Clinical Cancer Research Bridging the Lab and the Clinic in Cancer Medicine
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Saijo, Y.
Right arrow Articles by Nukiwa, T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Saijo, Y.
Right arrow Articles by Nukiwa, T.
Clinical Cancer Research Vol. 5, 1203-1209, May 1999
© 1999 American Association for Cancer Research

Cancer Biology, Immunology, Cytokines

Autologous High-Killing Cytotoxic T Lymphocytes against Human Lung Cancer Are Induced Using Interleukin (IL)-1ß, IL-2, IL-4, and IL-6: Possible Involvement of Dendritic Cells1

Yasuo Saijo2, Xin Hong, Masashi Tanaka, Ryushi Tazawa, Shu Qin Liu, Kaoru Saijo, Tadao Ohno, Kaoru Koike, Kazuhiro Ohkuda, Ken Satoh and Toshihiro Nukiwa

Department of Respiratory Oncology and Molecular Medicine, Institute of Development, Aging, and Cancer, Tohoku University, Sendai 980-8575 [Y. S., X. H., M. T., R. T., Ke. S., T. N.]; RIKEN Cell Bank, The Institute of Physical and Chemical Research (RIKEN), Tsukuba Science City [S. Q. L., Ka. S., T. O.]; Miyagi Cancer Center, Sendai [K. K.]; and Miyagi Semine Hospital, Semine-cho, Miyagi [K. O.], Japan


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Although CTLs bear main immune responses in human tumors, stable CTL clones against human lung cancer have rarely been generated. Our previous study demonstrated efficient autologous CTL induction in human gastric cancer and glioblastoma by cytokine combination of interleukin (IL)-1ß (167 IU/ml), IL-2 (67 IU/ml), IL-4 (67 IU/ml), and IL-6 (134 IU/ml). In this study, we demonstrated successful induction of autologous stable CTLs in five of six patients with lung adenocarcinoma from mixed-lymphocyte tumor culture using this cytokine combination. All CTLs revealed potent and specific killing activity against autologous target cells (over 75% in CD8+ CTLs and over 50% in CD4+ CTLs at an E:T ratio of 10 for 24 h). Using a series of antibodies, CD8+ CTLs showed to recognize tumor-specific antigens of lung cancer cells through HLA class I. In the separate experiments, failure of CTL induction from monocyte-depleted peripheral blood mononuclear cells and appearance of cells with characteristics of dendritic cells from adherent peripheral blood mononuclear cells in the culture of the same concentration of IL-1ß, IL-4, and IL-6 indicated that CTLs can be efficiently generated by this cytokine combination via possible dendritic cell induction. This is the first study of an efficient and reproducible in vitro CTL induction against human lung cancer.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Lung cancer is the most common neoplasm leading to cancer death in a number of developed countries. The majority of NSCLCs3 are diagnosed unresectable, are relatively resistant to currently available treatment modalities, and, thus, are poor prognosis (1) . Immunotherapy has been investigated as a new treatment modality using LAK cells or TILs. These adoptive immunotherapies have been effective on several cancers, but have failed to demonstrate direct antitumor activity in cases of NSCLC (2) . Tumor-specific CTLs specifically lyse target cells and show more potent cytotoxic activities than LAK cells or TILs (3, 4, 5, 6, 7) . Generation of autologous CTLs against human lung cancer, however, has been described in only a limited number of studies despite extensive testing of CTL induction (8 , 9) . No stable CTLs against lung adenocarcinoma has been generated.

Liu et al. (10) and Tsurushima et al. (11) have previously demonstrated that autologous CTL with high tumor specificity and high cytotoxicity could be induced from MLTC in cytokine combination of IL-1ß, IL-2, IL-4, and IL-6. We attempted to induce autologous CTL in patients with lung cancer using this cytokine combination and succeeded in induction of autologous CTLs with specific and high cytotoxicity in five of six cases. We also demonstrated that this cytokine combination could induce CTL effectively through possible generation of DCs in MLTC.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Patients and Establishment of Lung Cancer Cell Lines
All research followed the tenets of the Declaration of Helsinki. Of 47 surgically resected NSCLC specimens, six lung adenocarcinoma cell lines (IDACA-1, IDACA-2–1, IDACA-3, IDACA-4, IDACA-5, and IDACA-11) were established using previously described methods (12) . IDACA-2–2 was established as a subline of IDACA-2–1, which shows different morphology and shorter doubling time. These cell lines were maintained in MEM containing 10% fetal bovine serum for at least 6 months. Serological HLA class I typing was performed using PBMCs from the original patients by a microcytotoxicity assay (One Lamda, Canoga Park, CA). For CTL induction from monocyte-depleted PBMCs, IDACA-11 was established and examined.

