TY - JOUR
T1 - Separate signaling events control TCR downregulation and T cell activation in primary human T cells
AU - van der Donk, Lieve E. H.
AU - Ates, Louis S.
AU - van der Spek, Jet
AU - Tukker, Laura M.
AU - Geijtenbeek, Teunis B. H.
AU - van Heijst, Jeroen W. J.
N1 - Funding Information: We would like to thank Prof. Dr. Noam Zelcer for the kind gift of plasmids lentiCRISPR v2, pMDLg/pRRE, pRSV-Rev, and pMD2.G, as well as for useful discussion.?Lieve E. H. van der Donk, Louis S. Ates, Jet van der Spek, and Jeroen W. J. van Heijst were supported by the Netherlands Organization for Scientific Research (Vidi grant 91717305 to Jeroen W. J. van Heijst). Louis S. Ates is furthermore supported by a PostDoc stipend of the Amsterdam Infection and Immunity Institute and Teunis B. H. Geijtenbeek is funded by the European Research Council (advanced grant 670424). Funding Information: We would like to thank Prof. Dr. Noam Zelcer for the kind gift of plasmids lentiCRISPR v2, pMDLg/pRRE, pRSV‐Rev, and pMD2.G, as well as for useful discussion. Lieve E. H. van der Donk, Louis S. Ates, Jet van der Spek, and Jeroen W. J. van Heijst were supported by the Netherlands Organization for Scientific Research (Vidi grant 91717305 to Jeroen W. J. van Heijst). Louis S. Ates is furthermore supported by a PostDoc stipend of the Amsterdam Infection and Immunity Institute and Teunis B. H. Geijtenbeek is funded by the European Research Council (advanced grant 670424). Publisher Copyright: © 2020 The Authors. Immunity, Inflammation and Disease published by John Wiley & Sons Ltd Copyright: Copyright 2021 Elsevier B.V., All rights reserved.
PY - 2021/3
Y1 - 2021/3
N2 - 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.
AB - 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.
KW - CD4 T cells
KW - T-cell activation | T-cell receptor
KW - TCR downregulation
KW - protein tyrosine kinases
UR - http://www.scopus.com/inward/record.url?scp=85097878475&partnerID=8YFLogxK
U2 - https://doi.org/10.1002/iid3.383
DO - https://doi.org/10.1002/iid3.383
M3 - Article
C2 - 33350598
SN - 2050-4527
VL - 9
SP - 223
EP - 238
JO - Immunity Inflammation and Disease
JF - Immunity Inflammation and Disease
IS - 1
ER -