Separate signaling events control TCR downregulation and T cell activation in primary human T cells

Introduction: T-cell antigen receptor (TCR) interaction with cognate peptide:MHC complexes trigger clustering of TCR:CD3 complexes and signal transduction. Triggered TCR:CD3 complexes are rapidly internalized and degraded in a process called ligand-induced TCR downregulation. Classic studies in immortalized T-cell lines have revealed a major role for the Src family kinase Lck in TCR downregulation. However, to what extent a similar mechanism operates in primary human T cells remains unclear. Methods: Here, we developed an anti-CD3-mediated TCR downregulation assay, in which T-cell gene expression in primary human T cells can be knocked down by microRNA constructs. In parallel, we used CRISPR/Cas9-mediated knockout in Jurkat cells for validation experiments. Results: We efficiently knocked down the expression of tyrosine kinases Lck, Fyn, and ZAP70, and found that, whereas this impaired T cell activation and effector function, TCR downregulation was not affected. Although TCR downregulation was marginally inhibited by the simultaneous knockdown of Lck and Fyn, its full abrogation required broad-acting tyrosine kinase inhibitors. Conclusions: These data suggest that there is substantial redundancy in the contribution of individual tyrosine kinases to TCR downregulation in primary human T cells. Our results highlight that TCR downregulation and T cell activation are controlled by different signaling events and illustrate the need for further research to untangle these processes.

Automated summary

The interaction between T-cell antigen receptor (TCR) and its cognate peptide:MHC complexes triggers clustering of TCR:CD3 complexes and subsequent signal transduction. While the classic studies have shown a major role for Src family kinase Lck in TCR downregulation, the extent of this mechanism in primary human T cells remains unclear. Knockdown experiments in primary human T cells revealed redundancy in the contribution of individual tyrosine kinases to TCR downregulation, highlighting the need for further research to understand the complex signaling events controlling TCR downregulation and T cell activation.