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Reducing T-cell receptor expression

June 1st, 2020

Watch the video where Alexander Zhu '21 presents his research on reducing T-cell receptor expression in T cells using RNAi and CRISPR-dCas9.

 

Adaptive immunity enables the human body to generate an immune response to specific pathogens. An important step in this process involves the scanning of the surfaces of antigen-presenting cells (APCs) by T cells. Such interactions are mediated by T-cell receptors (TCRs), which recognize and bind antigens presented on APCs. Various mechanisms have been proposed for how TCR binding leads to T-cell activation.

 

Understanding the mechanism requires TCR recruitment and triggering to be characterized at the level of individual proteins. In order for individual molecules to be visualized using TIRF microscopy, TCR expression must be reduced. RNA-mediated interference (RNAi) has been used to “knock down” TCR levels, but the effects of CRISPR-dCas9 on TCRs have never been studied before. The purpose of this study was to determine whether RNAi and CRISPR-dCas9, alone and in combination, could be used to reduce TCR expression. Oligonucleotides were generated and inserted into the pHR-sin-U6 plasmid. This plasmid was introduced into Jurkat T-cells using lentivirus transduction, and the TCR expression of 30,000 cells was measured using flow cytometry. We found that targeting the CD3ε chain through RNAi and CRISPR-dCas9 reduced TCR expression by 81.3% and 84.9% respectively. Combining these methods did not reduce TCR levels further, however.

 

These results suggest that RNAi and CRISPR-dCas9 can both effectively lower TCR expression, but these strategies do not act synergistically or additively when used together. Future studies should investigate the mechanism by which RNAi and CRISPR-dCas9 fail to act in conjunction and probe their effects on other CD3 chains.

 

Alexander Zhu is mentored by Dr Simon Davis, Weatherhall Institute of Molecular Medicine, University of Oxford.

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