Analysis of pathogenesis and establishment of novel diagnostic strategy of oral mucosal diseases by T cell receptorbased analysis

这项研究旨在确定口腔粘膜疾病中的疾病特异性T细胞受体(TCR)，并通过检测疾病患者中是否存在表达TCR的疾病特异性T细胞来建立诊断策略。1)疾病特异性TcR基因的鉴定：聚合酶链式反应分析显示，限制性Vβ基因家族在口腔鳞癌患者的肿瘤浸润性淋巴细胞中经常被使用。进一步的单链构象多态性分析发现，在一些Vβ基因家族中，T细胞克隆型的寡克隆性扩增。2)口腔鳞癌患者外周血中识别肿瘤相关抗原肽的T细胞的诱导：口腔鳞癌患者外周血单个核细胞与13种肿瘤相关抗原肽…体外培养然后检测它们的多肽特异性细胞毒性T淋巴细胞(CTL)活性。结果表明，SART-1、SARI-2、ART4、ART4、SARI、ART4、SARI-2、SARI-2和ART4_&LT；诱导了抗SART-1、SARI-2、ART4和ART4的CTL活性的患者，也可诱导出抗其他多肽的CTL活性。3)口腔鳞癌患者皮损中免疫调节分子的表达：采用免疫组织化学方法检测皮损中各种免疫调节分子的表达。结果，在CTL抗肿瘤相关抗原肽的患者中，观察到SCC细胞上CD80和ICAM-1的异常表达。相反，在没有用任何多肽诱导CTL活性的患者中，观察到肿瘤相关抗原RCASI在SCC细胞上的异常表达。肿瘤相关抗原肽sARI-1、sART-2、sART-2、sART_2、sART_2、s但也指出，必须重视肿瘤细胞的抗原性和肿瘤逃逸宿主免疫系统的机制。新的诊断策略也正在建立，以量化各种口腔粘膜疾病患者的病变或外周血中疾病特异性T细胞的存在。较少

This research was addressed to identify a disease-specific T cell receptor (TCR) in oral mucosal diseases and to establish a diagnostic strategy by examining the presence of disease-specific T cells expressing the TCR in patients with the diseases. Results obtained in this research were as follows :1)Identification of disease-specific TCR genes : PCR-based analysis revealed that restricted Vβ gene families are frequently used in tumor-infiltrating lymphocytes of patients with oral squamous cell carcinoma (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.2)Induction of T cells recognizing tumor-associated antigenic peptides from peripheral blood of patients with oral SCC : Peripheral blood mononuclear cells from patients with oral SCC were cultured in vitro with 13 kinds of tumor-associated antigenic peptides, … More 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-1_<690>, SARI-2_<93>, and ART4_<75>. CTL activities against other peptides also tended to be inducible from patients in whom CTL activities against SART-1_<690>, SARI-2_<93>, and ART4_<75> were induced. In contrast, in 40% of the patients examined, no CTL activity was induced with any peptides.3)Expression of immune-regulating molecules in the lesions of patients with oral SCC : Expression of, various immune-regulating molecules in the lesions 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 SCC cells was observed. In contrast, an aberrant expression of tumor-associated antigen RCASI on SCC cells was observed in patients in whom no CTL activity was induced with any peptides. Apoptotic T cells were frequently observed around SCC cells expressing RCAS1, suggesting RCASI plays an important role in the tumor escape mechanism from the host immune system.This research indicated that tumor-associated antigenic peptides including SARI-1_<690>, SART-2_<93>, and ART4_<75> are 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 antigenecity of tumor cells and the tumor escape mechanism from the host immune system must be paid attention. Novel diagnostic strategy to quantify the presence of disease-specific T cells in the lesions or peripheral blood from patients with various oral mucosal diseases is being also established. Less