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*
Division of Clinical Onco-Immunology, Ludwig Institute for Cancer Research, University Hospital, Lausanne, Switzerland;
Ludwig Institute for Cancer Research, Lausanne Branch, University of Lausanne, Epalinges, Switzerland;
Research Unit, Department of Surgery, University of Basel, Basel, Switzerland;
Multidisciplinary Oncology Center, University Hospital, Lausanne, Switzerland; and
¶
Institute of Molecular Medicine, Nuffield Department of Medicine, John Radcliffe Hospital, Headington, Oxford, United Kingdom
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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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These findings have opened new possibilities for Ag-specific immunotherapy of melanoma. The more direct approach consists of the immunization of melanoma patients with the appropriate MHC class I allele with synthetic peptides corresponding to the defined melanoma-derived MHC class I-restricted Ags. However, the immunogenicity of short synthetic peptides corresponding to CTL epitopes in patients may be limited; on the one hand, even those peptides that bind MHC class I molecules with high affinity are generally weak immunogens, probably due to their susceptibility to rapid degradation by serum proteases (4, 5, 6), thus requiring repeated administration of relatively high doses (7, 8). On the other hand, although administration of CTL peptides in adjuvant generally results in expansion of specific CTL, experimental evidence for tolerance induction in this experimental setting has also been reported (9).
For these reasons there is a clear interest in designing alternative immunization protocols for the induction of CTL responses directed against melanoma-associated Ags. One attractive strategy consists of using recombinant viral vectors encoding the sequences of interest either as full genes or as minigenes as immunogens (10, 11, 12, 13, 14, 15, 16). Recombinant viral vectors encoding full-length differentiation Ags have to date shown limited immunogenicity in both mice and humans (10, 11, 17). Different factors may account for this result. These are 1) the existence of a high degree of tolerance to self Ags, 2) suboptimal processing and presentation of the relevant sequences following infection with recombinant vaccinia viruses (rVV),3 and/or 3) the fact that immunodominant peptides from self Ags often bind to MHC with low affinity and are poorly recognized by specific T cells.
We and others have recently identified differentiation Ag-derived peptide analogues carrying single or multiple amino acid substitutions that have improved immunogenicity compared with the parental sequences (18, 19, 20). In particular, we have described Melan-A26–35 immunodominant peptide analogues that exhibit improved binding to HLA-A2, form stable HLA-A2 peptide complexes, and are recognized with improved efficiency by Melan-A-specific CTL. We have also shown that Melan-A26–35 analogues are more efficient than parental Melan-A peptides at inducing Melan-A-specific CTL responses both in vitro and in vivo (21, 22). One of these sequences contains a single leucine to alanine substitution at position 2 of the Melan-A26–35 decapeptide sequence (peptide A27L) (19).
In the present study we have analyzed the impact of introducing the A27L mutation into rVV encoding Melan-A-derived sequences. We observed that introduction of the A27L mutation into a rVV encoding the Melan-A26–35 minigene resulted in a strongly increased immunogenicity in vitro and in vivo. However, when the mutation was introduced into the Melan-A full gene sequence, we observed specific lysis of infected cells but no induction of Melan-A-specific CTL. These findings suggest that both qualitative (the sequence of the peptide Ag generated), and quantitative (the efficiency of Ag processing) factors can be crucial for the generation of efficient recombinant virus vectors. In addition, they indicate that recombinant viruses encoding optimized minigene sequences from tumor Ags may be more effective than minigenes encoding parental sequences for the generation of anti-cancer immune responses.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The generation of the plasmid pGFP/Ub (ubiquitin) that served as
the vector for all constructs used in this study has been described
previously (20), and details are available upon request.
The open reading frame coding for the full-length human Melan-A protein
was obtained by PCR amplification and was inserted between the
SacII and AvaI sites of pGFP/Ub (Fig. 1
, construct I). Constructs II–IV were
obtained by annealing complementary synthetic oligonucleotides.
