Japanese Journal of Clinical Oncology 31:311-317 (2001)
© 2001 Foundation for Promotion of Cancer Research
Generation of Autologous Tumor-specific T Cell Clones From a Patient With Adenosquamous Carcinoma of the Lung
Department of Surgery II, School of Medicine, University of Occupational and Environmental Health, Kitakyushu, Japan
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONAcknowledgmentsREFERENCES |
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Background: Adenosquamous carcinoma of the lung is not a common cancer, but its prognosis is worse than that of adenocarcinoma or squamous cell carcinoma. Therefore, new therapeutic strategies need to be developed to treat this type of lung cancer. Recently, vaccination using tumor antigens which are recognized by cytotoxic T lymphocytes (CTL) has been applied mainly to melanoma patients. We therefore attempted to establish T cell clones specific for autologous tumor cells (AT) from a patient with adenosquamous carcinoma in order to analyze the specific immune responses against AT.
Methods: A lung adenosquamous carcinoma cell line was established from a resected tumor obtained from a 72-year-old patient. Regional lymph node lymphocytes were stimulated weekly with CD80-transfected AT to induce CTL. The CTL activities were assessed by a standard 51Cr release assay and by cytokine release.
Results: We succeeded in inducing an AT-specific CTL line. Using a limiting dilution method, eight T cell clones were established. AT-specific activity was observed in three CD8+ T cell clones and one CD4+ T cell clone out of the eight clones tested. Anti-HLA class I and anti-HLA-B/C mAbs inhibited IFN-
production from the AT-specific CD8+ clones co-cultured with AT, thus indicating the restriction element to be HLA-B*5201 or HLA-Cw*1202. In contrast, the CD4+ T cell clone recognized AT in an HLA class II-restricted manner.
Conclusions: These results are the first demonstration of a successful induction of AT-specific T cell clones from a patient with lung adenosquamous carcinoma. It may therefore supply a possible way to apply specific immunotherapy to this type of lung cancer.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONAcknowledgmentsREFERENCES |
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Adenosquamous carcinoma of the lung is an uncommon entity. It is defined as a tumor composed of squamous cell carcinoma and adenocarcinoma components of at least 20% each based on a microscopic examination, according to the general rules for the clinical and pathological recording of lung cancer as established by the Japan Lung Cancer Society (1). Its frequency is reported to be 0.4–4.2% of lung cancers (2–5). Lung cancer has one of the worst prognoses compared with tumors occurring in other organs. In lung cancers, the prognosis of adenosquamous carcinoma is reported to be poorer than that of adenocarcinoma and squamous cell carcinoma (5). We therefore have to create new therapeutic strategies against lung cancer including adenosquamous carcinoma.
In the 1990s, numerous human tumor antigens were identified, mainly in melanoma, since the identification of the MAGE gene (6,7). Moreover, synthetic peptides derived from identified tumor antigens have been used as vaccines to treat patients with malignant melanoma (8–11). Clinical trials using these peptide vaccines are now under way and some of them have been reported to be tolerable and effective, especially against malignant melanoma. The development of methods of immunological monitoring remains a major issue regarding the vaccination of patients with melanoma in order to understand the mechanism of tumor regression (12,13).
However, the identification of tumor antigens in malignancies has been limited to only a few cancers, including mainly melanoma, because of difficulties in establishing tumor cell lines from cancer tissues and in inducing cytotoxic T lymphocytes (CTL). We previously reported the successful induction of tumor-specific CTL from a patient with adenocarcinoma, which thus indicated the presence of tumor antigens in lung adenocarcinoma recognized by autologous CTL (14). In this paper, we report the establishment of a lung adenosquamous carcinoma cell line and the generation of autologous tumor (AT)-specific T cell clones in order to understand the tumor-specific immune responses which arise in patients with this type of lung cancer.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONAcknowledgmentsREFERENCES |
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Culture Medium (CM)
The CM consisted of RPMI 164 (GIBCO-BRL, Grand Island, NY) supplemented with 10% heat-inactivated fetal calf serum (Equitech-Bio, Ingram, TX), 10 mM HEPES, 100 units/ml penicillin G and 100 mg/ml streptomycin sulfate (14).
