Japanese Journal of Clinical Oncology 30:117-121 (2000)
© 2000 Foundation for Promotion of Cancer Research
Analysis of Individual Specific Cytotoxic T Lymphocytes for Two MAGE-3-derived Epitopes Presented by HLA-A24
1Department of Otorhinolaryngology, Kumamoto University School of Medicine, Kumamoto and 2Allergy and Immunology Center of Kyushu, Kumamoto, Japan
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Background: The human MAGE-3 gene encodes tumor-specific antigens that are recognized by cytotoxic T lymphocytes (CTLs) and expressed in a high percentage of various malignant tumors. Of the five MAGE-3-derived CTL epitopes identified to date, two nonapeptides (TFPDLESEF and IMPKAGLLI, designated MAGE-3.A24a and MAGE-3.A24b, respectively) can be expressed on the tumor surface by binding to the HLA-A24 molecule, which is the most frequent HLA class I molecule in Asian populations. To compare the immunogenecities of the two peptides, individual specific CTL lines were generated for each peptide (MAGE-3.A24a and MAGE-3.A24b).
Methods: Peripheral blood mononuclear cells (PBMCs) from four HLA-A24+ healthy donors were stimulated in vitro with autologous dendritic cells pulsed with MAGE-3.A24a, MAGE-3.A24b or both and were subsequently cultivated with a cytokine combination including interleukin-2.
Results: We succeeded in generating peptide-specific CTL lines in two of the four donors. The two CTL lines showed similar cytolytic levels against three MAGE-3+/HLA-A24+ cancer cell lines and also target cells pulsed with the corresponding peptide. Cytolytic activities were blocked by either anti-CD8 or anti-HLA-A24 monoclonal antibodies.
Conclusions: The results suggest that MAGE-3.A24a and MAGE-3.A24b peptides have equal potential in inducing MAGE-3-specific and HLA-A24-restricted CTLs.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The MAGE gene family, containing at least 12 different genes, is expressed in various histological types of tumor but not in normal cells except for germ line cells of the testis (1). The MAGE-3 gene in this family is expressed in a high percentage of several types of tumor including squamous cell carcinoma of the head and neck (SCCHN) (2–4).
In Europe, one MAGE-3-derived cytotoxic T lymphocyte (CTL) epitope, the MAGE-3.A1 peptide, which can bind to the HLA-A1 molecule, was used as a cancer vaccine for 39 HLA-A1+ melanoma patients whose tumors expressed the MAGE-3 gene. Of the 25 patients who completed the course, seven manifested an objective clinical response with two showing long-term complete response (5). This previously reported clinical effectiveness of MAGE-3.A1 peptide encouraged us to extend MAGE-3.A1 peptide immunization to various malignant tumors and melanomas. However, less than 1% of the majority of Asians including Japanese expresses HLA-A1, in contrast to approximately 28% of Caucasians (6–7). Another two CTL epitopes derived from MAGE-3 can bind to HLA-A2 (MAGE-3.A2 peptide) and HLA-B44 (MAGE-3.B44 peptide), which are expressed by 42.4 and 14% of Japanese, respectively (8–10). Although HLA-A2 is frequently expressed in both Asians and Caucasians, the usefulness of the MAGE-3.A2 peptide for specific immunotherapy is controversial. Valmori et al. (11) suggested a limited usefulness of the MAGE-3.A2 peptide on the basis of their finding that MAGE-3.A2-specific CTLs induced from PBMCs of A2+ melanoma patients failed to recognize MAGE-3+/HLA-A2+ melanoma cells. To extend the possibility of immunotherapy with MAGE-3 antigen, we recently identified MAGE-3-derived peptide recognized by HLA-A24-restricted CTLs using PBMCs of HLA-A24+ healthy donors because HLA-A24 is one of the most frequently expressed molecules in Japanese (12). Another group has also succeeded in generating CTLs specific for a different MAGE-3-derived epitope from an HLA-A24+ healthy donor (13). Identification of these CTL epitopes opens up the possibility of clinical applications of these peptides as cancer vaccines for patients with MAGE-3+/HLA-A24+ tumors. Before clinical application of these peptides, several issues should be addressed. These include (1) which peptide is immunodominant in generating MAGE-3-specific and HLA-A24-restricted CTL, (2) whether a precursor CTL specific for the respective peptides exists in PBMC or tumor-infiltrating lymphocytes of HLA-A24+ cancer patients whose tumor express the MAGE-3 gene and (3) what kind of adjuvant should be used in combination with these peptides.
