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Immunology |
1 INSERM487, Institut Gustave Roussy, Villejuif;
2 Unité d’Immunité Cellulaire Antivirale, Institut Pasteur, Paris; and
3 Immuno-Designed Molecules, Paris, France
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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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EphA2 (Eck) belongs to the Eph receptors family. The Eph family contains at least 14 members that are type I transmembrane glycoproteins (9) . Upon cell-cell contact, Eph receptors interact with their ligands called ephrins. Glycosylphosphatidyl inositol-linked ephrin-A ligands bind to EphA receptors and transmembrane ephrin-B ligands bind to EphB receptors (9 , 10) . Eph-ephrin signaling mediates contact-dependent cell interactions involved in the spatial organization of neurons and neural crest cells, embryogenesis, tissue patterning, and blood vessels formation (9 , 10) .
Strikingly, EphA2 is overexpressed in several human cancers such as breast, colon, lung, prostate, gastric carcinoma, and metastatic melanoma but not in nonmalignant breast, prostate, and skin lesions (11, 12, 13, 14, 15) . EphA2 overexpression correlates with malignant transformation (14) , poor prognosis (16 , 17) , metastatic progression (18) , and p53-mediated apoptosis (19) . Interestingly, EphA2 has been detected in tumor neovasculature and was demonstrated to play a role in tumor angiogenesis (20, 21, 22) . The large number of human cancers expressing the antigen and the association with tumor vascularization and metastatization prompt EphA2 as an appealing target for tumor immunotherapy.
In this article, we show that EphA2 is target of antitumor CD8+ T lymphocytes, and we describe two HLA-A*0201-restricted epitopes that induce specific CD8+ T cells able to recognize tumors of various origins.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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1 and 2 domains of HLA-A*0201 allele and the 3 and intracellular domains of Db allele) linked by its NH2 terminus to the COOH terminus of the human ß2m by a 15-amino acid peptide arm.
Peptides and Plasmids.
EphA258 (IMNDMPIYM) and EphA2550 (VLAGVGFFI) peptides were synthesized by Synt:em (Nîmes, France). The EphA2 cDNA was kindly provided by Dr. Elena Pasquale (The Burnham Institute, La Jolla, CA). The HHD construct was described previously (23)
.
Cells and Peptides.
The murine RMAS/HHD cells were described previously (23)
. The HLA-A*0201-expressing human tumor cells were: T2 (deficient in TAP1 and TAP2 transporters), MCF-7 (breast cancer), CACO-2 (colon cancer), 1355 (lung cancer), and M113 (melanoma) kindly provided by Dr. Francine Jotereau (Nantes, France); SAOS (sarcoma) and HIEG (renal cell carcinoma) kindly provided by Dr. Eric Angevin (Villejuif, France); and LNCaP and DU-145 (prostate cancer). Cells were grown in RPMI 1640 (Invitrogen, Paisley, United Kingdom) supplemented with 10% FCS.
Measurement of Peptide RA to HLA-A*0201.
The protocol used has been described previously (24)
. Briefly, T2 cells were incubated with various concentrations of peptides ranging from 100 to 0.1 µM overnight and then stained with BB7.2 mAb (25)
to quantify the surface expression of HLA-A*0201 allele. For each peptide concentration, the HLA-A*0201-specific staining was calculated as the percentage of staining obtained with 100 µM of the reference peptide HIVpol589 (IVGAETFYV). The RA was determined as: RA = (concentration of each peptide that induces 20% of HLA-A*0201 expression/concentration of the reference peptide that induces 20% of HLA-A*0201 expression).
Assessment of Peptide/HLA-A*0201 Complex Stability.
