Rapid cloning, expression, and functional characterization of paired ?? and ?? T-cell receptor chains from single-cell analysis.

ABSTRACT: 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 ??TCR 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 ?? TCRs and report the cloning and expression of a TRGV9/TRDV2 receptor that is activated by zoledronic acid. The functional activity of these ?? and ?? TCRs can be characterized in a novel reporter cell line (Nur77-GFP Jurkat 76 TCR?(-)?(-)) 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.

SUBMITTER: Guo XZ 

PROVIDER: S-EPMC4729322 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature