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.