As previously described (24)
, briefly T2 cells were incubated overnight with 100 µM of each peptide. Cells were then incubated with Brefeldin A (Sigma, St. Louis, MO) at 10 µg/ml for 1 h, washed, incubated at 37°C during 0, 2, 4, or 6 h in the presence of Brefeldin A (0.5 µg/ml), and then stained with BB7.2 mAb. For each time point, peptide induced HLA-A*0201 expression was calculated as: mean fluorescence of peptide preincubated T2 cells - mean fluorescence of T2 cells treated in similar conditions in the absence of peptide. DC50 was defined as the time required for the loss of 50% of the HLA-A*0201/peptide complexes stabilized at t = 0.
Western Blot Analysis of EphA2 Expression by Tumor Cells.
Cellular samples were lysed with lysis buffer [150 mM NaCl, 1% Triton X-100, 50 mM Tris-HCl (pH 7.4), 0.1% SDS, and 0.5% NP40] supplemented with Complete Protease Inhibitor mixture (Boehringer Mannheim, Mannheim, Germany). Fifty µg of protein were loaded on 10% SDS-Polyacrilamide Gel Electrophoreses (Bio-Rad, Hercules, CA) and transferred to nitrocellulose membrane (Amersham Pharmacia, Buckinghamshire, United Kingdom). Detection of EphA2 was done with rabbit polyclonal anti-EphA2 antibody (sc-924; Santa Cruz Biotechnology, Santa Cruz, CA) and goat-antirabbit IgG, (H+L) peroxidase conjugated (Pierce, Rockford, IL). The housekeeping protein, actin, was detected using goat polyclonal antiactin antibody (sc-1615)-horseradish peroxidase (Santa Cruz Biotechnology). Membranes were then reveled with enhanced chemiluminescence kit (Amersham Pharmacia) and exposed to photosensitive film (Amersham Pharmacia).
Generation of CTL in HHD Mice.
HHD mice received s.c. injections of 100 µg of peptide emulsified in incomplete Freund’s adjuvant in the presence of 140 µg of the I-Ab-restricted HBVcore128 (TPPAYRPPNAPIL) T-helper epitope. Eleven days later, 5 x 107 spleen cells were stimulated in vitro with peptide (10 µM). On day 6 of culture, the bulk populations were tested for specific cytotoxicity. Upon response, CTL lines were established by weekly in vitro restimulations with 2 x 107-irradiated spleen cells in the presence of 1–0.1 µM peptide and 50 IU/ml IL-2 (Proleukin; Chiron Corp. Emeryville, CA).
Generation of CTL from Human PBMCs.
PBMCs were collected by leukapheresis from healthy HLA-A*0201 volunteers. Dendritic cells were produced from adherent cells (2 x 106 cells/ml) cultured for 7 days in the presence of 500 IU/ml granulocyte macrophage colony-stimulating factor (Leucomax; Schering-Plough, Kenilworth, NJ) and 500 IU/ml IL-4 (R&D Systems, Minneapolis, MN) in complete medium [RPMI 1640 supplemented with 10% heat-inactivated human AB serum, 2 µM L-glutamine (Invitrogen) and antibiotics]. On day 7, dendritic cells were collected and pulsed with 40 µg/ml peptide in the presence of 3 µg/ml ß2m (Sigma) for 4 h at 20°C and then irradiated (4200 rad). CD8+ T cells were isolated by positive selection with immunomagnetic beads (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer instructions. A total of 0.5 x 106 CD8+ T cells were cocultured with 0.25 x 105 dendritic cells in a final volume of 0.5 ml/well in a 48-well plate in the presence of 10 ng/ml IL-7 (R&D Systems). Human IL-10 (R&D Systems) at 10 ng/ml was added the next day, and 30 IU/ml human IL-2 (Proleukin; Chiron Corp.) were added on day 2. Seven and 14 days after the primary stimulation, CD8+ T cells were restimulated with irradiated adherent cells pulsed with 10 µg/ml peptide in the presence of 3 µg/ml ß2m. Human IL-10 (10 ng/ml) and IL-2 (50 IU/ml) were added 24 and 48 h later, respectively. Seven days after the second restimulation individual wells from the cultures were tested for peptide specific cytotoxicity on peptide loaded T2 cells in the presence of cold K562 cells (hot/cold target ratio 1:33 ratio). Cells from positive wells (>10% peptide specific lysis) were tested for intracellular IFN-
production upon tumor cell stimulation.