Patients and Cell Lines
A lung adenosquamous carcinoma cell line designated A529L was established from a specimen obtained from a 72-year-old man who underwent a left upper lobectomy with a combined resection of the chest wall in our department. The patient’s pathological stage was T3N2M0, stage IIIA according to the general rules for the clinical and pathological recording of lung cancer as established by the Japan Lung Cancer Society (1). The tests for identification of HLA genotypes of the patient and tumor cell lines used in this study were performed by Shionogi Co. (Osaka, Japan) and the results are shown in Fig. 4. Epstein–Barr virus transformed B cells (EBV-B) were produced from this patient by an infection of peripheral blood mononuclear cells with supernatant from the EBV producer line B95.8. As indicated in Fig. 4, A529L showed each allelic loss in HLA-A, -B and -C loci as compared with EBV-B. A529L did not express CD80 on their surfaces (data not shown). K562 is an erythroleukemia cell line lacking MHC class I expression on the cell surface and is sensitive to natural killer cell cytotoxicity. A110L, RERF-LC-AI and PC9, lung adenocarcinoma cell lines, were described previously (14) and A110L and RERF-LC-AI expressed exactly the same HLA class I genotypes as A529L. All cell lines were maintained in CM. Tumorigenesis of the A529L was confirmed by transplantation of the cells into a SCID mouse (C.B-17 scid/scid, Charles River, Tokyo, Japan).
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Transfection of CD80 into Tumor Cells
A529L were transfected with 2 µg of the pBj/hCD80 plasmid containing human CD80 gene (kindly provided by Dr M. Azuma, National Children’s Medical Research Center, Department of Immunology) (15) using a lipofectin reagent (GIBCO-BRL) according to the instruction manual. This cell line was selected with G418 (0.8 mg/ml) for more than 1 month. The successful induction of CD80 in G418-resistant cells was confirmed by flow cytometry following staining with FITC-conjugated antihuman CD8 (PharMingen, San Diego, CA) and viable cells were then expanded to a continuous cell line.
Isolation of Lymphocytes
Regional lymph nodes from patient A529 were obtained at the time of surgery. Each lymph node was divided into two parts for the histological diagnosis and for this study. The latter part of each lymph node was squeezed between a pair of glass slides in Hanks’ balanced salt solution (HBSS) and then passed through a gauze filter. The cells were washed twice with HBSS and then resuspended in CM (14). RLNL were frozen in a deep-freezer at –130°C until use.
Induction of CTL Clones
RLNL were rapidly thawed and stimulated with solid-phase anti-CD3 mAb (Ortho Pharmaceutical, Raritan, NJ) for 48 h and expanded in CM containing 50 units/ml recombinant interleukin-2 (rIL-2) (kindly donated by Takeda Chemical Industries, Osaka, Japan) in 24-well plates (Iwaki Glass, Tokyo, Japan) for 14 days at 37°C in a 5% CO2 atmosphere as previously reported (14). Subsequently, the lymphocytes were stimulated with irradiated (100 Gy) CD80-transfected autologous tumor cells weekly at a tumor-to-lymphocyte ratio of 1:10 in CM with 50 units/ml rIL-2 for 5 weeks. CTL activities of the bulk CTL line thus obtained were assessed at 7 days after five successive stimulations.
To generate T cell clones, limiting dilution was performed from the bulk CTL line by the following method. The cells were seeded at 0.1, 0.3, 1 or 3 cells per well in 96-well U-bottomed plates (Iwaki Glass) and were stimulated under the culture conditions as follows; CM with irradiated CD80-transfected AT (5 x 103/well) and EBV-B established from allogeneic PBMC (5 x 104/well) as feeder cells in the presence of IL-2 (50 units/ml), IL-4 (5 ng/ml) (Serotec) and IL-7 (5 ng/ml) (Genzyme Techne). Next, irradiated tumor cells and feeder cells were added to each well for the restimulation of lymphocytes once per week with CM containing IL-2, IL-4 and IL-7.
