| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
,§
Departments of
*
Surgery,
Chemistry, and
Microbiology, The Beirne Carter Center for Immunology, and
§
Department of Pathology, University of Virginia, Charlottesville, VA 22908
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
A number of gene products have been identified and characterized as sources of immunogenic peptides in human melanoma. These include the MAGE, BAGE, and GAGE gene families (5, 6) and the melanocytic tissue differentiation proteins Pmel-l7/gp100, MART-1/Melan-A, and tyrosinase (7, 8, 9, 10). The tyrosinase gene encodes two peptide sequences that have been demonstrated to be epitopes for two different HLA-A*0201+ CTL clones, and an HLA-B44-restricted tyrosinase peptide has also been reported as a CTL epitope (11, 12, 13). In addition, tyrosinase encodes an epitope recognized by class II MHC-restricted melanoma-reactive CD4+ T cell clones (14). Most of the progress in identifying peptide epitopes for melanoma-reactive CTL has focused on HLA-A*0201-restricted responses; however, over half of the patients do not express HLA-A*0201 (15), and some tumors may lose expression of certain MHC molecules as an immune escape mechanism (16, 17, 18, 19). Thus, it is important to identify immunodominant peptide epitopes for CTL restricted by common MHC molecules other than HLA-A*0201. Two peptide epitopes, derived from MAGE-1 and MAGE-3, have been identified as being presented by HLA-A1 (5, 20, 21), and we have recently reported a Pmel-17/gp100-derived epitope restricted by HLA-A3 (22). However, most of the CTL epitopes for MHC molecules other than HLA-A*0201 remain to be identified.
Pmel-17/gp100, MART-1/Melan-A, and tyrosinase proteins are expressed in the vast majority of human melanomas (8, 23, 24). With the demonstration that peptides from each of these function as shared epitopes for HLA-A*0201-restricted melanoma-reactive CTL, we hypothesized that additional peptides derived from these proteins function as epitopes for melanoma-reactive CTL restricted by other common class I MHC molecules. In the present report we describe reactivities of a panel of six HLA-A1+ CTL lines against these melanocytic tissue differentiation Ags. We have identified a peptide epitope derived from tyrosinase that is recognized by at least two distinct CTL lines, and we have characterized some features of this peptide that have significant implications for its use in clinical tumor vaccine trials and will probably have implications for other MHC-associated peptides epitopes.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The human melanoma cell lines DM6 and DM331 were provided by Drs. H. F. Seigler and T. L. Darrow (Duke University, Durham, NC). The Na8 Mel and Na8 Mel+Tyr cell lines were gifts from Vincent Brichard and Thierry Boon. Na8 Mel is a tyrosinase-negative human melanoma line, and Na8 Mel+Tyr was derived from transfection of Na8 Mel with the tyrosinase cDNA 123.B2 (7). The human melanoma line SkMel24 was obtained from the American Type Culture Collection (Rockville, MD). The human melanoma cell lines VMM12, VMM14, and VMM15 and the fresh tumor cell digests VMM21 and VMM40 were obtained from patients at the University of Virginia (Charlottesville, VA). Immunohistochemical staining of these lines with S-100, HMB-45, and vimentin Abs was characteristic of melanoma. EBV-transformed B lymphoblastoid cell lines (B-LCL)4 were generated from melanoma patients by incubating PBMC in 1 ml of EBV-containing supernatant (from B-958 cells) for 1 h at 37°C and then culturing in RPMI/PHA. The HLA class I transfectants of the C1R cell line (C1R-A1, C1R-A3, C1R-B7, and C1R-B8) were provided by Dr. Peter Cresswell (Yale University, New Haven, CT). The 143B (tk-) and CV-1 lines used in the propagation of vaccinia virus were obtained from the American Type Culture Collection. HLA typing was performed by microcytotoxicity assay on autologous lymphocytes when available (GenTrak, Inc., Plymouth Meeting, PA; Olympus Corp., Lake Success, NY; One Lambda, Canoga Park, CA). Expression of HLA-A1 and -A3 by tumor cells was confirmed by staining with the mAbs HA-A1 (One Lambda, Canoga Park, CA) and GAP-A3 (provided by P. Cresswell). Melanoma-reactive CTL from peripheral blood lymphocytes, from tumor-involved nodes, or from tumor-draining nodes were generated in vitro by repeated stimulation with autologous tumor cells following the detailed protocols previously reported (22, 25). The tissue culture medium used for the human cell lines was RPMI 1640 (Sigma Chemical Co., St. Louis, MO) supplemented with 10% FCS, glutamine, and antibiotics, hereafter referred to simply as RPMI.
