NEW ASSAY FOR TCR DIVERSITY IN TMJ RHEUMATOID ARTHRITIS

激活的T细胞似乎是发病机制中的中心元素 类风湿性关节炎(RA)。这个 负责T细胞激活的机制尚不清楚。 如果T细胞按照常规机制被激活，那么 特异性免疫原(S)应通过T细胞抗原识别 受体(TCR)。涉事的免疫原(S)和TCR；S都没有 在分子水平上被定义。我们假设一个占主导地位的 在体内激活的人中可以发现TCR可变序列的形式 TMJ T淋巴细胞。这项提案的主要目标是分析 表达的多样性TCRα/βV区核苷酸序列 体内激活的T细胞，并鉴定其核苷酸序列 任何主要的受体。为此，我们将选择六个合适的 我们可以从TMJ RA患者体内获得活化的滑膜T细胞 细胞。我们将开发一种新的检测方法，它结合了“连接物锚定” 聚合酶链式反应(LA-PCR)和变性梯度凝胶电泳法(DGGE) 定量评估TCR V区序列的多样性。这些 细胞将通过LA-PCR+DGGE进行分析。优势受体 凝胶上鉴定的物种将被克隆和测序。发展 这项测试将允许解决克隆，并可能在 未来需要鉴定患者特有的致病T细胞克隆。 作为未来的努力，这些主要受体的独特区域将 在重组系统中表达，并获得特异性的单抗 将会被生成。主要T细胞克隆的鉴定， 而单抗的发展可能会直接导致重要的 医学和科学应用。特异性的单抗 主要的TCR可能被证明是有用的新的诊断和 监测疾病活动，并可能用于特定的免疫治疗。

The activated T-cells appear to be a central element in the pathogenesis of temporomandibular joint (TMJ) rheumatoid arthritis (RA). The mechanisms responsible for the activation of the T cells are not known. If the T cells are activated according to conventional mechanisms, then specific immunogen(s) should be recognized through the T-cell antigen receptor (TCR). Neither the immunogen(s), nor the TCR;s involved have been defined at the molecular level. We hypothesize that a predominant form of TCR variable sequence can be found among the in vivo activated TMJ T lymphocytes. The primary objective of this proposal is to analyze the diversity TCR alpha/beta V-region nucleotide sequences expressed by the in vivo activated T cells, and characterize the nucleotide sequences of any predominant receptors. To this end, we will select six suitable TMJ RA patients from which we can obtain in vivo activated synovial T- cells. We will develop a novel assay which combines "linker-anchored" PCR (LA-PCR) and Denaturing Gradient Gel Electrophoresis (DGGE) to quantitatively estimate the diversity of TCR V-region sequences. These cells will be analyzed by the LA-PCR plus DGGE. Predominant receptor species identified on the gel will be cloned and sequenced. Development of this assay would allow resolution of the clonality, and might in the future be needed to identify patient-specific pathogenic T cell clones. As a future effort, unique regions of these predominant receptors will be expressed in a recombinant system, and monoclonal antibodies specific for them will be generated. Identification of predominant T cell clones, and development of monoclonal antibody could lead directly to important medical and scientific applications. Monoclonal antibodies specific for predominant TCR might prove useful as new assays for diagnosis and for monitoring disease activity, and potentially in specific immunotherapy.