人IFN-α基因修饰NK细胞系的建立以及人IFN-α/人IL-15基因修饰NK细胞抗肿瘤功能机制研究
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摘要
研究目的
恶性肿瘤严重危害人类健康,其死亡人数约占世界总死亡人数的四分之一,且死亡率常年居高不下。目前用于恶性肿瘤治疗的手段主要包括手术、化疗、放疗、内分泌治疗和射频消融治疗等,由于缺乏特异性和靶向性,在杀伤肿瘤细胞的同时,机体正常细胞的功能也遭到破坏,从而导致患者免疫功能受损,生存质量下降。近来,随着免疫学、分子生物学和细胞生物学的交融和发展,以机体免疫细胞为主体的细胞免疫治疗,以其强大的抗肿瘤作用和较低的毒副作用,逐步成为肿瘤治疗研究的新方向。
自然杀伤(Natural killer,NK)细胞作为机体天然免疫系统中不可或缺的组成部分,是机体抵抗肿瘤和病毒感染细胞的第一道天然防线,也是连接天然免疫和获得性免疫之间的桥梁。基于NK细胞的抗肿瘤免疫治疗以其独特的抗肿瘤效果引起人们的高度重视,现己进入临床试验阶段,主要包括白体NK细胞的活化,同种异体NK细胞的输注,体外扩增NK细胞的输注以及NK细胞系的过继转输等。由于体外NK细胞的纯化和大规模培养仍具有较高的技术难度,NK细胞系以其易于体外扩增和大规模培养、具有较强的细胞毒作用等优点,克服了自体以及同种异体NK细胞治疗的局限性,具有广阔的应用前景。NK细胞系在体外生长状态良好,能够通过基因修饰等手段增强抗肿瘤活性。
在众多活化NK细胞的细胞因子中,IFN-α占有非常重要的地位。IFN-α属于I型干扰素家族,广泛应用于造血系统肿瘤和某些实体肿瘤的治疗中,并且能够通过下调癌基因的表达,诱导肿瘤抑制基因进而影响肿瘤细胞的功能,还能够促进免疫细胞,比如T细胞,DC,NK细胞的分化和活化,介导IFN诱导的抗肿瘤免疫活性。IFN-α现已被广泛应用于DC的基因修饰,使其适应肿瘤过继免疫治疗的要求
白细胞介素-15(IL-15)是由Grabstein等于1994年在猴肾表皮细胞系cv21/EBVA的培养上清中发现,早期被定义为T细胞生长因子,在自体内环境的稳定,调节天然免疫和获得性免疫方面发挥多重作用。IL-15与IL-2在功能上有许多相似之处,均可以诱导CD34+的细胞分化为NK细胞,但IL-15在NK细胞的分化和功能上发挥了更为重要的作用,实验证实,在IL-15Rα-1-和IL-15-/-小鼠中,NK细胞无法正常发育、成熟并发挥功能。
本研究选用了两种对NK细胞发育、分化、功能等起关键作用的细胞因子IL-15和IFN-α,对NK细胞系NKL进行基因修饰。建立了hIFN-a基因修饰的NKL细胞,证实修饰后的细胞能够有效抑制肝癌的生长,并探讨了作用机制。前期实验中建立了hIL-15基因修饰的NKL细胞系,并证实了其在体外对肝癌细胞的杀伤效果,本研究进一步确证了hIL-15修饰后NKL细胞在体内对肝癌的治疗效果,并初步探讨了其作用机制。同时研究了hIL-15基因修饰后的NKL细胞对白血病的抑制效果和作用机制。结果证实,修饰后的NKL细胞具有更为显著地抗肿瘤作用,更加符合肿瘤过继免疫治疗的要求。
研究方法
首先,我们建立了hIFN-a基因修饰的NKL细胞,并研究其对肝癌的抑制作用和杀伤机制。通过PT-PCR方法从外周血PBMC中扩增hIFN-α1基因全长,插入pSecTagB真核表达载体中；通过电穿孔技术将hIFN-a基因转染入NKL细胞中；通过RT-PCR方法检测hIFN-a基因转染后,NKL细胞IFN-a的mRNA水平,和肝癌细胞上凋亡相关分子Fas、Mcl-1、Bcl-xl的表达水平；通过RNAi技术,下调HepG2细胞上Fas的表达水平；通过Real-time PCR检测NK细胞杀伤相关分子的mRNA水平；通过流式细胞术,检测NK细胞杀伤相关分子和HepG2细胞Fas的蛋白水平；通过MTT法检测NK细胞对肝癌细胞系以及原代肝癌细胞的杀伤；通过抗体阻断实验,检测IFN-γ和TNF-α在NKL-IFNa细胞对HepG2细胞杀伤过程中的作用；利用皮下注射和腹腔注射两种不同荷瘤方式建立裸鼠荷瘤模型,通过检测荷瘤小鼠瘤体积和小鼠生存期来验证NKL-IFNα细胞的治疗效果。
