CRISPR/Cas9-mediated knockout of DGK improves anti-tumor activities of human T cells

The efficacy of T cell therapy is inhibited by various tumor-associated immunosuppressive ligands and soluble factors. Such inhibitory signals turn specific T cell signaling pathways on or off, impeding the anti-cancer functions of T cells. Many studies have focused on PD-1 or CTLA-4 blockade to invigorate T cell functions through CD28/B7 signaling, but obtaining robust clinical outcomes remains challenging. In this study, we use CRISPR/Cas9 to potentiate T cell function by increasing CD3 signaling via knockout of diacylglycerol kinase (DGK), an enzyme that metabolizes diacylglycerol to phosphatidic acid (PA). Knockout of DGK augmented the effector functions of CAR-T cells in vitro via increased TCR signaling. DGK knockout from CAR-T cells rendered them resistant to soluble immunosuppressive factors such as TGF-β and prostaglandin E2 and sustained effector functions under conditions of repeated tumor stimulation. Moreover, DGK knockout caused significant regression of U87MGvIII glioblastoma tumors through enhanced effector functions in a xenograft mouse model. Collectively, our study shows that knockout of DGK effectively enhances the effector functions of CAR-T cells, suggesting that CRISPR/Cas9-mediated knockout of DGK could be applicable as part of a multifaceted clinical strategy to treat solid cancers.

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