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Divisions of
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Experimental Oncology D,
Immunohematology, and
Surgical Oncology B, Istituto Nazionale Tunori, Milan, Italy
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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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In the present paper we describe the identification of two novel T cell epitopes derived from tyrosinase-related protein-2 (TRP-2)3 and gp100 differentiation proteins. These two peptides, being restricted by HLA-Cw8 allele, represent novel epitopes that may potentially be used to bypass the down-modulation of the HLA-A locus (2, 3). Being derived from distinct proteins, their simultaneous use as immunogens may also override the epitope loss variants involving the gp100 and TRP-2 differentiation Ags. In the melanoma patient studied, a strong immunity against these novel HLA-Cw8 epitopes was detected both at the local tumor site (i.e., TIL) and in the PBL obtained 96 mo after the surgical resection of metastatic lymph nodes. Together, the data suggest that TRP-2 and gp100 Ags may lead to a systemic immunization in melanoma patients and underline an important role for alleles of the HLA-C locus in the antimelanoma immune response.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The melanoma lines used in this study were isolated from primary or metastatic lesions obtained from surgical specimens of patients admitted for surgery to the Istituto Nazionale Tumori (Milan, Italy). Melanoma cultures were established from single cell suspensions and were maintained in RPMI 1640 with 10% FCS (Biological Industries, Beit Haemek, Israel). Normal human melanocytes FM723 and FM529 were provided by Dr. M. Herlyn (Wistar Institute, Philadelphia, PA), while 822CLO was purchased from Clonetics (Palo Alto, CA). All melanocytes were cultured in melanocyte growth medium (Clonetics).
CTL lines and clones
Antimelanoma CTL were established from T lymphocytes isolated from tumor-invaded lymph nodes (tumor-associated lymphocytes (TAL)) of melanoma patient 15392 (HLA typed as A*0301, B*40012, B*1402, C*0602, and C*0802). A total of 31 independent T cell clones recognizing the autologous tumor in an HLA class I-restricted fashion were isolated by a limiting dilution procedure (6). To confirm the clonality and to classify the clones with identical TCR specificity, the TCR repertoire of each clone was examined by reverse transcribed PCR using a panel of TCR BV- and TCR AV-specific primers as previously described (7).
By in vitro stimulation with autologous tumor, two CTL lines, TAL15392 and LNL15392, were established from TAL and noninvaded lymph nodes (LNL). respectively. Their TCRs were also analyzed.
All CTL were cultured with autologous tumor cells and 300 IU/ml of
human rIL-2 (EuroCetus, Amsterdam, The Netherlands). CTL expressed a
CD3+, CD8+, CD4-, or TCR 
ß
phenotype, as assessed by flow cytometry with mAb (data not shown).
Transfection of COS-7 cells and their recognition by CTL clones
Transfections were conducted using the DEAE-dextran-cloroquine
method and transfected COS-7 cells were assayed for the ability to
induce IFN-
production by CTL clones TB254 and TB327 as previously
described (8). The amount of IFN- released was determined by ELISA
(MABTECH, Stockholm, Sweden).
cDNA encoding BAGE, GAGE, MAGE, and Melan-A/MART-1 were provided by Prof. Thierry Boon (Ludwig Institute, Brussels, Belgium), cDNA encoding TRP-2 were supplied by Dr. Paul Robbins (National Institutes of Health, Bethesda, MD), and GP75/TRP-1 and tyrosinase were provided by Dr. Alan Houghton (Memorial Sloan-Kettering Cancer Center, New York, NY).
IFN- release
Lymphocytes were seeded (5 x 103 in 50 µl
for CTL clones or oligoclonal T cell lines, 5 x 104
for bulk T cells) in 96-well U-bottomed plates with
104/well melanoma cells in a final volume of 0.1 ml of RPMI
1640/10% pooled human serum supplemented with 60 IU/ml of human rIL-2
(EuroCetus, Amsterdam, The Netherlands). Controls were performed by
incubating T cells and stimulators with medium alone. Plates were
centrifuged for 5 min at 400 x g and incubated for
18 h at 37°C, then supernatants were collected, and
their IFN- content was determined by ELISA (MABTECH).
