A High-Throughput Strategy for T-Cell Receptor Cloning and Expression

Abstract

Expression of T-cell receptor (TCR) genes is a critical step for TCR characterization and epitope identification. The recent interest in using specific TCRs for cancer immunotherapy has further increased the demand for practical and robust methods to rapidly clone and express TCRs. We show that a recombination-based cloning protocol facilitates simple and rapid transfer of the TCR transgene into different expression systems. In this protocol, we first constructed all the human TRAV and TRBV genes into individual plasmid. To clone any TCR, we only need to ligate a short CDR3 fragment to its corresponding V gene plasmid using Golden Gate cloning. This strategy significantly improves the efficiency of individual TCR cloning and mutagenesis, providing a flexible high-throughput method for TCR analysis and TCR-mediated therapeutics.

Keywords: Lentivirus package; TCR expression; TCR high-throughput cloning; Transfection.

Figure 1:. A streamlined approach for reconstructing TCRs and expressing.

Dominant TCR clonotypes are individually cloned by variable plus constant chain plasmid library components and an on-demand oligonucleotide encoding the CDR3 regions, TCRs are expressed in Jurkat-J76 cells with an NFAT-luciferase construct. Image was created using BioRender.com.

Figure 2.. Gibson assembly protocol.

Figure 3.. Designing and cloning the CDR3α/β oligos into TRAVC/TRBVC backbone.

Figure 4:. TCR expression was analyzed by flow cytometry.