Novel method to detect T clonality and the antigen specificity

为了确定抗原特异性T细胞反应的存在并跟踪这些反应的变化，评估某些T细胞是否克隆性地聚集在淋巴细胞群体中被认为是有用的。为此，我们建立了一种结合T细胞受体β链转录本的逆转录聚合酶链式反应和单链构象多态性分析T细胞克隆性的新方法。利用这种方法，我们从异质T细胞群体中获得了一种涂片状的DNA电泳图。另一方面，单个T细胞克隆在适当的Vbeta扩增中表现出一条带，而在异质淋巴细胞群体中积累的T细胞克隆被鉴定为背景涂片模式中的一条带。如果在体外或体内用抗原刺激淋巴细胞群，在背景涂片中发现有几个不同的条带。因此，该方法可以监测T细胞克隆反应的动态变化。对包括自身免疫性疾病、恶性疾病和移植在内的几种免疫疾病的分析表明，抗原特异性T细胞免疫反应参与了这些疾病。此外，利用一条SSCP凝胶的重复性，我们现在可以比较不同样本中累积的T细胞克隆的同一性，而不需要对每个克隆进行核苷酸测序。因此，这些信息可以解释宿主体内发生的均匀或稳定的免疫反应，也表明这些反应在体内发挥着重要作用。因此，上述新的T细胞克隆性分析被认为在免疫学的各个领域中研究T细胞免疫反应是有用的。

To identity the existence of antigen-specific T cell responses and to follow the changes of these reactions, it is considered useful to evaluate whether certain T cells clonally accumulate in the lymphocyte population. For this purpose, we have established a novel method to analyze T cell clonality using a combination of reverse transcriptase-polymerase chain reaction of T cell receptor beta chain transcripts and single-strand conformation polymorphism (SSCP). Using this method, we obtained a smear-like pattern of electrophoresed DNA from the heterogeneous T cell population. On the other hand, a single T cell clone exhibits a band in appropriate Vbeta amplification and an accumulatedT cell clone in a heterogeneous lymphocyte population is identified as a band in the background smear pattern. If a lymphocyte population was stimulated by an antigen either in vitro or in vivo, several distinct bands were found to be generated in the background smear. Thus, the dynamic changes of T cell clonal responses could be monitored with this method. Analyzes of several immunological disorders, including autoimmune diseases, malignant disorders, andtransplantation, revealed the involvement of antigen-spesific T cell immune responses in these disorders. Furthermore, taking advantege of the reproduciblemobility of a band of SSCP gel, we are now able tocompare identities of the accumulated T cell Clones in different samples without the need for nucleoptidesequencing of each clone. Such information can thus elucidate the occurrence of uniform or stable immunological reactions in the host and also suggests that these reactions play an important role in vivo. Therefore, taken togeter, the above described novel T cell clonality analysis is considered to be useful in studying the T cell immune responses in various fields of immunology.