Analysis of pathogenesis and establishment of novel diagnostic strategy of oral mucosal diseases by T cell receptor-based analysis

本研究旨在鉴定在口腔鳞状细胞癌（SCC）抗肿瘤T细胞及其靶抗原上表达的T细胞受体（TCR），特别是参与抗肿瘤T细胞活化和/或失活的肿瘤相关抗原。本研究结果如下：1)免疫调节分子在口腔鳞状细胞癌中的表达：用免疫组织化学方法检测各种免疫调节分子在口腔鳞状细胞癌细胞中的表达情况。因此，在CTL具有抗肿瘤相关抗原肽活性的患者中，我们观察到CD80和ICAM-1在口腔SCC细胞上的异常表达。相比之下，在没有任何肽诱导CTL活性的患者中，观察到肿瘤相关抗原RCAS1在口腔SCC细胞上的异常表达。在表达RCAS1的口腔SCC细胞周围经常观察到凋亡T细胞，提示RCAS1在肿瘤逃避宿主免疫系统机制中发挥重要作用。2)进一步鉴定疾病特异性TCR基因及其靶抗原：基于pcr的分析显示，限制性Vβ基因家族在口腔鳞状细胞癌患者的肿瘤浸润淋巴细胞中经常使用。进一步基于sscp的分析发现，在一些Vβ基因家族中T细胞克隆型存在低克隆扩增。目前正在进行独特TCR基因的DNA测序和表达独特TCR的T细胞克隆的建立。用12种肿瘤相关抗原肽体外培养口腔鳞状细胞癌患者外周血单个核细胞，检测其肽特异性细胞毒T淋巴细胞（CTL）活性。结果表明，SART-l_<690 bb0最常诱导肽特异性CTL活性。相比之下，在40%的患者中，任何肽都没有诱导CTL活性。本研究提示肿瘤相关抗原肽SART-l_<690>在口腔鳞状细胞癌患者的诊断和随访中最有用，也可用于建立肽类疫苗的肿瘤免疫治疗。然而，也指出肿瘤细胞的抗原性和肿瘤逃避宿主免疫系统的机制必须引起重视。少

This research was addressed to identify T cell receptors (TCR) expressed on anti-tumor T cells and their target antigens, especially tumor-associated antigens involved in activation and/or inactivation of anti-tumor T cells in oral squamous cell carcinoma (SCC). Results obtained in this research were as follows:1) Expression of immune-regulating molecules by oral SCC: Expression of various immune-regulating molecules on oral SCC cells was immunohistochemically examined. As a result, in patients in whom CTL activities against tumor-associated antigenic peptides, an aberrant expression of CD80 and ICAM-1 on oral SCC cells was observed. In contrast, an aberrant expression of tumor-associated antigen RCAS1 on oral SCC cells was observed in patients in whom no CTL activity was induced with any peptides. Apoptotic T cells were frequently observed around oral SCC cells expressing RCAS1, suggesting RCAS1 plays an important role in the tumor escape mechanism from the host immune system.2) Ident … More ification of disease-specific TCR genes and their target antigens: PCR-based analysis revealed that restricted Vβ gene families are frequently used in tumor-infiltrating lymphocytes of patients with oral SCC. Further SSCP-based analysis found oligoclonal expansion of T cell clonotypes in some Vβ gene families. DNA sequencing of the unique TCR genes and establishment of T cell clones expressing the unique TCR are being performed. Peripheral blood mononuclear cells from patients with oral SCC were cultured in vitro with 12 kinds of tumor-associated antigenic peptides, and were then examine their peptide-specific cytotoxic T lymphocyte (CTL) activities. As a result, peptide-specific CTL activities were most frequently induced with SART-l_<690>. In contrast, in40% of the patients examined, no CTL activity was induced with any peptides.This research indicated that tumor-associated antigenic peptide SART-l_<690> is most useful in the diagnosis and follow-up of patients with oral SCC, and also for the establishment of cancer immunotherapy by peptide vaccination. However, it was also indicated that the antigenicity of tumor cells and the tumor escape mechanism from the host immune system must be paid attention. Less