Full Text] [PDF]
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D. Valmori, J.-F. Fonteneau, S. Valitutti, N. Gervois, R. Dunbar, D. Lienard, D. Rimoldi, V. Cerundolo, F. Jotereau, J.-C. Cerottini, et al. Optimal activation of tumor-reactive T cells by selected antigenic peptide analogues Int. Immunol., December 1, 1999; 11(12): 1971 - 1980. [Abstract] [Full Text] [PDF]
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F. Wang, E. Bade, C. Kuniyoshi, L. Spears, G. Jeffery, V. Marty, S. Groshen, and J. Weber Phase I Trial of a MART-1 Peptide Vaccine with Incomplete Freund's Adjuvant for Resected High-Risk Melanoma Clin. Cancer Res., October 1, 1999; 5(10): 2756 - 2765. [Abstract] [Full Text] [PDF]
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D. E. Speiser, M. J. Pittet, D. Valmori, R. Dunbar, D. Rimoldi, D. Lienard, H. R. MacDonald, J.-C. Cerottini, V. Cerundolo, and P. Romero In Vivo Expression of Natural Killer Cell Inhibitory Receptors by Human Melanoma–specific Cytolytic T Lymphocytes J. Exp. Med., September 20, 1999; 190(6): 775 - 782. [Abstract] [Full Text] [PDF]
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A. Anichini, A. Molla, R. Mortarini, G. Tragni, I. Bersani, M. Di Nicola, A. M. Gianni, S. Pilotti, R. Dunbar, V. Cerundolo, et al. An Expanded Peripheral T Cell Population to a Cytotoxic T Lymphocyte (CTL)-defined, Melanocyte-specific Antigen in Metastatic Melanoma Patients Impacts on Generation of Peptide-specific CTLs but Does Not Overcome Tumor Escape from Immune Surveillance in Metastatic Lesions J. Exp. Med., September 6, 1999; 190(5): 651 - 668. [Abstract] [Full Text] [PDF]
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M. J. Pittet, D. Valmori, P. R. Dunbar, D. E. Speiser, D. Lienard, F. Lejeune, K. Fleischhauer, V. Cerundolo, J.-C. Cerottini, and P. Romero High Frequencies of Naive Melan-A/MART-1–specific CD8+ T Cells in a Large Proportion of Human Histocompatibility Leukocyte Antigen (HLA)-A2 Individuals J. Exp. Med., September 6, 1999; 190(5): 705 - 716. [Abstract] [Full Text] [PDF]
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, 
), Me260 (
, 
), and T2 (
, •) by TILN from
HLA-A*0201 melanoma patients was measured in a 4-h
51Cr release assay in the absence (open symbols) or
presence (closed symbols) of exogenously added peptide
Melan-A26-35 (M10, 1 µM).
