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* Unit Institut National de la Santé et de la Recherche Médicale Unité 463, Nantes, France;
Faculté des Sciences de Nantes, Nantes, France; and
Unit of Skin Cancer, Centre Hospitalier Régional Hotel Dieu, Nantes, Place Alexis Ricordeau, Nantes, France
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Through a systematic screening of the specificity of 60 melanoma tumor-infiltrating lymphocyte (TIL) populations, we reported previously the recognition of 
20 new HLA-class I/peptide complexes, among which 6 were restricted by the HLA-B*3501 allele (Melan-A/MART-1, tyrosinase, gp-100, NY-ESO-1, and MAGE-A3/A6) (11). Because this HLA is one of the most frequently expressed HLA-B alleles (12), we sought to identify the B*3501-restricted epitopes recognized by these TIL populations and to look for the efficacy of their presentation by HLA-B35 melanoma cells.
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Materials and Methods |
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Melanoma cell lines were established from fragments of metastasic tumors or tumor-invaded lymph nodes. Mouse fibrosarcoma WEHI 164 clone 13, used for TNF production assays and COS-7 cells were obtained from T. Boon (Ludwig Institute for Cancer Research (LICR), Brussels, Belgium). COS-7 cells, WEHI 164 clone 13, and melanoma cells lines were cultured, as described before (11). The EBV-B-transformed cell line LAZ 338 was a gift from T. Hercend (Vertex Pharmaceutical, Abingdon, U.K.). The BM36.1, an HLA-B*3501 TAP-deficient B-EBV cell line previously described (13), was a gift from A. Ziegler (Universitatsklinikum Charite, Berlin, Germany).
T cell clones
T cell clones were derived by limited dilution culture of polyclonal TIL obtained from melanoma-invaded lymph nodes of stage III patients (American Joint Committee on Cancer). TIL clones were cultured, as described before (11).
Synthetic peptides
Wild-type and modified peptides were purchased from EPYTOP (Nîmes, France). Purity (>70%) was controlled by reversed-phase HPLC. Peptides were lyophilized, dissolved in DMSO at 10 mg/ml, and stored at -80°C.
HLA cDNAs
cDNAs coding for HLA-B*3501, HLA-B*3503, and HLA-B*3508 were obtained respectively in our laboratory from T. Boon (LICR) and from E. Zorn (Unité Institut National de la Santé et de la Recherche Médicale Unité 267, Villejuif, France). cDNAs coding for melanoma Ags MAGE-A3, MAGE-A6, tyrosinase, Melan-A/MART-1, and NY-ESO1/LAGE-2 were obtained from T. Boon. gp100 cDNA was cloned in our laboratory.
Construction of cDNA fragments
Tyrosinase fragments 1-1086 and 1-598 were kindly given by B. Van Den Eynde (LICR). cDNA restriction fragment of gp100 1156–1986(1156–1986) was generated by enzymatic digestion with KpnI. This fragment used, for expression, the internal ATG at position 1174. The other gp100 and tyrosinase cDNA fragments were obtained by PCR using specific oligonucleotides, and were cloned into pcDNA3 plasmid. Melan-A/MART-1 and NY-ESO-1 cDNA fragments were obtained by exonuclease III digestion. Briefly, plasmids encoding Melan-A/MART-1 and NY-ESO-1 were respectively opened with XbaI and ApaI or SpHI and NotI before digestion with exonuclease III. This treatment was performed with the Erase-a-base System (Promega, Madison, WI).
Transfection of COS-7 cells
Transfection was performed by the DEAE-dextran-chloroquine method (14, 15). Details of the procedure have been described previously (16).
Determination of relative affinity (RA) and stability of HLA-B*3501-identified peptides
BM36.1 cell line, an EBV-transformed lymphoma cell line, is deficient in TAP function and expresses HLA-B*3501 mRNA. These cells (105/ml) were incubated overnight with peptide concentrations ranging from 10-16 to 10-4 M in serum-free RPMI 1640 medium supplemented with 100 ng/ml of 
2-microglobulin at 37°C. Subsequently, cells were stained with the B1.23.2 Ab to measure surface expression of HLA-B/C. The HLA-B*3501-binding peptide 37F (LPFDFTPGY) was used as a reference peptide (17, 18). The RA is determined as: concentration of each peptide/concentration of the 37F peptide that induces 20% of maximal HLA B/C expression (19).
