【作者】 周志侠；

【导师】 张彩；

【作者基本信息】 山东大学 ， 药理学， 2012， 博士

【摘要】 研究目的恶性肿瘤是一种严重危害人类健康的常见病和多发病,其死亡人数约占世界总死亡人数的四分之一,且呈现逐年递增的趋势。虽然目前用于恶性肿瘤治疗的手段有很多,包括手术、放疗、化疗、内分泌治疗、中医中药治疗、热疗和射频消融治疗等,但他们大多都缺乏特异性和靶向性,不仅杀死肿瘤细胞还破坏正常细胞的功能,导致患者免疫功能严重低下,给患者带来极大的毒副作用。近年来,以机体免疫细胞,如树突状细胞(dendritic cells, DC)、巨噬细胞、自然杀伤(natural killer, NK)细胞和细胞毒性T淋巴细胞(cytotoxic T lymphocytes, CTL)等为主体,以现代免疫学、细胞生物学和分子生物学等前沿科学为基础的细胞免疫治疗,成为21世纪最新的抗癌生物科学技术。通常情况下,细胞免疫治疗以重组、合成或天然制剂(细胞因子,趋化因子,寡核苷酸,Imiquimod和CPG等)作为细胞活化的免疫调节剂,试图激发或重塑机体免疫系统来排斥或摧毁肿瘤细胞,最终实现消灭肿瘤和治愈癌症的目的。NK细胞是先天免疫系统中不可或缺的一部分,也是连接天然免疫和获得性免疫之间的桥梁。不仅具有强大的抗细菌、病毒和肿瘤的功能,还在移植排斥、超敏反应和自身免疫性疾病中发挥着重要的调节作用。一方面,NK细胞的杀伤作用取决于其细胞表面活化受体和抑制性受体对靶细胞上相应配体的结合,其中NK细胞介导的靶向感染自体细胞的杀伤还可以触发强大的抗原特异性T细胞免疫反应和体液免疫反应。NK细胞还可以通过自身来源的细胞因子Ⅱ型干扰素(interferon gamma, IFN-y)和肿瘤坏死因子(tumor necrosis factor, TNF),进一步促使初始T细胞向着Thl细胞类型的分化。而且这两种细胞因子还能诱导DC的成熟,促进DC的抗原提成能力,间接调控T细胞介导的获得性免疫应答。此外,NK细胞还能提升另一种抗原提呈细胞(antigen-presenting cell, APC)-巨噬细胞的抗菌效应,促使诱导型一氧化氮合酶(inducible nitric oxide synthase, iNOS)的产生；NK细胞还能通过分泌白细胞介素-22(interleukin-22, IL-22),诱导巨噬细胞吞噬溶酶体的融合而起到抑制胞内结核分枝杆菌生长的效应。另一方面,NK细胞的杀伤和增殖还受到细胞因子,诸如IL-2, IL-15, IL-12, IL-18和IL-21等的调控。而这些细胞因子通常是由其它免疫细胞,尤其是活化的APC,所分泌的。因此,相比于其他免疫细胞,NK细胞与APC之间的相互调控关系(cross-talk),在先天免疫和适应性免疫反应的发展中起着重要的影响,近年来引起了人们极大的兴趣和关注。Toll样受体(Toll-like receptors, TLRs)也是天然免疫系统中重要成员,能够泛特异性地识别病原体表面的病原相关分子模式(pathogen associated molecular patterns, PAMPs)。TLRs家族广泛表达于多种细胞,包括上皮细胞、内皮细胞、间皮细胞、成纤维细胞、中性粒细胞、T淋巴细胞、NK细胞,巨噬细胞和DC。其中,属于TLR9亚家族的、表达于细胞内体的TLR3、TLR7、TLR8和TLR9,不仅能够直接活化多种免疫细胞,尤其是和APC和NK细胞,还能诱导肿瘤细胞的凋亡和杀伤敏感性的增强。因此,TLR9亚家族是APC和NK细胞之间cross-talk启动的桥梁和纽带,而TLR9亚家族的激动剂成为以两种细胞单独或联合应用为基础的细胞免疫治疗的最佳免疫调节剂。本研究是以TLR3的激动剂PolⅠ:C, TLR7和(或)TLR8激动剂Imiquimod (R837)、Gardiquimod (GDQ)以及ssRNA40为研究对象,观察它们刺激的APC对NK细胞抗肿瘤作用的影响,进而从细胞因子和细胞与细胞间接触分子两方面着手,深入探讨APC与NK细胞之间的cross-talk。研究方法(1)免疫磁珠分选(MACS)小鼠脾脏NK细胞；(2)淋巴细胞分离液密度梯度离心法分离健康人外周血PBMCs;(3)重组细胞因子体外诱导人PBMCs来源DC；(4)流式细胞术技术检测NK分选效率和巨噬细胞提纯效率,以及小鼠NK细胞、人NKL细胞和PBMCs表面活化分子、胞内杀伤相关分子或细胞因子的表达水平；(5)实时定量PCR方法检测小鼠NK细胞、巨噬细胞、人NKL细胞和PBMCs中活化分子、活化性(或抑制性)受体及细胞因子等的mRNA水平；(6)51Cr释放法和MTT法检测小鼠NK细胞、人NKL细胞或PBMCs对肿瘤细胞的杀伤效应；(7)ELISA方法检测细胞培养上清中细胞因子的分泌水平；(8)RT-PCR方法检测NKL细胞和PBMCs中TLR7和TLR8的基因表达水平；(9)RNA干扰技术阻断小鼠巨噬细胞内TLR3和Qa-1的表达；(10)抗体中和实验阻断小鼠NK细胞中NKG2D的表达或抑制细胞上清中IL-15或IFN的分泌；(11)Western Blotting技术检测NKL细胞、DC和PBMCs中NF-κB信号通路的活化情况；(12)人HepG2裸鼠肿瘤异种移植模型的建立和治疗；(13)Transwell实验阻断小鼠NK细胞与巨噬细胞的接触,或NKL细胞与DC的接触。结果结果一：PolyⅠ:C诱导的巨噬细胞增强小鼠NK细胞的抗肿瘤活性1、PolyⅠ:C诱导的巨噬细胞能够增强NK细胞地对肿瘤细胞的杀伤作用,而非活化它的巨噬细胞PolyI:C处理的巨噬细胞能够明显活化NK细胞,促进NK细胞对肿瘤细胞YAC-1, Hepa1-6, MCA-38和H22的杀伤效应,而对PolyI:C处理的小鼠巨噬细胞,尤其对原代腹腔巨噬细胞不具备杀伤增强效应。2、NK细胞介导的肿瘤杀伤具有NKG2D依赖性PolyI:C诱导的巨噬细胞能够通过上调NK细胞表面NKG2D的表达,而增强NK细胞的抗肿瘤活性。3、PolyⅠ:C能够诱导巨噬细胞表面NKG2D配体的表达PolyⅠ:C能够显著上调巨噬细胞表面NKG2D配体RAE-1, H60和MULT-1的表达水平,并且存在着一定的剂量依赖性。4、PolyⅠ:C对NKG2D配体的上调作用存在TLR3依赖性si-TLR3作用于巨噬细胞后,PolyI:C对其表面NKG2D配体的诱导表达能力明显下降。5、Qa-1保护巨噬细胞免受NK细胞介导的杀伤相比于肿瘤细胞YAC-1,巨噬细胞表面抑制性配体的表达水平明显优于活化性配体,即Qa-1和NKG2A的相互作用能够保护巨噬细胞免受NK细胞的攻击。