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*
Ludwig Institute for Cancer Research, Lausanne Branch, University of Lausanne, Epalinges, Switzerland; and
Division of Clinical Onco-Immunology, Ludwig Institute for Cancer Research, Lausanne Branch, Centre Hospitalier Universitaire Vaudois, Lausanne, 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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The human Melan-A/MART-1 (Melan-A) Ag is a cell lineage-specific tumor Ag that has been independently identified by two groups 3, 4 . Like tyrosinase 5 , gp100 6, 7 , gp75 8 , and other lineage-differentiation Ags, Melan-A is expressed only in melanomas and normal melanocytes. Melan-A is expressed in nearly all melanoma tumor samples and in 60% of melanoma cell lines. Melan-A-specific CTL are frequently found in both PBMC and tumor-infiltrating lymphocytes (TIL)3 from HLA-A*0201 melanoma patients 3, 9 . Four HLA-A*0201-restricted peptides corresponding to Melan-A sequences in positions 27–35, 27–36, 26–35, and 32–40 have been defined and tested for their recognition by CTL lines or clones derived from TIL of melanoma patients and from PBMC of normal HLA-A*0201 donors 9, 10, 11, 12 . While the Melan-A26–35 peptide is recognized more efficiently than Melan-A27–35 by TIL of different melanoma patients, Melan-A27–36 is only poorly recognized and Melan-A32–40 is not recognized at all 11, 12 . These observations suggest that the Melan-A26–35 and Melan-A27–35 peptides are immunodominant in the context of the HLA-A*0201 allotype and thus are potential candidates for the development of melanoma-specific vaccine.
However, although the data on the relative immunogenicity of these two peptides in vitro are controversial 12, 13, 14, 15, 16 , they are poorly immunogenic in vivo 13, 17 . One explanation of the weak immunogenicity of these Melan-A-derived peptides could be their poor binding to HLA class I molecules 18 or the low stability of peptide/MHC complexes 19 . Indeed, the lack of one of the dominant anchor amino acid residues (leucine or methionine) at position 2 of both peptides and the presence of a negatively charged amino acid at position 1 of peptide Melan-A26–35 20 render these two peptides relatively poor binders to HLA-A*0201, although the decapeptide Melan-A26–35 binds slightly more efficiently than the nonapeptide Melan-A27–35 11 . In addition, it has been shown that the complexes formed between HLA-A*0201 and these two peptides are unstable, with a t1/2 lower than 1 h at 37°C 12 .
The improvement of binding capacity and/or peptide/MHC complex
stability may be achieved by substituting anchor residues of the
original poor binder peptide with optimal amino acid residues 11, 12, 21, 22 . It has been reported that gp100-derived peptide analogues with
better binding capacity than their parental counterparts became more
immunogenic in terms of in vivo CTL priming and in vitro recognition by
specific gp100 CTL derived from melanoma patients 21, 22 . In the case
of Melan-A, single amino acid substitutions at position 1 (E
Y or
EF) or 2 (AL or AM) of the natural decapeptide
Melan-A26–35 greatly enhanced binding to HLA-A*0201,
stability of peptide/HLA-A*0201 complexes, and efficiency of
recognition by Melan-A-specific human CTL 11, 12 . Furthermore,
Melan-A peptide analogues are more potent than the natural peptides to
generate tumor-reactive CTL upon in vitro stimulation of PBMC from
melanoma patients 12 . However, the relative immunogenicity of these
peptide analogues compared with the natural ones in vivo remains
unknown. Clearly, an evaluation of the efficiency of peptide analogues
as immunogens in vivo would be useful in terms of designing potent
cancer vaccines for humans.
