T CELL RECEPTOR REPERTOIRE AND SUPERANTIGENS IN RHEUMATOID ARTHRITIS

大量证据表明，T细胞在类风湿关节炎的发病机制中起重要作用。我们目前研究的主要目标是确定类风湿关节炎患者滑液中克隆性CD4+T细胞扩增所表达的T细胞受体(TCR)的特征，并确定这些克隆的特异性。通过克隆扩增表达的TCR在T细胞杂交瘤中表达，以确定这些T细胞对滑膜抗原的特异性。此外，我们一直在使用共价II类MHC-胶原多肽和软骨糖蛋白多肽复合体和四聚体对患者滑膜和血液中的特异性T细胞进行染色。尽管滑膜T细胞受体序列具有明显的异质性，但我们注意到，在一个关节中发现的TCRB序列中，有20%-30%也在第二个关节中表达，但在同一个体的外周血T细胞中不表达。对TCRB互补决定区3(CDR3)序列的分析表明，在两个或更多的关节中存在大的克隆性扩张。对TCRβ链和β链谱系的持续分析显示，单个患者内的克隆之间有显著的同源性，强烈提示由相同的滑膜抗原选择。总之，这些研究表明，在这种疾病中，相当大比例的滑膜CD4+T细胞已经被常规抗原选择和扩增。在最近的资助期间，已经产生了表达RA滑膜TCR的杂交瘤，我们一直在筛选对候选滑膜抗原和多肽的反应。未发现对滑膜抗原的反应。然而，我们已经扩增了CD4+滑膜T细胞，它们似乎对Epstein-Barr病毒抗原和可能的丙型肝炎抗原有反应。人类白细胞抗原DR4-II型胶原共价复合体和四聚体已经被生产出来，并被证实可以染色针对DR4呈现的正确多肽(CII p259-271)的杂交瘤。检查患者和对照组的滑液、刺激血液和滑液样本的研究正在进行中。

Considerable evidence indicates that T cells are important in the pathogenesis of rheumatoid arthritis. The major goals of our current studies are to characterize the T cell receptors (TCRs) expressed by clonal CD4+ T cell expansions in the synovial fluid of patients with rheumatoid arthritis and to determine the specificity of these clones. The TCRs expressed by these clonal expansions are expressed in T cell hybridomas in order to determine the specificity of these T cells for synovial antigens. In addition, we have been using covalent class II MHC-collagen peptide and cartilage glycoprotein peptide complexes and tetramers to stain for specific T cells in synovium and blood of patients. Despite marked heterogeneity of the synovial T cell receptor sequences, we noted that 20-30% of the TCRB sequences found in one joint were also expressed in a second joint but not in peripheral blood T cells of the same individual. Analysis of TCRB complementarity determining region 3 (CDR3) sequences showed the presence of large clonal expansions present in two or more joints. Continued analysis of the TCR beta-chain and beta-chain repertoire showed remarkable homology among clones within an individual patient, strongly suggesting selection by the same synovial antigen. Together, these studies suggest that a significant proportion of synovial CD4+ T cells have been selected and expanded by conventional antigens in this disease. In the recent funding period, hybridomas expressing RA synovial TCRs have been generated, and we have been screening for responses to candidate synovial antigens and peptides. No responses to synovial antigens have been found. However, we have expanded CD4+ synovial T cells that appear to react to Epstein-Barr virus antigens and possibly hepatitis C antigens. HLA DR4-collagen type II covalent complexes and tetramers have been produced and verified to stain hybridomas specific for the correct peptide (CII p259-271) presented by DR4. Studies examining synovial fluid and stimulated blood and synovial fluid samples from patients and controls are in progress.