C. Shirakawa1, T. Suzuki1, S. Kimura1, S. Shichi1, H. Kitamura2, A. Taketomi1 1Hokkaido University Graduate School, Gastroenterological Surgery1, Sapporo, Hokkaido, Japan 2Toyo University, Biomedical Engineering Faculty Of Life Sciences, Kawagoe, SAITAMA, Japan
Introduction:
Diacylglycerol kinase (DGK) is a plasma membrane lipid mediator convertase that phosphorylates diacylglycerol (DG) to phosphatidic acid (PA). DGK regulates cellular functions by control of intracellular DG and PA levels. One of the DGK isoforms, DGKζ, is known to negatively regulate T cell responses through downregulation of DG signaling; inhibition of DGKζ may have anti-tumor effects via activation of anti-tumor immune responses.
Methods:
Mouse liver cancer and metastatic liver cancer models were generated using C57BL/6 wild-type and DGKζ gene-deficient (DGKζKO) mice, the fluorescent protein-transfected cell lines Hepa1-6 and MC38. Liver tumor volume was assessed by the amount of fluorescence by in vivo imaging system and Hematoxylin-Eosin staining. Immunocompetent cell dynamics in the liver tumor area were analyzed by flow cytometry (FCM) and immunohistochemical staining (IHC). siRNA (siRNA-DGKζ KD) was used to investigate the effect of DGKζ on the immunomodulatory capacity of C57BL/6 mouse bone marrow-derived dendritic cells (BMDC). Next, to examine the effect of siRNA-DGKζ KD BMDC on T cell function, cytokine concentrations were analyzed by ELISA after co-culture with T cells.
Results:
DGKζKO mice showed inhibition of tumor growth in the liver; FCM increased the percentage of NK1.1+ and CD11c+ positive cells in the tumor area of DGKζKO mice; IHC similarly showed an increase in CD11c positive cells infiltrating the tumor area. Compared to controls, siRNA-DGKζ KD treatment decreased DGKζ gene expression levels in BMDCs and increased IFN-β gene induction levels in BMDCs stimulated with the innate immune adjuvant poly I:C, a Toll Like Receptor 3 stimulus. Antigen-specific IL-2 and IFN-γ levels were increased in DGKζKD BMDCs after co-culture with OVA antigen-specific T cells, CD8-positive T cells and CD4-positive T cells.
Conclusion:
In vitro experiments using liver tumor models and BMDCs suggest that the degradative function of the DGKζ gene activates T cell function via DC activation. These anti-tumor immune activation leads to enhanced anti-tumor effects. Furthermore, it was suggested that DCs are induced around tumors and their activation enhances anti-tumor effector cells. The results of this study suggest that DGKζ may be a new target for liver tumor control.