To address these questions, we have chosen an experimental model of
human class I transgenic mice. Human class I transgenic mouse models
have been successfully used to determine the immunogenicity of human
CTL peptides derived from viral or tumor-associated Ags 23, 24, 25 and to
identify or predict human CTL epitopes 26, 27, 28, 29 . Data gathered from
these studies showed that the immunogenicity of peptides in vivo is
well correlated with the in vitro human CTL activity. Most of the
mentioned reports used HLA-A*0201/Kb transgenic mice. These
transgenic mice express a chimeric MHC class I molecule composed of
human HLA-A*0201 
1 and 2 domains and mouse Kb 3,
transmembrane and cytoplasmic domains 23 . The substitution of the
3 domain of A2.1 molecule with a mouse counterpart provides suitable
interaction sites for the mouse CD8 coreceptor and might also favor
class I MHC function through its binding to the mouse
ß2-microglobulin. Altogether, chimeric class I molecule
transgene expression leads to an increased HLA-A*0201-restricted CTL
responsiveness in mice 30 .
In the present study, the in vivo immunogenicity of human Melan-A natural peptides and peptide analogues was evaluated in HLA-A*0201/Kb transgenic mice. A homologue of the human Melan-A gene is endogenously expressed in the mouse 31 . The mouse putative protein contains 113 amino acids with 68.8% identity to the human one. The two overlapping HLA-A*0201-restricted epitopes map to residues 25–33 and 24–33, which are highly homologous to the human Melan-A27–35 and Melan-A26–35 peptides, respectively, with only one amino acid difference (human Melan-A26–35 = EAAGIGILTV, mouse Melan-A24–33 = EAAGIGILIV). It is conceivable that the endogenous expression of these Melan-A peptides in HLA-A*0201/Kb transgenic mice could influence the TCR repertoire available against human natural Melan-A peptides. Our results demonstrate that human natural peptides were relatively poorly immunogenic, while peptide analogues with modified binding/stability features are more potent immunogens than their parental peptides in vivo. In addition, Melan-A peptide-specific CTL generated in transgenic mice cross-recognized not only the natural Melan-A peptides, but also recognized human melanoma cells in an HLA-A*0201-restricted Melan-A-specific manner.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Mouse EL-4 cells transfected with HLA-A*0201/Kb gene (EL-4.A2/Kb transfectants) 32 were kindly provided by Dr. Linda Sherman (Scripps Clinic and Research Foundation, La Jolla, CA) and maintained in DMEM supplemented with 1% HEPES, 1% strepto-penicillin, 10% heat-inactivated FCS, and 0.5 mg/ml G418. Human melanoma cell lines Me 290 and Me 260 were established at the Ludwig Institute for Cancer Research, Lausanne Branch, from surgically excised melanoma metastases, as described 12 . Human melanoma cell line NA8 3 was kindly provided by Dr. Francine Jotereau (Institut National de la Santé et de la Recherche Médicale, Nantes, France). All human melanoma cells were maintained in RPMI 1640 medium supplemented with 10% FCS.
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 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. The HLA-A*0201-restricted natural and peptide
analogues used in this study are listed in Table I
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Breeding pairs of HLA-A*0201/Kb transgenic mice (Line 6) 23 were kindly provided by Harlan Sprague-Dawley (Indianapolis, IN). Different immunization protocols were used. An IFA-priming protocol described elsewhere 33 was used to generate strong CTL responses in mice. Briefly, HLA-A*0201/Kb transgenic mice were preinjected s.c. with an emulsion of IFA at the base of the tail in a total volume of 50 µl. Three weeks later, these mice were immunized s.c. with Melan-A peptides emulsified in IFA. Ten days after injection, mice were sacrificed and lymphocyte suspensions from draining lymph nodes (LN) were prepared for in vitro stimulation with the relevant peptides. In some experiments, mice were directly immunized (without IFA prepriming) with Melan-A peptides emulsified in SBAS2 adjuvant (provided by Dr. S. Cayphas of SmithKline Beecham, R&D Systems, Rixensart, Belgium). SBAS2 is a mixture of a natural saponin, QS21, and of a detoxified form of LPS, the monophosphoryl lipid A, in an oil water emulsion 34 .
Generation of specific mouse CTL by in vitro stimulation
LN cells (4–5 x 106) were cultured with 2–5 x 105 irradiated (10 krad) EL-4 A2/Kb cells, prepulsed with 1–10 µM of stimulating peptides for 1 h at 37°C, in 24-well cell culture plates in 2 ml of DMEM medium supplemented with 10 mM HEPES, 50 µM ß 2-ME, 10% FCS, and EL-4 cell culture supernatant containing 30 U/ml of IL-2. After one or two rounds of weekly stimulation, the cultured cells were tested for cytolytic activity.