Cytotoxic Assay.
Targets were labeled with 100 µCi of 51Cr for 60 min, plated in 96-well V-bottomed plates (3 x 103 cell/well) and pulsed with peptides (1 µM) at 37°C for 2 h. Effectors were then added and incubated at 37°C for 4 h. One hundred µl of supernatant were collected and radioactivity measured in a gamma counter. Percentage of specific lysis was determined as: (experimental release - spontaneous release)/(maximal release - Spontaneous Release) x 100.
Peptide Processing Assay on Transfected COS Cells.
A total of 2.2 x 104 simian COS-7 cells were plated in flat-bottomed 96-well plates in DMEM (Invitrogen) 10% FCS. Eighteen h later, cells were transfected with 100 ng of DNA plasmid with DEAE Dextran (Sigma). After 4 h, PBS +10% DMSO were added for 2 min. Transfected COS cells were incubated in DMEM +10% FCS during 40 h and then used to stimulate murine CTL in a TNF- secretion assay.
TNF- Secretion Assay.
Transfected COS cells at day 4 or 105 tumor cells were used as stimulating cells. They were plated in 50 µl of RPMI +10% FCS and, when necessary, incubated with 10 µM peptide for 2 h at 37°C. A total of 5 x 104 T cells were then added in 50 µl of RPMI +10% FCS and incubated for 6 h. Each condition was tested in triplicates. Fifty µl of the supernatant were collected for quantification of TNF- using the WEHI-164c13 cells as described previously (26)
.
Intracellular Detection of IFN-.
A total of 5 x 104 T cells were incubated with 105 peptide loaded T2 cells or with 105 tumor cells in the presence of 20 µg/ml Brefeldin A at 37°C. Six h later, they were stained with phycoerythrin-conjugated anti-CD8 mAb (Caltag Laboratories, Burlingame, CA) in PBS for 25 min at 4°C and fixed with PBS 4% Paraformaldeide (Sigma). Cells were then permeabilized with PBS +0.5% BSA +0.2% saponin (Sigma) and stained with adenomatous polyposis coli-conjugated anti-IFN- mAb (PharMingen, Mississauga, Ontario, Canada) for 25 min at 4°C. Cells were analyzed on a FACSCalibur (Becton Dickinson, Mountain View, CA) flow cytometer.
Cancer Patient PBMC Restimulation.
PBMCs were incubated in a 96 U-bottomed plate (2 x 105 cells/well) in the presence of 10 µM peptide and 20 IU/ml IL-2 in AIM-V medium (Invitrogen). Half of the media was changed every other day with fresh one containing IL-2 at 20 IU/ml. At day 9 of culture, pools of 12 wells were tested for the presence of peptide-specific CD8+ T cells by staining with the appropriate HLA-A*0201/peptide tetramer.
HLA-A*0201/Peptide Tetramer Staining of PBMCs.
