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

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.