Cytotoxicity assay
The 51Cr release cytotoxic assay was performed as previously described (9), and an E:T cell ratio of 20:1 was always used if not otherwise specified. The significance of lysis was established using the Student-Newman-Keuls multiple range test at p = 0.01. mAb directed to HLA determinants were used to inhibit cytotoxicity. They included W6/32 (which recognized HLA-A, -B, and -C, monomorphic determinant) and B1.23.2 (anti-HLA-B and -C) (16). The significance of inhibition of cytotoxicity seen in the presence of mAbs was evaluated using the Student-Newman-Keuls multiple range test (p = 0.01).
Antigenic peptides
All peptides were purchased from PRIMM (Milan, Italy). All peptides were >90% pure as assessed by analytical HPLC and mass spectrometry; peptide stock solutions were made at 1–4 mg/ml in pure DMSO.
Generation of peptide-specific T cells
Peptide-specific T cells were induced in vitro as previously described (10). PBMC were separated from PBL of patient 15392, obtained 96 mo after operation, by centrifugation on Ficoll-Paque gradients and were used as fresh or cryopreserved samples. After each round of peptide stimulation, the specificity of T lymphocyte lines was evaluated weekly using as stimulators an autologous lymphoblastoid cell line (LCL) or HLA-C*0802-transfected 722.221 cells pulsed with the specific or an irrelevant peptide. Specificity was evaluated by both cytokine release and cytotoxic assay. The recognition of autologous melanoma was also assessed in the presence or the absence of anti-HLA-class I mAb w6/32.
Epitope reconstitution assay
To analyze peptide recognition, different amounts of the
relevant peptide were added to microwells containing 5 x
103 LCL15392 in a final volume of 100 µl. Peptide loading
was allowed to proceed for 2 h at 26°C before effector cells
were added to give a final E:T cell ratio of 1:1. Supernatants were
collected after 18 h, and the IFN- content was determined using
an ELISA (MABTECH).
DNA sequence analysis
DNA sequencing was performed with the T7 sequencing kit (U.S. Biochemical Corp., Cleveland, OH) using specific oligonucleotides.
Progressive deletion of cDNA encoding TRP-2 and gp100 Ags
The TRP-2 cDNA-coding region was cloned in the EcoRI
site of the expression plasmid pcDNA3. This plasmid was digested with
BglII and self ligated, obtaining an in-frame cDNA with a
deletion of 252 bp. gp100 cDNA was cloned in the
NotI/BstXI sites of pcDNA3 plasmid. By complete
HindIII digestion, a gp100 deletion mutant was created that
contained the first 462 bp. These plasmids were opened with
XhoI and ApaI digestion, and progressive 3'
deletions were produced by exonuclease III treatments using the
Erase-a-Base System (Promega, Madison, WI). After ligation, the
plasmids were electroporated in the DH5 Escherichia coli
bacteria, selected with ampicillin. Clones were isolated, and plasmid
DNA was evaluated for the presence of the cDNA insert and transfected
in COS-7 cells together with the HLA-C*0802 cDNA. Each plasmid DNA was
carefully evaluated for the length of the cDNA insert, and each insert
was sequenced.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The CTL lines and clones used in this study and their TCR
composition are summarized in Table I
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These effectors were derived from TAL and LNL of melanoma patient 15392
(A*0301, B*40012, B*1402, C*0602, C*0802) and selected for HLA class
I-restricted recognition of the autologous tumor.
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To identify the HLA allele(s) involved in tumor recognition and to
initially define the epitopes recognized by our CTL, a panel of normal
and neoplastic cells of the melanocytic lineage sharing some HLA
alleles with the autologous tumor was screened for the susceptibility
to lysis and for the ability to induce IFN- release (data not shown)
by TB327, TB254, LNL15392, and TAL15392. As shown in Table II, melanoma cell lines expressing the
HLA-B14 and -Cw8 alleles were lysed by all CTL lines and clones tested.
