A discrete affinity-driven elevation of ZAP-70 kinase activity initiates negative selection

Abstract

Context: Although ZAP-70 is required for T-cell development, it’s unclear how this kinase controls both positive and negative selection.

Objective and methods: Using OT-I pre-selection thymocytes and a panel of peptide major histocompatibility complex (pMHC) ligands of defined affinity, the recruitment, phosphorylation and activity of ZAP-70 was determined at the interface with antigen-presenting cells (APCs).

Results: pMHC ligands promoting negative selection induce a discrete elevation of ZAP-70 recruitment, phosphorylation and enzymatic activity in the thymocyte:APCs interface.

Discussion: The quantity of ZAP-70 kinase activity per cell is a key parameter controlling the fate of a developing thymocyte since partial inhibition of ZAP-70 kinase activity converted negative into positive selection. Surprisingly, the amount of ZAP-70 enzymatic activity observed during negative selection is not controlled by differential phosphorylation of the ZAP-70 protein but rather by the total amount of T-cell receptor and co-associated ZAP-70 recruited to the thymocyte:APC interface.

Conclusions: These data provide evidence that a burst of ZAP-70 activity initiates the signaling pathways for negative selection.

Keywords::

• Negative selection
• thymic development
• TCR signaling

Acknowledgements

The authors would like to thank B. Hausmann for FTOC experiments; E. Wagner for animal husbandry; and D. Fabbro, P. Drueckes and J. Trappe, (Novartis Institutes for Biomedical Research, Basel) for profiling the ZAP-70 inhibitor in various kinase assays. The authors thank D. Naeher for comments on the manuscript and T. Hoefer for fruitful discussions.

Declaration of interest

The work is supported by grants from the Swiss National Science Foundation, Sybilla (EU FP7) and National Institutes of Health (AI074074) European Research Council (TerraIncognita). Gerhard Zenke is an employee of Novartis Pharma AG, Basel.