For stability analysis, BM36.1 cells were incubated overnight with 10-4 M of each peptide, then incubated with brefeldin A (10 µg/ml) for 1 h to block cell surface expression of newly synthesized HLA-B*3501 molecules, washed, and incubated at 37°C for 0, 30 min, 1 h, 2 h, 4 h, or 6 h. Subsequently, cells were stained with the B1.23.2 Ab to evaluate the HLA-B/C molecule expression. The dissociation complex (DC50) was determined as the time required for the loss of 50% of the HLA-B*3501/peptide complexes stabilized at t = 0 h.
RT-PCR for tyrosinase, MAGE-A3, MAGE-A6, and NY-ESO-1 Ags
Total RNA was extracted by the guanidinium-cesium chloride procedure. Reverse transcription was performed, as previously described (11). PCR amplification was then performed on 50 ng of the cDNA with PCR buffer, 1.5 mM MgCl2 (Life Technologies, Corgy Pontoise, France), 0.8 mM dNTP mix, 1 µM primers, and 0.1 U of Taq polymerase (Life Technologies-BRL) in a final water volume of 25 µl. Primers and PCR cycles are detailed in Table I. A total of 10 µl of PCR products was size fractionated on a 1% agarose gel. Expected lengths were 604 bp for Melan-A, 372 bp for gp100, 383 bp for tyrosinase, 615 bp for NY-ESO-1, 804 bp for MAGE-A3, and 288 bp for MAGE-A6.
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A total of 104 TIL clones was stimulated in duplicate cultures by target cells: melanoma cells (3 x 104), transfected COS-7 cells (48 h later), or BM36.1 cells loaded with 10 µM of peptide. Culture supernatants were harvested 6 h later and tested for TNF content by a biological assay, as previously described (16). To enhance the cell surface expression of HLA molecules, melanoma cells were preincubated 48 h in medium containing 500 U/ml of IFN-
(Tebu, Paris, France).
Cytotoxicity assay
Target cells were labeled with 100 µCi Na51CrO4 (Oris Industrie, Gif-sur-Yvette, France) for 1 h at 37°C before loading with synthetic peptides for 20 min. A total of 103 target cells was then mixed with 104 effector T cells (in a final volume of 100 µl), and radioactivity was measured, 4 h later, on a beta plate counter (EG&G Wallac, Evry, France).
Generation of Melan-A-specific CTL from HLA-B*3501 PBL
PBL were isolated from leukapheresis from healthy HLA-B*3501 volunteers. Dendritic cells (DC) were generated from adherent cells cultured for 7 days in the presence of 500 IU/ml GM-CSF and 100 IU/ml IL-4 (Abcys, Paris, France) in complete RPMI medium. On day 7, maturation agents, poly(I:C) (Sigma-Aldrich, Oakville, Canada) at 100 µg/ml and TNF-
(Abcys) at 10 ng/ml, were added in the culture for 48 h. Mature DC were loaded with peptides (5 x 10-5 M), in presence of 5 µg/ml of 2-microglobulin, at 37°C in serum-free medium for 2 h, and then irradiated (35 Gy). A total of 3 x 106 CD4-depleted autologous PBL was then stimulated by 3 x 105 peptide-pulsed DC, in complete medium supplemented with 1000 U/ml IL-6 and 5 ng/ml IL-12 (Abcys). Irradiated DC were added again twice, at 7-day intervals in medium containing 10 U/ml IL-2 (Chiron, Amsterdam, The Netherlands) and 5 ng/ml IL-7 (Abcys). Seven days after the third stimulation, the specificity of stimulated culture was tested by intracellular IFN- labeling.
