Effects Of Valproic Acid On The Antitumor Cytotoxicity Of NK Cells And Its Mechanisms

Background:Natural killer(NK) cells are a critical component of the innate immune system and play an essential role in the fight against tumor development and viral infection. Although representing only approximately 10-15% of all lymphocytes in the peripheral blood, NK cells can directly lyse tumor cells without prior sensitization as is commonly required for T lymphocytes. Because of high morbidity and high mortality rate, malignant tumor has become a serious threat to human health, and a major issues of social and economic development in our country. With the development of tumor immunology and more perfect of biotechnology, there is growing interest in the potential of exploring adoptive cellular immunotherapy to treat tumors by restoring immunity in cancer patients. Unfortunately, NK cells from the-peripheral blood of cancer patients have significantly reduced cytotoxic activity, probably due to interference through the membrane KIR receptors that interact with autologous major histocompatibility complex(MHC) antigens. Similarly, tumor infiltrating NK cells freshly isolated from patients are not cytotoxic to autologous tumors. Consequently, infusions of patients’ autologous NK cells have not shown significant clinical benefit to eradicate tumors. Several drugs can augment the function of NK cells, including cytokines and antibody drugs that target PD1, KIR, and tumor antigens. After activation and expansion with cytokines, NK cells may readily lyse tumor cells that express self-MHC molecules. As demonstrated recently, T cells can be engineered to express a chimeric antigen receptor(CAR) that recognizes a spe-cific antigen molecule on tumor cells. This approach has demonstrated potent clinical efficacy as it could induce remissions in patients with advanced leukemia. However, application of the CAR-T cell therapy to other cancers has been challenging, primarily due to life-threatening cytokine-release syndromes, antigen- nonspecific toxicities to normal tissues, and low transduction efficiency of primary T cells by viral vectors.Accumulating evidence has demonstrated the presence of epigenetic mechanisms that control tumor immunity. For example, tumor cells escape immune destruction by epigenetically suppressing immune genes, including MHC class II, CD40, MHC class I, components of the class I peptide presentation pathway(TAP1, TAP2, LMP2, LMP7, ERp57 and Tapasin), B7-1/2, NKG2 D ligands and certain tumor antigens. Similarly, epigenetic approaches involving DNA methylation and histone modifications have been explored as a means to regulate the expression of key immune system-related genes, thus modifying the development of the immune responses. However, it is unclear whether these epigenetic approaches can be employed to boost the antitumor therapy mediated by NK cells. Objectives:In this study, we sought to augment the antitumor activity of NK cells using epigenetic approaches. Four small molecules that have been known to promote epigenetic reprogramming were tested for their ability to enhance the activity of NK cells in order to further improve the anti-tumor ability of NK cells, and enhance the curative effect of NK cell in adoptive immunotherapy treatments. Methods:(1) Cytotoxicity of NK cell treated with different drugs was determined using the calcein release assay at various effector: target(E:T) ratios.(2) We used FACS to measure apoptosis, the expression of CD107 a and NKG2 D in the VPA-treated cells. IFN-γ production was assayed using the Human IFN-γ ELISA Kit according to the manufacturer’s instructions.(3) Western blotting was used to measure the expression of NKG2 D and STAT5 pathway.(4) PD-1 m RNA, PD-L1 m RNA and NKG2 D m RNA were tested for the expression level in VPA-treated and untreated group by RT-PCR.(5) We analyzed the DNA methylation status of NKG2 D initiation by BSP, and tested the histone methylation status of NKG2 D initiation by CHi P in order to explore the mechanism of NKG2 D epigenetic regulation.Results:(1) The thyroid hormone T3 slightly increased the specific lysis of K562, but the difference was not statistically significant. Vit-C and 5-Aza-C did not affect the ability of NK cells lyse tumor cells. However, the histone deacetylase inhibitor VPA significantly reduced NK cell cytotoxicity to K562、Jurkat、Hep G2 at all effector: target(E:T) ratios tested(P<0.05). We also found that removal of the cytokine(IL-2) and anti-CD3 monoclonal antibody in the medium during the exposure to small compounds did not significantly impact the role of small compounds in NK cells. The cytotoxicity of VPA-treated NK cells against K562 cells was reduced in a concentration-dependent manner. We also examined that NK cells would resume their tumor-killing activity after the withdrawal of VPA.(2) After VPA treatment for 24 h, the percentage of PI+/Annexin-V+ cell increased slightly(7.35%, 8.38%, 10.6%, 13.1%) as compared with the PBS control(5.15%). These data suggest that the induction of apoptosis may be involved in a small portion, if not all, of the attenuated cytotoxicity in NK cells.(3) We found impaired degranulation as demonstrated by the reduced CD107 a expression after treatment with VPA. These results indicate that VPA treatment not only induces NK cells apoptosis but also impairs the production of other molecules essential for the lytic activity of cytotoxic NK cells.(4) IFN-γ secretion decreased in NK cells treated with VPA in a dose-response manner, suggesting that histone acetylation significantly impaired cytokine production in association with attenuated cytotoxicity of NK cells. Similarly, treatment of NK cells with VPA also downregulated the activity of the STAT5 interferon pathway.(5) We quantitated the expression of PD-1 and PD-L1 in VPA-treated NK cells. In PBS-treated NK cell controls, both PD-1 and PD-L1 were constitutively expressed at very low levels. After treatment with 2 m M VPA, however, we found that both PD-1 and its ligand PD-L1 were significantly upregulated, suggesting a role of the PD-1 inhibitory pathway in VPA-mediated suppression of NK cell cytotoxicity.(6) There was a significant dose-dependent downregulation of NKG2 D expression on the cell surface, as measured by the percentage of NKG2 D positive cells and the Mean Fluorescence Intensity(MFI). RT-PCR results revealed that the decline of NKG2 D m RNA expression was VPA concentration-dependent. We also observed a similar decline of NKG2 D proteins using Western blot following the treatment of VPA. Collectively, these data suggest that VPA-mediated downregulation of NKG2 D may be an important mechanism underlying the suppression of lytic capacity of NK cells.(7) There was a slight increase in DNA methylation in the NKG2 D promoter in VPA-treated NK cells. These results suggest that DNA methylation contributes to only a small fraction of the down-regulation of the NKG2 D gene. We observed a significant increase in H3K9me2 levels at the NKG2 D locus in VPA-treated NK cells as compared with untreated NK cells, suggesting that the increase of H3K9me2 in the NKG2 D gene promoter may correlate with the decreased expression of NKG2 D receptor on the cell surface of NK cells.Conclusions:(1) The histone deacetylase inhibitor valproic acid(VPA), however, inhibited NK cell lytic activity against leukemic cells in a dose-dependent manner.(2) Pretreatment using VPA reduced IFNγ secretion, impaired CD107 a degranulation, and induced apoptosis by activating the PD-1/PD-L1 pathway.(3) VPA downregulated the expression of the activating receptor NKG2D(natural-killer group 2, member D) by inducing histone K9 hypermethylation and DNA methylation in the gene promoter.