Production of recombinant vaccinia viruses expressing full-length versions of the melanocyte differentiation proteins Pmel-17/gp100, tyrosinase, and MART-1/Melan-A
The full-length Pmel-17 cDNA in pcDNA1/neo (Invitrogen, San Diego, CA) was provided by Dr. S. N. Wagner, University of Essen (Essen, Germany). A tyrosinase cDNA was provided by Thierry Boon, Ludwig Institute (Brussels, Belgium). The MART-1/Melan-A gene was PCR cloned and sequenced in our laboratories from the human melanoma line DM6 using standard PCR conditions and the primers (5' to 3') GCG CGG TAC CCT GAC CCT ACA AGA TGC CA and GCG CAA GCT TGT CTC AGG TGT CTC GCT GGC. Each of these three gene sequences was subcloned into the vaccinia cloning vector pSC11.3 (26) adjacent to the vaccinia P7.5 early/late promoter and sequenced to verify proper insertion. Recombinant vaccinia viruses were then produced from these modified vectors using standard methods (27). Purified viral stocks were titrated and tested for proper expression of these three Ags using HLA-A*0201-restricted CTL reactive against the Pmel-17/gp100, MART-1/Melan-A, and tyrosinase epitopes YLEPGPVTA, AAGIGILTV, and YMDGTMSQV, respectively. These CTL lysed nonmelanoma target cells infected with the vaccinia recombinants at levels comparable to the levels of lysis of HLA-A*0201-positive tumor cells, while uninfected targets or targets infected with a vaccinia encoding an irrelevant Ag were not lysed (data not shown).
Synthetic peptides
Peptides were synthesized by standard F-moc chemistry using a Gilson model AMS422 peptide synthesizer (Gilson, Middleton, WI). Biologically active peptides identified at initial screening were purified to >98% by reverse phase HPLC on a Vydac C-4 column with 0.05% trifluoroacetic acid/water and an acetonitrile gradient and then re-evaluated in cytolysis assays.
Cytotoxicity assays
Cell-mediated lysis of target cells was determined using a standard 4-h 51Cr release assay, as described previously (25). Transient expression of individual melanoma differentiation Ags in nonmelanoma cells was accomplished by infecting target cells with recombinant vaccinia viruses using 50 plaque-forming units (titrated on CV-1)/cell for 5 to 7 h at 37°C before 51Cr labeling. Peptide-pulsed targets were generated by diluting peptides in RPMI and preincubating with 51Cr-labeled target cells for 2 h before the addition of CTL. Assay wells containing peptide and target cells but no CTL were used as controls to rule out toxicity of the peptides themselves. Positive controls for specific cytolytic activity consisted of autologous tumor or allogeneic melanoma tumor matched for MHC class I expression. Negative controls consisted of target cells infected with irrelevant recombinant vaccinia viruses or mock infected. Cold target inhibition assays were performed as follows. Labeled or unlabeled target cells were preincubated with peptides (20 µg/ml) for 1 h and then washed. T cells were then preincubated with unlabeled (cold) targets for 1 h before incubation with labeled (hot) targets for 4 h.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Melanoma-reactive CTL lines were generated from human melanoma
patients and tested for reactivity against autologous and allogeneic
melanomas and autologous and allogeneic B-LCL following transient
expression of the melanocyte differentiation proteins Pmel-17/gp100,
MART-1/Melan-A, and tyrosinase. These CTL lines were tested as soon as
residual NK and lymphokine-activated killer activities were low, and
reactivity against autologous melanoma could be demonstrated, typically
at 25 to 40 days in culture. As shown in Table I
, reactivity against tyrosinase-derived
epitopes was observed for five of the six HLA-A1+ CTL
lines. The one HLA-A1+ CTL line that did not demonstrate
reactivity against tyrosinase was derived from a patient whose tumor
line, DM331, does not express tyrosinase or any other defined
melanocyte differentiation
proteins.5 Thus,
tyrosinase-derived epitopes were recognized by CTL from all
HLA-A1-positive patients whose tumors express tyrosinase.
|
Of the HLA-A1+ CTL lines recognizing tyrosinase
epitopes, VMM12 CTL and VMM15 CTL lines were sufficiently available to
permit detailed analysis. To determine which of the class I MHC alleles
was responsible for presentation of tyrosinase epitopes to these two
CTL lines, the tyrosinase-vaccinia constructs were evaluated in C1R
cells transfected with one of the class I MHC alleles expressed on the
patient’s tumor. Following infection with the recombinant vaccinia
virus expressing full-length tyrosinase, the HLA-A1 transfectant of C1R
was recognized and lysed by VMM12 CTL and VMM15 CTL, but HLA-A3, -B7,
and -B8 C1R transfectants were not recognized under identical
conditions (Fig. 1). Thus, HLA-A1 is a
restriction element for the recognition of tyrosinase by CTL lines
VMM12 and VMM15.
|
, lysis
depended on the expression of HLA-A1 and the expression of tyrosinase.