As previously reported (9), LNL15392 also recognized HLA-A3-positive
melanoma cells lines. This pattern of recognition was confirmed by the
release of IFN- (data not shown). Comparing the pattern of
reactivity and taking into consideration the TCR specificity, at least
two distinct epitopes presented by HLA-B14 or -Cw8 molecules could be
identified; the first was defined by TB254 and TAL15392 (which share
TCRVA8S1J50 and TCRVB21S1J2S7), and the second was defined by TB327 and
LNL15392 (which share TCRVA24S1J26 and TCRVB3S1J2S3; Table II). In
addition, the first epitope is likely to derive from lineage-related
proteins, since cultured HLA-B14+, Cw8+
melanocytes 822CLO were killed by TB254 and TAL15392. Conversely, in
three independent experiments the 822CLO cell line was never recognized
by TB327 or LNL15392, indicating that the epitope of these effectors
was not present on the melanocytes tested here. However, a single
melanocyte cell line was available for testing, and therefore, no
conclusions on the nature of the epitope recognized by TB327 and
LNL15392 can be drawn.
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). These experiments
clearly indicate the involvement of HLA-B or -Cw8 alleles as
restriction molecules for TB327 and TB254. Unfortunately, a further
definition of the role of each of the two HLA alleles was not possible,
since mAbs with blocking activity distinguishing the HLA-B or -C locus
alleles were not available. In addition, no melanomas separately
expressing HLA-B14 or -Cw8 molecules were found in our panel of lines.
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A number of melanoma T cell-defined Ags have been recently
described (11, 12). Since these antigenic proteins could potentially
generate HLA-Cw8 or HLA-B14 binding peptides, we assessed the ability
of our CTL to recognize known Ags such as Melan-A/MART-1; tyrosinase;
gp100; TRP-1 and -2; MAGE-1, -2, -3, and -4; BAGE-1 and -2; and GAGE-1,
-2, -3, -4, -5, and -6. To this end, COS-7 cells were transfected with
HLA-B*1402 or HLA-C*0802 cDNA alone or together with cDNA encoding each
of the tumor Ags described above. Each transfectant was then evaluated
for its ability to elicit IFN- release by the antitumor CTL. The
screening was conducted using CTL clones representative for each TCR
specificity, namely TB327 and TB254. No recognition occurred when tumor
Ags were cotransfected in COS-7 cells with the HLA-B*1402 molecule
(data not shown). Conversely, as shown in Fig. 2, IFN- production was strongly
induced in TB254 by COS transfected with HLA-C*0802 cDNA and gp100,
while the pattern of reactivity of the TB327 clone clearly indicated
the recognition of a TRP-2-derived peptide presented by HLA-Cw8 (Fig. 2). These data confirm that two distinct antitumor responses were
detectable within T cells derived from tumor-infiltrated lymph nodes
and clearly indicate the role of HLA-Cw8 as a presenting molecule for
the immunogenic peptides. Combining this finding with the observation
that the melanoma 624.28 expressing a different HLA-Cw8 subtypes was
recognized by CTL (Table I
), it is likely that the processing and
presentation to our CTL of TRP-2 and gp100 epitopes may occur equally
for both HLA-C*0801 and -C*0802 subtypes.
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To identify the TRP-2- and gp100-encoded peptides presented by
HLA-Cw8 allele to TB327 and TB254, TRP-2 and gp100 variants were
generated and tested for their ability to be recognized by the specific
CTL upon cotransfection in COS-7 with cDNA encoding HLA-C*0802. TRP-2
cDNA with a deletion of 252 nucleotides between two internal
BglII sites led to an in-frame transcript still containing
the TB327-defined epitopes (Fig. 3); for
gp100, the cDNA containing the first 462 nucleotides created by
HindIII digestion was still recognized by TB254 in
transfection experiments (Fig. 4).