In the present study, we investigated peptides that are immunodominant in generating MAGE-3-specific and HLA-A24-restricted CTLs.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cell Lines
Cells of the major histocompatibility complex (MHC) class I-negative lymphoblastoid cell line C1R transfected with a cDNA clone encoding HLA-A*2402 (C1R-A*2402) were maintained in RPMI-1640 medium (Gibco-BRL, Gaithersburg, MD) supplemented with 10% fetal bovine serum (FBS) (Gibco-BRL) and 0.15 mg/ml of hygromycin B (Wako Pure Chemical Industries, Tokyo, Japan) and were used as target cells for cytotoxic assay. Tongue cancer cell lines HSC-3 (JCRB0623; HLA-A*2402/0201) and HSC-4 (JCRB0624; HLA-A*2402/3101) and colon carcinoma cell line WiDr (CCL218; HLA-A24+) were obtained from the Japanese Cancer Research Bank (Tokyo, Japan). These cells and erythroleukemia cell line K562 were cultured in RPMI-1640 medium supplemented with 10% FBS, L-glutamine and antibiotics.
Monoclonal Antibodies and Cytokines
Anti-CD8 monoclonal antibody (MAb) (mouse IgG1) and anti-HLA-A24 MAb (IgG2b) recognizing HLA-A23/24 were purchased from Beckton Dickinson (Mountain View, CA) and One Lambda (Canoga Park, CA), respectively. FITC-rabbit anti-mouse IgG was purchased from Zymed (San Francisco, CA). Cytokines used in this study were obtained from the following sources: human recombinant IL-1
and IL-6, Genzyme (Boston, MA); human recombinant GM-CSF, R&D Systems (Minneapolis, MN); and IFN-
, Endogen (Woburn, MA). Human recombinant IL-2, IL-4 and IL-12 were kindly provided by Takeda Chemical Industries (Osaka, Japan), Ono Pharmaceutical (Osaka, Japan) and Yamanouchi Pharmaceutical (Tokyo, Japan) respectively.
Flow Cytometric Analysis
Tumor cell lines incubated with and without IFN- (1000 U/ml) for 48 h were evaluated for their expression of HLA-A24 molecules by flow cytometry. These cell lines were incubated with anti-HLA-A24 MAb for 30 min on ice, washed twice and then incubated with FITC-conjugated rabbit antibodies to IgG for another 30 min on ice. After two additional washes, cells were analyzed on a FACScan (Beckton Dickinson). All incubations and washes were performed in phosphate-buffered saline (PBS) containing 0.1% NaN3 and 2% FBS.
MAGE-3-derived Synthetic Peptides
Two MAGE-3-derived peptides, 97TFPDLESEF105 and 195IMPKAGLLI203 (designated MAGE-3.A24a and MAGE-3.A24b, respectively), were synthesized using Fmoc methodology, purified by HPLC and sequences confirmed by mass spectral analysis (14). The lyophilized peptides were dissolved in DMSO at a concentration of 2 mg/ml and stored at –80°C.
