Establishment and application of a novel T cell clonality analysis using single-strand conformation polymorphism of T cell receptor messenger signals.
To identify 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 the appropriate VP amplification and an accumulated T 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. Analyses of several immunological disorders, including autoimmune diseases, malignant disorders, and transplantations, revealed the involvement of antigen-specific T cell immune responses in these disorders. Furthermore, taking advantage of the reproducible mobility of a band of SSCP gel, we are now able to compare identities of the accumulated T cell clones in different samples without the need for nucleotide sequencing 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 together, 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.
Yamamoto
K
K
Institute of Medical Science, St. Marianna University, Beppu, Japan.
Masuko-Hongo
K
K
Tanaka
A
A
Kurokawa
M
M
Hoeger
T
T
Nishioka
K
K
Kato
T
T
D001327:Autoimmune Diseases / Q000276:immunology; D016026:Bone Marrow Transplantation / Q000276:immunology; D002999:Clone Cells; D003001:Cloning, Molecular / Q000379:methods*; D004195:Disease Models, Animal; D005260:Female; D006801:Humans; D016246:Lymphocytes, Tumor-Infiltrating / Q000276:immunology; D010051:Ovarian Neoplasms / Q000276:immunology; D016133:Polymerase Chain Reaction / Q000379:methods*; D018807:Polymorphism, Single-Stranded Conformational; D011247:Pregnancy / Q000276:immunology; D012333:RNA, Messenger / Q000032:analysis*; D012194:RNA-Directed DNA Polymerase; D011948:Receptors, Antigen, T-Cell / Q000235:genetics*; D015398:Signal Transduction / Q000235:genetics*; D013601:T-Lymphocytes / Q000276:immunology*; D014594:Uterine Neoplasms / Q000276:immunology
D012333:RNA, Messenger; D011948:Receptors, Antigen, T-Cell; D012194:RNA-Directed DNA Polymerase