其次,我们验证了hIL-15基因修饰的NKL细胞对肝癌的治疗作用,并探讨其作用机制。利用皮下注射和腹腔注射两种不同荷瘤方式建立裸鼠荷瘤模型,通过检测荷瘤小鼠脾重、肝重、瘤重、脾脏淋巴细胞数和小鼠生存期来验证NKL-IL15细胞的治疗效果；通过ELISA检测NK细胞上清中IFN-y和TNF-a的分泌水平；通过流式细胞术检测NK细胞杀伤相关分子NKp80、颗粒酶B、Perforin、TRAIL和HepG2细胞上NKG2D配体的表达水平；通过细胞计数法检测与NKL-IL15细胞共培养后,HepG2细胞的增殖能力；通过MTT法检测NK细胞对肝癌细胞的杀伤活性；通过抗体阻断实验,检测NKG2D、IFN-y和TNF-a在NKL-IL15细胞对HepG2细胞杀伤过程中的作用。
最后,我们探讨了hIL-15基因修饰的NKL细胞对白血病的治疗作用。通过MTT法检测NK细胞对白血病细胞系以及原代白血病细胞的杀伤活性；通过ELISA检测NK细胞上清中IFN-y和TNF-a的分泌水平；通过流式细胞术检测NK细胞杀伤相关分子NKp80、颗粒酶B、Perforin和K562细胞上NKG2D配体的表达水平；通过通过细胞计数法检测与NKL-IL15细胞共培养后,K562细胞的增殖能力；通过抗体阻断实验,检测NKG2D、IFN-y和TNF-a在NKL-IL15细胞对K562细胞杀伤过程中的作用；利用皮下注射和腹腔注射两种不同荷瘤方式建立NOD/SCID鼠荷瘤模型,通过检测荷瘤小鼠瘤体积、瘤重和小鼠生存期来验证NKL-IL15细胞的治疗效果。
结果
结果一：hIFN-a基因修饰NKL细胞系的建立及其抗肝癌功能与机制研究
1.构建hIFN-a基因修饰的NKL细胞系我们已经成功建立了hIFN-a基因修饰的NKL细胞系,NKL-IFNa细胞能够分泌大量的IFN-α。
2IFN-a基因修饰能够增强NKL细胞对HCC的杀伤活性NKL-IFNa细胞对肝癌细胞系HepG2、H7402细胞和原代肝癌细胞的杀伤活性显著增强。特别是,IFN-α基因修饰亦能增强NKL细胞对HBV+肝癌肿瘤细胞HepG2.2.15细胞的杀伤效果。
3.IFN-α基因修饰增强NKL细胞的活性hIFN-α基因修饰能够上调NKL细胞中杀伤相关分子TNF-α、IFN-γ、perforin、Granzyme B和FasL的表达,从而增强NKL细胞对HCC的细胞毒性。
4.NKL-IFNa细胞诱导的Fas表达能够增强对HCC的杀伤敏感性HCC表面Fas的表达在NKL-IFNa细胞介导的细胞毒性反应中起到主导作用。阻断HepG2细胞表面Fas的表达,会使NKL-IFNa细胞所介导的杀伤效果显著下降。
5NKL-IFNa细胞通过分泌TNF-a和IFN-y诱导HCC细胞上Fas的表达阻断TNF-α和IFN-γ能够降低HepG2表面Fas的表达,进而抑制了NK细胞介导的细胞毒性作用。说明,NKL-IFNa细胞对HCC的杀伤是Fas依赖的,TNF-α和IFN-γ介导的。
6.IFN-α基因修饰能够增强NKL细胞在小鼠体内抑制肝癌的效果NKL-IFNα细胞能够在腹腔荷瘤和皮下荷瘤小鼠模型中,抑制小鼠体内肝癌细胞的生长,延长小鼠的生存期。
结果二：hIL-15基因修饰的NKL细胞系在小鼠体内抗HCC作用及机制研究
1.在小鼠荷瘤模型中,IL-15基因修饰的NKL细胞具有显著抗HCC作用NKL-IL15细胞治疗能够有效抑制腹腔和皮下荷瘤两种荷瘤小鼠模型中肝癌细胞在荷瘤小鼠体内的生长,延长小鼠的生存期,发挥良好的治疗效果。
2.IL-15基因修饰能够增强NKL细胞对HCC细胞的应答IL-15基因修饰后的NKL细胞,对HepG2细胞的刺激更加敏感,能够上调杀伤相关分子的表达,从而增强对HCC的杀伤效果。
3.IL-15基因修饰的NKL细胞能够抑制HCC细胞的增殖IL-15基因修饰的NKL细胞能够通过分泌细胞因子,抑制HCC的增殖,增强抗肿瘤功效。
4.IL-15基因修饰的NKL细胞通过分泌TNF-α和IFN-γ上调HCC细胞上NKG2D配体的表达NKL-IL15细胞通过分泌TNF-a和IFN-y上调HCC细胞上NKG2D配体的表达,进而增强HCC对NK细胞的杀伤敏感性。