Analysis of T cell responses by measurement of IFN- production
A total of 105 stimulated PBL was incubated 6 h with 2 x 105 stimulator cells (peptide-pulsed BM36.1 cells or melanoma cells) in 200 µl of RPMI 1640 containing 10% FCS and 10 µg/ml brefeldin A (Sigma-Aldrich). For intracytoplasmic IFN- staining, cells were fixed for 10 min, washed, and stored at 4°C until staining. Negative controls were performed with unpulsed BM36.1 cells or B35-negative melanoma cell line. Lymphocytes were stained for 30 min at room temperature with the anti-human IFN- mAb (BD PharMingen, San Diego, CA), at the concentration of 5 µg/ml in PBS containing 0.1% BSA and 0.1% saponin (Sigma-Aldrich). After two washes, cells were incubated with F(ab')2 of goat anti-mouse IgG (Bio-Atlantic, Nantes, France). After staining, cells were resuspended in PBS, and 104 events were analyzed on a FACScan flow cytometer using CellQuest software (BD Biosciences, Grenoble, France). T cell responses were considered significant when the fraction of IFN--positive cells was greater than 0.2% (after subtraction of the background obtained on control cells).
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Results |
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Melanoma TIL, specific for Melan-A, gp100, tyrosinase, NY-ESO-1, and MAGE-A3/A6 Ags in the HLA-B35 context, had been evidenced in a previous study (11). We now used TIL-derived clones to identify the recognized epitopes. Fig. 1A shows the reactivity of the TIL clones to COS-7 cells cotransfected by the MAA and the restricting HLA-B35 allele.
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treatment. Melanoma lines M125 and M140, lacking spontaneous expression of HLA-B*3501, were also recognized by these clones upon IFN- treatment.
The gp100 (Fig. 1C)- and NY-ESO-1 (Fig. 1E)-specific clones respectively recognized one of three and one of two melanoma lines (M147 and M47) that expressed the Ags and the HLA-B*35 spontaneously. IFN- treatment increased the efficiency of Ag recognition of these cell lines and induced that of the two cell lines lacking spontaneous expression of HLA-B*3501 (M125 and M140). As shown by RT-PCR, the two cell lines M47 and M131 that failed to be recognized by the gp100-specific clone expressed this Ag at a low level (Fig. 1C).
Therefore, all the epitopes recognized by HLA-B35 TIL clones were efficiently presented by most melanoma cell lines that spontaneously expressed the HLA and the MAA.
Identification of MAA-derived peptides recognized by TIL clones from HLA-B35 melanoma patients
To pinpoint the cDNA regions coding for each epitope, COS-7 cells were cotransfected by both HLA-B35 cDNA and various Ag-encoding cDNA fragments. As shown in Fig. 2, the C-terminal ends of the specific peptides were encoded by: the Melan-A cDNA fragment 94–119 bp, the gp100 cDNA fragment 1199–1601 bp, the tyrosinase cDNA fragment 936–975 bp, and the NYESO-1 cDNA fragment 259–339 bp.
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The Melan-A-specific CTL clone recognized three overlapping Melan-A peptides (Table II). The peptide 26–35 (EAAGIGILTV) was the most efficiently recognized with a half-maximal lysis of 5 x 10-10 M (Fig. 3 and Table III). This peptide was also recognized by a TIL clone from another melanoma patient (data not shown).
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As shown in Table II, the tyrosinase-specific clone recognized three overlapping peptides: the 14-mer peptide 309–322, the 13-mer peptide 309–321, and the 12-mer peptide 309–320. Deletion of the phenylalanine at the COOH-terminal end or of the threonine at the N-terminal end of the 12-mer strongly reduced the recognition by the CTL (Table II). The 12-mer peptide was the best fitting peptide, with a half-maximal lysis at 2 x 10-10 M (Fig. 3 and Table III). Curiously, the tyrosinase nonapeptide 312–320, previously shown to be recognized by a TIL clone in the B*3501 context (20), failed to be recognized by our CTL clone (Table II and Fig. 3).
The MAGE-A3/A6-specific clone M171.8C recognized the MAGE-A3/B*3501-restricted epitope EVDPIGHLY, previously described by the group of T. Boon (21), with a half-maximal lysis at 10-12 M. This clone also reacted with the MAGE-A6 peptide EVDPIGHVY, albeit with a considerably lower efficacy (half-maximal lysis at 10-8 M) (Fig. 3 and Table III).
