Histone acetylation is an epigenetic mechanism that regulates the expression of various genes, such as natural killer group 2, member D (NKG2D) ligands. These NKG2D ligands are the key molecules that activate immune cells expressing the NKG2D receptor. It has been observed that cancer cells overexpress histone deacetylases (HDACs) and show reduced acetylation of nuclear histones. Furthermore, HDAC inhibitors are known to upregulate the expression of NKG2D ligands. Humans have 18 known HDAC enzymes that are divided into four classes. At present, it is not clear which types of HDAC are involved in the expression of NKG2D ligands. We hypothesized that specific types of HDAC genes might be responsible for altering the expression of NKG2D ligands. In this study, we monitored the expression of NKG2D ligands and major histocompatibility complex (MHC) class I molecules in lung cancer cells which were treated with six selective HDAC inhibitors and specific small interfering RNAs (siRNAs). We observed that treatment with FK228, which is a selective HDAC1/2 inhibitor, also known as Romidepsin, induced NKG2D ligand expression at the transcriptional and proteomic levels in two different lung cancer cell lines. It also caused an increase in the susceptibility of NCI-H23 cells to NK cells. Silencing HDAC1 or HDAC2 using specific siRNAs increased NKG2D ligand expression. In conclusion, it appears that HDAC1 and HDAC2 might be the key molecules regulating the expression of NKG2D ligands. These results imply that specifically inhibiting HDAC1 and HDAC2 could induce the expression of NKG2D ligands and improve the NK cell-mediated anti-cancer immunity.

Background Transforming growth factor beta (TGF-β) is a typical immuno-inhibitory cytokine and highly secreted by lung cancer cells. It was supposed that its immunosuppressive effects to NK cell might be related with the altered expression of activating and inhibitory molecules in lung cancer cells. In this study, we examined the expression of NKG2DLs, PD-L1 and PD-L2 in lung cancer cells after treatment of TGF-β and a TGF-β inhibitor, Galunisertib (LY2157299). Results TGF-β reduced the level of surface proteins of five NKG2DLs without altered transcription levels in lung cancer cells. Galunisertib reversed the effect of TGF-β on the expression of NKG2DLs. Since MMP inhibitors, MMPi III and MMP2 inhibitor I, restored the reduced expression of NKG2DLs after treatment of TGF-β, it was thought that TGF-β induced the expression of MMP2 which facilitated the shedding of the NKG2DLs in cancer cells. However, the expression of PD-L1, L2 were not changed by treatment with TGF-β or Galunisertib. Conclusions Therefore, inhibition of TGF-β might reverse the immunosuppressive status on immune cells and restore NK cell mediated anticancer immune responses by upregulation of NKG2DLs in cancer cells.

Pancreatic cancer is difficult to diagnose at the initial stage and is often discovered after metastasis to nearby organs. Gemcitabine is currently used as a standard treatment for pancreatic cancer. However, since chemotherapy for pancreatic cancer has not yet reached satisfactory therapeutic results, adjuvant chemotherapy methods are attempted. It can be expected that combining immune cell therapy with existing anticancer drug combination treatment will prevent cancer recurrence and increase survival rates. We isolated natural killer (NK) cells and co-cultured them with strongly activated autologous peripheral blood mononuclear cells (PBMCs) as feeder cells, activated using CD3 antibody, IFN-r, IL-2, and γ-radiation. NK cells expanded in this method showed greater cytotoxicity than resting NK cells, when co-cultured with pancreatic cancer cell lines. Tumor growth was effectively inhibited in a pancreatic cancer mouse xenograft model. Therapeutic efficacy was increased by using gemcitabine and erlotinib in combination. These findings suggest that NK cells cultured by the method proposed here have excellent anti-tumor activity. We demonstrate that activated NK cells can efficiently inhibit pancreatic tumors when used in combination with gemcitabine-based therapy.

Since ionizing radiation has showed the dramatic effect to kill the cancer cells through direct DNA damage as well as triggering anti-cancer immune responses including induction of NKG2D ligands, it has used for long time to treat many cancer patients. However, it has been known that radiotherapy might promote the remnant cancer cells to escape immune system and metastasis. One of the suggested ways of immune evasion is induction of a ligand for programmed death-1 (PD-L1) in head and neck cancer, bladder cancer and lung cancer cells which engages the receptor, programmed death-1 (PD-1) in immune cells. PD-1/PD-L1 axis transduces the inhibitory signal and suppresses the adaptive immunity. However, their role in innate immunity remains poorly understood. Therefore, we investigated whether ionizing radiation could change the expression of PD-L1 in malignant melanoma cells and the receptor, programmed death-1 (PD-1), in NK-92 cells. Surface PD-L1 levels on melanoma cells were increased by ionizing radiation in a dose-independent manner but the level of PD-L1 was not changed significantly in NK-92 cells. Radiation-induced PD-L1 suppressed the activity of the NK-92 cells against melanoma cells despite of upregulation of NKG2D ligands. Furthermore, activated NK cells had high level of PD-1 and could not kill PD-L1+ melanoma cells effectively. When we used PD-L1 inhibitor or silenced PD-L1 gene, inhibited PD-1/PD-L1 axis reversed the activity of the suppressed NK cells. Through these results, we supposed that PD-1/PD-L1 blockade could enhance the immune responses of NK cells against melanoma cells after radiotherapy and might overcome the PD-L1 mediated radioresistance of cancer cells.

Natural killer (NK) cells play a crucial role in early immune defenses against transformed cells and are used in the therapeutic management of cancer. However, it is difficult to sufficiently obtain high purity activated NK cells for clinical application. The function of NK cells is dependent on the balance of activating and inhibitory signals. Strong and diverse stimuli are required to increase the function of NK cells. Radiotherapy modulates the expression of various immunomodulatory molecules that recruit and activate NK cells. NK cell-mediated antibody-dependent cellular cytotoxicity is one of the most potent cytotoxic effects of NK cells against target cancer cells. To generate activated and irradiated autologous peripheral blood mononuclear cells (PBMCs), cytokine and monoclonal antibody stimulation followed by ionizing radiation was performed in the present study. The expanded NK cells were cultured for 21 days using activated/irradiated autologous PBMCs. Colorectal cancer cells (SW480 and HT-29) were used to analyze the expression of NK group 2D ligands and EGFR by radiation. The cytotoxicity of radiation plus NK cell-based targeted therapy against colorectal cancer cell lines was analyzed using flow cytometry. Activated and irradiated PBMCs exhibited significantly increased expression of various activating ligands that stimulated NK cells. In total, >10,000-fold high-purity activated NK cells were obtained, with negligible T-cell contamination. To confirm the antitumor activity of the NK cells expanded by this method, the expanded NK cells were treated with cetuximab, radiotherapy, or a combination of cetuximab and radiotherapy in the presence of human colorectal cancer cells. Expanded NK cells were effective at targeting human colorectal cancer cells, particularly when combined with cetuximab and radiotherapy. Thus, in the present study, a novel method for high-purity activated NK cell expansion was developed using activated and irradiated PBMCs. In addition, combined radiotherapy and antibody-based immunotherapy with expanded NK cells may be an effective strategy to enhance the efficiency of treatment against colorectal cancer.