5.NKL-IL15细胞杀伤功能的增强依赖于NKG2D的识别和TNF-α的分泌NKG2D的识别和TNF-α的分泌,在NKL-IL15介导的HCC杀伤过程中起到非常重要的作用。
结果三：hIL-15基因修饰NKL细胞的抗白血病功能及机制研究
1.hIL-15基因修饰能够增强NKL细胞对白血病细胞的杀伤功能hIL-15基因修饰能够增强NKL细胞对白血病细胞系和原代白血病细胞的杀伤功能。
2.IL-15基因修饰的NKL细胞能够抑制白血病细胞的增殖,诱导白血病细胞上NKG2D配体的表达IL-15基因修饰的NKL细胞能够通过分泌细胞因子抑制白血病细胞的增殖,同时诱导白血病细胞表面NKG2D配体的表达,促进了NKL细胞对白血病细胞的杀伤。
3.IL-15基因修饰促进了NKL细胞的活化IL-15基因修饰能够增强NKL细胞杀伤相关受体和分子的表达,进而增强NKL细胞对白血病细胞的杀伤功能。
4.IL-15基因修饰NKL细胞杀伤功能的增强依赖于NKG2D和IFN-y NKG2D的识别和IFN-y的分泌,在NKL-IL15介导的白血病细胞杀伤过程中起到非常重要的作用。
5.IL-15基因修饰能够增强NKL细胞在小鼠体内的抗肿瘤效果NKL-IL15细胞能够抑制小鼠体内白血病细胞的生长,延长小鼠的生存期。
6.NKL-IL15细胞能够活化hPBMC,增强其对白血病细胞的杀伤NKL-IL15细胞具有对hPBMC的活化作用。
结论
1.构建hIFN-α基因修饰NKL细胞,在体内和体外实验中均证实其对肝癌的抑制作用,且NKL-IFNa细胞通过分泌TNF-α和IFN-y诱导HCC细胞上Fas的表达,依赖Fas/FasL途径发挥细胞毒作用。
2.验证前期构建的hIL-15基因修饰的NKL细胞,在小鼠体内对肝癌的抑制作用,其杀伤作用依赖于NKG2D的识别和’TNF-α的分泌。
3.探讨hIL-15基因修饰的NKL细胞对白血病的治疗作用,初步研究其杀伤机制是依赖于NKG2D和IFN-γ。
基因修饰的NK细胞系,可以通过细胞因子IL-15和IFN-α的修饰,增强细胞毒作用,使其适用于肿瘤的过继免疫治疗。基因修饰NK细胞系的应用,能够为疾病治疗带来新的思路。
Object
As a frequently occurring disease, malignant tumors have a great threat to human health. The death of malignant tumors accounts for about1/4of total deaths in the world, and shows an increasing trend in death rate. There are many treatment methods for malignant tumors, including surgery, chemotherapy, radiotherapy, endocrine therapy and radiofrequencyablation therapy, but most of them are lack of specificity and targeting, as they could not only kill tumor cells but also destroy normal cells, leading to a serious decline in immune function of tumor patients. In recent years, immunotherapy based the cutting-edge science of 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, immunotherapy is attempting to stimulate the immune system to reject and destroy tumors.