As shown on Fig. 4, the NY-ESO-1-specific TIL clone efficiently recognized the peptides 92–104 and 94–102 (Table II and Fig. 3). The best fitting one was clearly the nonamer 94–102 (half-maximal lysis: 10-12 M) (Fig. 3 and Table III).
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With the perspective to use the identified peptides for immunotherapy, their RA and stability for the HLA-B*3501 molecule were addressed, as described in Materials and Methods. We can distinguish two main groups: peptides having both a high affinity (RA <3) and a good stability (>6 h), and peptides having both low affinity (RA >3) and stability (<6 h). All the peptides efficiently recognized by specific TIL clones belonged to the first group, with the exception of Melan-A, which belongs to the second group (Table III).
Identification of Melan-A peptide analogues of improved affinity and stability for HLA-B35 molecule
Inefficient binding to HLA-B35 of the Melan-A peptide might be due to the lack of the main anchor residues in this peptide. Anchor residues, i.e., P at position 2 and/or Y at the N-terminal end (22), were therefore introduced to enhance the affinity of this peptide. As shown in Fig. 4, the peptides EAAGIGILTY (EAA10Y) and EPAGIGILTY (EPA10Y) exhibited both a high RA and stability for HLA-B*3501 molecule. Furthermore, these two peptides induced half-maximal lysis by the clone at concentrations respectively 50 and 10 times lower than the natural peptide. In contrast, single amino acid-substituted peptide EPAGIGILTV (EPA) did not show improved properties compared with the natural decamer.
Melan-A peptide analogues, EAA10Y and EPA10Y, are more efficient than the natural peptide in inducing the growth of Melan-A-specific CTL
CD4-depleted PBL from two HLA-B35 healthy donors were stimulated by autologous DC pulsed with the Melan-A peptide 26–35, and with the modified analogues EAA10Y and EPA10Y. Representative data obtained in one patient are shown in Fig. 5A. Stimulation by the natural peptide induced the growth of Melan-A-specific CTL in 13 of 48 microcultures, while modified peptides induced the growth of higher fractions of specific T cells in 36 of 48 microcultures for EAA10Y, and 42 of 48 microcultures for EPA10Y (Fig. 5A). Interestingly, microcultures stimulated by the Melan-A-modified peptides were also strongly reactive against the natural Melan-A peptide 26–35 (Fig. 5B). As shown in Fig. 5C, most microcultures reactive against the natural Melan-A peptide also reacted against a HLA-B35 melanoma cell line expressing the Melan-A Ag.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The B*3501-restricted Melan-A/MART-1 epitope was the peptide 26–35, also presented in the HLA-A*0201 context (Fig. 6). Because the same TIL clone also recognized this peptide in HLA-B*3503 and B*3508 contexts, this peptide could therefore be presented in at least four different HLA class I contexts. In vitro stimulations of HLA-B35 healthy donor PBL yielded Melan-A-specific T cells in a large proportion of culture wells (Fig. 5, and data not shown). This suggests that a B*3501-restricted repertoire as large as the A*0201-restricted one could exist for this peptide. Nonetheless, as shown previously for the HLA-A*0201 context (23), this peptide did not bind efficiently to the HLA-B*3501 molecule, due to a lack of appropriate anchor residues. Hence, we designed two modified peptides, EAA10Y and EPA10Y, by introducing anchor amino acids at positions 2 and/or 10. Those peptides bind more efficiently to the HLA-B*3501 and were more efficiently recognized by the specific TIL clone than the natural peptide. Moreover, in vitro generation of tumor-reactive CTL from HLA-B*3501 PBL stimulated by these peptide analogues was much more efficient than that observed with the natural peptide (Fig. 5). Because Melan-A is expressed by 90% of primary melanomas and 80% of metastatic ones, the modified Melan-A peptides are good candidates for peptide-based vaccine trials in HLA-B*35 melanoma patients.
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The previously described B*3501-restricted tyrosinase epitope 312–320 (20) was not recognized by our tyrosinase-specific clone (Table II and Fig. 2). The longer tyrosinase peptide 309–320 was the peptide best recognized by the specific CTL clone. It is also the peptide that bound the most stably to the HLA-B35 molecule. Therefore, it is likely the natural epitope, although two longer peptides, 309–322 and 309–321, were also recognized efficiently (Table I). Other examples of 11-mer T cell epitopes have been described (27, 28). Because this peptide encompasses the previously described B*3501-restricted tyrosinase epitope 312–320 (20), it also looks an interesting candidate for peptide-based vaccination of HLA-B35 patients.
