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细胞是否在淋巴细胞群中克隆积累被认为是有用的。为此，我们建立了一种利用T细胞受体β链转录物的逆转录聚合酶链反应和单链构象多态性（SSCP）相结合的新方法来分析T细胞的克隆性。使用这种方法，我们从异质T细胞群中获得了电泳DNA的涂片样图案。另一方面，单个T细胞克隆在适当的v β扩增中表现出条带，而在异质淋巴细胞群体中积累的T细胞克隆在背景涂片模式中被识别为条带。如果淋巴细胞群受到抗原的刺激，无论是在体外还是在体内，在背景涂片中发现几个不同的条带。因此，该方法可以监测T细胞克隆反应的动态变化。对几种免疫疾病的分析，包括自身免疫性疾病、恶性疾病和移植，揭示了抗原特异性T细胞免疫反应在这些疾病中的作用。此外，利用SSCP凝胶带的可重复性迁移性，我们现在能够比较不同样品中积累的T细胞克隆的身份，而无需对每个克隆进行核核苷酸测序。因此，这些信息可以阐明宿主体内均匀或稳定的免疫反应的发生，并表明这些反应在体内发挥重要作用。因此，综上所述，新的T细胞克隆分析被认为对研究免疫学各个领域的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.