Natural killer (NK) cells are a key component of the innate immune system, and characterized by a strong cytolytic activity against various types of tumor cells and virus-infected cells via non-major histocompatibility complex (MHC)-restricted and non-T-cell receptor (TCR)-restricted mechanisms. NK cell-based immunotherapy has been in development, and can potentially be implemented through the administration of cytokines or immunomodulatory drugs to activate endogenous NK cells, or the adoptive transfer of induced alloreactive NK cells developed from allogeneic stem cells, ex vivo expanded autologous NK cells or donor-derived allogenic NK cells. By comparison, there are some limitations as to the efficacy of the reinfusion of ex vivo expanded autologous or allogeneic NK cells.Clinical use of human permanent NK cell lines would overcome some of the limitations, which are more cytotoxic and can be easily expanded and maintained invitro without contamination by other lymphocytes.
IFN-a, a pleiotropic type I IFN (Interferon), is one of the most important cytokines which activates NK cells. IFN-a is extensively used in the treatment of patients with hematological malignancies or certain solid tumors, such as melanoma and renal carcinoma. IFN-a not only affects tumor cell function, for example by downregulating oncogene expression and inducing tumor suppressor genes, but can also promote the differentiation and activity of host immune cells, including T cells, dendritic cells and NK cells, resulting in IFN-a-induced antitumor immunity. IFN-a has also been widely used in the gene-modification of DCs (Dendritic cells) for cancer immunotherapy.
IL-15is the major physiologic growth factor responsible for NK cell ontogeny; additionally, it is a potent regulator for NK cell proliferation, survival and cytolytic activity, and there are no functional NK cells in IL-15Rα-/-and IL-15-/-mice.
On this basis, we used electroporation technology to genetically modify NKL cells with the human IFN-a gene and human IL-15gene, and explored the potential of these gene-modified NKL cells for adaptive immunotherapy.
Methods
Firstly, we established human IFN-a gene-modified NKL cells and explored the potential of NKL-IFNa cells for HCC adaptive immunotherapy. Electroporation, RT-PCR, Flow cytometry, ELISA, MTT, RNAi methods were performed for gene-modification, molecular expression, cytokines secretion, cytotoxicity assays and mechanism discussion. Different therapeutic schedules were investigated in a xenograft model of HCC in nude mice, to assess the application of gene-modified NKL cells in adoptive cellular immunotherapy.
Secondly, to further assess the applicability of NKL-IL15cells in adoptive cellular immunotherapy, in present study, we further investigated their natural cytotoxicity against HCC in vivo. HCC xenograft nude mice were established by intraperitoneal or subcutaneous injection with HepG2cells, and then treated with irradiated NKL cells. Flow cytometry, ELISA, cell counting and MTT methods were performed for molecular expression, cell proliferation and cytotoxicity assays.
At last, to further confirm the applicability of NKL-IL15cells in leukemia, we evaluated the efficiency of NKL-IL15cells against leukemia. Leukemia xenograft NOD/SCID mice were established by intraperitoneal or subcutaneous injection with K562cells, and then treated with irradiated NK cells. Flow cytometry, ELISA, cell counting and MTT methods were performed for molecular expression, cell proliferation and cytotoxicity assays.
Results
Part1:hIFN-alpha gene modification augments human natural killer cell line anti-human hepatocellular carcinoma function
1.Establishment of the hIFN-a gene-modified NKL cell line We had successfully established a hIFN-α gene-modified NKL cell line.
2.IFN-a gene-modification enhances NKL cell-mediated natural cytotoxicity against human hepatocarcinoma cells The enhanced natural cytotoxicity of NKL-INFa cells against human HCC cells is mediated directly by IFN-a acting on the NKL cells, rather than IFN-a acting on the tumor cells.
3.IFN-a gene-modification increases the activation of NKL cells hlFN-a gene modification altered the expression of cytotoxicity-associated genes, which in turn enhanced the natural cytotoxicity of NKL cells against HCC cells.
4.Elevated Fas on HCC cells contributes to the augmented sensitivity of HCC cells to NKL-IFNa-mediated cytolysis Fas expressed on HCC cells plays a critical role in NKL-IFNa cell-mediated cytolysis.
5.TNF-a and IFN-y secreted by NKL-IFNa cells induce the expression of Fas on HCC cells Cytokines produced by IFN-a gene-modified NKL cells, such as TNF-a and IFN-y, are also involved in NKL-IFNa cell-mediated cytolysis, possibly by inducing increased expression of Fas on the target cells.
6.IFN-a gene-modified NKL cells exert an augmented anti-tumor effect in a xenograft model of HCC in nude mice Treatment with NKL-IFNa cells is effective in suppressing the tumor growth and prolonging the survival time of mice bearing HCC xenograft tumors.