Phycoerythrin-conjugated HLA-A*0201/peptide tetramers were synthesized by ProImmune (Oxford, United Kingdom). Cells were stained with tetramers (5 ng/ml) in PBS +0.5% human AB serum for 1 h at room temperature, washed once in the same buffer, stained with antihuman CD3-FITC (Caltag Laboratories) and antihuman CD8-APC (PharMingen) mAbs for 30 min at 4°C followed by flow cytometry acquisition.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), fulfilling the characteristics of immunogenic peptides (24)
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, demonstrating that both peptides are immunogenic in vivo. We established CTL lines specific for EphA258 (mCTL58) and EphA2550 (mCTL550) from responding mice and tested their efficiency to recognize the cognate peptides. mCTL58 and mCTL550 lines exhibited high avidity with a half maximal lysis obtained with 10 and 20 nM of peptide, respectively (Fig. 1B)
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1 and 2 domains of HLA-A*0201 allele and the 3 and intracellular domains from H-2Db allele; Ref. 23
) and for EphA2 antigen. CTL activation was measured by TNF- secretion. mCTL58 and mCTL550 were activated by COS cells coexpressing HHD and EphA2 but not by COS cells expressing only HHD or EphA2 (Fig. 2A)
. These results show that EphA258 and EphA2550 are naturally processed epitopes. However, the COS-7 model ensures a very high level of antigen expression, which probably is not reached in tumor cells. To validate that EphA258 and EphA2550 are efficiently presented by human tumor cells, we stimulated mCTL58 and mCTL550 with the HLA-A*0201+ EphA2+ CACO-2 cell line (Ref. 13
and Figs. 3, A and B
) and measured TNF- secretion. Two negative controls (LNCaP, HLA-A*0201+ EphA2- and DU-145, HLA-A*0201- EphA2+; Ref. 13
; Fig. 3, A and B
) were also included. Both mCTL lines were stimulated by CACO-2 but not by LNCaP nor by DU-145 cells (Fig. 2B)
. Because our aim was to identify tumor epitopes expressed by a variety of human cancers, we asked whether these peptides were efficiently presented by cell lines derived from EphA2-expressing tumors of different origins. Western blot analysis revealed that the HLA-A*0201-positive HIEG (renal cell carcinoma), 1355 (lung carcinoma), and SAOS (sarcoma) cell lines expressed EphA2 (Fig. 3, A and B)
. We then used the HIEG, 1355, SAOS, CACO-2, and LNCaP tumor cells to stimulate mCTL58 and mCTL550. Both CTL lines responded to the EphA2+ HIEG, 1355, CACO-2, and SAOS but not to the EphA2- LNCaP cells (Fig. 3C)
. For both mCTL lines, the strongest TNF- response was obtained by HIEG and SAOS. These data, taken together, indicate that EphA258 and EphA2550 are naturally processed and presented by a variety of human tumor cells of different histological origins.
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-producing cells upon activation. Results in Fig. 4
confirm the peptide specificity of hCTL58 and hCTL550. CD8+ T cells were activated by T2 cells loaded with the corresponding EphA2 peptides but not by T2 cells loaded with an irrelevant peptide. Importantly, hCTL58 and hCTL550 responded to the HLA-A*0201+ EphA2+ CACO-2 and 1355 but not to the HLA-A*0201+ EphA2- LNCaP, M113, and MCF-7 nor to the HLA-A*0201- EphA2+ DU-145 tumor cells (Fig. 5)
, confirming that EphA258 and EphA2550 are presented by human tumor cells associated with HLA-A*0201.
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and data not shown). In contrast, after one in vitro restimulation, tetramer-positive CD8+ T cells appeared in one of five patients tested. In fact, in two independent cultures of PBMCs of patient VAV (prostate cancer), 0.15% of CD8+ T cells were stained with the HLA-A*0201/EphA258 tetramer (Fig. 6)
, suggesting that in this patient, EphA258-specific CD8+ T cells were in vivo amplified, although to a level that can only be detectable after in vitro restimulation.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-actinin) or nonmutated (G250, GnTV, RAGE) with a restricted expression to some tumors; and finally tumor/testis antigens (MAGE-A, NY-ESO-1, BAGE, GAGE; for review see Ref. 27
). A particular interest has been recently focused to another group of antigens that are nonmutated proteins such as hTERT (6)
, HER-2/neu (28)
, and Survivin (7
, 8)