Generation of CTLs Responsive to Lung Cancer Cells
PBMCs from five patients whose cell lines had been established as IDACA-1, -2–1, -3, -4, and -5 were isolated by Lymphoprep (Nycomed Pharma AS, Oslo, Norway). CTL induction was performed by a slight modification of Liu et al. (10) . Briefly, PBMCs were first stimulated with irradiated (30 Gy) autologous tumor cells at an E:T ratio of 10 in AIMV medium (Life Technologies, Inc., Rockville, MD) supplemented with 5% autologous serum and with IL-1ß (167 IU/ml), IL-2 (67 IU/ml), IL-4 (67 IU/ml), and IL-6 (134 IU/ml). The medium was changed every 3 days until the lymphocytes began to grow. After induction, these CTLs were maintained in culture medium containing the same concentration of IL-2, and one-tenth of the concentrations of IL-1ß, IL-4, and IL-6 described above. CTLs were restimulated weekly with nonirradiated live autologous cancer cells. After 2 months, these CTLs were analyzed for cytotoxicity and surface markers.

Determination of Surface Markers of Tumor Cells and CTLs
Tumor cells were stained with FITC-conjugated anti-HLA-A B C antibody (W6/32; DAKO Japan, Tokyo, Japan) or FITC-conjugated anti-HLA-DP DQ DR antibody (DAKO Japan). Surface phenotypes of CTLs were determined by flow cytometry after staining with FITC- or PE-conjugated antibodies against CD3, CD4, CD8, CD16, and TCR{alpha}ß (Becton Dickinson, Mountain View, CA). Flow cytometry was performed using a FACScan (Becton Dickinson).

Cytotoxic Activities
Cytotoxic activity was determined by 51Cr releasing assay (13) and crystal violet staining assay (14) , both after 6 and 24 h. Although 51Cr releasing with a 6-h incubation is widely used as the standard assay, 51Cr releasing with a 24-h incubation is also performed. In our previous study, we showed that the two different methods yield comparable results under a 24-h incubation (10) .

Blocking of Cytotoxicities by MAbs
51Cr-labeled target cells were pretreated with anti-HLA class I (DAKO Japan) or anti-HLA class II MAb (50 µg/ml; DAKO Japan) at 37°C for 1 h. Effector cells (CTLs) were incubated with anti-CD3, -CD4, or -CD8 MAb (50 µg/ml; Ancell, Bayport, MN) at 4°C for 1 h. Lysis of target cells with or without MAbs was determined by a 6-h 51Cr releasing assay.

Induction of DCs from PBMCs
PBMCs were cultured in AIMV medium containing 5% autologous serum, allowing to adhere to plastic dishes. Nonadherent cells were removed after 2 h, and adherent cells were subsequently cultured for 7 days with IL-1ß (167 IU/ml), IL-4 (67 IU/ml), and IL-6 (134 IU/ml), or with GM-CSF (1000 units/ml) and IL-4 (500 units/ml; Ref. 15 ). Surface markers of DCs, such as CD1a, CD11c, CD83, HLA class I, HLA class II, and B7–1 were determined by flow cytometry.

CTL Induction from Monocyte-depleted PBMCs
Nonadherent PBMCs (monocyte-depleted PBMCs) were used for MLTC with target cells (IDACA-11) in the cytokine combination of IL-1ß, IL-2, IL-4, and IL-6. In addition, mixture of monocyte-depleted PBMCs and DCs induced by GM-CSF and IL-4 or by IL-1ß, IL-4, and IL-6, as described above, were used for MLTC. Induction of CTL was monitored by inverted microscopy and counting cell number of lymphocytes.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Patient Profiles, HLA Typing, HLA Expression on Tumor Cells, and CTL Induction
Table 1Citation summarizes the patient profiles, serological HLA class I types, tumor cell lines, and results of CTL induction. The tumor cell lines used for CTL induction expressed HLA class I only, except IDACA-2–1 and the subline IDACA-2–2, which expressed trace amounts of HLA class II antigen (data not shown). CTLs began to grow after 1 week of MLTC, and they continued growing by repeated stimulation with live target cells (Fig. 1)Citation . Only IDACA-2–1 could not induce autologous CTL (Fig. 1Citation ; Table 1Citation ). CTLs expanded from 5 x 106 to 1 x 109 in number after 1 month of induction. After a 2-month culturing of CTLs, phenotypic analyses revealed that CTLs were CD4+ dominant or CD8+ dominant (Table 2)Citation . As a result, CD8+ CTLs were induced in IDACA-1, -3, -4, and -5, whereas CD4+ CTLs were induced in IDACA-2–2 (Fig. 2)Citation . Although CTL inductions of IDACA-2–1 and -2–2 were performed twice independently, only IDACA-2–2 repeatedly induced CD4+ CTLs.


