Analysis of MAGE-3 derived synthetic peptide as a human lung cancer antigen recognized by cytotoxic T lymphocytes.

BACKGROUND: The human MAGE-3 gene was originally discovered in melanoma cells that encode tumor antigens, and has been reported to be expressed in various types of tumors, including lung cancer, but not in normal tissues other than testis or placenta. Our aim in this study was to clarify whether HLA-A2 restricted MAGE-3 peptide (FLWGPRALV) could be a lung cancer antigen recognized by cytotoxic T lymphocytes (CTL). METHODS: MAGE-3-derived peptide-specific CTL were induced from the peripheral blood mononuclear cells (PBMC) of HLA-A0201-positive healthy donors and the regional lymph node lymphocytes (RLNL) of HLA-A2-positive patients with lung cancer by multiple stimulations with peptide-pulsed HLA-A0201-positive antigen-presenting cells. RESULTS: Lymphocytes stimulated with MAGE-3 peptide exhibited specific lysis of Epstein-Barr virus-transformed B cells (EBV-B) pulsed with MAGE-3 peptide, but not with control peptide derived from influenza matrix protein, erbB-2, or wild type p53. Specific activity for MAGE-3-presenting targets was found after the second stimulation, and increased depending on the number of stimulations. The peptide-specific activity was inhibited by the addition of monoclonal antibodies against MHC class I and HLA-A2. Such CTL also recognized tumor cell lines expressing both HLA-A2 and MAGE-3 in an MHC class I-restricted manner, but did not recognize tumor cell lines that did not express HLA-A2 or MAGE-3. CONCLUSION: These results suggested the MAGE-3 peptide could be a potential target of specific immunotherapy for HLA-A2 patients with lung cancer.

Generation of autologous tumor-specific T cell clones from a patient with adenosquamous carcinoma of the lung.

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 (51)Cr 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-gamma 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.

Unfavorable prognosis of patients with non-small cell lung carcinoma associated with HLA-A2.

BACKGROUND: HLA class I molecules present antigenic peptides to cytotoxic T lymphocytes and, thus, play an important role in immune surveillance. Since 1970s there have been many reports of an increased frequency of one or more HLA haplotype in association with autoimmune disease, and malignancy. We studied types of HLA class I antigens in 204 resected non-small cell lung carcinoma (NSCLC) patients and also examined its correlation with clinicopathologic features and prognosis. METHOD: Serological typing for HLA class I antigens was performed using a microcytotoxicity test. The disease-free survival curves were calculated by the Kaplan-Meier method and then compared using the Logrank test. Multivariate analysis was carried out by Cox's proportional hazard method. RESULTS: The difference in disease-free survival time between the HLA-A2 present group and A2 absent group was significant (P = 0.040). The 3-year disease-free survival rate of all patients was 44% in HLA-A2 present group and 66% in A2 absent group. When a comparison was made within the group with stage I, expression of HLA-A2 was the only independent factor that affected survival time by multivariate analysis (P = 0.0457). CONCLUSIONS: Expression of HLA-A2 was considered as one of the unfavorable prognostic factors in NSCLC patients. Our results suggested expression of HLA-A2 in NSCLC patients was one of the mechanisms of escape from immune surveillance.

Successful induction of tumor-specific cytotoxic T lymphocytes from patients with non-small cell lung cancer using CD80-transfected autologous tumor cells.

Cytotoxic T lymphocytes (CTL) against human lung cancer cells are difficult to induce by a conventional method using tumor cell stimulation probably due to an insufficiency of tumor antigens (TA) or costimulatory molecules such as CD80. We, therefore, investigated the potential of CD80-transfected tumor cells as stimulators of the in vitro induction of autologous tumor-specific CTL from regional lymph node lymphocytes in patients with lung cancer. Five non-small cell lung cancer cell lines (two adenocarcinomas, 1 squamous cell carcinoma, 1 large cell carcinoma and 1 adenosquamous cell carcinoma) were established from surgical specimens and were successfully transduced with a plasmid constructed with expression vector pBj and human CD80 cDNA, using a lipofection method. CD80-transfected tumor cells (CD80-AT) significantly augmented the proliferation of autologous lymphocytes from all cases as compared with non-transfected tumor cells (AT). AT-stimulated lymphocytes from 4 out of 5 cases did not show any cytotoxicity against AT; however, lymphocytes stimulated with CD80-AT exhibited substantial cytotoxicity against parental AT in all 5 cases tested. AT-stimulated lymphocytes derived from only one out of 5 cases showed major histocompatibility complex (MHC)-class I-restricted cytokine production in response to AT, while the MHC-class I-restricted responses were found in CD80-AT-stimulated lymphocytes from 4 out of 5 cases. These results indicate that CD80 on tumor cells could be a beneficial costimulatory molecule to elicit CTL against lung cancer, and also show that TA recognized by CTL was frequently expressed on lung cancer cells.