In particular, there is no lysis of the tyrosinase-negative melanoma
Na8 Mel, but there was significant lysis of the tyrosinase-transfected
cell line Na8 Mel+Tyr. Both Na8 Mel and Na8 Mel+Tyr express HLA-A1
(data not shown). The melanoma line DM331, which is HLA-A1+
and tyrosinase-, is not lysed, whereas the
HLA-A1+/tyrosinase+ melanomas HT144 and VMM14
are lysed. VMM12 tumor cells variably lose expression of HLA-A1 and are
not lysed in this assay, but have been lysed by VMM15 CTL in other
assays when HLA-A1 is expressed (data not shown). DM6 is a
tyrosinase+/HLA-A1- melanoma and is not lysed.
In several other assays, SkMel24, another
tyrosinase-/HLA-A1+ melanoma, was not lysed by
this CTL line (data not shown). Thus, tyrosinase expression was both
necessary and sufficient for lysis of HLA-A1+ target cells
by VMM15 CTL.
|
To identify the HLA-A1-restricted peptide epitope(s) recognized by
VMM12 CTL and VMM15 CTL, we scanned the tyrosinase protein sequence for
peptides matching described HLA-A1 binding motifs (30, 31, 32, 33, 34). For
nine-residue peptides, these motifs can be summarized as follows:
threonine, serine, or methionine at position 2; aspartate, glutamate,
alanine, or serine at position 3; and tyrosine at the carboxyl terminus
(position 9). Also included in this evaluation were peptides containing
more than nine residues. Since the C-terminal tyrosine residue appears
to be invariant for HLA-A1 binding peptides, we predicted that longer
peptides would retain the C-terminal tyrosine. In a previous study of
HLA-A*0201-restricted epitopes, we identified a post-translational
modification of tyrosinase resulting in the conversion of an asparagine
residue in an N-linked glycosylation consensus sequence into
aspartate (12). Therefore, in addition to testing the wild-type version
of potential HLA-A1 binding peptides that contained N-linked
glycosylation sites (-N-x-[S/T]-), we also tested variants of these
peptides with aspartate (D) substituted for asparagine (N). With these
considerations, peptides matching this motif and ranging in length from
8 to 12 amino acids were synthesized and tested for their ability to
reconstitute the epitope recognized by VMM15 CTL. None of the first set
of peptides tested had activity; so additional peptides synthesized
included a number that fit the binding motif loosely: containing only
threonine or serine at position 2, only an acidic residue at position
3, or only the C-terminal tyrosine. A total of 119 peptides were
synthesized and evaluated. In one experiment, 3 of 48 peptides tested
were recognized: KCDICTDEY, DAEKCDICTDEY, and EKCDICTDEY, all of which
contained the core nonamer sequence KCDICTDEY (Fig. 3). These three peptides containing the
KCDICTDEY sequence will, collectively, be referred to as
KCDICTDEY-related peptides.
|
These synthetic KCDICTDEY-related peptides were further purified
by HPLC and evaluated in a dose titration assay (Fig. 4). Recognition of C1R-A1 cells pulsed
with each of these peptides was indistinguishable by VMM15 CTL, and at
higher peptide concentrations, targets pulsed with each of these three
peptides were recognized at levels comparable to those of the
autologous tumor cells (Fig. 4). Similarly, we found that lysis of
target cells pulsed with DAEKCDICTDEY (50 µg/ml) can be inhibited by
competition with the wild-type VMM15 tumor cells in a cold target
inhibition assay (data not shown). Thus, the epitope reconstituted by a
KCDICTDEY-related peptide corresponds to the epitope presented
naturally on VMM15 tumor cells. However, the concentration of peptide
required for reconstitution was high, with half-maximal lysis observed
at 2 µg/ml (
2 µM). These peptides are distinguished from most
other CTL epitopes by containing two cysteine residues. Since most
other defined epitopes for melanoma-reactive CTL can be reconstituted
at nanomolar concentrations or lower (10, 21, 35), this raised the
question of whether reconstitution of the A1-associated epitope by
these KCDICTDEY-related peptides was complicated by modifications at
the sulfhydryl groups.
|
By direct evaluation of peptides eluted from HLA-A1 molecules, it was not possible to determine definitively which of the KCDICTDEY-related peptides was naturally processed and presented in association with HLA-A1 (data not shown), suggesting that they are present in low quantity or have been modified. Subsequent studies thus addressed potential modifications of both the nonamer KCDICTDEY and the dodecamer DAEKCDICTDEY. Spontaneous modification of free cysteine residues by disulfide bond formation will occur in medium containing cystine (C-C) or glutathione, such as RPMI or human serum (36). Thus, we examined the effect of substituting serine or alanine for one or both cysteine residues in the synthetic KCDICTDEY-related peptides in an effort to prevent the formation of disulfide bonds.