View this table:
[in this window]
[in a new window]
  		Table 1 Characteristics of established lung cancer cell lines: designated tumor cell lines, patient profiles, and CTL induction

 


View larger version (19K):
[in this window]
[in a new window]
  		Fig. 1. Cumulative growth curves of PBMCs stimulated with irradiated autologous tumor cells. PBMCs (5 x 106) were mix-cultured with irradiated (30 Gy) autologous adenocarcinoma cells (IDACA-1, IDACA-2–1, and IDACA-2–2), at an E:T ratio of 10, in AIMV medium containing 5% autologous serum, IL-1ß (167 IU/ml), IL-2 (67 IU/ml), IL-4 (67 IU/ml), and IL-6 (134 IU/ml). The medium was changed, and PBMCs were counted every 3 days. Autologous target cells: {triangleup}, IDACA-1; •, IDACA-2–1; {circ}, IDACA-2–2. Note that PBMCs mix-cultured with IDACA-2–1 did not expand, despite the fact that another autologous cell line (IDACA-2–2) yielded CTL-2–2.

 

View this table:
[in this window]
[in a new window]
  		Table 2 Surface phenotype and cytotoxic activity against autologous target cells

 


View larger version (183K):
[in this window]
[in a new window]
  		Fig. 2. Photomicrographs taken through an inverted microscopy demonstrated strong cytotoxicity of CTLs. After a 12-h preincubation of target cells (1 x 105/well) in a 6-well culture plate, CTLs were added to the adherent target cells at an E:T ratio of 10. Photomicrographs were taken through inverted microscopy after 6- and 24-h incubations. Left, mix-culture of CTL-1 (CD8+) and autologous target cells (IDACA-1). A, tumor cells alone; B, 6-h incubation; C, 24-h incubation. Right, mix-culture of CTL-2–2 (CD4+) and autologous target cells (IDACA-2–2). D, tumor cells alone; E, 6-h incubation; F, 24-h incubation.

 
Cytotoxic Activity of CTLs
Although all CTLs showed specific killing activity for autologous target cells, CTL-1 (CD8+) revealed stronger cytotoxicity than CTL-2–2 (CD4+; Fig. 3, A and BCitation ). At an E:T ratio of 10, CTL-1 (CD8+) attached to the autologous target cells and had lysed almost all of them after a 24-h incubation (Fig. 2A)Citation . Even at an E:T ratio of 1, half of the target cells were lysed after 24-h incubation (Fig. 3A)Citation . All of CD8+ CTLs lysed autologous target cells entirely after a 48-h incubation.



View larger version (24K):
[in this window]
[in a new window]
  		Fig. 3. Cytotoxicities of CD8+ CTL and CD4+ CTL. Cytotoxic activities were determined by 6- and 24-h incubations at different E:T ratios. A 51Cr releasing assay was used for 6- and 24-h incubations, as described in "Materials and Methods." A, CTL-1 (CD8+): {circ}, autologous target cells with a 6-h incubation; •, autologous target cells with a 24-h incubation; {triangleup}, Daudi cells with a 6-h incubation; {blacktriangleup}, K562 cells with a 6-h incubation. B, CTL-2–2(CD4+): {circ}, IDACA-2–2 cells with a 6-h incubation; •, IDACA-2–2 cells with a 24-h incubation; {circ}, IDACA-2–1 cells with a 6-h incubation; •, IDACA-2–1 cells with a 24-h incubation; {triangleup}, Daudi cells with a 6-h incubation; {blacktriangleup}, K562 cells with a 6-h incubation.

