Introduction

Wiskott--Aldrich Syndrome patients feature defects in T cell activation and proliferation, but also in megakaryopoiesis and platelets. We discovered that diacylglycerol kinase alpha (DGKα) constrains T-cell activation, and its activity is limited by a pathway comprising SAP (an adaptor mutated in Duncan’s disease) and the WAS protein (WASp). We propose that, in the context of SAP or WASp deficiency, there is excessive DGKα activity contributing to diseases phenotypes.

Methods

In T cells, we triggered immune synapse (IS) formation among Jurkat triple reporter and superantigen-loaded Raji cells. Jurkat triple reporter cells allow for the quantification of NFAT, NFκB, and AP-1 activity induction using flow cytometry.

Similarly, we silenced WAS in the erythroleukemia cell line (HEL) and induced differentiation through PMA treatment measuring cell elongation, spread, and CD41 induction.

Results

IS analysis confirmed that SAP- and WASp-deficient cells have a reduced capacity to form IS and, surprisingly, DGKα inhibition further reduces it, underling an important role of DGKα in shaping IS. SAP- and WASp-deficient cells also show a defect in NFAT and AP-1 signaling pathways, whereas NFκB activity is unaffected. DGKα inhibition rescues both NFAT and AP-1 defects but does not interfere with NFκB.

In megakaryocytes, DGKα dampers thrombopoiesis and platelet aggregation. DGKα inhibition rescued the elongation defect of WASp-deficient HEL cells, but was not able to restore CD41 induction.

Conclusions

Thus, in the absence of SAP or WASp, excessive DGKα activity consumes diacylglycerol, perturbing signaling. Indeed, DGKα inhibition is required for NFAT and AP-1 activation, and this can be compensated by DGK inhibitors.

Similarly, excessive megakaryocyte DGKα activity in WAS reduces cytoskeletal remodelling, and this is also restored by DGK inhibitors.

All of these observations suggest that inhibiting DGKα activity could represents a novel therapeutic approach for WAS.