Heterogeneous response patterns of alveolar macrophages from patients with lung cancer by stimulation with interferon-gamma.

BACKGROUND: Macrophages are considered to play an important role in the host defense against malignant tumors. In this study, cytotoxic activity of alveolar macrophages (AM) derived from 32 patients with lung cancer was investigated. METHODS: AM were aseptically obtained by lavage from resected lung and subsequently tested for cytolytic activity against QG56, a lung squamous cell line, following treatment with recombinant interferon-gamma (IFN-gamma). RESULTS: In seven patients (21.9%), AM showed no cytotoxicity even though AM were incubated with IFN-gamma. In 20 (62.5%), AM showed substantial cytotoxicity in response to IFN-gamma in a dose-dependent manner. In the other five (15.6%), relatively strong cytotoxicity was observed even without preincubation with IFN-gamma. Such a heterogeneous profile of the cytotoxicity of AM might be a reflection of various activated states of AM since the potential of cytotoxicity and that of IL-1 secretion were almost parallel. Both IFN-gamma dependent and -independent cytotoxicity were partially blocked either by anti-tumor necrosis factor-alpha (TNF-alpha) antibody or by the inhibitor of nitric oxide synthesis. However, those activities were completely abrogated by both treatments. Since the supernatant of AM culture exhibited TNF-alpha-mediated but not NO-mediated cytolysis, TNF-alpha could mediate a bystander killing whereas NO acts in close contact with tumor cells. CONCLUSION: The AM have anti-tumor cytotoxicity in lung cancer although the cytolytic potential is heterogeneous and that the tumor lysis by AM is mediated by both TNF-alpha and NO production.

Short-course immunosuppression using FK506 for rat tracheal allografts.

BACKGROUND: A minimizing immunosuppression after a tracheal allotransplantation is desirable. METHODS: We examined the usefulness of a short-course of immunosuppression after tracheal allotransplantation in rat. Each transplant consisting of a 5-ring segment was heterotopically implanted into the omentum. Four animals underwent a syngeneic transplantation and thus served as controls (Group A). Thirty animals underwent an allogeneic transplantation and were randomly classified into 4 groups as follows: No immunosuppression (Group B, n=6), treatment with 0.5 mg/kg of Tacrolims (FK506) (Group C, n=8), 1.0 mg/kg of FK506 (Group D, n=8), and 1.5 mg/kg of FK506 (Group E, n=8). Different doses of FK506 were administered intramuscularly for only three consecutive days after heterotopic tracheal allotransplantation. The serum levels of FK506 were then investigated 3, 7, 14, 21, and 28 days after transplantation in groups C, D, and E. All rats were killed 28 days after transplantation and then the implanted tracheae were harvested, and evaluated histologically. RESULTS: All animals survived for the protocol period. The graft morphology of Group E was significantly better than that of groups B, C, and D regarding both macro- and microscopy, and also showed the same findings as that of Group A, except for low-grade mononuclear cell infiltration. Only in Group E, the FK506 blood level was maintained at over 0.5 ng/ml, which is the lowest detectable limit in this assay, until 21 days after transplantation. CONCLUSIONS: We thus conclude that 1.5 mg/kg of FK506 which was administered for only three consecutive days after surgery may be used to maintain the morphology of tracheal allografts in rats for 28 days after transplantation.

Tumor-infiltrating B-cell-derived IgG recognizes tumor components in human lung cancer.

Tumor-infiltrating lymphocytes consist predominantly of T cells, whereas B cells, plasma cells, and natural killer cells are observed with different degrees of frequency. We investigated the nature of tumor-infiltrating B lymphocytes (TIB) in human lung cancer. First, to examine the ability of immunogloblin production by TIB, cancer tissues were subcutaneously transplanted in severe combined immunodeficient mice, and the murine serum was examined for the concentration of human immunogloblin. Human IgG (huIgG) was detected in the serum of all 12 mice engrafted with lung cancer tissues. huIgM was almost undetectable. The levels of huIgG reached a peak approximately 6 weeks after engraftment and gradually decreased but were detectable until 20 weeks postengrafment. Serum from a large cell carcinoma-engrafted mouse reacted with a protein of 60 kDa derived from lung cancer cell lines (PC-9, Sq-1) and autologous tumor cells but did not react with cell lysates of normal lung tissue. Serum from an adenocarcinoma-engrafted mouse reacted with two proteins, 33 and 55 kDa, derived from lung cancer cell lines (PC-9, Sq-1, A549) and autologous tumor cells but did not react with the lysate of normal lung tissues. These results suggest that B cells infiltrating lung cancer tissues produce IgG that recognizes common tumor-specific antigen.

