A T‐cell reporter platform for high‐throughput and reliable investigation of TCR function and biology

• Published in: Clinical & translational immunology 2020, Vol.9 (11), p.e1216-n/a
• Main Authors: Müller, Thomas R, Schuler, Corinna, Hammel, Monika, Köhler, Amelie, Jutz, Sabrina, Leitner, Judith, Schober, Kilian, Busch, Dirk H, Steinberger, Peter
• Format: Article
• Language: English
• Subjects: adoptive T‐cell therapy; Antigens; Avidity; Biology; Cas9; Cell lines; Cloning; CRISPR; Flow cytometry; Histocompatibility antigen HLA; Investigations; Lymphocytes; Lymphocytes T; Major histocompatibility complex; Original; Peptides; reporter T‐cell line; T cell receptors; TCR biology; TCR functional avidity; TCR gene editing; Test systems
• ISSN: 2050-0068; 2050-0068
• DOI: 10.1002/cti2.1216

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. A highly sensitive triple parameter T‐cell receptor (TCR) reporter cell line was adapted for high‐throughput testing of transgenically expressed TCRs by CRISPR/Cas9‐mediated deletion of the endogenous TCR and introduction of the CD8αβ co‐receptor. Using 59 CMV‐specific TCRs, we demonstrated a high similarity with primary T cells in terms of multimer staining, functional avidity and CD8‐dependence. In conclusion, we present a T‐cell reporter platform excellently suited for high‐throughput functional preclinical testing of TCRs.