组蛋白去乙酰化酶抑制剂对人γδT细胞生物学特性的调控作用及机制研究

发布时间：2018-02-27 00:32

本文关键词： γδT细胞组蛋白去乙酰化酶抑制剂免疫调节杀伤 Notch信号通路　出处：《浙江大学》2015年博士论文　论文类型：学位论文

【摘要】：γδT细胞作为T细胞中的一个亚群,其识别抗原的方式与αβT细胞不同,γδT细胞通过(major histocompatibility complex, MHC)限制性方式识别肿瘤相关抗原,发挥免疫调控作用。目前,不少研究报道了γδT细胞及其亚群在感染、自身免疫性疾病以及抗肿瘤免疫监视中发挥重要的作用,因此,这类细胞应用于临床的肿瘤免疫治疗有着光明前景。尽管近年来γδT细胞在临床上治疗难治耐药的恶性肿瘤取得一定进展,但是大部分恶性肿瘤患者对这类细胞无反应,或者缓解后复发。本实验室前期的体外研究也发现急性髓系白血病细胞(acute myeloid leukemia, HDAC的nRNA表达水平无显著变化,HDAC6和HDAC10的mRA表达较未处理的稍降低,有少部分HDAC家族成员,如HDAC9和HDA感。因此,γδT细胞的临床应用仍然阻碍重重。 γδT细胞的活化依赖于其表面受体对抗原的识别,活化后的γδT细胞产生生物学效应,分泌细胞因子或发挥细胞毒效应。但是,对γδT细胞杀伤不敏感的肿瘤细胞大多由于表面抗原无法识别或发挥负调控机制,导致丫δT细胞未能活化。令人兴奋的是,研究者发现了一些药物,如双膦酸类化合物可作为类似抗原的刺激物,促进γδT细胞增殖和活化,增强抗肿瘤活性。因此,采用药物调控γδT细胞免疫反应并促进杀伤功能的方案可能更易于提高恶性肿瘤的免疫治疗效果。由于对γδT细胞活化和抗肿瘤机制的认识非常有限,目前可用于调控γδT细胞生物学特性、激活其功能的药物属于未知的领域。那么,是否有合适的抗肿瘤药物可作为促进γδT细胞功能的理想药物呢?只有探索和寻找合适的药物并深入研究其对γδT细胞的调控机制,才能有效提高γδT细胞在抗肿瘤免疫治疗中的效果。我们实验室前期研究发现去甲基化药物地西他滨可促进调节性γδT细胞(regulatory γδT,γδTreg)细胞增殖,并增强对移植物抗宿主病(graft-versus-host disease, GVHD)的抑制效应。其他研究者发现,组蛋白去乙酰化酶抑制剂可导致T细胞激活,CD8+T细胞以及NK细胞表面激活型受体受组蛋白乙酰化调控。更多的研究表明,T细胞的功能性基因与表观遗传修饰密切相关,而组蛋白去乙酰化酶抑制剂在临床多用于治疗T细胞相关的血液系统肿瘤。鉴于此,我们推测组蛋白去乙酰化酶抑制剂可作为调控γδT细胞功能的理想药物。在第一部分研究中,本课题采用组蛋白去乙酰化酶抑制剂LBH589处理人γδT细胞,研究结果发现低浓度的LBH589(5nM)对γδT细胞的扩增无显著影响,如克隆形成能力、γδT细胞占外周血单个核细胞比例以及γδT细胞绝对数量均未出现明显变化；但较高浓度或高浓度的LBH589(5nM)则显著抑制γδT细胞扩增,γδT细胞克隆形成数量减少,绝对数量明显下降,但在外周血单个核细胞中的比例无显著下降。随LBH589浓度增加,γδT细胞表面活化分子CD69和CD25表达水平均未发生改变,各处理组γδT细胞免疫表型均大部分为CD45RA-/CD27-的效应记忆型细胞。与杀伤功能相关的活化性受体NKG2D (natural killer group2, member D)高表达于γδT细胞,并且不随LBH589浓度增加而改变。我们还检测γδT细胞表达IFN-γ的能力,结果发现LBH589并未对IFN-γ的表达产生影响。为进一步研究LBH589对γδT细胞的细胞毒性效应影响,我们选择对γδT细胞不敏感的AML细胞株作为靶细胞。LBH589预处理γδT细胞后以不同效靶比对AML细胞株进行杀伤,发现在较高或高浓度的LBH589(≥10nM)预处理后,γδT细胞对原先不敏感的AML细胞株(HL-60)杀伤效应增强,具有显著统计学差异。但对另一型的AML细胞株(KG-1)的杀伤效应无增强作用。研究结果提示LBH589对γδT细胞的扩增有显著抑制效应,但是对其表面活化分子以及免疫表型、细胞因子表达无明显影响。与抑制增殖相反的是,LBH589可显著增强γδT细胞的杀伤功能。因此,本研究首次发现组蛋白去乙酰化酶抑制剂促进γδT细胞的细胞毒性效应,并且不会影响其活化状态及细胞因子表达,该结果可为提高血液系统恶性疾病患者采用过继输注或激活自体γδT细胞行免疫治疗的疗效提供实验室数据支持。 γδT细胞表面活化型受体与抗原识别后发出信号,传递至胞内,激活一系列信号传导通路,如丝裂原活化蛋白激酶(mitogen-activated protein kinase, MAPK)通路、磷脂酰肌醇3-激酶(phosphatidylinositol3-kinase, PI3K)通路,此外,Notch信号通路也被发现参与γδT细胞的活化和杀伤作用。但是究竟哪条通路与γδT细胞杀伤AML细胞的功能相关?LBH589增强γδT细胞细胞毒效应与这些信号通路是否有关,或者有另外的信号通路参与这一过程?对此,我们进行深入研究。第二部分研究发现,LBH589并不抑制γδT细胞的组蛋白去乙酰化酶(histone deacetylase, HDAC)的mRNA表达水平,HDAC蛋白表达也无显著影响。随着LBH589处理γδT细胞时间延长或处理浓度增加,ERK、JNK、Akt等蛋白均未发生磷酸化,并且JNK总蛋白表达水平还随着时间增加而降低,但ERK上游蛋白Raf-1随时间延长持续高表达；我们另外发现Notch2蛋白表达显著升高,但其下游转录因子RBP Jκ表达无显著改变。Notch抑制剂可逆转LBH589对γδT细胞杀伤增强的效应,提示Notch信号通路与γδT细胞杀伤功能相关。此外,我们发现γδT细胞凋亡通路的蛋白表达水平未见明显改变,提示LBH589对γδT细胞扩增的抑制不是通过促进其凋亡,可能是通过其他生存、增殖相关信号通路,调控γδT细胞的生存和凋亡过程。因此,本研究表明Notch通路被LBH589激活后,参与调节γδT细胞的杀伤效应。Notch通路可作为γδT细胞发挥抗肿瘤功能作用的触发点,提高γδT细胞的免疫治疗效果。综上所述,我们的研究首次发现γδT细胞杀伤血液系统恶性肿瘤的效应可通过组蛋白去乙酰化酶抑制剂获得提高,并且不会影响免疫调节功能,Notch信号通路参与组蛋白去乙酰化酶抑制剂对γδT细胞的调控作用,研究其具体机制可为优化血液系统恶性疾病的免疫治疗提供新策略。
[Abstract]:Gamma delta T cells as T cells in a subpopulation of different antigen recognition and alpha beta T cells, T cells (major histocompatibility complex MHC, by way of limiting) recognition of tumor associated antigen, immune regulation. At present, many studies have reported the gamma delta T cells and its subsets in infection, autoimmune diseases and anti tumor immune surveillance play an important role, therefore, this kind of cells used in clinical tumor immunotherapy has bright prospects. Although some progress in recent years of gamma delta T cells in the clinical treatment of refractory resistant malignant tumor, but the majority of patients with malignant tumors do not respond to this type of cells or relapse after remission. In our previous study also found that acute myeloid leukemia cells (acute myeloid leukemia, HDAC nRNA expression level did not change significantly, HDAC6 and HDAC10 mRA expression Not treated slightly, with a small number of members of the HDAC family, such as HDAC9 and HDA. Therefore, the clinical application of gamma delta T cells is still obstructing.