 
Although CTL-2–2 (CD4+) attached to the target cells in the manner of CTL-1 (Fig. 2B)Citation , less than half of IDACA-2–2 cells were lysed by CTL-2–2 after a 24-h incubation (Figs. 2F and 3B)Citation . Longer culturing did not increase the cytotoxicity of CTL-2–2 (50.3 ± 6.5% at 24 h versus 56.4 ± 7.3% at 48 h; E:T ratio of 10). CTL-2–2 (CD4+) also revealed significant, but less, cytotoxicity against IDACA-2–1 (Fig. 3B)Citation , compared with IDACA-2–2. All other CD8+ CTLs demonstrated over 75% specific cytotoxicities (Table 2)Citation .

Cytotoxicity of these CTLs against allogeneic target cells are examined by 6-h 51Cr releasing assay (Table 3)Citation . Although CTL-5 lysed HLA-A locus-matched target cells (IDACA-2–1 and -2–2) only, CTL-1 showed a high level of lysis against not only HLA-A locus-matched cells (IDACA-2–1 and -2–2), but also HLA-C matched cells (IDACA-4). CTL-4 showed a high level of lysis against only IDACA-1, the HLA-C locus of which was matched. None of the allogeneic tumor cells were specifically lysed by CTL-3. Because these CTLs were cultured in bulk, CTL-1 may contain both HLA-A locus- and HLA-C locus-restricted CTLs recognizing tumor antigen. Cytotoxicity of CTL-2–2 (CD4+) against allogeneic tumor cells was not matched for restriction of HLA class I. In contrast, no significant cytotoxicities against autologous fibroblasts, K562 cells, or Daudi cells were observed (Fig. 3, A and BCitation ; Table 3Citation ).


View this table:
[in this window]
[in a new window]
  		Table 3 Cytotoxicities of CTLs against allogenic tumor and normal cells

 
Over a 6-month culturing, CTL-1 (CD8+) maintained strong and specific cytotoxicity (85.6 ± 3.2% at 2 months versus 82.8 ± 4.5% at 6 months; E:T ratio of 10) by weekly stimulation of tumor cells.

Characterization of CTLs Using Antibodies
The cytotoxic activity of CTL-1 (CD8+) was inhibited by anti-CD3 and -CD8 antibodies, but not by anti-CD4 antibody. Anti-HLA class I, but not HLA class II antibody, inhibited CTL-1 cytotoxic activity against target cells (Fig. 4)Citation . Although the killing ability of CTL-2–2 (CD4+) was blocked by anti-CD3 antibody, only partial blocking was observed by anti-CD4 antibody. Neither HLA class I nor HLA class II antibody inhibited CTL-2–2 activity (Fig. 4)Citation .



View larger version (37K):
[in this window]
[in a new window]
  		Fig. 4. Inhibition of cytotoxicities by MAbs related with antigen recognition. CTLs were pretreated with MAbs (anti-CD3, CD4, and CD8) at 4°C for 1 h. Target cells were pretreated with MAbs (anti-HLA class I, HLA class II) at 37°C for 1 h. Cytotoxic activities were determined by a 6-h 51Cr releasing assay at an E:T ratio of 10. Cytotoxicity on cells not treated with antibody was determined as 100%. Concentrations of MAbs were 50 µg/ml. {square}, CD4+ CTL; , CD8+ CTL.

 
Involvement of DCs for CTL Induction
To study whether DCs are involved for CTL induction, monocytes, DC precursor cells, were depleted from PBMCs when PBMCs were used for MLTC. As shown in Fig. 5Citation , CTLs could not be induced from monocyte-depleted PBMCs. However, addition of the cells generated by IL-1ß, IL-4, and IL-6 from monocytes into monocyte-depleted PBMCs could induce CTLs as well as addition of DCs generated by GM-CSF and IL-4. The cells from adherent PBMCs cultured with IL-1ß, IL-4, and IL-6, but without IL-2, for 7 days showed characteristic morphology of DCs as induced by GM-CSF and IL-4, under phase contrast microscopic observation (Fig. 6A and B)Citation , whereas no DCs were induced by IL-2 alone or without cytokine stimulation (Fig. 6, C and D)Citation . May-Giemsa staining of the nonadherent cells also confirmed these observations. In addition, the cells generated by this cytokine combination contained markers typical of immature DCs as induced by GM-CSF and IL-4. They were positive for HLA class I, HLA class II, B7–1, CD1a, CD4+, and CD11c+, but negative for CD3, CD8, CD19, and CD83 (specific marker of mature DCs; Fig. 7Citation ). The cytokine combination of IL-1ß, IL-2, IL-4, and IL-6 as used in the CTL induction, thus, generated immature DCs while lymphocytes proliferated due to the addition of IL-2 (Fig. 6E)Citation . These morphological and functional studies indicated that the cytokine combination generated CTLs efficiently via induction of DCs.