The synthetic nonamer with the wild-type sequence (KCDICTDEY)
induced half-maximal lysis at 2 to 20 µM. However, when the cysteine
closer to the N-terminus (position 2) was replaced with serine or
alanine (KSDICTDEY or KADICTDEY),
half-maximal lysis was observed after incubation of targets with 100-
to 1000-fold lower concentrations (2 and 20 nM, respectively; Fig. 5A). On the other hand,
peptides with alanine or serine substituted for the cysteine distal to
the N-terminus, at position 5 (KCDISTDEY or
KCDIATDEY) were not discernibly more effective than the
wild-type peptide. Nonamer peptides with alanine or serine substituted
for both cysteine residues were actually less effective than the
wild-type peptide.
|
B). On the other hand, alanine substitution
for the cysteine closer to the C-terminus, at position 8
(DAEKCDIATDEY), prevented recognition at every
concentration tested. These data demonstrate that the more C-terminal
cysteine (residue 247 of tyrosinase) is critical to the formation of
the CTL epitope, raising the possibility that spontaneous modifications
of that residue by disulfide bond formation may be integral to the
actual epitope.
Because of the similar effects on CTL recognition observed with amino
acid substitutions for the more N-terminal cysteine residue in both the
dodecamer DAEKCDICTDEY and the nonamer KCDICTDEY, and because of
the similar dose-titration curves observed for CTL recognition (Fig. 4), we hypothesized that residues 243 to 251 (KCDICTDEY) of both
peptides adopt a similar conformation when bound to HLA-A1. A cold
target inhibition assay was performed with peptide-pulsed target cells
to test that hypothesis. As shown in Figure 6, unlabeled target cells pulsed with the
dodecamer effectively inhibited lysis of 51Cr-labeled
targets pulsed with the nonamer, at a level equivalent to the
inhibition induced by cold targets pulsed with the nonamer itself.
Recognition of labeled targets pulsed with the dodecamer
DAEKSDICTDEY was similarly inhibited by unlabeled targets
pulsed with the nonamer KSDICTDEY (data not shown).
These data suggest that the conformation of the peptide-MHC complex
within the region recognized by the TCR of VMM15 is, therefore, similar
or identical for the nonamer and dodecamer peptides.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The naturally processed epitope may be a nonamer or may be as long as a dodecamer. Peptides containing 12 residues are uncommon among described class I MHC-associated peptides (37), and we are unaware of previously reported examples of dodecamer peptides representing CTL epitopes. Dodecamer peptides have been identified in association with cells containing Ag-processing defects, where a TAP-independent pathway has been implicated in their processing and presentation (38). Also, in cells with normal Ag-processing machinery, dodecamers associated with HLA-A1 and HLA-A11 have been described (37). A dodecamer from cytochrome c oxidase that is associated with HLA-A1 contains putative anchor residues at positions 3 and 12 (YTDYGGLIFNSY) (37). Thus, that peptide is predicted to bind to the HLA-A1 molecule with a kink between residues 3 and 12 to allow for the extra length. The synthetic DAEKCDICTDEY peptide, however, could potentially bind to HLA-A1 in either of two conformations. While the tyrosine residue at the C terminus is almost certain to function as the C-terminal anchor residue, the motif requirement for an acid residue at position 3 could be satisfied either by the glutamic acid (E) at position 3 or by the aspartic acid (D) at position 6. In the former case, the central portion of the peptide would have to be kinked and would extend out of the plane of the binding groove, a conformation that could be stabilized or induced by a disulfide bond between the two cysteine residues. If, however, the aspartic acid residue at position 6 functions as an anchor residue, the three N-terminal residues (DAE) would be expected to extend up and out of the binding groove, while the rest of the peptide interacts in the groove in a conformation resembling that of the nonamer peptide.
If the dominant conformation of the dodecamer is kinked, then it would be anticipated that CTL recognition would be sensitive to the peptide structure between residues 5 and 12, and that CTL recognizing that peptide would not cross-react with target cells pulsed with the synthetic nonamer lacking the N-terminal DAE residues. However, CTL reactive against the dodecamer were equally reactive against both the decamer EKCDICTDEY and the nonamer KCDICTDEY. While these studies were performed with a CTL line, which probably contained distinct CTL subpopulations, our cold target inhibition assays provide evidence that both the nonamer and dodecamer peptides are recognized by the same CTL. We have also observed that dodecamers containing glycine substituted for alanine or glutamate at positions 2 and 3 were recognized at levels comparable to those of the wild-type peptide (data not shown). Therefore, the nonamer and dodecamer peptides probably exist in similar conformations when bound to HLA-A1, such that the three amino-terminal residues of the dodecamer should extend out of the binding groove, and residues 4 through 12 would be expected to lie flat, in a manner analogous to the configuration expected for the nonamer peptide.
