Identifying ZAP-70 kinase-dependent and -independent functions in T cells using a chemical genetic approach (35.5)

Abstract The tyrosine kinase ZAP-70 activates signals proximal to the TCR and is critical for the activation of downstream signal pathways. To identify which T cell functions require ZAP-70 kinase activity, our lab has generated a novel mouse that expresses an analog-sensitive ZAP-70 mutant that can be inhibited specifically by an analog of the Src family kinase inhibitor PP1. This titratable, chemical genetic approach gives us the ability to rapidly and specifically inhibit ZAP-70 activity in primary mouse T cells. We asked whether naïve and effector responses upon TCR stimulation required ZAP-70 activity. TCR induced calcium mobilization, upregulation of CD69, and proliferation were highly dependent on ZAP-70 activity. Similarly, effector cytokine production by primed CD4+ and CD8+ cells was ZAP-70 kinase-dependent. Inhibition of ZAP-70 activity also severely impaired CD8+ cytolytic activity. In contrast to conventional CD4+ and CD8+ T cells, we observed that CD4+ CD25+ Treg suppress CD4+ CD25- T cell proliferation, even in the presence of the ZAP-70 inhibitor. Thus, while conventional T cell activation and function are ZAP-70 kinase dependent, CD4+ CD25+ Treg suppressive function appears to be ZAP-70 kinase-independent. Our results imply that an inhibitor of the WT form of ZAP-70 could be effective for the attenuation of T cell-mediated pathology.