View larger version (16K):
[in this window]
[in a new window]
  		Fig. 5. Growth curves of monocyte-depleted-PBMCs with or without DCs stimulated with irradiated autologous tumor cells. Monocyte-depleted PBMCs (5 x 106) with or without DCs (5 x 105) generated by GM-CSF+IL-4 or IL-1ß, -4, and -6 were mix-cultured with irradiated autologous tumor cells (IDACA-11). {circ}, complete PBMCs; •, monocyte-depleted PBMCs; {square}, monocyte-depleted PBMCs with DCs generated by GM-CSF and IL-4; {blacksquare}, monocyte-depleted PBMCs with DCs generated by IL-1ß, -4, and -6.

 


View larger version (142K):
[in this window]
[in a new window]
  		Fig. 6. Generation of DCs from PBMCs by cytokine combination. Adherent PBMCs were cultured for 7 days with various cytokine combinations. Photomicrographs were taken after 7 days. A, cells cultured with IL-1ß (167 IU/ml), IL-4 (67 IU/ml), and IL-6 (134 IU/ml); B, cells cultured with GM-CSF (1000 IU/ml) and IL-4 (500 IU/ml); C, cells cultured with IL-2 (67 IU/ml); D, cells cultured without cytokine; E, cells cultured with IL-1ß, IL-2, IL-4, and IL-6; Note that cells in A, B, and E were nonadherent or easily detached from the dish, but cells in D were tightly attached to the dish.

 


View larger version (18K):
[in this window]
[in a new window]
  		Fig. 7. Cytofluorographic analysis of DCs grown in the presence of IL-1ß, -4, and -6. The cells were stained with a panel of antibodies, as described in "Materials and Methods." Contaminating lymphocytes were excluded by light scatter properties.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Immune Responses against Tumor Cells
The basic concepts of cancer immunology have been derived largely from studies of tumor rejection in tumor-bearing syngeneic mice (16) . Although a variety of effector cells (including natural killer cells, TILs, LAK cells, T lymphocytes, macrophages, and neutrophils) are recognized to contribute to the surveillance for developing tumors and the control of tumor progression, CTL response is thought to play a key role in the elimination of tumor cells (17) .

In humans, immune responses to melanoma have been well documented. To date, many CTLs to melanoma were induced, and tumor antigens recognized by those CTLs have been identified (3 , 4 , 18) . In contrast to melanoma, reports of induction of autologous CTLs in other tumors, including lung cancer, have been limited (5, 6, 7, 8, 9 , 19) . Our previous studies on induction of autologous CTLs in patients with gastric cancer (10) and glioblastoma (11) led us, in this study, to generate stable CTLs against lung cancer by using the cytokine combination of IL-1ß, IL-2, IL-4, and IL-6. This resulted in induction of stable CTLs in five of six cases, and these CTLs showed high specificity and potent cytotoxicity against autologous target cells.

Priming of CTL responses requires the presentation of the relevant tumor antigen by professional antigen-presenting cells capable of providing costimulation (20) . DCs are only natural professional antigen-presenting cells (21) . DCs are now efficiently generated by GM-CSF and IL-4 from PBMCs (15) . Although IL-2 has been the primary cytokine used among in vitro systems of CTL induction (18) , addition of IL-2 alone cannot induce DCs from PBMCs, as we demonstrated. In our system, however, DCs were generated in the lower concentration of cytokines compared with GM-CSF and IL-4. When monocytes were depleted from PBMCs, induction of CTL was failed. Furthermore, IL-1ß can augment the stimulatory capacity of DCs against T cells (22) . Generation and functional enhancement of DCs from PBMCs may explain the effective CTL induction by this cytokine combination.