Regardless of the precise length and conformation of the peptides bound
to the MHC, the -KCDICTDEY-related peptides are unusual in containing
two cysteine residues. In general, sulfhydryl groups on cysteine
residues are susceptible to derivatization with sulfhydryl groups on
other biologic molecules such as cystine or glutathione, both of which
are present in human serum and RPMI (36). Especially since formation of
disulfide bonds is favored at mildly basic pH and in the presence of
oxygen, both the in vivo setting (pH 7.4, pO2 40–100 mm
Hg) and the standard culture conditions in vitro would favor
modification of cysteine residues with exposed side chains. To assess
the roles of the two cysteine residues in the KCDICTDEY-related
peptides with respect to T cell recognition, a series of experiments
was performed comparing the naturally occurring peptides with peptides
containing amino acid substitutions. Substitution of serine or alanine
for the more N-terminal cysteine prevents potential modifications of
that residue, including formation of an internal disulfide bond. After
making such a substitution, both the dodecamer and the nonamer were
capable of reconstituting CTL epitopes at concentrations 2 to 3 logs
lower than the unsubstituted peptides. Thus, we hypothesize that
disulfide bonds involving this more N-terminal cysteine residue may
negatively affect binding of exogenous peptide to the HLA-A1 molecule,
and that in the naturally processed peptide, the cysteine residue at
that position (position 2 of KCDICTDEY, residue 244 of tyrosinase) is
not modified, perhaps being protected within the binding groove of
HLA-A1. Similarly, the more C-terminal cysteine would be anticipated to
point upward and to have an exposed side chain. In cystine- and
glutathione-rich medium, such as RPMI (in vitro) or human serum (in
vivo), the more C-terminal cysteine may well be modified by
cysteinylation or by derivatization with another molecule containing a
sulfhydryl group. T cell recognition of that epitope may depend on such
a modification. Indeed, as shown in Figure 5, recognition by VMM15 CTL
is acutely sensitive to the presence of a cysteine residue at this
position (KSDICTDEY vs
KSDISTDEY).
Peptides load onto MHC molecules in the oxidative environment of the endoplasmic reticulum (ER), where modification of cysteine residues by reversible formation of disulfide bonds would be expected (39). However, at least one enzyme in the ER, protein disulfide isomerase, can catalyze dissociation of disulfide bonds (40). We predict that those peptides not containing internal disulfide bonds or other modifications of the more N-terminal cysteine are those capable of binding to the nascent MHC molecules for presentation at the cell surface. Whether there are specific molecular chaperones favoring this process in the ER remains to be demonstrated.
Aside from what happens during normal Ag processing and presentation, synthetic peptides free in solution are susceptible to modifications of cysteine residues by formation of disulfide bonds, especially in the presence of oxygen or in slightly basic conditions (e.g., exposed to air or in vivo). Because of the proximity of the two cysteine residues, the likelihood of interaction between them probably exceeds the likelihood of interaction with another molecular species in solution. In fact, intramolecular disulfide bonds between cysteine residues in short peptides containing the sequence -CXYC- are highly favored at concentrations up to 200 µM in neutral pH solutions and are facilitated by air oxidation of the cysteine residues (28). The resulting cyclized form of KCDICTDEY-related peptides may not be capable of binding to the HLA-A1 molecule or, if bound, would probably result in a molecular conformation different from that of the epitope recognized by CTL. The fact that cyclization and other cysteine modifications occur readily may well explain the high concentration of KCDICTDEY-related peptides required for sensitization of C1R-A1 target cells in vitro and would imply similar results in vivo if a tumor vaccine using the wild-type peptide were administered.
On the other hand, peptides containing substitutions of serine or
alanine for the more N-terminal cysteine induced half-maximal lysis of
target cells at 1 to 20 nM, which compares favorably to peptide
concentrations required for reconstitution of the MAGE-1 epitope
EADPTGHSY on HLA-A1 (half-maximal lysis at 10 nM) (5) and the MART-1
peptide AAGIGILTV on HLA-A*0201 (half-maximal lysis at 1–100 nM)
(35). We believe that replacement of the more N-terminal cysteine
probably enables purified peptides to bind to cell surface MHC
molecules in a manner that more accurately mimics the configuration of
peptides binding to nascent MHC molecules in the ER. Ideally, we would
like to be able to evaluate directly the HLA-A1 binding affinity for
the KCDICTDEY-related peptides and for the substituted peptides.