Assessment of in vitro cytolytic activity
Cytolytic activity of specific CTL was determined in a 51Cr release assay. Target cells were labeled with 51Cr for 1 h at 37°C in the presence or absence of tested peptides, then washed and coincubated with effector cells at the indicated lymphocyte to target cell ratio in V-bottom 96-well plates in a total volume of 200 µl DMEM medium. In the Ag titration experiments, labeled target cells were incubated in the presence of various concentrations of peptide for 15 min before the addition of the effector cells. Chromium release was measured in 100 µl of supernatant harvested after 4–6 h of incubation at 37°C. The percentage of specific lysis was calculated as following: % specific lysis = [(experimental release - spontaneous release)/(total release - spontaneous release)] x 100.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the first set of experiments, groups of
HLA-A*0201/Kb transgenic mice were first injected with IFA.
Three weeks later, these mice were immunized s.c. with the relevant
Melan-A peptides listed in Table I
in the presence of a Th peptide P30
derived from tetanus toxin 947–967 35 . Ten days after immunization,
the draining LN cells were stimulated in vitro with the immunizing
peptides. After one round of in vitro stimulation, strong CTL responses
to Melan-A analogues were observed in each peptide analogue-immunized
mice, while no specific response was detected in the natural Melan-A
peptide groups (Fig. 1A).
After the second round of stimulation, CTL activity specific for human
Melan-A27–35 and Melan-A26–35 natural peptide
became detectable and the CTL activity against peptide analogues was
maintained or even increased further (Fig. 1B). No CTL
response against natural peptides or peptide analogues was obtained
with lymphocytes coming from nonimmunized mice regardless of the number
of in vitro stimulations (data not shown). In similar experiments
performed with mouse natural Melan-A peptides, no CTL response was
detected even after three rounds of in vitro stimulation (data not
shown). These results indicate that the human Melan-A natural peptides
or peptide analogues tested are immunogenic in vivo and that peptide
analogues are more potent at inducing specific CTL responses than their
parental counterparts. However, the inability to induce CTL against
mouse peptides suggests that the T cell repertoire specific for these
endogenous peptides has been tolerized in the mouse.
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, CTL
specific for the Melan-A26–35 and
Melan-A27–35 natural peptides were generated after
immunization with all analogues tested, although CTL obtained from
Melan-A26–35 E26A, A27L-immunized mice displayed a
slightly lower natural peptide-specific response. These results
indicate that the analogues used in our study are more immunogenic than
the Melan-A natural peptides in vivo and that CTL generated are able to
cross-react with natural Melan-A peptides.
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Since Melan-A peptide analogues were able to elicit specific CTL
responses, the subsequent experiments were designed to examine the
requirement of a Th peptide for efficient priming with Melan-A
peptides. This would be important for simplifying the Ag preparation
and vaccination procedure. Groups of HLA-A*0201/Kb
transgenic mice were immunized with either Melan-A26–35
natural peptide or Melan-A26–35 A27L peptide analogue, in
presence or absence of the Th peptide P30, in IFA emulsion or mixed in
SBAS2 adjuvant 34 . As shown in Fig. 3,
in the case of immunization with Melan-A26–35 natural
peptide, a specific CTL response could be induced only when Th peptide
P30 was coinjected in IFA and not in SBAS2. In contrast, immunization
with Melan-A26–35 A27L peptide analogue formulated in
either adjuvant-induced specific CTL responses, regardless of the
absence of P30, confirming the strong immunogenicity of this peptide
analogue (Fig. 3, A and B, lower
panels). These results indicate that SBAS2 adjuvant is as equally
effective as IFA for relatively good immunogenic peptides and raise the
possibility to replace IFA by a safer adjuvant for clinical trials.