6、巨噬细胞对NK细胞的活化作用具有IL-15和IFN-β依赖性PolyⅠ:C上调巨噬细胞分泌细胞因子的水平,尤其是IL-15和IFN-p.当IL-15和IFN-p的分泌被中和后,NK细胞的活化、细胞因子的分泌和杀伤功能均显著下降。7、细胞与细胞间分子接触在巨噬细胞对NK细胞的活化过程中起重要作用Transwell培养体系中,NK细胞表面CD69和NKG2D以及胞内IFN-y的表达水平均有所下降。结果二：TLR7/TLR8激动剂介导的DC/NK cross-talk对人肝癌的免疫治疗1、TLR7/TLR8激动剂能够直接活化人NK细胞TLR7激动剂R837, TLR7/8激动剂GDQ和TLR8激动剂ssRNA40能够直接活化人NK细胞系NKL和新鲜分离的人PBMCs中原代NK细胞,尤其是GDQ。2、TLR7/TLR8激动剂增强NK细胞IFN-γ和TNF-α的分泌能力R837, GDQ和ssRNA40能够上调NKL分泌炎性细胞因子IFN-γ和TNF-α。3、TLR7/TLR8激动剂诱导NK细胞表面NKG2D和NKp80的表达水平GDQ可以促进NK细胞表面NKG2D的表达水平；三种激动剂均能够诱导NKp80的表达上调。4、TLR7/TLR8激动剂协同树突状细胞促进NK细胞的活化DC协同激动剂能够更加显著地上调NKL细胞表面CD69和CD25的表达水平、增强NKL细胞对HepG2细胞的杀伤功能、上调胞内杀伤相关基因的表达水平,以及IFN-γ的胞内表达和分泌水,尤其是DC协同GDQ刺激组。5、GDQ协同DC增强NK细胞的体内抗肿瘤作用以腹腔荷瘤方式建立了HepG2裸鼠异种移植肿瘤模型,用不同刺激的NKL细胞进行治疗。结果表明,TLR7/TLR8激动剂活化的NKL细胞可以明显抑制HepG2肿瘤的体内生长,尤其是GDQ、DC和NKL联用组。6、TLR7/TLR8激动剂协同NK细胞促进DC的成熟和细胞因子的分泌三种激动剂协同NK能够明显促进DC表面成熟分子CD1a、CD11c、CD83和CD86的表达水平,诱导其Th1类细胞因子IFN-α、IFN-β、IL-12p35、IL-15和IL-18,以及炎性因子IL-6的分泌,而对IL-1β和IL-10没有影响。7、DC介导的NK细胞的活化依赖于Ⅰ型干扰素和IL-12的分泌混合培养细胞中IFNR的阻断可以明显削弱NK细胞表面表面CD69和CD25的表达水平、胞内杀伤相关基因的表达水平,以及细胞上清中IFN-γ的分泌水平。与此同时,相比于激动剂或IL-12单独刺激组,激动剂和IL-12的协同刺激能够更加显著地促进NK细胞表面CD69和胞内IFN-γ的表达水平。8、DC与NK细胞间的分子接触参与了DC介导的NK细胞的活化在Transwell条件下,NK细胞表面CD69的表达水平有所下降,同时,细胞上清中IFN-γ的分泌水平也明显降低。NK细胞与DC之间的活化性受体NKG2D和NKp80及其配体、协同刺激分子CD40-CD40L和LFA-1-ICAM-1、以及趋化因子CCL2和CCL4及其受体可能参与了两种细胞之间的相互调控。结论及意义本研究证实了TLR激动剂介导的APC与NK细胞间的cross-talk能够增强NK细胞的抗肿瘤效应。一方面,TLR3激动剂PolyⅠ:C刺激的巨噬细胞可以增强小鼠NK细胞的抗肿瘤作用,其机制是：PolyⅠ:C可以诱导小鼠巨噬细胞NKG2D配体的表达,以及细胞因子IL-15、IL-12和IFN-β等的分泌,从而促进NK细胞的活化和IFN-γ的分泌；而巨噬细胞自身却因为高表达抑制性受体NKG2A的配体Qa-1而免受活化NK的攻击。另一方面,TLR7和(或)TLR8激动剂介导的DC与NK细胞间的cross-talk可以增强人类NK细胞的抗肿瘤作用,其机制是：尽管TLR7激动剂R837、TLR8激动剂ssRNA40和TLR7/8激动剂GDQ均可以直接诱导人NK细胞的活化,而DC的参与能够更加显著地诱导NK细胞抗肿瘤效应的增强,这在HepG2人肿瘤裸鼠异种移植模型的体内试验中也得到了进一步证实；而DC对NK细胞功能的增强主要依赖于其所分泌的大量Thl型细胞因子,尤其是IFN-α/β和IL-12。此外,活化性受体NKG2D和NKp80及其配体,协同刺激分子CD40-CD40L和LFA-1-ICAM-1,以及趋化因子CCL2和CCL4及其受体等细胞与细胞间接触分子,有可能参与了NK/DC之间的cross-talk。本研究明确了APC对NK细胞的活化作用以及此过程中所需要的细胞因子和细胞与细胞间接触,这为深入探讨APC/NK cross-talk的分子机制提供了新理论基础和思路。本研究提示PolyⅠ:C、R837、GDQ和ssRNA40均可以作为抗肿瘤的有效药物,或许可以作为疫苗或免疫佐剂与其他抗肿瘤药物联合应用；而TLR激动剂、APC和NK细胞三者的联合应用有可能成为一种细胞治疗方式,应用于抗肿瘤、抗病毒和自身免疫性疾病的临床治疗和研究。更多还原

【Abstract】 ObjectMalignant tumor, as a frequently occurring and common disease, has a great threat to human health; the death of it accounts for a quarter of total deaths in the world, and shows an increasing trend year by year. There are many treatment methods for malignant tumor, including surgery, radiotherapy, chemotherapy, endocrine therapy, Chinese medicine treatment, hyperthermia and radiofrequency ablation therapy, but most of them are lack of specificity and targeting, as they not only can kill tumor cells but also destroy normal cells, leading to a serious decline in immune function of patients. In recent years, immunotherapy based the cutting-edge science of modern immunology, cell biology and molecular biology, has become the latest anti-cancer biological science and technology