CTLs against Human Lung Cancer
In earlier studies, CTLs were generated by MLTC from NSCLC patients and showed specific and highly potent cytotoxicity (23, 24, 25) . In these cases, the MLTC culturing periods were short (<1 month), and the phenotypes of CTLs were a mixture of CD4+ and CD8+. Although Kurnick et al. (25) generated TILs from lung adenocarcinoma tissues, neither tumor specificity nor the role of MHC molecules was demonstrated. Nevertheless, these studies provided substantial evidence of a cellular immune response to human lung cancers. When Seki et al. (26) induced HLA-A locus-restricted CTLs from TILs in NSCLC, the tumor-specific cytotoxicity only lasted for 1 month.

In contrast, stable CTL clones previously have been established in only two cases of lung squamous cell carcinoma (8 , 9) . These two CTL clones showed highly specific cytotoxicity against autologous target cells and were identified as MHC class I-restricted CD8+ CTLs. In the present study, CTLs were all proved to be specific to autologous target cells. Furthermore, our CTLs were able to maintain tumor specificity at least 6 months and reached as many as 1 x 109 cells in number after 1 month, a concentration approximately sufficient for practical use in immunotherapy.

The cytotoxicities of CD8+ CTLs were inhibited by anti-HLA class I antibody, suggesting that CD8+ CTLs are HLA class I-restricted and, thus, may recognize tumor antigen presented in HLA class I molecules (27) . Although the HLA loci restriction of CD8+ CTLs were not fully studied, all of the CD8+ CTLs showed HLA-A- and/or -C-restricted cytotoxicity.

The CD4+ CTL was induced in IDACA-2–2 only and showed tumor-specific, but weaker, cytotoxicity. Responses of CD4+ CTLs against tumor cells have been reported recently (28, 29, 30) . Interestingly, CD4+ CTLs were induced repeatedly in independent experiments from IDACA-2–2, but not from IDACA-2–1, although both cell lines were proved to be derived from one patient by genomic analysis (31) .

In conclusion, we reproducibly generated stable CTLs in five of six patients with lung adenocarcinoma by the cytokine combination. The mechanism of efficient CTL induction may be attributed to induction of DCs from PBMCs.


   ACKNOWLEDGMENTS
 
We thank the Ohtsuka Pharmaceutical Co., Ltd. for providing recombinant IL-1ß, the Shionogi Pharmaceutical Co., Ltd. for providing IL-2, the Ono Pharmaceutical Co., Ltd. for providing IL-4, and the Kirin Brewery Co., Ltd. for providing IL-6.


   FOOTNOTES
 
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.

1 Supported by a Grant-in-Aid from the Ministry of Education, Science, Sports and Culture, Japan, and by the Public Trust Haraguchi Memorial Cancer Research Fund. Back

2 To whom requests for reprints should be addressed, at Department of Respiratory Oncology and Molecular Medicine, Institute of Development, Aging, and Cancer, Tohoku University, 4-1 Seiryomachi Aobaku, Sendai 980-8575, Japan. Phone: 81-22-717-8539; Fax: 81-22-717-8549; E-mail: yasosj{at}idac.tohoku.ac.jp Back

3 The abbreviations used are: NSCLC, non-small cell lung cancer; LAK, lymphokine-activated killer; TIL, tumor-infiltrating lymphocyte; MLTC, mixed-lymphocyte tumor culture; IL, interleukin; DC, dendritic cell; PBMC, peripheral blood mononuclear cell; 51Cr, 51Chromium releasing assay; GM-CSF, granulocyte-macrophage colony-stimulating factor; MAb, monoclonal antibody. Back