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The Melan-A peptide-specific CTL were then assessed for their
ability to recognize each of the Melan-A substituted or natural
peptides. Dose-response curves were generated for each combination of
CTL/antigenic peptide. Fig. 4 illustrates
the Ag dose titration curves obtained by specific CTL generated against
Melan-A26–35 natural peptide (Fig. 4A) or
Melan-A26–35 A27L peptide analogue (Fig. 4B),
respectively. Importantly, Melan-A26–35 A27L-specific CTL
efficiently cross-recognized both natural Melan-A peptide and peptide
analogues. Similar results were obtained by using CTL specific for the
other analogues tested. As shown in Table II, the concentration of peptide required
to sensitize the target cells for lysis by each CTL line is variable,
as reflected by the peptide concentration required to achieve 50% of
maximal target cell lysis (8–5000 nM). Interestingly, the immunizing
peptides were not always the most efficient peptides to sensitize the
lysis of target cells by the corresponding CTL line. In fact, among the
seven Melan-A peptides tested, the two peptide analogues,
Melan-A26–35 A27L and Melan-A26–35 E26A,
A27L, were the most potent peptides for inducing lysis of
EL-4.A2/Kb cells by all of the CTL tested, requiring a
peptide concentration 20- to 700-fold lower than
Melan-A26–35 natural peptide. Melan-A26–35
E26A, Melan-A26–35 E26F, and Melan-A26–35
E26Y peptide analogues were slightly more efficient (2- to 40-fold)
than Melan-A26–35 natural peptide to induce the lysis of
target cells, while the nonapeptide Melan-A27–35 was about
5- to 50-fold less efficient. Altogether, the above data demonstrate a
clear cross-reactivity of CTL generated against Melan-A natural or
peptide analogue. They also suggest the existence of a defined
hierarchy in the antigenicity of this group of peptides that appears to
be independent of the immunizing peptide.
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To determine whether the Melan-A peptide-specific CTL generated
from HLA-A*0201/Kb transgenic mice were able to recognize
Melan-A Ag endogenously processed and presented on the cell surface, we
chose the HLA-A*0201-positive and Melan-A-expressing human melanoma
cell line Me 290 as target for the cytolytic assay. As shown in Fig. 5A, CTL induced by
immunization with human natural Melan-A peptide or peptide analogues in
transgenic mice were able to lyse Me 290 cells. No further increase of
target cell lysis was observed when the relevant peptides were added to
Me 290 cells, indicating that maximal tumor reactivity of CTL was
already attained. The tumor lysis was due to the specific recognition
of Melan-A endogenous peptide in the context of HLA-A*0201, since CTL
recognized neither HLA-A*0201-positive Melan-A-negative human melanoma
cell line NA8, unless the relevant peptides were added, nor
HLA-A*0201-negative human melanoma cells Me 260 even in the presence of
peptides (Fig. 5A). Surprisingly, we also observed that the
lysis of Me 290 by the mouse CTL was blocked efficiently by addition of
anti-mouse CD8 mAb, while anti-mouse CD4 or CD28 mAbs did not
influence the CTL activity. Similar results were observed when
EL-4.A2/Kb cells pulsed with the relevant peptide were used
as targets (Fig. 5B). These results suggest that the lysis
of Ag-expressing targets by mouse anti-Melan-A CTL is CD8-dependent
and that the mouse CD8 seems to interact efficiently with the human
HLA-A2 class I molecule.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The consistency of our results with those of the in vitro stimulating capacity of Melan-A peptides for human lymphocytes highlights the relevance of HLA-A*0201/Kb transgenic mice as a model to predict the immunogenicity of human Melan-A peptides and to select within several candidates the most efficient one. Indeed, an extensive overlap between mouse and human CTL repertoires specific for various HLA-A2-restricted Ags has been suggested 37 , even if some diversity has been found in TCR usage in response to the influenza matrix peptide 38 . In addition, Ag processing and presentation machinery in transgenic mice and human cells are sufficiently similar to allow presentation of the same epitopes 18 .
