Objective: Transgenic re-expression enables unbiased investigation of T-cell receptor (TCR)-intrinsic characteristics detached from its original cellular context. Recent advancements in TCR repertoire sequencing and development of protocols for direct cloning of full TCRαβ constructs now facilitate large-scale transgenic TCR re-expression. Together, this offers unprecedented opportunities for the screening of TCRs for basic research as well as clinical use. However, the functional characterisation of re-expressed TCRs is still a complicated and laborious matter. Here, we propose a Jurkat-based triple parameter TCR signalling reporter endogenous TCR knockout cellular platform (TPRKO) that offers an unbiased, easy read-out of TCR functionality and facilitates high-throughput screening approaches. Methods: As a proof-of-concept, we transgenically re-expressed 59 human cytomegalovirus-specific TCRs and systematically investigated and compared TCR function in TPRKO cells versus primary human T cells. Results: We demonstrate that the TPRKO cell line facilitates antigen-HLA specificity screening via sensitive peptide-MHC-multimer staining, which was highly comparable to primary T cells. Also, TCR functional avidity in TPRKO cells was strongly correlating to primary T cells, especially in the absence of CD8αβ co-receptor. Conclusion: Overall, our data show that the TPRKO cell lines can serve as a surrogate of primary human T cells for standardised and high-throughput investigation of TCR biology.      «

Objective: Transgenic re-expression enables unbiased investigation of T-cell receptor (TCR)-intrinsic characteristics detached from its original cellular context. Recent advancements in TCR repertoire sequencing and development of protocols for direct cloning of full TCRαβ constructs now facilitate large-scale transgenic TCR re-expression. Together, this offers unprecedented opportunities for the screening of TCRs for basic research as well as clinical use. However, the functional characterisati...     »