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). Therefore, the immunogenic peptide should be partially
contained inside the region spanning between amino acid residues
393–397. For gp100 Ags (Fig. 4), the deletion mutants ending at amino
acid 78 fully contained the TB254 epitope, while recognition was lost
when the cDNA was a single amino acid shorter, clearly indicating that
the isoleucine in position 78 was essential for recognition by TB254. Identification of the TB327 and TB254 epitopes
To identify the antigenic peptide recognized by TB327, each amino
acid of the TRP-2397–393 fragment was considered the last
amino acid of a putative peptide starting nine positions upstream. The
resulting four nonamers were synthesized and screened for their ability
to induce IFN- release by TB327 when pulsed on the autologous LCL.
The ANDPIFVVL peptide showed a strong stimulating activity with a level
of IFN- released in the medium similar to that achieved with tumor
stimulation. We then synthesized the two octameric peptides contained
in the immunogenic nonamer as well its two decameric forms obtained
with an elongation of one amino acid in the NH2 or COOH
termination. Only the decameric form with an additional residue in
NH2 position, AANDPIFVVL, was still recognized by TB327
(Table III). The nonamer and decamer,
ANDPIFVVL and AANDPIFVVL, were then compared for their abilities to
sensitize the autologous LCL or the 721.221 line transfected with
HLA-C*0802 cDNA to recognition by TB327 over a wide range of peptide
concentrations. Both peptides were equally efficient in conferring
recognition, and half-maximal IFN- production was achieved at a
concentration between 1–10 nM (Fig. 5A).
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). We reasoned that the
isoleucine at position 78 was the last amino acid of the immunogenic
peptide. We then synthesized peptides starting 8, 9, or 10 amino acids
upstream this crucial isoleucine and evaluated their ability to induce
IFN- release by the TB254 when pulsed on autologous LCL. All three
peptides were able to reconstitute the TB254 epitope equally well when
used at a concentration of 1000 nM (Table III). In a titration
experiment, however, the octamer and nonamer peptides were more
efficient than the decamer in inducing IFN- release by TB254, and
half-maximal stimulation was achieved at 1–10 nM (Fig. 5B). In vitro immunogenicity of peptides and induction of peptide-specific responses from PBL of a melanoma patient
The previous data showed that the nonameric, the decameric, and,
in the case of gp100-derived peptide, the octameric forms containing a
common core sequence were able to reconstitute the epitope of their
respective CTL. This suggested that at least at the effector phase the
TCR of these effectors may display a relative degree of degeneracy. To
evaluate whether all the identified peptides had a similar capacity for
driving and selecting in vitro specific T cells, the PBL of melanoma
patient 15392, obtained 96 mo after surgery, were cultured with
autologous PBMC pulsed with the different forms of TRP-2 or gp100
peptides. After the first round of in vitro peptide restimulation, the
specificity of each T cell culture was evaluated weekly by monitoring
the cytotoxic activity or the IFN- release upon exposure to
autologous LCL or to HLA-C*0802-transfected 722.221 cells (data not
shown), alone or pulsed with specific or irrelevant peptides. The
TRP-2-derived nonamer and decamer were both able to generate
peptide-specific T cells, although they differ in the number of
restimulations required to achieve specificity. In fact, CTL induced by
ANDPIFVVL recognized the stimulating peptide at the second week of
culture by either IFN- release or cytotoxic activity (Fig. 6, A and C), while
at least 1 additional week was needed for the decamer AANDPIFVVL to
select and expand specific T cells (Fig. 4, B and
D).
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, A–C). However, a
hierarchy in the immunogenic capacity could be detected also for the
gp100-related peptides. In fact, the octamer SNDGPTLI was able to
generate T cells displaying a specific cytotoxicity toward a target
pulsed with the relevant peptides as early as the first week of culture
(Fig. 7F). Generally, cytokine release (Fig. 6, A
and B; Fig. 7, A, B, and D)
when compared with cytotoxicity (Fig. 6, C and D;
Fig. 7, D–F) showed a better sensitivity in the detection
of the specific response. Together, these data indicate that with
similar, but not identical, efficiency, all the different forms of
peptides induced specific T cells starting from PBL of a melanoma
patient and that the generated T cells equally recognized the peptide
used for the induction as well as all the other related forms
containing the same core sequence. In fact, the T cells generated by
the nonameric form of TRP-2 recognized the nonamer as well the decamer
and vice versa, and the same occurred for gp100-related peptides. These
data confirm with a bulk T cell population the cross-recognition noted
at the clonal level by the analysis of TB254 and TB327 reactivities. In
addition, the fast kinetics observed for the generation of
peptide-specific T cells suggests that in the melanoma patient here
analyzed a systemic immunity against these epitopes was generated
in vivo.