The activation of T cells depends on their surface receptors for antigen recognition, activation of gamma delta T cells to produce biological effects, the secretion of cytokines or exert cytotoxic effects. However, the gamma delta T cells is not sensitive to the tumor cell surface antigen mostly due to unrecognized or play a negative regulatory mechanism, leading to ya Delta T cells did not activate. Exciting, researchers found that some drugs, such as bisphosphonates as a stimulant similar antigen, promote the proliferation of T cells and activation, enhanced antitumor activity. Therefore, the drug regulation of gamma delta T cell immune response and promote the cytotoxic function scheme may be more to improve the effect of immunotherapy of malignant tumors. The understanding of gamma delta T cell activation and anti tumor mechanism is very limited, currently available for regulation of gamma delta T cells biological characteristics, drug activation of its function belonging to the unknown Then, is there any suitable antitumor drug as an ideal drug to promote the function of gamma delta T cells? Only by exploring and finding the appropriate drugs and further studying the regulatory mechanism of the gamma delta T cells can we effectively improve the effect of gamma delta T cells in anti-tumor immunotherapy.
Our previous study found that demethylation drug decitabine can promote the regulation of gamma delta T cells (regulatory gamma delta T, gamma delta Treg) cell proliferation, and enhance of graft-versus-host disease (graft-versus-host disease, GVHD). The inhibitory effect of other researchers found that histone deacetylase inhibitors can lead to the activation of T cell, CD8+T cell and NK cell surface receptor activation by histone acetylation. Many studies suggest that functional genes of T cells with epigenetic modifications are closely related, and histone deacetylase inhibitors used in clinical treatment of T cell related hematological malignancies. In view of this we speculate that the ideal drug, histone deacetylase inhibitors can be used as the control function of gamma Delta T cells.
In the first part of the study, this paper uses the histone deacetylase inhibitor LBH589 treated human gamma delta T cells, the results showed that low concentration of LBH589 (5nM) had no significant effect on the amplification of gamma delta T cells, such as clone formation ability of gamma delta T cells accounted for significant changes were not found in peripheral blood nuclear cell proportion and absolute number of gamma delta T cells; but high concentration or high concentration of LBH589 (5nM) inhibited gamma delta T cells, T cells clone formation decreased, the absolute number decreased significantly, but in peripheral blood mononuclear cells in proportion with the concentration of LBH589 decreased significantly. Increased gamma activation molecules CD69 and CD25 surface expression of delta T cells were not changed, each group of gamma delta T cell phenotype were mostly effector memory CD45RA-/CD27- cells. The activation of NKG2D receptor associated with the cytotoxicity of killer (NATURAL group2, memb Er D) is highly expressed in gamma delta T cells, and does not change with increasing LBH589 concentration. We also detect the ability of gamma delta T cells to express IFN- gamma, and it is found that LBH589 has no effect on IFN- gamma expression.