Ligation of these fragments into pGFP/Ub led to the generation of
plasmids directing the expression of the reference protein GFP-Ub and
the Melan-A fragments spanning aa 27–35 (construct II), aa 26–35
(construct III), or aa 26–35 with an A to L substitution at position
27 (construct IV). A fragment spanning the Melan-A open reading frame,
in which the codon for amino acid A27 was substituted for one encoding
L, was obtained by PCR amplification and was inserted between the
BstEII and AvaI of construct I, resulting in
construct V. The sequence of the critical regions in all constructs was
confirmed by DNA sequencing. Wild-type vaccinia virus (Copenhagen)
strain was a gift from Dr. R. Drillien, (Strasbourg, France). The DNA
fragments spanning GFP-Ub and the Melan-A sequences of constructs I–V
were isolated and inserted into the plasmid pKT1030, placing them under
the control of a mutant vaccinia virus promoter P 7.5
(23). For each construct, the cassette containing the
promoter and insert was subcloned into plasmid pKT 1401. This plasmid
contains flanking sequences homologous to the I4L locus of vaccinia
virus. Recombination into the I4L locus of vaccinia virus was achieved
following the transient dominant gpt selection protocol on CV-1
(American Type Culture Collection, Manassas, VA; CCL 70) and BSC-40
(derivative of BSC-1, American Type Culture Collection CCL26) cells
(14). The rVV encoding the
MAGE-3271–279 peptide was generated by the same
procedure. The MAGE-3 sequence was derived from pGFP/Ub-MAGE-3
described previously (24). After plaque purifications the
recombinant viruses were PCR screened, amplified, and semipurified on
sucrose cushions. Where indicated, the virus was
psoralen-long-wave-UV-inactivated before infection of APC as previously
described (23).
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Tumor cell lines were maintained in culture as previously described (19). The human melanoma cell line Na-8 MEL was provided by Dr. F. Jotereau (Unit 211, Institut National de la Santé et de la Recherche Médicale, Nantes, France). Melanoma cell line Me 290 (HLA-A*0201 and Melan-A+) has been previously described (19). In Ag recognition assays we used the CTL line LAU 203 EAA as effector. This CTL line was derived after in vitro stimulation of CD8+ highly enriched lymphocytes from melanoma patient LAU 203 with the parental decapeptide Melan-A26–35, followed by isolation of A2/Melan-A tetramer-positive cells by tetramer-guided cell sorting and further mitogen-driven expansion (21). In CTL induction experiments CD8+ lymphocytes were positively selected by magnetic cell sorting from PBMC of HLA-A*0201 melanoma patients using a miniMACS device (Miltenyi Biotec, Sunnyvale, CA). From the CD8- fraction CD14+ cells were then similarly selected and used as APCs. After infection with recombinant or wild-type viruses at 10 multiplicity of infection during 2 h, CD14+ cells were extensively washed, irradiated (3000 rad), and mixed with CD8+ responder T cells at a 1:1 ratio in 2 ml of CTL medium containing human rIL-2 (100 U/ml; Glaxo, Geneva, Switzerland; provided by Dr. M. Nabholz, ISREC, Epalinges, Switzerland), and human rIL-7 (10 ng/ml; donated by Sanofi Recherche, Labege, France). Where indicated, synthetic peptides were added at a final concentration of 1 µM. For mouse experiments, the murine EL-4 cell line transfected with the HLA-A*0201/Kb gene (EL-4 A2/Kb transfectants) was provided by Dr. Linda Sherman (Scripps Clinic and Research Foundation, La Jolla, CA). HLA-A*0201/Kb transgenic mice (line 6) were provided by Harlan Sprangue Dawley (Indianapolis, IN). Two- to three-month-old mice were immunized i.p. with 107 PFU rVV encoding the Melan-A minigene 26–35, Melan-A minigene 26–35 A27L, or an irrelevant Ag. Three weeks later, T cells were purified from spleens by passage on nylon wool columns. Purified T cells (2 x 106) were then cultured with 2 x 105 irradiated (10,000 rad) EL-4 A2/Kb cells previously pulsed with peptide Melan-A26–35 A27L (1 µM) for 1 h at 37°C in 12-well cell culture plates in 2 ml of DMEM/10% FCS containing 30 U/ml of IL-2 as previously described (22). Cultures were tested for peptide-specific cytolytic activity after two rounds of weekly stimulation.