Based on the close correlation between the immunogenicity of peptides and their binding affinity and stability of peptide/MHC complexes 18, 19 , several authors have tried to modify residues involved in MHC binding without altering peptide-TCR contact residues. As a consequence, the amount of peptide-MHC complexes displayed at the surface of APCs in vivo might be increased, resulting in enhanced priming of specific CTL. In mouse models, the efficiency of vaccination with modified CTL peptides has been demonstrated for viral protection 39 , and the immunization of mice with a high affinity H-2Db-binding CTL epitope derived from human papillomavirus induced peptide-specific CTL able to lyse tumor cells in vitro and to protect mice from tumor challenge in vivo 40 . Improved in vitro and in vivo induction of melanoma-reactive CTL with peptides from the gp100 melanoma Ag modified at HLA-A*0201-binding residues has been reported in humans 21, 41 . It has also been shown that the Melan-A26–35 A27L peptide analogue is more immunogenic than the natural decapeptide in vitro in humans 12 . In this study, we show that substituted peptides at position 2 (A27L), at position 1 (E-26F, E-26Y, and E-26A), or at positions 1 and 2 (E26A A27L), defined as good binders to HLA-A2 molecules 11, 12 , are also more immunogenic than the natural peptides in vivo. Moreover, the immunization of HLA-A*0201/Kb transgenic mice with Melan-A26–35 A27L peptide analogue is efficient enough to generate CTL responses in the absence of P30 helper epitope, thereby simplifying the vaccine preparation.
It should be emphasized that besides the critical role of binding affinity and stability of peptide/MHC complexes, the immunogenicity of peptides also depends on the available T cell repertoire. Since the melanoma-associated Ags studied are differentiation Ags endogenously expressed in normal melanocytes, we cannot exclude that the T lymphocytes specific for their derived peptides have been tolerized 30 . During negative selection, T cells specific for dominant peptides and/or expressing a high avidity of recognition are deleted 42 , whereas those specific for subdominant peptides and/or expressing a low avidity of recognition may remain in the periphery 43 . The weaker activity of low avidity T cells can be restored in vitro by adding exogenous IL-2 44 . However, several reports using other transgenic mouse models have shown that specific T lymphocytes may be allowed to go to the periphery and remain immunologically naive or ignorant to the transgene protein 45, 46 . In agreement with the hypothesis of active T cell tolerance against self Ag, we have observed the unresponsiveness to mouse natural Melan-A peptides in HLA-A*0201/Kb mice. This unresponsiveness is not likely due to the immunization protocol or adjuvant effect since vaccination of mice with mouse Melan-A recombinant adenovirus or vaccinia virus also failed to generate mouse Melan-A-specific CTL in contrast to the vaccination with recombinant vaccinia viruses encoding human Melan-A 31 . Interestingly, we have observed that immunization with mouse Melan-A peptide analogues can induce CTL response in those mice (data not shown). Altogether, these data indicate that the modification of natural Melan-A peptides allows to recruit more efficiently the responding CTL precursors remaining in the periphery and to circumvent the specific CTL tolerance againt natural peptides derived from self Ag.
It has been reported that peptide-based vaccines were able to induce strong protective CTL-mediated immunity against tumors in animal models 40, 47, 48, 49, 50, 51, 52 . Interestingly, in our model, CTL induced against Melan-A peptide analogues are able to recognize the natural peptides and to lyse human melanoma cells, and thus might be involved in tumor regression. However, a recent report showed that immunization of melanoma patients with modified gp100 peptides, which are more immunogenic than the natural peptide in vivo, is not accompanied by significant cancer regression 41 . It is possible that Melan-A-specific CTL precursors, which remain in the periphery and are a priori of low avidity 43 , might only be suboptimally activated under the above-mentioned vaccination conditions in vivo, leading to a CTL response quantitatively insufficient for tumor regression. Consistent with this hypothesis, it has been shown that coinjection of gp100-modified peptides together with IL-2 leads to significant tumor regression in a high proportion of immunized melanoma patients 41 . An alternative explanation could be the induction of CTL unresponsiveness following peptide vaccination. In this connection, vaccination with a CTL epitope derived from the early region 1A of the human adenovirus type 5 (Ad5E1A) induced Ad5E1A-specific CTL tolerance, leading to enhanced outgrowth of Ad5E1A-transformed tumors in mice 53 . Different mechanisms have been suggested to account for the tolerance caused by peptide vaccination, including the route of immunization, the dose of peptide 55 , and the effect of certain adjuvants 53, 54, 55 . In this respect, the use of HLA-A2/Kb transgenic mouse model is crucial to determine the potentiality of immunity rather than tolerance induced by peptide-based vaccines. As essential to discriminate these contrasting outcomes will be the ability to follow the fate of specific CTL over time during the course of immunization and of tumor evolution. A promising tool to accomplish this has recently become available that consists of fluorescent A2/Melan-A tetramers 56 .