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At different time points of in vitro culture, all the T cell lines
generated by peptide stimulation were analyzed for the ability to
recognize the autologous tumor Me15392 in an HLA class I-restricted
fashion by a cytotoxic assay. Data are reported in Table IV as the percentage of lysis achieved by
the effectors on melanoma cells incubated with or without the
anti-HLA class I-specific Ab W6/32. The two cell lines generated by
the decameric peptides, namely by gp10069–78 and
TRP-2386–395, killed the autologous tumor predominantly in
an MHC-unrestricted fashion as evaluated by the lack of inhibition of
lysis in the presence of W6/32. Conversely, the TRP-2 nonamer peptide
induced CTL with a restricted tumor lysis at the second week of culture
simultaneously with the acquisition of peptide specificity (Fig. 6, A and C). CTL lines generated by the octameric
and nonameric gp100 peptides displayed similar behavior (Table IV). For
these effectors the detection of peptide specificity by a cytotoxic
assay at the first and second weeks, respectively, correlated with
specific tumor recognition. The analysis of the data obtained
for these TRP and gp100 peptides in terms of ability to reconstitute
the epitope of anti-tumor-specific CTL clones and to generate
peptide-specific T cells also recognizing the melanoma led to the
conclusion that TRP-2386–395 can be considered
the optimal TRP-2 peptide. Conversely, gp10070–78 and
gp10071–78 displayed similar activities, with a preference
for the octamer gp10071–78 showing faster kinetics for the
induction of tumor-specific T cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To date, the majority of the antitumor reactivity against the differentiation proteins was restricted by the alleles of the HLA-A locus; only occasionally was the HLA-B locus involved. In addition, no evidence was ever provided for the ability of these differentiation Ags to generate HLA-C-restricted epitopes (11, 12). In general, the B and C loci have received less attention, probably due to the fact that they were reported to be expressed on melanoma cells with a lower density than the HLA-A locus (2, 16, 17, 18). However, when the amount of HLA molecules was compared between melanoma and the normal melanocytes, a similar level of expression was detected at least for the B locus, suggesting that expression of the different HLA loci was lineage related (19). Our data, together with the previous findings of GAGE, BAGE, and MAGE genes (20, 21, 22) clearly indicate that the alleles encoded by HLA-C genes may have a central role in a T cell-mediated antitumor response despite their generally low expression.
Although not enough data are available to conclusively define the binding motif, if any, for peptides presented by HLA-Cw8 molecules, by screening all the synthetic peptides derived from the gp100 or TRP-2 deletion mutants conferring recognition to the melanoma-specific CTL clones, we found that no variation was admitted at the acidic end for both epitopes. The leucine in position 78 or the isoleucine in position 395 for gp100 and TRP-2, respectively, are conserved as aliphatic residues, and they may function as the C-terminal anchor amino acid for the binding to HLA molecules. Isoleucine in the terminal COOH position has also been reported for the HLA-Cw8-presented peptide derived from gp120 protein of HIV (23). In addition, comparing our peptides and the other two epitopes described (23), asparagine in positions 1 or 3 represent an additional conservative feature.