Flow cytometric immunofluorescence analysis
Tetramers were synthesized as previously described (25). As the antigenic peptide, the Melan-A26–35 A27L analogue (ELAGIGILTV), which has a higher binding stability to HLA-A*0201, was used. This results in a much more efficient generation of stable tetramer preparations compared with parental peptides. The Melan-A26–35 A27L analogue also has a higher T cell antigenicity and immunogenicity than the natural Melan-A decapeptide EAAGIGILTV or the nonapeptide AAGIGILTV (19). We have recently extensively validated the use of Melan-A A27L-containing tetramers for the visualization and isolation of Melan-A-specific, tumor-reactive CTL from either tumor-infiltrated lymph nodes or peptide-stimulated PBMC of melanoma patients (21, 25). Cells were stained with tetramers (200 ng/sample) in 20 µl of PBS/2% FCS for 20 min at room temperature, then 20 µl of anti-CD8FITC mAb (Becton Dickinson, Basel, Switzerland) was added, and cells were incubated for an additional 30 min at 4°C. Cells were washed once in the same buffer and analyzed by flow cytometry. Data analysis was performed using CellQuest software (Becton Dickinson).
Cytotoxicity and TNF-
release assays
Cytotoxic activity was measured using standard 4-h chromium
release assays as previously reported (19). NA8 melanoma
cells (HLA-A*0201+
Melan-A-) were used as target cells. For mouse
experiments chromium release assays were similarly performed using EL-4
A2/Kb transfectants as target cells. Transient
transfection of NA8-MEL cells used in a TNF- release assay was
measured as previously described (24).
The IFN-
release assay
CD8+ lymphocytes were enriched, stimulated
with rVV, and then cocultured (2 x 105) with either
NA8-MEL or Me 290 melanoma cells at a lymphocyte to stimulator cell
ratio of 2/1 in 96-well round-bottom microplates in 200 µl of
RPMI/10% FCS containing 20 U/ml human IL-2. After overnight incubation
at 37°C, IFN- production was measured in the culture supernatant
using an ELISA as previously described (26).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Vectors encoding either full-length or fragments of Melan-A were
developed based on the previously described UPR constructs (24, 27). Ub is a 76-aa polypeptide that is naturally synthesized as
a linear fusion product either to itself or to other cellular proteins
(28). The UPR constructs exploit this finding and consist
of linear fusion products in which Ub is located between a reference
protein (here the GFP) and the protein of interest (Fig. 1
).
Cotranslational cleavage by Ub-specific proteases after the last
residue of Ub yields two independent proteins in equimolar amounts, the
protein of interest and the reference protein. It is noteworthy that
the use of Ub fusion also bypasses the need for a Met residue at the
N-terminus of minigene products, thus allowing direct generation of the
relevant peptides. The rVVs containing UPR constructs were generated as
described in Materials and Methods.
Infection of Melan-A- melanoma cells with rVV encoding the mutated, but not the parental, Melan-A26–35 minigene results in maximal lysis by Melan-A-specific CTL
To determine whether infection with rVV vectors coding for
parental or mutated Melan-A sequences would render the
Melan-A- HLA-A2+ tumor
line Na-8 MEL susceptible to killing by Melan-A-specific tumor-reactive
CTL, we performed the experiment illustrated in Fig. 2A. We used the polyclonal
monospecific CD8+ T cell line LAU 203 EAA as
effector cells (21). The fine specificity of Ag
recognition of line LAU 293 EAA is similar to that of the majority of
Melan-A-specific CTL previously described (19, 20).
Infection of Na-8 MEL cells with rVV encoding the Melan-A gene protein,
or the nona- or decapeptide minigene sequences resulted in low, but
significant, levels of lysis by the Melan-A-specific CTL line. In
contrast, infection with rVV carrying the
Melan-A26–35 A27L analogue minigene sequence
resulted in efficient lysis at levels comparable to those obtained in
the presence of saturating amounts of exogenously added synthetic
parental peptide Melan-A26–35 or
Melan-A26–35 A27L analogue (data not shown).
Similar results were obtained after transient transfection of NA8-MEL
cells with UPR/Melan-A constructs using measurement of TNF-
secretion by specific CTL (data not shown).