in the21st century. Regarding immune effector cells as main body’s, such as lymphocytes, macrophages, dendritic cells (DC), natural killer (NK) cells, cytotoxic T lymphocytes (CTL), or the combination of them, immunotherapy is attempting to stimulate the immune system to reject and destroy tumors. In some cases, the immune effector cells need active agents referred to as immunomodulators, which are a diverse array of recombinant, synthetic and natural preparations, such as cytokines, chemokines, oligodeoxynucleotides, Imiquimod, or CpG.NK cells are integral components of the innate immune system and are characterized by their strong cytolytic activity against tumors and virus-infected cells. NK cells also regulate innate and adaptive immune responses through secretion of immunoregulatory cytokines and cell-to-cell contact. Activated NK cells promote DC mature and cytokines production through production of gamma interferon (IFN-y) and tumor necrosis factor-α (TNF-α), leading to the induction and regulation of immune responses. In addition to DC, NK cells can strongly prompt the generation of inducible nitric oxide synthase (iNOS) from macrophages, resulting in the enhancement the antimicrobial effect of macrophages that also belong to antigen-presenting cells (APC). Moreover, NK cells induce the fusion of lysosomal in macrophages by secreting interleukin-22(IL-22), to inhibit the growth of intracellular Mycobacterium tuberculosis. Usually, the actions of NK cells are thought to be mediated by the complex interactions between inhibitory and activating signals sent by cell surface receptors following ligation. In many cases, cytokines, such as IL-2, IL-15, IL-12, IL-18and IL-21, produced by other immune cells, and particularly activated APC, can also play important roles in the regulation of NK cell activity. Therefore, a great deal of interest and information has emerged with respect to the crosstalk between APC and NK cell, in contrast to the interactions between NK cells and other innate immune cells.Toll-like receptors (TLRs) are receptors of the innate immune system that directly recognize conserved pathogen structures, and they also conserved receptors that recognize a variety of pathogen-break down products. TLRs are widely expressed on many cells, including epithelial cells, endothelial cells, mesothelial cells, fibroblasts, neutrophils, T lymphocytes, monocytes, NK cells, macrophages, and DC. Among the family of TLRs, TLR3, TLR7, TLR8and TLR9which belong to the TLR9subfamily that expressed in endosomal compartments, not only can directly activate a variety of immune cells, particularly APC and NK cells, but also induce tumor cell apoptosis and enhance their sensitive to effector cells. Therefore, TLR9subfamily can serve as the functional bridges and links of the cross-talk between