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Updated: Mar 21, 2026
Streamlined Single Cell TCR Isolation and Generation of Retroviral Vectors for In Vitro and In Vivo Expression of Human TCRs
Published on: September 10, 2017
A novel, rapid and efficient method of cloning functional antigen-specific T-cell receptors from single human and
Hiroshi Hamana1, Kiyomi Shitaoka1, Hiroyuki Kishi1
1Department of Immunology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama, 930-0194, Japan.
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.