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B, prolonged infection with the various
inactivated rVV did not result in improved CTL recognition. Melan-A-specific tumor-reactive CTL are efficiently induced upon stimulation of PBMC from melanoma patients with autologous APCs infected with rVV encoding the mutated, but not the parental, Melan-A26–35 minigene
Highly enriched CD8+ T cells isolated from
PBMC of melanoma patient LAU 56 were stimulated with autologous APCs
infected with rVV encoding either parental or analogue Melan-A
sequences. As a control, CD8+ T cells were also
stimulated with uninfected autologous APCs alone or in the presence of
synthetic parental Melan-A nona- or decapeptides. Cultures were tested
14 days after stimulation for the presence of
CD8+ A2/Melan-A tetramer+
cells. A2/Melan-A tetramer+ T cells were detected
in freshly isolated CD8+ T cells from patient LAU
56 at a frequency of 0.08% (not shown). This frequency did not
significantly vary after the 2-wk culture period in the absence of
Ag-specific stimulation (Fig. 3). The
frequency of A2/Melan-A tetramer+
CD8+ T cells was not increased over pre-existing
values upon infection with rVV encoding parental Melan-A sequences as
either a full gene or a minigene. In contrast, infection with rVV
encoding the Melan-A26–35 A27L analogue sequence
resulted in a potent expansion of A2/Melan-A
tetramer+ CD8+ T cells
(98-fold increase), superior to the expansion obtained upon stimulation
with 1 µM of the parental peptide Melan-A26–35
(36-fold increase) and similar to that obtained upon stimulation with
the peptide Melan-A26–35 A27L (126-fold
increase). Similar experiments performed with
CD8+-enriched PBMC from three additional melanoma
patients yielded similar results. Nevertheless, some variation was also
found, as patient LAU 97 responded to rVV encoding the Melan-A A27L
analogue sequence less efficiently than after stimulation with the
corresponding synthetic peptide (7- vs 48-fold expansion). Patient LAU
203 also responded to stimulation with rVV containing the parental
minigene, although weakly compared with the response obtained after
stimulation with the rVV containing the mutated minigene (5.7- vs
130-fold expansion). We have recently documented the functional
specificity of A2/Melan-A tetramer+ T cells found
in PBMC cultures from melanoma patients (20). To further
document the ability of Melan-A-specific CTL obtained by stimulation
with rVVs to specifically recognize Melan-A-expressing melanoma cells
we tested IFN- release upon stimulation with Melan-A-expressing and
nonexpressing melanoma cell lines. Background levels of IFN- were
released by the cultures following stimulation with the
Melan-A-negative cell line NA8-MEL. However, significant amounts of
IFN- were produced by the cultures containing increased numbers of
A2/Melan-A tetramer+ cells upon stimulation with
the Melan-A-positive melanoma cell line Me 290. These levels directly
correlated with the percentage of A2/Melan-A
tetramer+ cells present in the culture
(Table I).
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To evaluate the impact of the enhanced stimulatory capacity of rVV
carrying the Melan-A26–35 A27L mutated minigene
on the in vivo immunogenicity of our recombinant viral vectors, we
immunized A2/Kb transgenic mice with rVV carrying
either the parental or the A27L mutated
Melan-A26–35 minigene. Highly enriched T cells
obtained from the spleen of A2/Kb transgenic mice immunized with rVV
encoding the parental Melan-A26–35 sequence
exhibited low to undetectable peptide-specific CTL activity after two
rounds of in vitro stimulation. This specific CTL activity was similar
to that obtained from A2/Kb transgenic mice immunized with an
irrelevant rVV (rVV MAGE-3) and to that of naive mice included as
internal controls (Fig. 4). In contrast,
high levels of peptide-specific lysis were detected for splenic T cells
obtained from mice immunized with rVV carrying the A27L mutated 26–35
minigene.