Induction of Peptide-specific CTLs
PBMCs from four HLA-A24+ healthy donors were obtained by centrifugation on Ficoll-Hypaque gradients. HLA-A typing of PBMCs was done by using FACScan with anti-HLA-A24 MAb. Dendritic cells (DCs) were generated from PBMCs using a modification of the method reported by Sallusto and Lanzavecchia (15). Briefly, PBMCs were resuspended at 10 x 106 cells/ml in AIM-V medium (Gibco-BRL) and distributed in T-75 flasks (Falcon, Becton Dickinson). After 2 h of incubation, the non-adherent cells were removed by gentle washing and AIM-V medium containing GM-CSF (1000 U/ml) and IL-4 (10 ng/ml) were added. After 7 days of incubation, non-adherent DCs were harvested and used as antigen-presenting cells (APCs). The DCs were resuspended in AIM-V medium containing 40 µg/ml peptide (MAGE-3.A24a, MAGE-3.A24b or both) and incubated at 37°C for 4 h. Subsequently, the peptide-pulsed DCs were irradiated (3000 rad) and washed. At day 0, 2 x 106 PBMCs and 2 x 105 peptide-pulsed DCs were co-cultured in 24-well tissue culture plates in a final volume of 2 ml AIM-V medium supplemented with 5% human AB serum, IL-6 (125 IU/ml) and IL-12 (1 ng/ml). On day 7 and weekly thereafter, responder cells were restimulated with peptide-pulsed autologous adherent PBMCs in AIM-V medium supplemented with IL-1 (10 U/ml), IL-2 (10 IU/ml), IL-4 (1 ng/ml) and IL-6 (125 IU/ml). These cells were prepared as follows: irradiated 4 x 106 PBMC were incubated for 2 h in a final volume of 1 ml/well AIM-V medium containing 10 µg/ml of each peptide in a 24-well plate and then non-adherent cells and excess peptide-containing medium were gently removed. To the 24-well plate containing peptide-pulsed autologous adherent PBMCs, the responder lymphocytes were added in a cytokine-supplemented medium. After five rounds of stimulation, responder cells were tested for their specificity.
Cytotoxicity Assays
Chromium release assays were performed as described previously(12). Briefly, target cells (1 x 106) were labeled with 51Cr for 1 h. Effector cells were added to 2 x 103 target cells in triplicate wells of V-bottomed microtiter plates (Limbro, Flow Laboratories, McLean, VA) in a final volume of 0.2 ml. For the Ab-blocking assays, effector cells or 51Cr-labeled target cells were preincubated with each MAb for 20 min. The labeled target cells were plated at a concentration of 2 x 103 cells/well and the effector cells were added to each well at an E:T ratio of 20:1. After 4 h of incubation, the supernatant was harvested and chromium release was measured. Percentage specific lysis was calculated from the average of triplicate wells as [(E – S)/(M – S)] x 100%, where E is the experimental release, S is the spontaneous release and M is the maximum release.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Induction of CTLs Specific for MAGE-3-derived Peptides in HLA-A24+ Individuals
Two MAGE-3-derived peptides, MAGE-3.A24a and MAGE-3.A24b, were tested for the potential to induce HLA-A24-restricted CTLs by stimulating PBMCs from four HLA-A24+ healthy donors with irradiated DCs pulsed with either MAGE-3.A24a or MAGE-3.A24b peptides. After five rounds of stimulation, responder cells effectively lysed the corresponding peptide-pulsed C1R-A*2402 cells in two of four HLA-A24+ donors but did not lyse either irrelevant peptide-pulsed or -unpulsed target cells. The results obtained in one donor are shown in Fig. 1 because the responder cells in the two HLA-A24+ donors showed similar results. Responder cells induced by stimulation of MAGE-3.A24a peptide effectively lysed MAGE-3.A24a pulsed target cells but did not lyse either MAGE-3.A24b peptide-pulsed or -unpulsed target cells or the K562 cell line (Fig. 1A). In addition, the cytotoxicities of these effector cells were blocked by either anti-CD8 or anti-HLA-A24 MAbs (Fig. 1A). These results show that the CTL lines were CD8 dependent and HLA-A24 restricted. Similarly, CTLs induced by MAGE-3.A24b peptide specific showed MAGE-3.A24b-specific cytotoxicity and the CTLs were also CD8 dependent and HLA-A24 restricted (Fig. 1B). Both the MAGE-3.A24a peptide-specific and MAGE-3.A24b peptide-specific CTLs exhibited similar cytolytic levels against the corresponding peptide-pulsed target cells.