APC and NK cells, and the agonists of TLR9subfamily are the best immunomodulators for the cells-based immunotherapy.In this study, TLR3agonist Poly Ⅰ:C and TLR7/TLR8agonists, Imiquimod, Gardiquimod, or ssRNA40were used to observe the APC-increased NK cells cytotoxicity against tumor cells. We also explored the cross-talk between APC and NK cells based on cytokine stimulation and cell-cell contact.Methods(1) Magnetic bead sorting (MACS) was used to isolate NK cells from spleen in mice;(2) PBMCs were isolated with Ficoll by density gradient centrifugation from human peripheral blood of healthy donor;(3) Dendritic cells were induced from human PBMCs with recombinant cytokines in vitro;(4) The separation efficiency of murine NK cells and purification efficiency of macrophages, the expression of activating molecules, lysis-associated molecules, receptors and intracellular cytokines of murine NK cells, human NKL cells or PBMCs were tested by flow cytometry;(5) The gene expression of activating molecules, lysis-associated molecules, receptors or intracellular cytokines of murine NK cells, NKL cells and PBMCs were tested by real-time quantitative PCR;(6) The cytotoxicity of murine NK cells, NKL cells and PBMCs was assayed by Cr release assay or MTT assay;(7) The level of cytokines in cell culture supernatants were determined by ELISA;(8) Conventional reverse transcription PCR was used to detect the mRNA expression of TLR7and TLR8in NKL cells and PBMCs;(9) RNA interference was used to knockdown the expression of TLR3and Qa-1in macrophages;(10) The antibody neutralization assay was used to block the expression of NKG2D, and the production of IL-12and IFN;(11) The activation of NF-κB signaling pathway was analyzed by Western Blotting;(12) The HepG2nude mice xenograft was established and treated intraperitoneally;(13) The cell to cell contact between NK cells and macrophages, or between NKL cells and DC was blocked by Transwell assay.ResultsPart1:Poly I:C-treated macrophages enhance mouse NK cells anti-tumor activity1. Poly Ⅰ:C-treated macrophages increase NK cell-mediated cytotoxicity to target tumor cells, but not to macrophages. NK cell cytotoxicity against YAC-1cells, Hepal-6cells, MCA38cells or H22cells, but not macrophages, was dramatically increased when NK cells were pre-incubated with Poly Ⅰ:C-treated macrophages.2. NK cell-mediated target cell lysis is NKG2D-dependent. Blockade of NKG2D alleviated the activation and cytotoxicity of NK cells, reduced the expression of perforin, FasL and TRAIL, as well as the secretion of IFN-γ by NK cells.3. Poly Ⅰ:C up-regulates expression of NKG2D ligands on macrophages. The NKG2D ligands RAE-1, H60and MULT-1on macrophages were significantly elevated in a dose-dependent manner following stimulation with Poly Ⅰ:C.4. Poly Ⅰ:C-induced up-regulation of NKG2D ligands