However, these peptides are so susceptible to modification in medium by
cyclization, dimerization, or polymerization that the effective
concentration of free peptide cannot be expected to match the total
measured concentration. Thus, existing methods for measuring binding
affinity of these peptides can be expected to be misleading, and
specialized methods for working with these peptides are required.
In summary, at least three peptides containing the KCDICTDEY sequence are capable of reconstituting an epitope for HLA-A1-restricted melanoma-reactive CTL. Modifications of the sulfhydryl group of the more C-terminal cysteine residue by cysteinylation or by derivatization with glutathione are predicted to occur in vitro and in vivo, and these modifications may well be required for T cell recognition. Modifications of the more N-terminal cysteine residue, however, are not believed to occur in vivo during normal processing and presentation. However, they may occur when the peptide is free in solution, and modifications such as intramolecular disulfide bond formation leading to cyclization of the peptide probably inhibit markedly the loading of that peptide on HLA-A1 at the cell surface.
As demonstrated by our data with the wild-type and substituted forms of KCDICTDEY-related peptides, cysteine residues can both positively and negatively affect MHC binding and/or T cell recognition, a finding corroborated by another recent study (29). This suggests that potential problems with using cysteine-containing peptides as immunogens may be overcome by modifications of those cysteine residues, where those modifications are based on an understanding of interactions with the medium components, with the MHC, and with the TCR. In this particular example, immunization with a peptide containing serine or alanine in place only of the more N-terminal cysteine may result in more effective loading onto MHC molecules of APC.
As more peptide epitopes for melanoma-reactive CTL are identified, there is growing evidence for the important role of those peptides derived from the melanocytic tissue differentiation proteins Pmel-17/gp100, MART-1, and tyrosinase. The biochemical interactions of these peptides with other molecules in vitro and in vivo may affect interactions with the MHC molecules and with the TCR. Design of peptide-based immune therapies must take into account these potential interactions, which are probably even more complex in vivo than they are in vitro.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Current address: University of Oxford, Molecular Immunology Group, Institute of Molecular Medicine, John Radcliffe Hospital, Oxford, United Kingdom OX3 9DU.
3 Address correspondence and reprint requests to Dr. Craig L. Slingluff, Jr., Department of Surgery, University of Virginia, P.O. Box 10005, Charlottesville, VA 22906. E-mail address:
4 Abbreviations used in this paper: B-LCL, Epstein-Barr virus-transformed B lymphoblastoid cell line; ER, endoplasmic reticulum.
5 C. L. Slingluff, Jr., T. A. Colella, D. D. Graham, J. C. A. Skipper, L. Brinkerhoff, D. J. Kittlesen, D. H. Deacon, N. L. Harthun, E. L. Huczko, T. L. Darrow, and V. H. Engelhard. Immune escape due to concordant loss of multiple melanocytic differentiation proteins may be overcome by CTL recognizing unique Ags. Submitted for publication.
Received for publication March 26, 1997. Accepted for publication November 6, 1997.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
This article has been cited by other articles:
|
|
 |
|
  M. Ostankovitch, M. Altrich-VanLith, V. Robila, and V. H. Engelhard N-Glycosylation Enhances Presentation of a MHC Class I-Restricted Epitope from Tyrosinase J. Immunol., April 15, 2009; 182(8): 4830 - 4835. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. L. Slingluff Jr., G. R. Petroni, K. A. Chianese-Bullock, M. E. Smolkin, S. Hibbitts, C. Murphy, N. Johansen, W. W. Grosh, G. V. Yamshchikov, P. Y. Neese, et al. Immunologic and Clinical Outcomes of a Randomized Phase II Trial of Two Multipeptide Vaccines for Melanoma in the Adjuvant Setting Clin. Cancer Res., November 1, 2007; 13(21): 6386 - 6395. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. R. Lindsey, L. Gritz, R. Sherry, A. Abati, P. A. Fetsch, L. C. Goldfeder, M. I. Gonzales, K. A. Zinnack, L. Rogers-Freezer, L. Haworth, et al. Evaluation of Prime/Boost Regimens Using Recombinant Poxvirus/Tyrosinase Vaccines for the Treatment of Patients with Metastatic Melanoma Clin. Cancer Res., April 15, 2006; 12(8): 2526 - 2537. