title: A novel, rapid and efficient method of cloning functional antigen-specific T-cell receptors from single human and mouse T-cells.
abstract: T-cell receptor (TCR) gene therapy is a promising approach for the treatment of infectious diseases and cancers. However, the paired cloning and functional assays of antigen-specific TCRα and TCRβ is time-consuming and laborious. In this study, we developed a novel, rapid and efficient antigen-specific TCR-cloning system by combining three technologies: multiplex one-step RT-PCR, transcriptionally active PCR (TAP) and luciferase reporter assays. Multiplex one-step RT-PCR with leader primers designed from leader peptide sequences of TCRs enabled us to amplify cDNAs of TCRα and β pairs from single T-cells with remarkably high efficiency. The combination of TAP fragments and HEK293T-based NFAT-luciferase reporter cells allowed for a rapid functional assay without the need to construct expression vectors. Using this system, we cloned human TCRs specific for Epstein-Barr virus BRLF-1-derived peptide as well as mouse TCRs specific for melanoma-associated antigen tyrosinase-related protein 2 (TRP-2) within four days. These results suggest that our system provides rapid and efficient cloning of functional antigen-specific human and mouse TCRs and contributes to TCR-based immunotherapy for cancers and infectious diseases.
authors.affiliation: Department of Immunology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama, 930-0194, Japan.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Hamana
authors.affiliation: Department of Immunology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama, 930-0194, Japan.
authors.forename: Kiyomi
authors.initials: K
authors.lastname: Shitaoka
authors.affiliation: Department of Immunology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama, 930-0194, Japan. Electronic address: immkishi@med.u-toyama.ac.jp.
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Kishi
authors.affiliation: Department of Immunology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama, 930-0194, Japan.
authors.forename: Tatsuhiko
authors.initials: T
authors.lastname: Ozawa
authors.affiliation: Department of Immunology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama, 930-0194, Japan.
authors.forename: Atsushi
authors.initials: A
authors.lastname: Muraguchi
mesh_terms: D000818:Animals; D019556:COS Cells; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular / Q000379:methods*; D057809:HEK293 Cells; D006801:Humans; D008222:Lymphokines / Q000235:genetics* / Q000276:immunology*; D051379:Mice; D011948:Receptors, Antigen, T-Cell / Q000235:genetics / Q000276:immunology; D016693:Receptors, Antigen, T-Cell, alpha-beta / Q000235:genetics* / Q000276:immunology*; D013045:Species Specificity; D013601:T-Lymphocytes / Q000276:immunology*
chemical_list: D008222:Lymphokines; D011948:Receptors, Antigen, T-Cell; D016693:Receptors, Antigen, T-Cell, alpha-beta; C057264:antigen-specific helper factors
keywords: Luciferase reporter assay; Multiplex one-step RT-PCR; T-cell receptor; Transcriptionally active PCR