is dependent on TLR3. When TLR3expression on macrophage was silenced by TLR3specific siRNA, Poly Ⅰ:C-mediated up-regulation of the three NKG2D ligands was abrogated significantly.5. Qa-1contributes to the protection of macrophages from NK cell-mediated cy to lysis. Qa-1in poly Ⅰ:C-treated or untreated primary macrophages are much higher than that in YAC-1cells, and anti-Qa-lb blockade or Qa-1-siRNA silence significantly increased the NK cell-mediated cytolysis against Poly Ⅰ:C-treated macrophage.6. Poly Ⅰ:C increases many cytokines production by macrophages. Poly I:C stimulation markedly increased the secretion of IL-15, IFN-β, IL-18and IL-12in both RAW264.7cells and BALB/c peritoneal macrophages supernatants, in particular IL-15and IFN-p.7. Activation of NK cells is dependent on IL-15and IFN-β produced by Poly Ⅰ:C-stimulated macrophages. Blocking either IL-15or IFN-P in Poly Ⅰ:C-stimulated macrophages supernatants decreased NK cells function.8. Cell-to-cell contact was involved in the crosstalk between NK cells and macrophages. In Transwell system, Poly Ⅰ:C-stimulated macrophages still promote the expression of CD69and NKG2D, as well as the production of IFN-γ by NK cells, but the up-regulation effect was not as strong as that in direct contact co-culture.Part2:Role of NK-DC cross-talk mediated by TLR7/TLR8agonist in human HCC immunotherapy1. TLR7/TLR8agonists activate human NK cells directly. R837, GDQ and ssRNA40could activate NKL cells and primary NK cells, and enhanced cytotoxicity to HepG2cells, particularly GDQ.2. TLR7/TLR8agonists induce the production of IFN-γ and TNF-α by NKL cells. The protein Levels of IFN-γ and TNF-α in NK cells were promoted by R837, GDQ and ssRNA40. 3. TLR7/TLR8agonists enhance the expression of NKG2D and NKp80on NKL cells. GDQ enhanced the expression of NKG2D, and all three agonists strongly up-regulated NKp80expression.4. DCs strongly enhance NK cells function in the present of TLR7/TLR8agonists. Although NK cells could be directly activated by these three agonists, changes in the activation status of the co-cultured NK cells were more significantly, particularly NKL cells treated with DCs and GDQ.5. DCs plus GDQ improve the antitumor effects of NK cells in vivo. The human HepG2liver carcinoma xenografts were established and treated with NKL cells. NKL plus DC and GDQ exerted a significantly greater inhibitory effect and delayed the growth of tumor nodules compared to other groups.6. Increase of mature maker expression and cytokine secretion by the DCs exposed to NKL cells and TLR7/TLR8agonists. The expression of co-stimulatory molecules CD1a, CD11c, CD83and CD86on DCs were up-regulated. IFN-α, IFN-β, IL-12p35, IL-15, and IL-18, pro-inflammation cytokines IL-1(3and L-6, but not the Th2type cytokine IL-10, in the co-culture supernatant were increased by exposure