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) or MAGE-3.A24b peptide (•) under peptide concentration 1 µM, unpulsed (
) or K562 cell line (
) (2 x 103 cells/well) were used as targets at the indicated E/T ratio. For the HLA-A24 blocking assay, C1R-A*2402 (2 x 103 cells/well) pulsed with peptide MAGE-3.A24a peptide(•) or MAGE-3.A24b peptide(
) were added to the corresponding peptide-specific CTL lines. To block CD8, the effector cells were preincubated with anti-CD8 MAb (open triangle) before the addition of the 51Cr-labeled cells and the corresponding peptide-pulsed C1R-A*2402. (C) Induction of peptide-specific CTLs by stimulating a mixture of MAGE-3.A24a peptide and MAGE-3.A24b peptide. Effector cells were induced from the PBMC of an HLA-A24+ healthy donor by five rounds of stimulation with autologous cells with a mixture of MAGE-3.A24a peptide and MAGE-3.A24b peptides. C1R-A*2402 (2 x 103 cells/well) pulsed with MAGE-3.A24a peptide (A mixture of MAGE-3.A24a and MAGE-3.A24b peptides, instead of each peptide alone, was also used for induction of peptide-specific CTLs. After five rounds of stimulation, responder cell lysis of both MAGE-3.A24a and MAGE-3.A24b peptides was significantly greater in pulsed target cells than in unpulsed target cells (Fig. 1C; p < 0.05). These results indicate that both peptide-specific CTLs were generated simultaneously in this condition, indicating that co-existence of the two peptides did not interfere with induction of each peptide-specific CTL.
Recognition of Cancer Cell Lines Expressing Both HLA-A24 Molecules and MAGE-3 Gene by the MAGE-3.A24a or MAGE-3.A24b Peptide-specific CTLs
Previous reports demonstrated that three cancer cell lines, HSC-3, HSC-4 and WiDr, were MAGE-3+/HLA-A24+ and that the first two were sensitive to MAGE-3.A24a-specific CTLs(12) and the third was sensitive to MAGE-3.A24b-specific CTLs (13). Therefore, to analyze cytolytic activity of MAGE-3.A24a- or MAGE-3.A24b-specific CTLs against MAGE-3+/HLA-A24+ cancer cell lines, we used these cancer cell lines (HSC-3, HSC-4 and WiDr) as target cells. As shown in Fig. 2, the expression of HLA-A24 molecules on these cell lines was confirmed by flow cytometry using anti-HLA-A24 MAb. As expected, pretreatment of these cancer cell lines with IFN- enhanced the expressions of HLA-A24 molecules on their cell surface. Both MAGE-3.A24a peptide-specific and MAGE-3.A24b-peptide specific CTLs significantly lysed these cell lines at an E/T ratio of 20 (p < 0.05) (Fig. 3 ). Among untreated cell lines, WiDr was the most sensitive to cytolysis by these two CTL lines. Moreover, pretreatment of these cancer cell lines with IFN- enhanced their sensitivities to cytolysis by both the MAGE-3.A24a peptide-specific CTLs (Fig. 3A) and the MAGE-3.A24b peptide-specific CTLs (Fig. 3B). Especially in both CTLs, HSC-4 pretreated with IFN- was twice as susceptible to lysis as HSC-4 not treated with IFN-. The killing activity of the CTL lines against HSC-3, HSC-4 and WiDr was abrogated by either anti-CD8 or anti-HLA-A24 MAbs. Additionally, CTL lines induced by a mixture of MAGE-3.A24a and MAGE-3.A24b peptides that consisted of MAGE-3.A24a peptide-specific and MAGE-3.A24b peptide-specific CTL lines showed similar levels of cytolysis against HSC-4 and WiDr to those of CTLs generated by stimulation with either MAGE-3.A24a or MAGE-3.A24b alone (Fig. 3C).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In the present study, using peptide-pulsed autologous DCs as APCs, we succeeded in generating separate CTL lines specific for MAGE-3.A24a and MAGE-3.A24b peptides from PBMCs of two of four HLA-A24+ healthy individuals. In these two, both MAGE-3.A24a peptide and MAGE-3.A24b peptide-specific CTLs were induced, but in the other two individuals, neither MAGE-3.A24a peptide- specific nor MAGE-3.A24b peptide-specific CTLs were induced. Hence the existence of a precursor CTL specific for each of the two MAGE-3 derived peptides was confirmed in two of four donors tested.