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The introduction of a single mutation was critical in determining
the immunogenicity of rVV vectors carrying the Melan-A decapeptide
minigene. To determine the impact of introducing the same mutation in
the full gene, we constructed and analyzed the corresponding rVV (Fig. 1, construct V). Infection of NA8-MEL cells with this rVV resulted in
efficient CTL recognition (Table II). However
stimulation of CD8+ T cells from patient LAU 56
with rVV carrying the Melan-A full gene A27L did not result in
expansion of A2/Melan-A tetramer+
CD8+ T cells significantly above background
levels. In addition, only low levels of TNF- were produced by the
CTL line upon stimulation with NA8-MEL cells transiently transfected
with the corresponding UPR/Melan-A construct. These results suggest
that infection with rVV carrying the Melan-A mutated full gene may lead
to Ag expression, but only at lower levels than rVV carrying the
mutated minigene and at levels not sufficient to stimulate the
expansion of specific CTL precursors.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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) was not sufficient to overcome the low immunogenicity of the
rVV encoding the Melan-A full gene. These results suggest that although
optimization of antigenic sequences can dramatically modify the
immunogenic properties of recombinant viral vectors encoding minigenes,
additional factors other than the antigenic sequence itself can
influence the immunogenicity of recombinant viral vectors encoding
full-length genes. Recombinant viral vectors encoding full-length melanocyte differentiation Ags represent, in principle, attractive vaccines. Indeed, this vaccination strategy is based on the endogenous processing and presentation of the whole Ag containing many potential T cell epitopes that could be presented in the context of each individual HLA allele, thus overcoming the need to characterize the patient’s HLA phenotype and immunogenic epitopes. One potential limitation for the use of recombinant viral vectors is the presence of neutralizing Abs either as a result of previous infection with a wild-type form of the vector or postimmunization. However, the availability of numerous vectors may now allow highly effective prime/boosting immunization protocols employing non-cross-reactive vectors. In addition, immunization with recombinant viral vectors could be alternated with other immunization strategies using recombinant viral vector-infected dendritic cells, synthetic peptides, or naked DNA. Unfortunately, vaccination attempts based on in vivo expression, processing, and presentation of whole differentiation Ags (including Melan-A), using recombinant technology, have to date failed to fulfill these expectations (16, 29).
One explanation for this failure could be the existence of a high degree of tolerance to self sequences derived from melanocyte differentiation Ags. The observation that immunization of C57BL/6 mice with recombinant adenovirus encoding human, but not murine, gp100 could protect mice from challenge with murine B16 melanoma (11), favors this hypothesis. However, in humans tolerance to melanocyte differentiation Ags may operate mainly by deleting high affinity specific T cells (30, 31). Indeed, CTL specific for melanocyte-derived differentiation Ags can often be found in both normal donors and melanoma patients. However, they generally recognize the antigenic peptide at concentrations much higher than those required for efficient recognition by T cells specific for virally derived Ags (20, 32). Nevertheless, because target cell lysis generally occurs at Ag densities lower than those required for full T cell activation (including cytokine production and proliferation), relatively low affinity T cells could still be instrumental in destroying Ag-expressing tumor cells, provided that they are efficiently activated and expanded through appropriate immunization protocols.
In this context, the use of modified antigenic peptides exhibiting
improved binding and antigenicity could be fundamental. Indeed,
enhanced peptide analogues of HLA-A2-restricted CTL epitopes derived
from gp100 have recently been shown to be more immunogenic that
parental sequences in HLA-A2 melanoma patients (8, 33). Although the antigenic Melan-A peptide originally described
by Kawakami et al. (34) was indeed the nonapeptide
Melan-A27–35, we have shown that the decapeptide
Melan-A26–35 is recognized more efficiently than
the nonapeptide Melan-A27–35 by the majority of
Melan-A-specific CTL (20, 35). Based on these results, we
designed the Melan-A26–35 A27L analogue
(ELAGIGILTV), which has a higher binding affinity and stability to
HLA-A*0201 as well as a higher T cell antigenicity and immunogenicity
than the natural Melan-A decapeptide EAAGIGILTV or the nonapeptide
AAGIGILTV (19). We have extensively investigated the CTL
response elicited both in vitro and in vivo by the
Melan-A26–35 A27L analogue (19, 21, 22) and found complete cross-reactivity of
Melan-A26–35 A27L analogue elicited CTL with
Melan-A parental peptide sequences. More importantly, analogue-induced
CTL could specifically and efficiently recognize endogenously expressed
Ag on Melan-A-expressing tumor cells. In the present study we further
assessed specific tumor Ag recognition by CTL lines obtained from A2
melanoma patients upon stimulation with either
Melan-A26–35 A27L analogue or autologous APC
infected with rVV encoding Melan-A26–35 A27L
(Table I). Thus, even though the
Melan-A26–35 A27L analogue is obviously not
present at the surface of Melan-A-expressing tumors, the immune
response elicited by stimulation with
Melan-A26–35 A27L efficiently cross-recognize
endogenously expressed Melan-A sequence(s).
