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

本研究旨在鉴定口腔黏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表达TCR的疾病特异性T细胞的存在来建立诊断策略。本研究结果如下：1)疾病特异性TCR基因的鉴定：基于pcr的分析显示，限制性Vβ基因家族在口腔鳞状细胞癌（SCC）患者的肿瘤浸润淋巴细胞中频繁使用。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。2)诱导T细胞识别口腔鳞状细胞癌患者外周血肿瘤相关抗原肽：体外培养口腔鳞状细胞癌患者外周血单个核细胞13种肿瘤相关抗原肽，检测其肽特异性细胞毒性T淋巴细胞（CTL）活性。结果表明，sari -1_<690>、SARI-2_<93>和ART4_<75>最常诱导肽特异性CTL活性。对sart -1 <690>、sari -2 <93>和art4 <75>的CTL活性诱导的患者，对其他多肽的CTL活性也倾向于诱导。相比之下，在40%的患者中，任何肽都没有诱导CTL活性。3)口腔鳞状细胞癌患者病变组织中免疫调节分子的表达：免疫组织化学检测病变组织中各种免疫调节分子的表达。因此，在CTL具有抗肿瘤相关抗原肽活性的患者中，CD80和ICAM-1在SCC细胞上的异常表达被观察到。相比之下，在没有任何肽诱导CTL活性的患者中，肿瘤相关抗原RCASI在SCC细胞上的异常表达被观察到。在表达RCAS1的SCC细胞周围经常观察到凋亡T细胞，提示RCASI在肿瘤逃避宿主免疫系统机制中发挥重要作用。本研究提示SARI-1_<690>、SART-2_<93>、ART4_<75>等肿瘤相关抗原肽可用于口腔SCC患者的诊断和随访，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。新的诊断策略也正在建立，以量化各种口腔粘膜疾病患者病变或外周血中疾病特异性T细胞的存在。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