We previously examined the cytotoxicity of MAGE-3.A24a-specific CTL against HSC-3 and HSC-4, but not against WiDr (12). On the other hand, the Japanese group that identified MAGE-3.A24b as a CTL epitope performed an experiment examining the cytotoxity of MAGE-3.A24b-specific CTL against WiDr, but not against HSC-3 and HSC-4 (13). In the present study, we tested the cytotoxicities of both MAGE-3.A24a-and MAGE-3.A24b-specific CTLs against these three cancer cell lines. These specific CTL lines lysed all of these MAGE-3+/HLA-A24+ cancer cell lines in a CD8-dependent and HLA-A24-restricted manner. These results suggest that these cell lines express both MAGE-3.A24a- and MAGE-3.A24b peptides presented by HLA-A24 molecules on the cell surface of the cancer cell lines.
Pretreatment of the MAGE-3+/HLA-A24+ cancer cell lines (HSC-3, HSC-4 and WiDr) with IFN- induced relatively high expression of HLA-A24 molecules on the surface of the target cells as compared with those not treated with IFN- and enhanced the susceptibility of these cells to lysis by the two CTL lines. These results suggest that MAGE-3.A24a and MAGE-3.A24b peptides are present at a low level on the untreated cancer cell surface.
The percentage specific lysis of MAGE-3.A24a peptide-specific CTLs against these target cells was similar to that of MAGE-3.A24b peptide-specific CTLs in target cells pretreated with and without IFN-.
A mixture of the two peptides also induced each peptide-specific CTLs with similar cytotoxic levels against both peptide-pulsed targets and MAGE-3+/HLA-A24+ cancer cell lines to those observed in the corresponding peptide-specific CTLs induced by stimulation of each peptide alone. Hence the co-existence of MAGE-3.A24a and MAGE-3.A24b peptides in culture medium did not interfere with the induction of each peptide-specific CTL. These results suggest that MAGE-3.A24a and MAGE-3.A24b peptides have equal potential in inducing MAGE-3 specific and HLA-A24 restricted CTL.
MAGE-3.A24a and MAGE-3.A24b have previously shown similarly high capacities to bind to the HLA-A24 molecule (12,13). This prior finding may explain the lack of significant difference between the abilities of the two peptides to induce corresponding peptide-specific CTLs in our present study.
In conclusion, our results confirmed that MAGE-3-specific and HLA-A24-restricted CTL can be generated by stimulation of the MAGE-3.A24a and/or MAGE-3.A24b peptides and suggested that the two peptides are equally available for specific immunotherapy against MAGE-3+/HLA-A24+ cancer cells.
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FOOTNOTES |
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+ For reprints and all correspondence: Masao Eura, Department of Otorhinolaryngology, Kumamoto University School of Medicine, 1-1-1 Honjo, Kumamoto 860-8556, JapanAbbreviations: CTL, cytotoxic T lymphocyte; PBMC, peripheral blood mononuclear cell; SCCHN, squamous cell carcinoma of the head and neck; MHC, major histocompatibility complex; MAb, monoclonal antibody; PBS, phosphate-buffered saline; DC, dendritic cell; APC, antigen presenting cell
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Received September 27, 1999; Accepted December 3, 1999.
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