We and others (19, 36) have also recently described analogues of the Melan-A27–35 nonapeptide. Substitution of A at position 2 of the nonapeptide with L resulted in highly increased binding to HLA-A2, but loss of recognition by specific CTL. We also designed a nonapeptide analogue bearing an L to A substitution at position 27. This peptide did not exhibit improved binding to A2, but was recognized by specific CTL more efficiently than Melan-A27–35 parental sequence. Thus, in this case the enhanced recognition can be exclusively attributed to an enhanced efficiency of recognition by the TCR (superagonist peptide) that might be expected to vary considerably between TCRs. For these reasons we decided not to use this analogue and choose the Melan-A26–35 A27L for further investigations.
In the present study we have shown that enhanced immunogenicity is also
obtained when the optimized sequence is endogenously expressed by APC
from a minigene construct. It is not immediately clear why introduction
of the same mutation in the context of the Melan-A full-length gene
does not lead to the same result. Previous studies had already
suggested that generation of Ag upon infection with recombinant viral
vectors carrying the Melan-A full-length gene was suboptimal and could
be improved by treatment of infected cells with sodium butyrate and
TNF- (11). In addition, in vitro induction of
Melan-A-specific CTL expansion from PBMC stimulated with recombinant
viral vectors carrying the Melan-A full-length gene has been obtained
only when potent professional APC, such as dendritic cells, were used
(15). In the present study only low levels of specific
target cell lysis and no induction of specific CTL expansion were
observed upon infection with rVV encoding the Melan-A parental
full-length gene.
Melan-A is a membrane protein expressed in melanosomes and endoplasmic reticulum (ER) (37). In the classical pathway for processing of MHC class I-associated peptides, proteins are degraded in the cytosol by cytosolic proteases such as the proteasome to generate peptides that are then transported into the ER where they bind nascent MHC class I molecules (38, 39). Because membrane proteins are normally cotranslationally translocated in the ER, they would, in principle, bypass the cytosolic proteases of this pathway of Ag processing. Thus, degradation of membrane proteins and, consequently, processing and presentation of membrane protein-derived epitopes most likely occurs through alternative degradation pathways that may involve partial or complete proteolysis in the ER (40, 41), such as mistranslation in the cytosol (42, 43) or, as recently shown for tyrosinase, reverse translocation of the intact protein from the ER to the cytosol for proteolysis (44). Although the degradation pathway of Melan-A has not yet been elucidated, it could be of relatively low efficiency. Alternatively, the suboptimal efficiency of Melan-A processing could be inherent to the Melan-A sequence itself. Indeed, the Melan-A immunodominant epitope is located in the putative transmembrane region of the protein, that because of its high hydrophobicity might be not easily accessible to proteases. In favor of this last hypothesis, we have recently observed that reciprocal transplantation of the Melan-A26–35 sequence into the region 270–278 of the MAGE-3 protein resulted in improved Ag recognition of transiently transfected target cells by Melan-A-specific CTL (D. Valmori and F. Lévy, unpublished observations).
In conclusion, the results illustrated in the present study suggest that recombinant viral vectors encoding the Melan-A26–35 A27L analogue sequence can be highly immunogenic and may represent promising tools for immunotherapy of melanoma. In addition, the elucidation of the molecular mechanisms that govern Ag processing of Melan-A protein could in the future contribute to improve the effectiveness of vaccines based on recombinant viral vectors encoding Melan-A full protein.
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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 of Clinical Onco-Immunology, Ludwig Institute, CHUV-BH 19-602, 1011 Lausanne, Switzerland. E-mail:
3 Abbreviations used in this paper: rVV, recombinant vaccinia viruses; Ub, ubiquitin; GFP, green fluorescence protein; UPR, ubiquitin/protein/reference; ER, endoplasmic reticulum.
Received for publication August 2, 1999. Accepted for publication November 1, 1999.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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 A. Lamikanra, Z.-K. Pan, S. N. Isaacs, T.-C. Wu, and Y. Paterson Regression of Established Human Papillomavirus Type 16 (HPV-16) Immortalized Tumors In Vivo by Vaccinia Viruses Expressing Different Forms of HPV-16 E7 Correlates with Enhanced CD8+ T-Cell Responses That Home to the Tumor Site J. Virol., October 15, 2001; 75(20): 9654 - 9664. [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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D. A. Peterson, R. J. DiPaolo, O. Kanagawa, and E. R. Unanue Cutting Edge: A Single MHC Anchor Residue Alters the Conformation of a Peptide-MHC Complex Inducing T Cells That Survive Negative Selection J. Immunol., May 15, 2001; 166(10): 5874 - 5877. [Abstract] [Full Text] [PDF]
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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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