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. M. Luiten, E. W.M. Kueter, W. Mooi, M. P.W. Gallee, E. M. Rankin, W. R. Gerritsen, S. M. Clift, W. J. Nooijen, P. Weder, W. F. van de Kasteele, et al. Immunogenicity, Including Vitiligo, and Feasibility of Vaccination With Autologous GM-CSF-Transduced Tumor Cells in Metastatic Melanoma Patients J. Clin. Oncol., December 10, 2005; 23(35): 8978 - 8991. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. J. Wettstein, N. D. Borson, J. G. Park, K. T. McNallan, and A. M. Reed Cysteine-Tailed Class I-Binding Peptides Bind to CpG Adjuvant and Enhance Primary CTL Responses J. Immunol., September 15, 2005; 175(6): 3681 - 3689. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. V. Yamshchikov, D. W. Mullins, C.-C. Chang, T. Ogino, L. Thompson, J. Presley, H. Galavotti, W. Aquila, D. Deacon, W. Ross, et al. Sequential Immune Escape and Shifting of T Cell Responses in a Long-Term Survivor of Melanoma J. Immunol., June 1, 2005; 174(11): 6863 - 6871. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 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]
|
|
|
|
|
|
C. L. Slingluff Jr, G. R. Petroni, G. V. Yamshchikov, S. Hibbitts, W. W. Grosh, K. A. Chianese-Bullock, E. A. Bissonette, D. L. Barnd, D. H. Deacon, J. W. Patterson, et al. Immunologic and Clinical Outcomes of Vaccination With a Multiepitope Melanoma Peptide Vaccine Plus Low-Dose Interleukin-2 Administered Either Concurrently or on a Delayed Schedule J. Clin. Oncol., November 15, 2004; 22(22): 4474 - 4485. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Tsukahara, Y. Nabeta, S. Kawaguchi, H. Ikeda, Y. Sato, K. Shimozawa, K. Ida, H. Asanuma, Y. Hirohashi, T. Torigoe, et al. Identification of Human Autologous Cytotoxic T-Lymphocyte-Defined Osteosarcoma Gene That Encodes a Transcriptional Regulator, Papillomavirus Binding Factor Cancer Res., August 1, 2004; 64(15): 5442 - 5448. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Savage, L. Gao, K. Vento, P. Cowburn, S. Man, N. Steven, G. Ogg, A. McMichael, A. Epenetos, E. Goulmy, et al. Use of B cell-bound HLA-A2 class I monomers to generate high-avidity, allo-restricted CTLs against the leukemia-associated protein Wilms tumor antigen Blood, June 15, 2004; 103(12): 4613 - 4615. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Sasada, H. Takedatsu, K. Azuma, M. Koga, Y. Maeda, S. Shichijo, H. Shoumura, T. Hirai, A. Takabayashi, and K. Itoh Immediate Early Response Gene X-1, a Stress-Inducible Antiapoptotic Gene, Encodes Cytotoxic T-Lymphocyte (CTL) Epitopes Capable of Inducing Human Leukocyte Antigen-A33-Restricted and Tumor-Reactive CTLs in Gastric Cancer Patients Cancer Res., April 15, 2004; 64(8): 2882 - 2888. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. W. Purcell and J. J. Gorman Immunoproteomics: Mass Spectrometry-based Methods to Study the Targets of the Immune Response Mol. Cell. Proteomics, March 1, 2004; 3(3): 193 - 208. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Harada, R. Gohara, S. Matsueda, A. Muto, T. Oda, Y. Iwamoto, and K. Itoh In Vivo Evidence That Peptide Vaccination Can Induce HLA-DR-Restricted CD4+ T Cells Reactive to a Class I Tumor Peptide J. Immunol., February 15, 2004; 172(4): 2659 - 2667. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. L. Slingluff Jr, G. R. Petroni, G. V. Yamshchikov, D. L. Barnd, S. Eastham, H. Galavotti, J. W. Patterson, D. H. Deacon, S. Hibbitts, D. Teates, et al. Clinical and Immunologic Results of a Randomized Phase II Trial of Vaccination Using Four Melanoma Peptides Either Administered in Granulocyte-Macrophage Colony-Stimulating Factor in Adjuvant or Pulsed on Dendritic Cells J. Clin. Oncol., November 1, 2003; 21(21): 4016 - 4026. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. R. Reynolds, A. Zeleniuch-Jacquotte, R. L. Shapiro, D. F. Roses, M. N. Harris, D. Johnston, and J.-C. Bystryn Vaccine-induced CD8+ T-cell Responses to MAGE-3 Correlate with Clinical Outcome in Patients with Melanoma Clin. Cancer Res., February 1, 2003; 9(2): 657 - 662. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. A. Haque, P. Li, S. K. Jackson, H. M. Zarour, J. W. Hawes, U. T. Phan, M. Maric, P. Cresswell, and J. S. Blum Absence of {gamma}-Interferon-inducible Lysosomal Thiol Reductase in Melanomas Disrupts T Cell Recognition of Select Immunodominant Epitopes J. Exp. Med., May 20, 2002; 195(10): 1267 - 1277. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Akilesh, M. E. Dudley, P. A. Eden, and D. C. Roopenian Efficient Chromosomal Mapping of a Methylcholanthrene- Induced Tumor Antigen by CTL Immunoselection J. Immunol., November 1, 2001; 167(9): 5143 - 5149. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. A. Haque, J. W. Hawes, and J. S. Blum Cysteinylation of MHC Class II Ligands: Peptide Endocytosis and Reduction Within APC Influences T Cell Recognition J. Immunol., April 1, 2001; 166(7): 4543 - 4551. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. L. Zarling, S. B. Ficarro, F. M. White, J. Shabanowitz, D. F. Hunt, and V. H. Engelhard Phosphorylated Peptides Are Naturally Processed and Presented by Major Histocompatibility Complex Class I Molecules in Vivo J. Exp. Med., December 18, 2000; 192(12): 1755 - 1762. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Nishizaka, S. Gomi, K. Harada, K. Oizumi, K. Itoh, and S. Shichijo A New Tumor-Rejection Antigen Recognized by Cytotoxic T Lymphocytes Infiltrating into a Lung Adenocarcinoma Cancer Res., September 1, 2000; 60(17): 4830 - 4837. [Abstract] [Full Text]
|
|
|
|
|
|
S. Gomi, M. Nakao, F. Niiya, Y. Imamura, K. Kawano, S. Nishizaka, A. Hayashi, Y. Sobao, K. Oizumi, and K. Itoh A Cyclophilin B Gene Encodes Antigenic Epitopes Recognized by HLA-A24-Restricted and Tumor-Specific CTLs J. Immunol., November 1, 1999; 163(9): 4994 - 5004. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
I. Drexler, E. Antunes, M. Schmitz, T. Wolfel, C. Huber, V. Erfle, P. Rieber, M. Theobald, and G. Sutter Modified Vaccinia Virus Ankara for Delivery of Human Tyrosinase as Melanoma-associated Antigen: Induction of Tyrosinase- and Melanoma-specific Human Leukocyte Antigen A*0201-restricted Cytotoxic T Cells in Vitro and in Vivo Cancer Res., October 1, 1999; 59(19): 4955 - 4963. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Valmori, M. J. Pittet, C. Vonarbourg, D. Rimoldi, D. Lienard, D. Speiser, R. Dunbar, V. Cerundolo, J.-C. Cerottini, and P. Romero Analysis of the Cytolytic T Lymphocyte Response of Melanoma Patients to the Naturally HLA-A*0201-associated Tyrosinase Peptide 368-376 Cancer Res., August 1, 1999; 59(16): 4050 - 4055. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Yang, M. Nakao, S. Shichijo, T. Sasatomi, H. Takasu, H. Matsumoto, K. Mori, A. Hayashi, H. Yamana, K. Shirouzu, et al. Identification of a Gene Coding for a Protein Possessing Shared Tumor Epitopes Capable of Inducing HLA-A24-restricted Cytotoxic T Lymphocytes inCancer Patients Cancer Res., August 1, 1999; 59(16): 4056 - 4063. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. W. Rohrer, A. L. Barsoum, D. L. Dyess, J. A. Tucker, and J. H. Coggin Jr. Human Breast Carcinoma Patients Develop Clonable Oncofetal Antigen-Specific Effector and Regulatory T Lymphocytes J. Immunol., June 1, 1999; 162(11): 6880 - 6892. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Castelli, P. Tarsini, A. Mazzocchi, F. Rini, L. Rivoltini, F. Ravagnani, F. Gallino, F. Belli, and G. Parmiani Novel HLA-Cw8-Restricted T Cell Epitopes Derived from Tyrosinase-Related Protein-2 and gp100 Melanoma Antigens J. Immunol., February 1, 1999; 162(3): 1739 - 1748. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Kawakami, P. F. Robbins, X. Wang, J. P. Tupesis, M. R. Parkhurst, X. Kang, K. Sakaguchi, E. Appella, and S. A. Rosenberg Identification of New Melanoma Epitopes on Melanosomal Proteins Recognized by Tumor Infiltrating T Lymphocytes Restricted by HLA-A1, -A2, and -A3 Alleles J. Immunol., December 15, 1998; 161(12): 6985 - 6992. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Hudrisier, J. Riond, H. Mazarguil, and J. E. Gairin Pleiotropic Effects of Post-translational Modifications on the Fate of Viral Glycopeptides as Cytotoxic T Cell Epitopes J. Biol. Chem., October 5, 2001; 276(41): 38255 - 38260. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. Jager, S. Gnjatic, Y. Nagata, E. Stockert, D. Jager, J. Karbach, A. Neumann, J. Rieckenberg, Y.-T. Chen, G. Ritter, et al. Induction of primary NY-ESO-1 immunity: CD8+ T lymphocyte and antibody responses in peptide-vaccinated patients with NY-ESO-1+ cancers PNAS, October 24, 2000; 97(22): 12198 - 12203. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
