CRISPR/Cas9-Mediated Knockout of DGK Improves Antitumor Activities of Human T Cells

Cancer Res. 2018 Aug 15;78(16):4692-4703. doi: 10.1158/0008-5472.CAN-18-0030. Epub 2018 Jul 2.


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 anticancer 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. 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 TGFβ 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.Significance: This novel study demonstrates efficient ablation of diacylglycerol kinase in human CAR-T cells that leads to improved antitumor immunity and may have significant impact in human cancer immunotherapy. Cancer Res; 78(16); 4692-703. ©2018 AACR.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • CD28 Antigens / genetics
  • CD28 Antigens / immunology
  • CRISPR-Cas Systems / genetics
  • CRISPR-Cas Systems / immunology
  • CTLA-4 Antigen / antagonists & inhibitors
  • CTLA-4 Antigen / immunology
  • Cell Line, Tumor
  • Diacylglycerol Kinase / genetics*
  • Diacylglycerol Kinase / immunology
  • Gene Knockout Techniques
  • Humans
  • Immunotherapy, Adoptive*
  • Ligands
  • Lymphocyte Activation / immunology*
  • Mice
  • Programmed Cell Death 1 Receptor / antagonists & inhibitors
  • Programmed Cell Death 1 Receptor / immunology
  • Receptors, Antigen, T-Cell / genetics
  • Receptors, Antigen, T-Cell / immunology
  • Signal Transduction
  • T-Lymphocytes / immunology*
  • Transforming Growth Factor beta / genetics
  • Transforming Growth Factor beta / immunology
  • Xenograft Model Antitumor Assays


  • CD28 Antigens
  • CTLA-4 Antigen
  • Ligands
  • PDCD1 protein, human
  • Programmed Cell Death 1 Receptor
  • Receptors, Antigen, T-Cell
  • Transforming Growth Factor beta
  • Diacylglycerol Kinase