, which are frequently overexpressed in a large variety of tumors. These TAgs can be used for broad spectrum tumor immunotherapy. The tyrosine kinase EphA2, which belongs to the Eph family receptors, is also overexpressed in a high percentage of tumors of various origins such as breast, ovarian, lung, prostate, gastric cancer, and sarcomas (11, 12, 13, 14, 15)
. In addition, it is involved in tumor metastatization and is expressed at a higher level in metastasis than in primary tumors (11
, 14)
. Finally, EphA2 is involved in tumor angiogenesis and expressed by tumor neovasculature (20)
. Therefore, an EphA2-specific CTL response would particularly target tumor cells with high metastatic potential and tumor vascular endothelial cells, thus preventing tumor dissemination and growth. In this study, we describe two HLA-A*0201-restricted epitopes (EphA258 and EphA2550) that derive from the EphA2 protein and that are targets of antitumor CD8+ T cells. These epitopes have a high affinity for HLA-A*0201, trigger CTL in vivo in the HLA-A*0201-transgenic HHD mice and in vitro in healthy humans. In this study, we also present data suggesting an in vivo amplification of EphA258-specific CD8+ T cells in prostate cancer patients. Although melanoma, prostate, and lung cancer patients presented frequencies of CD8+ T cells specific for peptides EphA258 and EphA2550 comparable with the one detected on HLA-A*0201 healthy donors when assessed by ex vivo staining with the appropriate tetramer, on one prostate patient (VAV), the in vitro restimulation of PBMCs with peptide revealed the presence of EphA258-specific CD8+ T cells. This observation may be particularly important for peptide-based cancer immunotherapy. It provides the rational for therapeutic vaccination of cancer patients. However, a phenotypic and functional activity characterization of these in vivo-amplified CD8+ T cells remains to be evaluated.
As with the majority of universal TAgs, EphA2 is expressed at low level by normal tissues such as lung, kidney, skin, ovaries, and even the thymus (29 , 30) . This raises two questions that are common for all of the ubiquitously expressed antigens: (a) is there tolerance to EphA2, and if there is, how can we recruit in vivo anti-EphA2 CTL with high avidity; and (b) what is the risk of autoimmunity after vaccination with EphA2 CTL epitopes? These questions are being currently addressed in the HHD mouse model by using EphA2 epitopes that, unlike EphA258 and EphA2550, are shared by human and murine EphA2 sequences. However, the fact that we can induce CTL from human healthy donors after a reduced number of restimulations and, importantly, that these CTL are amplified in vivo in cancer patients suggest that the EphA2-specific CTL repertoire is not completely tolerized.
Several other epitopes have been described from TAgs of ubiquitous phenotype, namely HER-2/neu and hTERT (28) . The inclusion of epitopes of these antigens in a polyepitopic construct would be highly favorable to the development of vaccine strategies that would trigger polyspecific responses against a large number of tumors. An additional advantage of these strategies is to diminish the risks of tumor escape by loss of the TAg (31) . The EphA2-derived epitopes, here identified, could therefore be used along with hTERT and HER-2/neu epitopes in polyspecific vaccines with broad application in tumor immunotherapy.
In conclusion, in this article, we identified two HLA-A*0201-restricted EphA2-derived epitopes that induce EphA2-specific antitumor CTL response. EphA2, which is expressed by tumors of various origins, could, therefore, be considered as new universal TAg.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Requests for reprints: Pedro Alves, INSERM U487, Institut Gustave Roussy, PR1, 39 rue Camille Desmoulins, 94805 Villejuif, France. Phone: 33-0-142114921; Fax: 33-0-142115288; E-mail: kostas{at}igr.fr
4 The abbreviations used are: TAg, tumor antigen; hTERT, human telomerase reverse transcriptase; ß2m, ß2 microglobulin; mAb, monoclonal antibody; IL, interleukin; RA, relative affinity; PBMC, peripheral blood mononuclear cell; TNF-, tumor necrosis factor . 
Received 5/ 3/03. Revised 8/18/03. Accepted 9/16/03.
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) or an irrelevant (
) peptide at a E/T ratio of 60:1. Results represent the mean ± SD of triplicates and were confirmed in three independent experiments. B, mCTL58 and mCTL550 cell lines were established as described in "Materials and Methods." They were tested against RMAS/HHD targets in the presence of various concentrations of EphA2 peptides at a E/T ratio of 30/1.
