TY - JOUR
T1 - A novel, rapid and efficient method of cloning functional antigen-specific T-cell receptors from single human and mouse T-cells
AU - Hamana, Hiroshi
AU - Shitaoka, Kiyomi
AU - Kishi, Hiroyuki
AU - Ozawa, Tatsuhiko
AU - Muraguchi, Atsushi
N1 - Publisher Copyright:
©2016 Published by Elsevier Inc.
PY - 2016/6/10
Y1 - 2016/6/10
N2 - 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.
AB - 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.
KW - Luciferase reporter assay
KW - Multiplex one-step RT-PCR
KW - T-cell receptor
KW - Transcriptionally active PCR
UR - http://www.scopus.com/inward/record.url?scp=84968911495&partnerID=8YFLogxK
U2 - 10.1016/j.bbrc.2016.05.015
DO - 10.1016/j.bbrc.2016.05.015
M3 - 学術論文
C2 - 27155153
AN - SCOPUS:84968911495
SN - 0006-291X
VL - 474
SP - 709
EP - 714
JO - Biochemical and Biophysical Research Communications
JF - Biochemical and Biophysical Research Communications
IS - 4
ER -