Received 6/15/98; revised 1/11/99; accepted 2/11/99.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Somerfield M. R. Clinical practice guideline for the treatment of unresectable non-small-cell lung cancer. J. Clin. Oncol., 15: 2996-3018, 1997.[Abstract]
  2. Kradin R. L., Kurnick J. T., Lazarus D. S., Preffer F. I., Dubinett S. M., Pinto C. E., Gifford J., Davidson E., Grove B., Callahan R. J., et al Tumor-infiltrating lymphocytes and interleukin-2 treatment of advanced cancer. Lancet, 1: 577-580, 1989.[Medline]
  3. Van Der Bruggen P., Traversari C., Chomez P., Lurquin C., De Plaen E., Eynde B., Knuth A., Boon T. A gene encoding an antigen recognized by cytolytic T lymphocytes on a human melanoma. Science (Washington DC), 254: 1643-1647, 1991.[Abstract/Free Full Text]
  4. Brichard V., Van Pel A., Wolfel T., Wolfel C., De Plaen E., Lethe B., Coulie P., Boon T. The tyrosinase gene codes for an antigen recognized by autologous cytolytic T lymphocytes on HLA-A2 melanomas. J. Exp. Med., 178: 489-495, 1993.[Abstract/Free Full Text]
  5. Peoples G. E., Goedegebuure P. S., Andrews V. R., Schoof D. D., Eberlein T. J. HLA-A2 presents shared tumor-associated antigens derived from endogenous proteins in ovarian cancer. J. Immunol., 151: 5481-5491, 1993.[Abstract]
  6. Letessier E. M., Heo D. S., Okarma T., Johnson J. T., Herberman R. B., Whiteside T. L. Enrichment in tumor-reactive CD8+ T-lymphocytes by positive selection from blood and lymph nodes of patients with head and neck cancer. Cancer Res., 51: 3891-3899, 1991.[Abstract/Free Full Text]
  7. Linehan D. C., Goedegebuure P. S., Peoples G. E., Rogers S. O., Eberlein T. J. Tumor-specific and HLA-A2-restricted cytolysis by tumor-associated lymphocytes in human metastatic breast cancer. J. Immunol., 155: 4486-4491, 1995.[Abstract]
  8. Slingluff C. L., Cox A. L., Stover J. M., Moore M. M., Hunt D. F., Engelhard V. H. Cytotoxic T-lymphocyte response to autologous human squamous cell cancer of the lung: epitope reconstitution with peptides extracted from HLA-Aw68. Cancer Res., 54: 2731-2737, 1994.[Abstract/Free Full Text]
  9. Yoshino I., Goedegebuure P. S., Peoples G. E., Parikh A. S., DiMaio J. M., Lyerly H. K., Gazdar A. F., Eberlein T. J. HER2/neu-derived peptides are shared antigens among human non-small cell lung cancer and ovarian cancer. Cancer Res., 54: 3387-3390, 1994.[Abstract/Free Full Text]
  10. Liu S. Q., Saijo K., Todoroki T., Ohno T. Induction of human autologous cytotoxic T lymphocytes on formalin-fixed and paraffin-embedded tumour sections. Nat. Med., 1: 267-271, 1995.[Medline]
  11. Tsurushima H., Liu S. Q., Tsuboi K., Yoshi Y., Nose T., Ohno T. Induction of human autologous cytotoxic T lymphocytes against minced tissues of glioblastoma multiforme. J. Neurosurg., 84: 258-263, 1996.[Medline]
  12. Stevenson H., Gazdar A. F., Phelps R., Linnoila I., Ihde D. C., Ghosh B., Walsh T., Woods E. L., Oie H., O’Connor T., et al Tumor cell line established in vitro: an independent prognostic factor for survival in non-small-cell-lung cancer. Ann. Intern. Med., 113: 764-770, 1990.
  13. Wunderlich J., Shearer G. Induction, and measurement of cytotoxic T lymphocyte activity. Current Protocol in Immunology, 1991.
  14. Saotome K., Morita H., Umeda M. Cytotoxicity test with simplified crystal violet staining method using microtitre plates and its application to injection drugs. Toxic In Vitro, 3: 317-321, 1989.
  15. Sallusto F., Lanzavecchia A. Efficient presentation of soluble antigen by cultured human dendritic cells is maintained by granulocyte/macrophage colony-stimulating factor plus interleukin 4 and down-regulated by tumor necrosis factor a. J. Exp. Med., 179: 1109-1118, 1994.[Abstract/Free Full Text]
  16. Foley E. Antigenic properties of methylcholanthrene-induced tumors in mice of the strain of origin. Cancer Res., 13: 835-837, 1953.
  17. Kagi D., Ledermann B., Burki K., Zinkernagel R. M., Hengartner H. Molecular mechanisms of lymphocyte-mediated cytotoxicity and their role in immunological protection and pathogenesis in vivo. Annu. Rev. Immunol., 14: 207-232, 1996.[Medline]
