| As a subtype of T cell group, yST cells recognize the tumor-associated antigens independent of major histocompatibility complex (MHC) molecules. Recently, many studies have shown that y8T cells and their subgroups play an important role in infection, autoimmune diseases and tumor surveillance. Therefore, y8T cells have become ideal candidate for tumor immunotherapy. Although some patients with refractory malignancies have achieved comparable good outcomes by inducing or adoptive infusion of γδT cells in vivo, most of patients are nonresponders or relapse shortly after remission. Similarly, our lab has reported that very few type of acute myeloid leukemia (AML), e.g. kasumi-1cells (AML-M2) can be efficiently killed by γδT cells. However, there are still many subtypes of AML insusceptible to y8T cells. It is a challenge for the clinical application of y8T cells.Recognition of antigens by activating receptors on y8T cells can stimulate the activity of these cells, then the activated γδT cells exhibit their biological functions (e.g. cytokines release, cytotoxicity effect). In fact, the tumor cells which can not be recognized by γδT cells is not sufficient for γδT cells activation. And the evasion of tumor cells attributes to the inhibition of y8T cells proliferation and functions. Fortunately, researchers have found some drugs, such as nitrogen-containing bisphosphonates (NBP) can induce expansion of human V82T cells, stimulate their proliferation and enhance the anti-tumor effects. Consequently, taking advantage of drugs to regulate the immune responses and enhance the cytotoxicity of γδT cells might benefit for improving the efficacy of immunotherapy on malignancies. However, the limited knowledge of the mechanisms in γδT cells activation and cytotoxicity greatly impedes the exploration of drugs which can regulate the functions of these cells. Whether there is proper anti-tumor drugs for enhancing the functions of y8T cells? Only when the mechanisms of these drugs in regulation of γδT cells have been unraveled, the effects of application of γδT cells for anti-tumor immunotherapy can be improved.Our recent study revealed that hypomethylating agent decitabine increased the y8Treg cells proliferation while enhanced their inhibitory effects on regulating graft-versus-host disease (GVHD). Other studies also found that histone deacetylase inhibitor (HDACi) can facilitate the T cell activation, and histone acetylation controlled the transcription of the activating receptor natural killer group2, member D (NKG2D) gene in human NK and CD8+T cells. More and more researches showed that the epigenetic regulation closely associated with the functional genes in T cells. Additionally, HDACi is used for T cell-related hematological malignancies treatment. These data together indicate that HDACi may be applicable for regulating the functions of γδT cells.In the first part of our study, we used the HDACi (LBH589) to treat human γδT cells. The results show that low concentration of LBH589(<5nM) have no effects on the proliferation of γδT cells, such as clone formation, percentage of y8T cells in peripheral blood mononuclear cells (PBMCs) and the absolute number of y8T cells. However, higher or high concentration of LBH589(>5nM) significantly inhibit the expansion of γδT cells, especially the clone formation and the absolute number of γδT cells, except for the percentages of γδT cells in PBMCs. With the increase of LBH589concentration, there have no changes of CD69and CD25expression on γδT cells. Similarly, most of the phenotypes of γδT cells in each groups are effector memory cells (CD45RA-/CD27-). The NKG2D highly express on γδT cells regardless of the concentration of LBH589. We also examine the IFN-y expression on γδT cells, and we do not detect any changes in the percentage of IFN-y positive γδT cells among experimental groups.To further assess the influences of LBH589on cytotoxicity effects of γδT cells, we choose those AML cell lines as the target cells which can not be efficiently killed by γδT cells. After treated by LBH589, γδT cells were cocultured with AML cells in different effector:target ratios. Then we adopt the flow cytometry to test the lysis of AML cells. The results demonstrate that higher concentration of LBH589(>10nM) significantly enhance the cytolytic effects of γδT cells for killing HL-60cells. Unfortunately, another AML cell lines, KG-1cells still can not be efficiently killed by LBH589-treated y8T cells. These results suggest that the HDACi can restrain the proliferation of y8T cells without influencing their immunomodulation functions. Unlike the negative effects on expansion, it is observed that HDACi significantly enhance the cytotoxicity of γδT cells. The above data provides us with a better understanding of the roles of HDACi on γδT cells biological characteristics. Our work has provided supporting evidences for improvement of γδT cells anti-tumor effects.The activating receptors expressed on the surface of γδT cells recognize tumor-related antigen and generate signals, leading to activation of some intracellular signal transduction pathways, e.g. MAPK pathway and PI3K signaling pathway. In addition, a study demonstrate that the Notch pathway is involved in activation and cytotoxic effects of γδT cells. However, it is still not clear by which pathway involved in the cytotoxicity effects of γδT cells against AML cells. And it is not well understood whether the above mentioned pathways involved in the mechanisms of HDACi regulation on γδT cells, or whether there is the other pathways? We next sought to determine which signaling pathway participating in the effects of HDACi on γδT cells in the second part of this study. RT-PCR was used to test the changes of HDACs mRNA relative levels in γδT cells after treated with LBH589. The results showed that it has no change in HDACs mRNA levels. Western blot confirm that the LBH589has no effect on HDACs protein expression. Western blot analysis showed that with the exposure time of LBH589prolonged or concentrations increased, ERK or JNK were not be activated, and the total protein level of JNK decreased. Raf-1protein, the upstream of ERK, expressed in a high level with the time extending. Interestingly, we observed that the Notch2protein significantly raised. But RBP Jκ expression, the transcription factor downstream of Notch, did not change with time or concentration increased. And the Notch inhibitor can reverse the effects of LBH589on enhancing the cytolytic activity of γδT cells. Combined with the data of cytotoxicity test, it provides the evidence that the Notch signaling pathway may involve in regulation of y8T cells function. Apoptosis-related protein, e.g. Bax, Bcl-2and PARP have not been observed any change in protein expression level. Thus it indicates that the inhibition of proliferation of y8T cells by LBH589may not denpend on induction of cell apoptosis, but on the other signaling pathways which related to proliferation or survival. In this study, we have identified that Notch signaling pathway activated by LBH589can improve the cytolytic function of γδT cells. Hence, it is reasonable that the Notch signaling pathway may take as a trigger point in the application of y8T cells in anti-tumor immunotherapy.Overall, this is the first time to describe that the anti-tumor effects of y8T cells on hematological malignancies may be improved by HDACi without affecting their immuno-regulation functions. Notch signal pathway involves in the modulation of HDACi for promoting the cytotoxic effects of y8T cells. The mechanisms underlying the effects of drugs on y8T cells should be further studied, thus providing new therapeutic opportunities for treating hematological diseases. |
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