For the cytotoxic effect of LBH589 on the further study of gamma delta T cells, we choose the AML cell line is not sensitive to gamma delta T cells as target cells pretreated with.LBH589 gamma delta T cells after killing by different effector target ratio of AML cells were found in the high or high concentration of LBH589 (10nM) pretreatment after gamma delta T cells on AML cells was not sensitive (HL-60) enhanced the killing effect, the difference was statistically significant. But on another type of AML cell line (KG-1) of the killing effect of enhancing effect. The results suggest that the LBH589 of gamma delta T cells was significantly inhibited, but the the surface of activated molecules and immune phenotype, cytokine expression was not affected. In contrast with the inhibition of proliferation is that LBH589 can significantly enhance the gamma delta T cell killing function. Therefore, this is the first study found that histone deacetylase inhibitors promote cytotoxic gammadelta T cells Sex effect will not affect its activation state and cytokine expression. This result can provide laboratory data support for improving the efficacy of adoptive infusion or activation of autologous gamma delta T cells in patients with hematological malignancies.
The surface of gamma delta T cells activation receptor and antigen recognition after the signal transmitted to the intracellular, activate a series of signal transduction pathways, such as mitogen activated protein kinase (mitogen-activated protein, kinase, MAPK) pathway, phosphatidylinositol 3- kinase (phosphatidylinositol3-kinase, PI3K) pathway, in addition, Notch signal pathway were also found to be involved in gamma delta T cell activation and cytotoxicity. But what pathways and gamma delta T cells killing AML cell function? LBH589 enhanced gamma delta T cells and cytotoxic effect of these signaling pathways is related, or other signaling pathways involved in this process? In this regard, we conduct in-depth research.
The second part of the study found that LBH589 did not inhibit T cells of histone deacetylase (histone deacetylase, HDAC) mRNA expression, HDAC protein expression had no significant impact. With the LBH589 processing of gamma delta T cells prolonged or the increase of the concentration, ERK, JNK, Akt protein were not phosphorylated the expression of JNK, and total protein levels also decreased with time increasing, but upstream of the ERK protein Raf-1 with the prolonging of sustained high expression; we also found that Notch2 protein expression was significantly increased, but the downstream transcription factor RBP expression had no significant change J kappa.Notch inhibitor could reverse LBH589 of gamma delta T cells enhanced effect, suggesting that Notch signal pathway and gamma delta T cell killing function. In addition, we found that gamma delta T cells apoptosis protein expression level did not change significantly, suggesting that inhibition of LBH589 amplification of gamma delta T cells is not through the promotion of Apoptosis may be mediated by other survival, proliferation related signal pathway, survival and apoptosis regulation of gamma delta T cells. Therefore, this study suggests that the Notch pathway is activated by LBH589, is involved in the regulation of gamma delta T cell killing effect of.Notch pathway can be used as gamma delta T cells play the anti-tumor function of the trigger points. To improve the immune therapeutic effect of gamma delta T cells.
In summary, our study is the first to find the effect of gamma delta T cells of hematologic malignancies by histone deacetylase inhibitors have improved, and will not affect the immune function, the Notch signaling pathway is involved in regulation of histone deacetylase inhibitors of gamma delta T cells, to study its specific mechanism may provide a new strategy for the treatment of immune optimization of malignant hematological diseases.

【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R730.51

【共引文献】