TB254 and TB326 CTL clones were equally reactive against all the forms of their corresponding peptides, indicating that their TCRs could not distinguish the different forms of the nominal Ag. When bound to the HLA-Cw8 molecules, each antigenic related peptide probably exists in similar conformations with the alanine of the TRP-2386–395 as well the lysine and valine of the gp10069–78 stretching from the binding groove. This "tolerant" behavior of TCR has been previously reported for HLA-A1-restricted epitopes derived from tyrosinase (24). However, while TRP-2387–395, gp10070–78, and gp10069–78 drove the expansion of tumor-specific CTL, TRP-2386–395- and gp10069–78-generated T cells although peptide specific, were unable to recognize the tumor and displayed an HLA class I-unrestricted lysis of melanoma cells. The naturally processed peptides may include a decamer, and, especially in TAP-deficient cell lines, peptides containing 12 amino acids have been described (25). Although the evaluation by mass spectrometry of peptides eluted from HLA-Cw8 molecules will eventually determine which of the TRP-2 and gp100 peptides is naturally processed and presented in association with HLA-Cw8 allele, our data indirectly suggest that the decamer is probably not present on the cell surface of melanoma cells and discourage the usage of TRP-2 and gp100 decamers for peptide-based vaccines.
In addition to identify new epitopes, our data suggest that the HLA-Cw8-restricted gp100- and TRP-2-derived peptides evoked in vivo an immune response detectable at the tumor site and in the peripheral blood of the analyzed patient. T cells recognizing the gp100 and TRP-2 peptides were likely to be already expanded at the tumor site, and this expansion could reflect an in vivo Ag-specific recognition. This conclusion relies on two different observations. First, a few weeks of in vitro culture of bulk TAL or LNL were necessary to the epitopes presented by the HLA-Cw8 molecules expressed on the tumor cells to drive the expansion and selection of monoclonal TRP-2 or gp100 peptide-specific T cell lines (namely TAL15392 and LNL15392). Second, the majority of the specific clones derived from TAL were directed against the gp100 peptide. Unfortunately, LNL were not cloned, and no further information is available on this issue. In addition to a local immunization, a strong systemic immunity against gp100 and TRP-2 peptides was detectable in the PBL of this patient obtained 96 mo after the surgical resection of a lymph node metastasis. In fact, the PBMC cultured with gp100271–228 or TRP-2387–396 peptide for 6 or 12 days, respectively, generated peptide-specific CTL recognizing the autologous tumor in an HLA class I-restricted fashion. These kinetics of induction of peptide-specific T cells are comparable to a secondary recalling in vitro response obtained in melanoma patients immunized with repeated in vivo injection of gp100-derived, HLA-A2-restricted epitope (15, 26). In fact, all the immunized patients developed a specific response within 10–13 days of in vitro culture with stimulating peptide, whereas none of the patients not receiving the vaccine developed an immune reaction against the gp100 epitopes. Together these data demonstrate the immunogenicity of the TRP-2 and gp100 peptides presented by HLA-Cw8 allele and suggest that in vivo priming leading to a systemic immunity against these epitopes may occur in melanoma patients.
In conclusion, we have defined two novel TRP-2 and gp100 epitopes presented by the HLA-Cw8 allele. Comparing the amino acid structure of these peptides, conserved residues probably favoring binding in the pocket of HLA molecules can be identified. Being restricted by an allele of an HLA-C locus and derived from Ags already containing epitopes presented by alleles of the HLA-A locus, these peptides could be useful for the construction of a polyvalent vaccine that exploits alternative HLA-peptide complexes as targets for the immune system. Due to the strong immunogenicity revealed by these peptides in the patient studied, their usage in peptide-based therapy remains a distinct possibility.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint request to Dr. Chiara Castelli, Division of Experimental Oncology D, Istituto Nazionale Tumori, via Venezian 1, 20133 Milan, Italy. E-mail address:
3 Abbreviations used in this paper: TRP-2, tyrosinase-related protein-2; TIL, tumor-infiltrating lymphocytes; TAL, tumor-associated lymphocytes; LNL, lymph node lymphocytes; CM, complete medium: LCL, lymphoblastoid cell line; TCRBV, TCR beta variable region; TCRAV, TCR alpha variable region.
Received for publication July 15, 1998. Accepted for publication October 8, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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chain of a WT31+ cytotoxic T lymphocyte clone which specifically recognizes HLA-A2 molecule. Scand. J. Immunol. 35:487.[Medline]
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