| Autor: |
Guo XZ; Department of Immunology, St. Jude Children's Research Hospital, Memphis, Tennessee, USA; Integrated Biomedical Sciences Program, Department of Microbiology, Immunology and Biochemistry, University of Tennessee Health Science Center, Memphis, Tennessee, USA., Dash P; Department of Immunology, St. Jude Children's Research Hospital , Memphis, Tennessee, USA., Calverley M; Department of Immunology, St. Jude Children's Research Hospital , Memphis, Tennessee, USA., Tomchuck S; Department of Bone Marrow Transplantation and Cellular Therapy, St. Jude Children's Research Hospital , Memphis, Tennessee, USA., Dallas MH; Department of Bone Marrow Transplantation and Cellular Therapy, St. Jude Children's Research Hospital , Memphis, Tennessee, USA., Thomas PG; Department of Immunology, St. Jude Children's Research Hospital, Memphis, Tennessee, USA; Integrated Biomedical Sciences Program, Department of Microbiology, Immunology and Biochemistry, University of Tennessee Health Science Center, Memphis, Tennessee, USA. |
| Abstrakt: |
Transgenic expression of antigen-specific T-cell receptor (TCR) genes is a promising approach for immunotherapy against infectious diseases and cancers. A key to the efficient application of this approach is the rapid and specific isolation and cloning of TCRs. Current methods are often labor-intensive, nonspecific, and/or relatively slow. Here, we describe an efficient system for antigen-specific αβTCR cloning and CDR3 substitution. We demonstrate the capability of cloning influenza-specific TCRs within 10 days using single-cell polymerase chain reaction (PCR) and Gibson Assembly techniques. This process can be accelerated to 5 days by generating receptor libraries, requiring only the exchange of the antigen-specific CDR3 region into an existing backbone. We describe the construction of this library for human γδ TCRs and report the cloning and expression of a TRGV9/TRDV2 receptor that is activated by zoledronic acid. The functional activity of these αβ and γδ TCRs can be characterized in a novel reporter cell line (Nur77-GFP Jurkat 76 TCRα(-)β(-)) for screening of TCR specificity and avidity. In summary, we provide a rapid method for the cloning, expression, and functional characterization of human and mouse TCRs that can assist in the development of TCR-mediated therapeutics. |