mAb (Monoclonal Antibody)
Hybridomas (HB-145, HB-95) were purchased from the American Type Culture Collection (ATCC, Rockville, MD). C7709.A2.6 (anti-HLA-A24) and B 1.23.2 (anti-HLA-B/C) were kindly donated by Dr P. G. Coulie (Cellular Genetics Unit, Université Catholique de Louvain, Brussels). The culture supernatants of ATCC HB-145 (IVA12; anti-HLA-DR, DP, DQ), HB-95 (W6/32; anti-HLA-A, B, C), C7709.A2.6 and B 1.23.2 were used for analyzing the HLA restriction of T cell clones.
FITC-conjugated Nu-TH/I (anti-CD4) was purchased from Nichirei (Tokyo, Japan). PE-conjugated Leu-2a (anti-CD8) was purchased from Becton & Dickinson (Mountain View, CA) for phenotypic analysis.
Cytotoxicity Assay
The cytotoxicity of lymphocytes was assessed by a standard 51Cr release assay. Briefly, the target cells were labeled for 90 min with 100 µCi of 51Cr at 37°C. Labeled cells were then washed twice before plating at 1000 cells/well. The effectors were plated in 96-well round-bottomed plates at an indicated effector/target (E/T) ratio and then were co-incubated with labeled targets for 4 h. Radionuclide release was duplicated using a gamma counter. The results are expressed as a percent specific lysis as determined by (experimental release cpm – spontaneous release cpm)/(maximum release cpm – spontaneous release cpm) x 100 (%). Spontaneous release was assessed by incubating target cells in medium alone and maximum release was determined in the presence of 2% Triton X.
Measurement of Cytokine Release by CTL
The cultured lymphocytes were incubated with the parental autologous tumor cells (5 x 105/ml) in CM containing IL-2 (25 units/ml) for 6 h and the amount of IFN- in the culture supernatant was measured using a Human Interferon Gamma ELISA Test Kit (Endogen) according to the instruction manual. In the blocking assay using mAbs, a 1:4 diluted culture supernatant of hybridomas was added into the co-culture of the CTL and AT. In order to determine the optimal concentration of the antibodies in this blocking assay, we performed a fluorescence analysis and measured the IFN- production with various titrations of the antibodies (data not shown). The hybridoma-derived mAbs did not affect the detection of IFN- in the preliminary experiments.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONAcknowledgmentsREFERENCES |
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Establishment of the Autologous Tumor Cell Line, A529L
A529L was generated from a resected specimen that had been diagnosed to be adenosquamous carcinoma of the lung. The primary tumor consisted of poorly differentiated adenocarcinoma and few squamous foci, but the tumor which extended to the chest wall mainly had pathological features of squamous cell carcinoma (Fig. 1A and B). The A529L cell line has been maintained in continuous culture for more than 1 year. To confirm tumorigenesis of the cell line, A529L cells were transplanted to a SCID mouse by subcutaneous injection. The tumor derived from the injection of A529L cells showed the features of poorly differentiated adenocarcinoma with many mitoses, which were very similar to those of the adenocarcinoma component in the primary lesion of patient A529 (Fig. 1C). Cytokeratin, which is a marker of epithelial cells, was strongly expressed on the transplanted A529L cells by use of immunohistochemical staining and these findings were consistent with an epithelial lineage (Fig. 1D). A529L expressed both HLA class II and class I at a substantial level by flow cytometory (data not shown). As shown in Fig. 2, transfectants expressed CD80 molecule on their surfaces.
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Induction of CTL Specific for Autologous Lung Adenosquamous Carcinoma Cells
The CTL line exhibited a substantial level of cytotoxic activity against AT (26% lysis at an E/T ratio of 40:1), but no lytic activity against autologous EBV-B or K562 after five successive stimulations with CD80 transfected AT (Fig. 3). On the other hand, lymphocytes stimulated with non-transduced AT (without CD80) showed almost undetectable cytotoxicity against AT (0.2% lysis at an E/T ratio of 20:1). These results indicated that CD80 transfected AT efficiently stimulated lymphocytes to induce tumor-specific CTLs.