to NKL cells and TLR7/TLR8agonists.7. DC induces NK cells activation in a Type ⅠIFN and IL-12-dependent manner. Blockade of type Ⅰ IFN pathway on DC and NKL cells in the co-culture system significantly attenuated CD69and CD25expression on NK cells which were also failed to maintain high levels of lysis-associated molecules. Recombinant IL-12plus agonists could increase CD69expression and IFN-y production.8. Cell-to-cell contact is critical for DC-mediated NK cells activation. DC in Transwell culture could still promote the expression of CD69and IFN-y secretion of NK cells, but the effect is not as strong as that in direct contact co-culture. Activating receptors NKG2D and NKp80and their ligands, co-stimulatory molecules CD40-CD40L and LFA-1-ICAM-1, and chemokines CCL2and CCL4and their receptors may involve in the NK/DC cross-talk.Conclusion In this study, we demonstrated that the cross-talk between APC and NK cells mediated by TLR agonists enhanced NK cells anti-tumor activity. On the one hand, macrophages treated by TLR3agonist, Poly I:C, enhanced mouse NK cell anti-tumor effect. Poly I:C induced the expression of NKG2D ligands on murine macrophages, and up-regulated the production of IL-15, IL-12, IFN-β and other cytokines, leading to the activation and IFN-y secretion of NK cells. However, macrophages escaped the attack from activated NK cells, because of the high expression of Qa-1, the ligand of inhibitory receptor NKG2A. On the other hand, the DC/NK cross-talk mediated by the TLR7/TLR8agonist improved the anti-tumor effect of human NK cells. Although the TLR7agonist R837, TLR8agonist ssRNA40, and TLR7/TLR8agonist GDQ could directly induce the activation of NK cells, the participation of DC further significantly promoted NK cell activation. And this phenomenon was further confirmed by the in vivo therapy of hepatoma in HepG2nude mice xenograft model. In addition, it was demonstrated that the promotions of TLR7/TLR8agonist-stimulated DC for NK cell activation were dependent on the secretion of Type Ⅰ IFN and IL-12, as well as the cell-cell contacts between NKG2D-NKG2DL, NKp80-AICL, CD40-CD40L, LFA-1-ICAM-1, CCR2-CCL2and CCR4-CCL4.This study strongly confirmed the enhancement function of NK cells by APC, and identified the need of cytokines and cell-cell contact between cells interaction. It provided theoretical foundations and new ideas for the further exploration of the molecular mechanisms in the APC/NK cross-talk. The present study suggests that Poly Ⅰ:C, R837, GDQ and ssRNA40can be used as vaccine adjuvant or combined with other anti-tumor agents for treatment of cancer. And the combination of TLR agonists, APC and NK cells may be served as a new form of cell-based immunotherapy used in the clinical application and investigation for anti-tumor, anti-viral and autoimmune diseases.更多还原