Concerning NY-ESO-1, we identified three overlapping peptides encompassed between aa 92 and 104 recognized by the TIL clone. Two of these (92–104 and 94–102) bound efficiently to the HLA-B*3501 molecule and were also recognized very efficiently by our TIL clone. Because the 94–102 peptide gives a better titration response and because it could be present as a contaminant peptide in 92–104 peptide preparation, this nonamer is most likely the natural epitope. Nonetheless, both peptides have interesting properties: the 94–102 is also presented in the HLA-B51 context (29) (Fig. 6), while the peptide 92–104 encompasses both this B51-restricted epitope and a HLA-Cw3-restricted epitope (30). In vitro PBL stimulation will be necessary to establish whether these two peptides are good candidates to stimulate melanoma-reactive CTL.
The MAGE-A3 peptide 168–176 described previously (21) was the peptide recognized by the MAGE-A3/MAGE-A6-specific TIL clone. This peptide had been identified as the target of MAGE-A3-specific PBL obtained after stimulation with DC infected with a virus coding for MAGE-A3 protein. Our results show the immunogenicity of this epitope in vivo. The same TIL clone also recognized the MAGE-A6 peptide 168–176, differing from the MAGE-A3 epitope by 1 aa in position 8, and a MAGE-A3-negative/MAGE-A6-positive HLA-B35 melanoma cell line. The MAGE-A6 peptide 168–176 is therefore a new B*3501-restricted melanoma epitope.
The present study extends to eight the number of known MAA epitopes shared by a majority of HLA-B*3501 melanoma tumors (20, 21, 31). Interestingly, several of these epitopes encompass shorter peptides that can be presented in the same or other common HLA contexts. This panel of melanoma epitopes represents an interesting pool to check the efficiency of multiepitope peptide-based vaccine protocols. Furthermore, the definition of many peptides presented by various HLA molecules allows the development of new tools for monitoring the induction of T cell responses in immunized patients. Indeed, identification of antigenic peptides is essential not only to increase the availability and efficacy of peptide-based vaccines, but also to monitor CTL responses in patients immunized with peptides or with other forms of Ags, such as full-length recombinant proteins or recombinant viruses.
Aside from these important implications for cancer immunotherapy, the observations made in this study confirm and extend previous findings concerning the ability of certain antigenic peptides to bind to more than one MHC class I gene product. It thus appears that this peptide behavior is more common than it had been anticipated previously. One may speculate that this might be the result of central tolerance that deleted T cells for high affinity MHC class I-binding peptides. Moreover, this recurrence of antigenic peptides being recognized in different MHC class I context may also reflect a bias in the Ag-processing machinery in tumor cells. Be it as it may, these findings should be taken into account when making prediction of MHC-binding peptides with currently used algorithms.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Nathalie Labarrière, Institut National de la Santé et de la Recherche Médicale Unité 463, 9 Quai Moncousu, 44093 Nantes cedex 1, France. E-mail address: nlabar{at}nantes.inserm.fr
3 Abbreviations used in this paper: TAA, tumor-associated Ag; DC, dendritic cell; MAA, melanoma-associated Ag; RA, relative affinity; TIL, tumor-infiltrating lymphocyte.
Received for publication July 10, 2003. Accepted for publication September 26, 2003.
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, TNF secretion by TIL clones in response to COS-7 cells cotransfected with cDNA encoding the HLA and the Ag. 
, TNF secretion by TIL clones in response to COS-7 cells transfected with the cDNA encoding the HLA molecule alone. Recognition of B35-melanoma cells by TIL clones specific for Melan-A (B), gp100 (C), tyrosinase (D), NY-ESO-1 (E), and MAGE-A3/A6 (F) Ags. TIL clones were incubated for 6 h at 37°C with melanoma cell lines at an E:T ratio of 1:3. Before biological assay, melanoma cell lines were either treated (
