The purpose of our present study is to identify a tumorspecific antigen capable of inducing a specific cellular immune response in lung cancer patients. We established a lung adenocarcinoma cell line, designated as F1121L, and induced tumor-specific CTL clone H1 from regional lymph node lymphocytes of patient F1121. CTL clone H1 lysed autologous tumor cells in an HLA-B*1507-restricted manner, but not autologous EBV-B, phytohemagglutinin-blast cells, and K562. The CTL clone also recognized allogeneic HLA-B*1501-or 1507-positive lung cancer cell lines in the HLArestricted manner. Using the CTL clone, we identified an antigen-coding gene by cDNA expression cloning technique. The gene consisted of 556 bp, including an open reading frame consisted of 113 amino acids, designated as Kita-kyushu lung cancer antigen 1 (KK-LC-1). A 9-mer peptide (KK-LC-1 76-84 ; RQKRILVNL) was identified as an epitope peptide. The genomic DNA of this antigen was located in chromosome Xq22. A reverse transcription-PCR analysis revealed that the mRNA of this gene was only expressed in the testis among normal tissues. It was expressed in 9 of 18 (50%) allogeneic non-small-cell lung cancer cell lines and in 40 of 100 (40%) non-small-cell lung cancer tissues. We thus identified a new tumor antigen-coding gene categorized as a cancer/germline gene by an autologous lung cancer and CTL system. The new cancer/germline gene was located in Xq22, which is apparently different from the locations of previously reported cancer/germline genes. (Cancer Res 2006; 66(9): 4922-8)

One of tumor escape mechanisms from the host's immunosurveillance system (i.e., a haplotype loss of HLA class I antigens) has been detected in various tumor cells. We hypothesize that the majority of tumor cells with normal HLA class I expression were attacked and eradicated by CTLs, and only a minority with an abnormal expression of HLA class I antigens could escape the host's immunosurveillance system. Using HLA class I-transfected tumor variants as stimulators in A904L lung cancer cell line, which has a haplotype loss of HLA class I antigens, both the transfected HLA-A26 and HLA-B39-restricted CTL lines were induced from autologous lymphocytes. However, only one HLA-B39-restricted CTL clone (CTL G3b) was established, and it was then used to identify the antigen. SGT1B [suppressor of G2 allele of SKP1 (SGT1), suppressor of kinetochore protein (SKP1)] was identified as the antigen recognized by CTL G3b. Further experiments using 13 subclones from a primary culture of A904L were found to confirm our above-mentioned hypothesis.

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We recently identified several Ags recognized by tumor-infiltrating B lymphocyte-derived Ab using SCID mice and a xenografted non-small cell lung cancer system. One of these identified Ags was mutated p53 with a point mutation resulting in the alteration of codon 158 from Arg to Leu. The aim of this study was to ascertain whether cellular immunity against mutated p53 exists in the same patient together with humoral immunity. Two different nona peptides (mutated p53150 and p53155 peptides), including a mutated amino acid derived from p53, were synthesized according to the binding motif of HLA class I of the established cancer cell line A904L from the patient. Mediastinal lymph node lymphocytes of the patient were stimulated weekly with the peptides. The mutated p53155 peptide-stimulated lymphocytes showed specific cytotoxicity against both autologous EBV-transformed B cells pulsed with mutated p53155 peptide and A904L. The mutated p53155 peptide-specific CTL clone in an HLA-Cw*0702 restriction was established and analyzed for its TCR usage. Clonotypic PCR using CDR3-specific primers was applied to the tumor tissue containing the tumor-infiltrating lymphocytes. The specific amplification of PCR was found in the tumor tissue. These results demonstrated that not only B lymphocytes producing specific Ab against the p53 protein, but also CTL against mutated p53, expressed in autologous lung cancer cells exist in the tumor tissue. This approach may allow us to better understand the mechanisms of T and B cell immunity against the same tumor Ag in cancer patients.

Cytokines produced by tumor cells may have various effects on antitumor immune responses and tumor growth. In the present study, the cytokine production of 31 lung cancer cell lines was evaluated, while any correlation with the histological type, the induction of tumor‐specific cytotoxic T lymphocytes (CTL) in vitro, and angiogenesis and the infiltration of inflammatory cells in tumor tissues were also examined. Production of interleukin (IL)‐1α, IL‐1β, IL‐4, IL‐6, IL‐8, IL‐10, tumor necrosis factor (TNF)‐α, granulocyte macrophage colony stimulating factor (GM‐CSF), granulocyte colony stimulating factor, transforming growth factor (TGF)‐β and vascular endothelial growth factor (VEGF) in the culture supernatant was measured using enzyme‐linked immunosorbent assay. Each cytokine was produced in a substantial number of the tumor cell lines. In particular, IL‐6, IL‐8, TGF‐β and VEGF were produced in 18 (55%), 29 (94%), 31 (100%) and 28 (90%) of 31 cell lines, respectively. However, neither IL‐4 nor TNF‐α was produced at all by any tumor cell line. TGF‐β production was significantly higher in adenocarcinoma than in squamous cell carcinoma (P = 0.03). Immunohistochemical staining revealed the magnitude of macrophage infiltration, and angiogenesis in surgically resected tumor tissue specimens correlated well with GM‐CSF and IL‐8 production from the corresponding cell lines. Among six lung cancer cell lines, CTL were induced in the three lung cancer cell lines that produced a lower amount of TGF‐β (<100 pg/mL). These findings suggested that TGF‐β produced by tumor cells could inhibit the induction of CTL in vitro. The present results suggest that the production of various cytokines from tumor cells might exert various paracrine effects both in vivo and in vitro. (Cancer Sci 2007; 98: 1048–1054)