Although IFA is theoretically efficient at enhancing the immunogenicity of peptides and proteins by activating costimulatory factors and mediating the production of cytokines, specific tolerance rather than immunity following injection of certain peptides in IFA has been reported 53, 55 . In addition, IFA is rather aggressive, frequently causing tissue injury as well as other side effects. Thus, safer and more potent adjuvants need to be found. In the present study, we have shown that SBAS2, which is well tolerated by humans 34 , is a potent alternative adjuvant of CTL response induction. Future studies taking advantage of the HLA-A*0201/Kb transgenic mouse model will enable the assessment of adjuvant activity of different preparations amenable for human use.
Although natural human melanoma cells, which lack the mouse MHC 3
domain, were proposed to be less optimal for recognition by
HLA-A*0201/Kb transgenic mouse CTL 29 , these cells were
efficiently specifically lysed by Melan-A peptide-specific CTL
generated in HLA-A*0201/Kb mice. Moreover, the addition of
anti-CD8 mAb completely blocked the lysis of target cells. Although
we cannot exclude the possibility that an inhibitory signal is induced
by the binding of anti-CD8 mAb to mouse CTL, these results suggest
the interesting possibility that mouse CD8 can efficiently interact
with human class I 3 domains. It should be possible to map precisely
the regions involved in these interactions.
Taken together, the previous observations with human lymphocytes and our current data indicate that peptide analogues derived from Melan-A protein and presented in the context of HLA-A*0201 molecule are attractive candidates for development of peptide-based vaccines and for immunotherapy of melanoma patients. These analogues are more immunogenic than natural peptides in vitro in humans and in vivo in HLA-A*0201/Kb mice and are able to induce CTL efficiently recognizing the natural peptides. The efficiency of Melan-A peptide-based vaccination in mediating protection against tumor growth is currently addressed in HLA-A*0201/Kb transgenic mice in tumor challenge experiments in vivo.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Isabelle Miconnet, Ludwig Institute for Cancer Research, Lausanne Branch, University of Lausanne, Chemin des Boveresses 155, 1066 Epalinges, Switzerland. E-mail address:
3 Abbreviations used in this paper: TIL, tumor-infiltrating lymphocyte; LN, lymph node.
Received for publication September 8, 1998. Accepted for publication December 14, 1998.
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References |
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3 domain of class I influence the magnitude of the xenogeneic response. J. Exp. Med. 170:1091.This article has been cited by other articles:
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  D. Escors, L. Lopes, R. Lin, J. Hiscott, S. Akira, R. J. Davis, and M. K. Collins Targeting dendritic cell signaling to regulate the response to immunization Blood, March 15, 2008; 111(6): 3050 - 3061. [Abstract] [Full Text] [PDF]
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T. N. J. Bullock, D. W. Mullins, T. A. Colella, and V. H. Engelhard Manipulation of Avidity to Improve Effectiveness of Adoptively Transferred CD8+ T Cells for Melanoma Immunotherapy in Human MHC Class I-Transgenic Mice J. Immunol., November 15, 2001; 167(10): 5824 - 5831. [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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R. P. M. Sutmuller, L. R. H. M. Schurmans, L. M. van Duivenvoorde, J. A. Tine, E. I. H. van der Voort, R. E. M. Toes, C. J. M. Melief, M. J. Jager, and R. Offringa Adoptive T Cell Immunotherapy of Human Uveal Melanoma Targeting gp100 J. Immunol., December 15, 2000; 165(12): 7308 - 7315. [Abstract] [Full Text] [PDF]
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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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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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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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