Chronic expanding hematoma in the chest.

We report the successful surgical treatment of chronic expanding hematoma in the chest. Four patients who had previously undergone artificial pneumothorax, thoracoplasty or tumor extirpation more than 30 years earlier recently became aware of a slowly growing mass. Chronic expanding hematoma which developed into very large masses over a long period of time were thus successfully resected. These patients are now all in good health with no recurrence after the operation. It is important to monitor such patients' laboratory data for hemostasis including the platelet cell counts, the % prothrombin time and the D-dimer, both before and immediately after operation, and the intraoperative bleeding volume.

Adoptive immunotherapy using specific cytotoxic T lymphocytes against human lung-cancer-engrafted severe combined immunodeficiency mice.

OBJECTIVE: We studied the ability of human lung-cancer-specific cytotoxic T lymphocytes to suppress the growth of human lung adenocarcinoma (PC-9) engrafted in severe combined immunodeficiency mice. METHODS: PC-9-specific cytotoxic T lymphocytes were generated by multiple stimulation with irradiated PC-9 cells of regional lymph node lymphocytes from lung cancer patients expressing the same human leukocyte antigen-A locus haplotype as PC-9 following expansion due to the administration of immobilized anti cluster of differentiation 3 mAb and interleukin-2. Cytotoxic T lymphocytes showed specific cytotoxicity against PC-9 cells in vitro. Severe combined immunodeficiency mice with a subcutaneous graft of PC-9 were treated with a PC-9-specific cytotoxic T lymphocyte by i.v. injection and/or with interleukin-2 by s.c. injection. RESULTS: Cytotoxic T lymphocyte treatment suppressed PC-9 graft growth significantly an effect, significantly enhanced when combined with interleukin-2 injection. To evaluate the in vivo specificity of anti-PC-9 cytotoxic T lymphocytes, each mouse was subcutaneously inoculated in the right flank with PC-9, and in the left flank with A549 or Sq-1. Cytotoxic T lymphocytes plus interleukin-2 treatment was found to suppress PC-9 growth selectively, but not A549 or Sq-1 growth. CONCLUSIONS: These results provide sufficient rationale for conducting further clinical trials on immunotherapy using cytotoxic T lymphocyte for lung cancer patients.

Effects of interleukin-12 on the induction of cytotoxic T lymphocytes from the regional lymph node lymphocytes of patients with lung adenocarcinoma.

Lung cancer-specific cytotoxic T lymphocytes (CTL) were induced by repeated stimulations of regional lymph node lymphocytes (RLNL) in lung cancer patients with either autologous or HLA-A-locus-matched tumor cells. To investigate the effect of interleukin-12 (IL-12), IL-12 was added during the stimulation of RLNL from HLA A24/adenocarcinoma patients with either autologous tumor cells or HLA A24-positive adenocarcinoma cells (PC-9) in combination with, or instead of interleukin-2 (IL-2), and then the cytotoxic activity, cytokine production and populations of the lymphocyte subsets were examined. The addition of IL-12, or the substitution of IL-2 by IL-12 was found to enhance the cytotoxic activity and the cytokine production (IFN-gamma, GM-CSF) of the CTL as compared with IL-2 alone. The cytotoxic activity and cytokine production were both partially inhibited by anti-MHC-class I monoclonal antibody. The CTL thus induced by IL-12 had a higher proportion of CD3+/CD56+ cells than the CTL induced with IL-2 alone. The positively selected CD8+/CD56- lymphocytes showed PC-9-specific cytotoxic activity, because the population did not show any cytotoxicity to K562 or A549 (HLA-A26/A30). However, the CD3+/CD56+ lymphocytes were cytotoxic to both PC-9 and K562. In conclusion, IL-12 is considered to be a useful cytokine for both the induction of lung-cancer specific CTL and the augmentation of non-MHC-restricted cytotoxicity against tumor cells, and may be applicable for adoptive immunotherapy using CTL.

Autologous tumor-specific cytotoxic T lymphocytes in a patient with lung adenocarcinoma: implications of the shared antigens expressed in HLA-A24 lung cancer cells.