  18. Boon T., Van Der Bruggen P. Human tumor antigens recognized by T lymphocytes. J. Exp. Med., 183: 725-729, 1996.[Free Full Text]
  19. Nakao M., Yamana H., Imai Y., Toh Y., Toh U., Kimura A., Yanoma S., Kakegawa T., Itoh K. HLA A2601-restricted CTLs recognize a peptide antigen expressed on squamous cell carcinoma. Cancer Res., 55: 4248-4252, 1995.[Abstract/Free Full Text]
  20. Banchereau J., Steinman R. M. Dendritic cells and the control of immunity. Nature (Lond.), 392: 245-252, 1998.[Medline]
  21. Nestle F. O., Alijajic S., Gilliet M., Sun Y., Grabbe S., Dummer R., Burg G., Schadendorf D. Vaccination of melanoma patients with peptide- or tumor lysate-pulsed dendritic cells. Nat. Med, 4: 328-332, 1998.[Medline]
  22. Romani N., Gruner S., Brang D., Kampgen E., Lenz A., Trockenbacher B., Konwalinka G., Fritsch P. O., Steinman R. M., Schuler G. Proliferating dendritic cell progenitors in human blood. J. Exp. Med., 180: 83-93, 1994.[Abstract/Free Full Text]
  23. Vose B. M., Bonnard G. D. Human tumour antigens defined by cytotoxicity and proliferative responses of cultured lymphoid cells. Nature (Lond.), 296: 359-361, 1982.[Medline]
  24. Vose B. M., Bonnard G. D. Specific cytotoxicity against autologous tumour and proliferative responses of human lymphocytes grown in interleukin 2. Int. J. Cancer, 29: 33-39, 1982.[Medline]
  25. Kurnick J. T., Kradin R. L., Blumberg R., Schneeberger E. E., Boyle L. A. Functional characterization of T lymphocytes propagated from human lung carcinomas. Clin. Immunol. Immunopathol., 38: 367-380, 1986.[Medline]
  26. Seki N., Hoshino T., Kikuchi M., Hayashi A., Itoh K. HLA-A locus-restricted and tumor-specific CTLs in tumor-infiltrating lymphocytes of patients with non-small cell lung cancer. Cell. Immunol., 175: 101-110, 1997.[Medline]
  27. Houghton A. N. Cancer antigens: immune recognition of self and altered self. J. Exp. Med., 180: 1-4, 1994.[Free Full Text]
  28. Kharkevitch D. D., Seito D., Balch G. C., Maeda T., Balch C. M., Itoh K. Characterization of autologous tumor-specific T-helper 2 cells in tumor-infiltration lymphocytes from a patients with metastatic melanoma. Int. J. Cancer, 58: 317-323, 1994.[Medline]
  29. Miyazaki A., Sato N., Takahashi S., Sasaki A., Kohama G., Yamaguchi A., Yagihash A., Kikuchi K. Cytotoxicity of histocompatibility leukocyte antigen-DR8-restricted CD4+ killer T cells against human autologous squamous cell carcinoma. Jpn. J. Cancer Res., 88: 191-197, 1997.[Medline]
  30. William N. S., Engelhard V. H. Identification of a population of CD4+ CTL that utilizes a perforin-rather than a Fas ligand-dependent cytotoxic mechanism. J. Immunol., 156: 153-156, 1996.[Abstract]
  31. Horn G. T., Richards B., Klinger K. W. Amplification of a highly polymorphic VNTR segment by the polymerase chain reaction. Nucleic Acids Res., 17: 2140 1989.[Free Full Text]



This article has been cited by other articles:


Home page
 Am. J. Respir. Cell Mol. Bio.Home page
Y. To, M. Dohi, K. Matsumoto, R. Tanaka, A. Sato, K. Nakagome, T. Nakamura, and K. Yamamoto
A Two-way Interaction between Hepatocyte Growth Factor and Interleukin-6 in Tissue Invasion of Lung Cancer Cell Line
Am. J. Respir. Cell Mol. Biol., August 1, 2002; 27(2): 220 - 226.
[Abstract] [Full Text] [PDF]

Home page
 JCOHome page
M. B. Atkins, M. T. Lotze, J. P. Dutcher, R. I. Fisher, G. Weiss, K. Margolin, J. Abrams, M. Sznol, D. Parkinson, M. Hawkins, et al.
High-Dose Recombinant Interleukin 2 Therapy for Patients With Metastatic Melanoma: Analysis of 270 Patients Treated Between 1985 and 1993
J. Clin. Oncol., July 1, 1999; 17(7): 2105 - 2105.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Saijo, Y.
Right arrow Articles by Nukiwa, T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Saijo, Y.
Right arrow Articles by Nukiwa, T.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online