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Generation of Various T Cell Clones Recognizing A529L
To investigate the mechanism of tumor recognition by the CTL, we made an attempt to generate CTL clones by limiting dilution. Eight clones which consisted of three CD8+ and five CD4+ T cell clones were thus derived in the culture condition of limiting dilution. A summary of their activities is listed in Table 1. Representative data on their cytotoxicity are also shown in Fig. 3. Three CD8+ T cell clones (Clones 1, 2 and 3) lysed AT (19–26% lysis at an E/T ratio of 30:1), but failed to lyse autologous EBV-B, K562 or HLA class I matched-allogeneic cell lines (A110L, RERF-LC-AI and PC9), as shown in Table 1 and Fig. 4.
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In order to define the HLA restriction of the T cell clones, they were assessed for cytokine production in response to AT in the presence of mAbs against HLA molecules (Table 1). CD8-positive Clone 3 produced IFN-
at more than 200 pg/ml in culture supernatant when co-cultured with AT but not with EBV-B or K562. Its production was completely inhibited by the addition of anti-HLA class I and anti-HLA-B/C mAbs but not by anti-HLA-A24 or anti-HLA class II. This pattern of the blocking effect was also observed in CD8-positive Clones 1 and 2 (Table 1). This observation indicates that these CD8+ T cell clones recognize tumor antigens that are presented by HLA-B*5201 or HLA-Cw*1202. CD4-positive Clone 4 produced IFN- (52 pg/ml) when stimulated with AT but not when stimulated with either autologous EBV-B or K562. Interestingly, the IFN- production by Clone 4 was completely abrogated only by anti-HLA class II antibody. This indicated that Clone 4 could be an autologous tumor-specific CD4+ T cell clone recognizing AT in an HLA class II-restricted fashion. CD4+ Clone 8 produced an IFN- in response to autologous EBV-B as well as AT. The IFN- production by Clone 8 was partially inhibited by a mAb against HLA class II, but not by mAbs against HLA class I, HLA-A24 or HLA-B/C, which was also observed in three other CD4+ clones (Clones 5, 6 and 7) (Table 1). This suggested that these CD4+ clones might recognize autologous antigens expressed on both AT and EBV-B presented by HLA class II molecules. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONAcknowledgmentsREFERENCES |
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The identification of tumor antigens recognized by autologous CTL led us to perform vaccine therapies utilizing several antigenic peptides derived from the MAGE family (16), MART-1 (17) and gp100 (18). Compared with malignant melanoma, several antigens recognized by CTL have been identified in lung cancer, for example, ART (19), MAGE, EF-2 (20),and cyclophilin B (21). Until now, knowledge about CTL against lung cancer has thus been limited. When classified according to the different cell types of lung cancer, AT-specific CTL have been reported to be generated from patients with small cell carcinoma (22,23), adenocarcinoma (14,24) and squamous cell carcinoma (23,25). There has been no report of CTL generated from patients with adenosquamous carcinoma of the lung.
In the present study, we succeeded in establishing an adenosquamous cancer cell line, A529L, from a surgical specimen. In order to induce autologous lung cancer-specific CTL, it is essential to establish tumor cell lines as stimulator cells against autologous lymphocytes. Even if such tumor cell lines are established, CTL have hardly been induced by conventional methods of tumor cell stimulation, probably owing to a shortness of either tumor antigens or co-stimulatory molecules such as CD80 (26). To resolve these difficulties, we modified the protocols of CTL induction. CD80-transfected autologous tumor cells were used as stimulators; this elevates the efficiency of induction of CTL from patients with lung cancer (27). A bulk CTL line specific for AT was induced by five successive stimulations with CD80-transfected AT. Thus, we obtained three AT-specific CTL clones and one AT-specific CD4+ T cell clone. The latter was established out of five CD4+ T cell clones. To our knowledge, this report is the first description that AT-specific T cell clones could be derived from the patient with adenosquamous carcinoma of the lung. All three AT-specific CD8+ CTL clones were restricted by HLA-B or -C in the blocking assay. The HLA genotype of AT was HLA-B*5201 and Cw*1202, thus suggesting that the HLA restriction of CTL clones was B*5201 or Cw*1202 and the expression of both is found in about 10% of all Japanese (28). Clones 1 and 2 failed to kill A110L and RERF-LC-AI possessing HLA-B*5201 and HLA-Cw*1202, which might suggest that CTL recognized a unique antigen expressed only on A529L. Since only two HLA class I-matched tumor cell lines were tested to investigate the possibility that CTL recognized shared antigens, transfection of HLA molecules into allogeneic tumor cell lines could help us determine the exact restriction element of the CTL clones if the antigens recognized by CTLs were shared antigens.