Human lung adenocarcinoma-specific cytotoxic T lymphocytes (CTL) were generated by multiple stimulations with autologous tumor cells (named A110L) from regional lymph node lymphocytes and tumor-infiltrating lymphocytes expanded by solid-phase anti-CD3 monoclonal antibody (mAb) and recombinant interleukin-2. The CTL lysed A110L but failed to kill either autologous B lymphocytes immortalized by the Epstein-Barr virus or K562. The killing activity of the CTL against autologous A110L was inhibited by anti-MHC class I mAb (W6/32), but not by anti-MHC class II mAb. The CTL produced interferon-gamma and GM-CSF in response to A110L and the production was completely blocked by the addition of anti-MHC class I mAb. The HLA type of the CTL was HLA-A2/A24, B52/B54, Cw1/-. Allele-specific deletion of HLA-A2 molecules was observed in A110L by staining with anti-HLA-A2 mAb. A partial blocking effect on the cytokine production from the CTL was also obtained with anti-CD8, and anti-HLA-A24 mAbs, but not with anti-MHC class II, anti-CD4 and anti-HLA-A2 mAbs. To analyze further the mechanism of antigen recognition by the CTL, the cross reactivity of the CTL against several HLA-A locus-matched (HLA-A24+) and mismatched allogeneic tumor cells (HLA-A24-) was investigated. The A110L-specific CTL showed a weak but significant cytotoxicity against some HLA-A24 positive lung cancer cell lines, such as Sq-1 (HLA-A11/A24, squamous cell carcinoma) and PC-9 (HLA-A2/A24, adenocarcinoma), but failed to kill HLA-A locus-mismatched allogeneic tumors. This cross reactivity of the CTL against Sq-1 and PC-9 was blocked by anti-MHC class I mAb. These results thus demonstrate that shared common tumor antigens might exist among lung cancer cells in the context of HLA-A24.

Induction of Tumor-Specific Cytotoxic T Lymphocytes from Regional Lymph Node Lymphocytes of Human Breast Cancer.

BACKGROUND: In this study we activated breast cancer-specific cytotoxic T lymphocytes (CTL) from regional lymph node lymphocytes (RLNL) of HLA-A2-positive patients with breast cancer. METHODS: Freshly isolated RLNL were stimulated with solid phase anti-CD3 monoclonal antibody followed by expansion with recombinant interleukin-2. Subsequently, the RLNL were stimulated with an irradiated HLA 0201 breast cancer cell line,MCF-7, at a responder/stimulator ratio of 10/1 once a week for 2 weeks. RESULTS: The cultured RLNL exhibited specific lysis against MCF-7 in all 5 HLA-A2-positive patients tested, but not in 2 HLA-A2-negative patients. Cytotoxicityagainst MCF-7 was substantially inhibited by addition of anti-HLA-A2 mAb. In 3 of 5 HLA-A2-positive patients, anti-MCF-7 CTL also exhibited a substantial levelof reactivity against PC-9, an HLA-A0206-positive lung adenocarcinoma cell line. Conversely, anti -PC-9-specific CTL were inducible by multiple stimulations ofRLNL with PC-9 cells in 2 of 3 patients. CONCLUSION: These results suggest that several common tumor antigens might exist among HLA-A2-positive breast cancers, some of which may be shared with lung adenocarcinomas.

The induction of cytotoxic T lymphocytes against HLA-A locus-matched lung adenocarcinoma in patients with non-small cell lung cancer.

To induce cytotoxic T lymphocytes (CTL) against non-small cell lung cancer (NSCLC) efficiently, the induction of CTL was attempted using HLA-A locus-shared allogeneic NSCLC cells. T cells derived from either tumor tissue specimens or the regional lymph nodes of patients with NSCLC were stimulated twice or three times with an HLA-A2/A24-positive NSCLC cell line (PC-9), and thereafter the cytotoxic activity was examined by 51Cr-release assay. In patients with HLA-A24/ adenocarcinoma, anti-PC-9 cytotoxicity was induced in all 6 patients tested. Anti-PC-9 cytotoxicity was induced in 2 out of 5 patients with HLA-A2 (A24-)/adenocarcinoma, in 2 out of 4 patients with HLA-A24/squamous cell carcinoma, and 1 of 2 patients with HLA-A2/squamous cell carcinoma. The cytotoxic activity was observed to kill PC-9 selectively, not other NSCLC lines, and the activity was substantially blocked by anti-MHC class I antibody, but not by anti-MHC class II antibody. The PC-9-specific CTL produced gamma-interferon in response to autologous tumor cells. These results indicated that the anti-PC-9 cytotoxicity was mediated by cytotoxic T lymphocytes that may recognize the T cell epitope(s) shared and presented by HLA-A2 and/or HLA-A24-positive NSCLC.

[Treatment with expandable metallic stent in patients with carcinomatous airway stenosis--evaluation of patient's quality of life].

The expandable metallic stent was used in 10 patients with carcinomatous airway stenosis. In all patients, the respiratory symptoms improved immediately after insertion of the stent. Eight of the 10 patients, performance status improved. Totally, the use of expandable metallic stent improved the quality of life for patients with carcinomatous airway stenosis. Complication was minimal. We conclude that expandable metallic stent for treatment of carcinomatous airway stenosis are useful in emergent cases.