Investigation of diacylglycerol kinase alpha role in lipid signalling during T cell activation

The diacylglycerol kinases (DGKs) are a conserved family of lipid enzymes able to convert diacylglycerol (DAG) into phosphatidic acid (PA). This reaction attenuates DAG intracellular levels, an important second messenger which regulates several signalling proteins. At the same time, also the product of DGK activity, PA, controls different cell functions such as cell growth, differentiation, and migration. Thus, DGK enzymes play a pivotal role within the cells, linking lipid metabolism to signalling functions. In T cells, DGKα and DGKζ isoforms play a major role as negative regulators of DAG-mediated T cell receptor (TCR) signalling, since their activity limits RasGRP1/Ras/MAPK pathway and interleukin 2 (IL-2) production. The aim of the research efforts described in this thesis is to explore the regulation of DGKα-mediated lipid signalling and the resulting biological effects during T cell activation. The project started from the observation that, upon T-cell receptor stimulation, the adaptor protein SAP mediates DGKα inhibition and this process is essential for a correct restimulation induced cell death (RICD) and cytokines production. However, the molecular pathway that links SAP to DGKα was poorly understood. To clarify the pathway and the mechanisms involved we explored the molecular mechanisms which lead to DGKα inhibition, focusing on the reported SAP interactors and specifically on the Wiskott-Aldrich syndrome protein (WASp). The results obtained demonstrate that WASp interacts with the DGKα recoverin homology domain through its homology 1 domain and this interaction leads to DGKα inhibition. In addition, the role of the adaptor protein NCK-1 and the small G protein CDC42 in this signalling was investigated, indicating an indirect WASp mediated role. A second research topic was the investigation of the DGKα promoted signalling, focusing on lipid binding and regulation by PA and other phospholipids on atypical PKCs, an important class of lipid regulated kinases. Finally, the biological relevance of WASp/DGKα was explored in the context of RICD and cytokine production in primary human T cells. While this pathway does not affect RICD, WASp silencing reduces IL-2 synthesis and DGKα inhibition restores this defect of WASp-deficient lymphocytes. In conclusion, we discovered a novel signalling pathway composed of SAP/CDC42 and NCK-1/WASp that, downstream to strong TCR stimulation, blocks DGKα activity resulting in a full cytokine response but minimally perturbs RICD. These findings could be relevant for the treatment not only of primary immunodeficiencies but also of other T cell hyporesponsive states such as anergy of tumour infiltrating lymphocytes.