In contrast to the generation of AT-specific CD8+ clones, an AT-specific CD4+ T cell (Clone 4) was also obtained from the same bulk CTL line. Clone 4 produced IFN- in response to AT but not EBV-B or K562. Its production was completely inhibited by anti-class II mAb, thus suggesting that a tumor antigen was presented by HLA class II on A529L. An autologous tumor, A529L, had an expression of HLA class II as well as HLA class I molecules by flow cytometry. Several investigators, including us, reported that primary tumors from patients with lung cancer expressed HLA class II by immunohistochemical staining (29–31). More recently, tumor antigens recognized by CD4+ T cells have been identified by the method of cDNA expression cloning (32–35). The generation of both CD8+ and CD4+ T cell clones recognizing autologous tumor cells is the first step towards the identification of antigens expressed on lung cancer, which can be used as a probe for the screening of the cDNA library. Taken together, the derivation of AT-specific CTLs and CD4+ T cell clone reflected the existence of precursors of AT-specific CTLs and CD4+ T cells in draining lymph nodes.
The most interesting point regarding the CTLs derived from an adenosquamous carcinoma is what these CTLs recognize. The primary tumor of patient A529 was composed of an adenocarcinoma component and a squamous cell carcinoma component. Which part do CTLs recognize? A529L cells inoculated in the SCID mouse showed similar histological features to those of the adenocarcinoma component of the primary region (Fig. 1). It is possible, therefore, that CTLs recognize a certain antigen expressed on the adenocarcinoma component. Interestingly, it has been reported that both components possessed biological characteristics in common by analyzing the DNA ploidy pattern (36), an X-chromosome-linked polymorphic marker (37) and genetic alteration of p53 (38). If the tumor antigen is identified, we could detect its expression in both components by analyzing micro-dissected primary tumor cells and could thus compare these two components with regard to antigenesities.
The prognosis of lung cancer is still poor even if a complete resection is performed. We therefore have to search for new therapies against lung cancer. We have demonstrated here that tumor-specific T cells existed even in a patient with advanced lung cancer as we found in this case. This result also indicates that RLNL from advanced cancer patients have the potential to recognize AT and to be activated as the effector cells. We should continue the present study and identify tumor antigens in order to analyze the immune response in patients with advanced cancer.
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Acknowledgments |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONAcknowledgmentsREFERENCES |
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This work was supported in part by Grant-in-Aid for Scientific Research on Priority Areas (C)(2) 12217149 and Grant-in-Aid for Scientific Research (C)(2) 11671348 from the Ministry of Education, Culture, Sports, Science and Technology. We thank Mrs Maki Uebayashi, Ms Kahoru Noda, Mrs Yumiko Hase and Mrs Miki Shimada for their expert help.
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FOOTNOTES |
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+ For reprints and all correspondence: Tomoko So, Department of Surgery II, School of Medicine, University of Occupational and Environmental Health, 1–1 Iseigaoka, Yahatanishi-ku, Kitakyushu 807-8555, Japan. E-mail: tomokoso@med.uoeh-u.ac.jp
Abbreviations: CTL, cytotoxic T lymphocyte; AT, autologous tumor; EBV-B, Epstein–Barr virus transformed B cell; RLNL, regional lymph node lymphocytes; CM, culture medium; E/T ratio, effector/target ratio; mAb, monoclonal antibody
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONAcknowledgmentsREFERENCES |
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Received December 18, 2000; accepted March 6, 2001.
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