DIACYLGLYCEROL KINASE ALPHA PROMOTES UBIQUITINATION AND CELL SURFACE DOWNREGULATION OF CHIMERIC ANTIGEN RECEPTOR

Loss of chimeric antigen receptor (CAR) surface expression emerged as a key determinant in CAR-T cell dysfunction. However, the underlying mechanisms directing CAR trafficking remain largely unknown. Current strategies to enhance the anti-tumour effectiveness of CAR-T cells involve targeting of negative regulators of T cell signalling, such as diacylglycerol kinase α (DGKα), which inhibit diacylglycerol (DAG)-driven TCR signalling by converting DAG to phosphatidic acid (PA). Although it is widely accepted that its kinase activity does play a role in limiting CAR-T cell function, several evidence suggest that it may act more upstream in the TCR signalling. Here, we showed that in DGKα depleted CAR-T cells, the engagement of tumour antigen result in a reduced loss of CARs from the cell surface compared to WT CAR-T cells. Reconstitution with both DGKα WT or kinase dead (KD) mutant reduced the amount of the receptor at the cell surface, suggesting a new kinase-independent function of DGKα in CAR regulation. Once internalized, CAR is saved from antigen-induced lysosomal degradation in DGKα KO CAR-T cells, due to a reduction of the E3 ubiquitin ligase Cbl recruitment to the receptor, and rather recycled back to the plasma membrane. Thus, DGKα is responsible for CAR ubiquitination, degradation and finally for the reduction of the CAR at the cell surface upon antigen stimulation. Together, these data indicate that DGKα couples antigen-induced T cell activation to the triggering of the negative feedback mechanism that lead to lysosomal degradation of the activated CAR.