Pharmacological inhibition of both DGKα and DGKζ is required for optimal T cell activation

Preclinical studies indicate that blocking diacylglycerol kinase (DGK) family members DGKα or DGKζ can improve antitumor immunity, prompting the development of clinical-stage, potent and selective small molecule inhibitors of DGKα and/or DGKζ. DGKα and DGKζ are the most widely expressed DGK family members by immune cells, and both enzymes convert the signaling lipid diacylglycerol (DAG) to phosphatidic acid. DAG is a critical second messenger downstream of T cell receptor (TCR) stimulation that promotes activation and effector function. Blocking DGKα or DGKζ activity enhances DAG-mediated signaling, potentiating immune cell activity. Because DGKα and DGKζ functionally overlap, we sought to compare the effects of pharmacological inhibition strategies targeting DGKα or DGKζ individually or simultaneously (DGKα/ζ) to determine which approach maximized immune cell activation. Evaluation of TCR downstream signaling using primary human and mouse cells revealed that dual DGKα/ζ inhibition promoted the greatest increase in cellular activity, including antigen-dependent cytokine production and tumor cell killing. In contrast, pharmacological inhibition of DGKζ alone had modest effects, and inhibition of DGKα alone had minimal bearing on TCR-mediated activity. Notably, loss of DGKα and DGKζ protein was observed following inhibitor treatment and may point to an additional mechanism of action for DGK targeting small molecule inhibitors. Finally, analysis of biopsies from patients with nonsmall cell lung cancer showed that tumor infiltrating lymphocytes expressed both DGKα and DGKζ and exhibited increased activation and cytokine production ex vivo upon DGKα/ζ coinhibition in conjunction with TCR stimulation, indicating that tumor infiltrating lymphocytes are sensitive to DGKα/ζ coinhibition. SIGNIFICANCE STATEMENT: This work directly compares pharmacological inhibition of DGKα and DGKζ, affirming that DGKα/ζ coinhibition is required to maximally increase TCR responses. Importantly, DGKα/ζ inhibition increased activation and cytokine production in both healthy T cells and tumor infiltrating lymphocytes.