Part2. hIL-15gene modified human natural killer cells exerted augmented anti-human hepatocellular carcinoma effect in vivo
1. Immunotherapy of IL-15gene-modified NKL cells in HCC xenograft mouse models The treatment of NKL-IL15cells were effective in suppressing tumor growth and prolonging the survival time of HCC bearing mice in different tumor-bearing models.
2. IL-15gene-modification promoted the activation of NKL cells IL-15gene-modified NKL cells showed more sensitive to HepG2cells stimulation, companied with the up-regulation of cytolysis-related molecules, thereby augmenting NKL cell cytotoxicity activity against HCC cells.
3. The products of IL-15gene-modified NKL cells decreased the proliferation of HCC cells IL-15gene-modified NKL cells could decrease the proliferation of HCC cells, and some cytokines produced by NKL-IL15might be involved in this process.
4. The expression of NKG2D ligands in HCC cells were up-regulated by TNF-a and IFN-y produced by IL-15gene-modified NKL cells TNF-a and IFN-y secreted by NKL-IL15cells promoted the expression of NKG2D ligands, which would increase the sensitivity of HCC to NK cell cytolysis.
5. The enhanced cytotoxicity of NKL-IL15cells was mainly dependent on NKG2D recognition and TNF-a secretion NKG2D recognition and TNF-a production were important for NKL-IL15cells-mediated cytolysis against HepG2cells. Part3. hIL-15gene modified human natural killer cells (NKL-IL15) anti-human leukemia function1. hIL-15gene-modification enhanced NKL cell-mediated natural cytotoxicity against human leukemia cells hIL-15gene modification of NKL cells could enhance the natural cytotoxicity against human leukemia cells.2. The products of IL-15gene-modified NKL cells decreased the proliferation of leukemia cells and induced the expression of NKG2D ligands in leukemia cells IL-15gene-modified NKL cells decreased the proliferation and up-regulated the expression of NKG2D ligands in leukemia cells by secreting some certain factors, which promoted the natural cytotoxicity of NKL cells against human leukemia cells.
3. IL-15gene-modification mediated the enhancement of NKL cell cytotoxicity was dependent on NKG2D and IFN-γ The high cytotoxicity mediated by NKL-IL15cells against K562cells depended on NKG2D and IFN-γ.
4. IL-15gene-modification improved the antitumor effects of NK cells in vivo The treatment of NKL-IL15cells was effective in suppressing tumor growth and prolong the survival time of leukemia-bearing mice.
5. NKL-IL15cells activated hPBMCs and enhanced the natural cytotoxicity against leukemia IL-15gene-modified NK cells could display active effects on hPBMCs cells.
Conclusion
Firstly, IFN-α gene-modified NKL cells could be suitable for the development of cell-based immunotherapeutic strategies for hepatocellular carcinoma.
Secondly, hIL-15gene-modified human natural killer cells exerted augmented anti-human hepatocellular carcinoma and anti-leukemia effect in vivo. The results suggested that hIL-15gene-modified NKL cells could be a promising new candidate for adoptive immunotherapy in the future.
We could gene-modified NK cell lines with cytokines which could induce the cytotoxicity effect to be suitable for the use of adoptive immunotherapy. The use of gene-modified NK cell lines may represent a novel and potential immunotherapeutic strategy in the future.
恶性肿瘤严重危害人类健康,其死亡人数约占世界总死亡人数的四分之一,且死亡率常年居高不下。目前用于恶性肿瘤治疗的手段主要包括手术、化疗、放疗、内分泌治疗和射频消融治疗等,由于缺乏特异性和靶向性,在杀伤肿瘤细胞的同时,机体正常细胞的功能也遭到破坏,从而导致患者免疫功能受损,生存质量下降。近来,随着免疫学、分子生物学和细胞生物学的交融和发展,以机体免疫细胞为主体的细胞免疫治疗,以其强大的抗肿瘤作用和较低的毒副作用,逐步成为肿瘤治疗研究的新方向。
自然杀伤(Natural killer,NK)细胞作为机体天然免疫系统中不可或缺的组成部分,是机体抵抗肿瘤和病毒感染细胞的第一道天然防线,也是连接天然免疫和获得性免疫之间的桥梁。基于NK细胞的抗肿瘤免疫治疗以其独特的抗肿瘤效果引起人们的高度重视,现己进入临床试验阶段,主要包括白体NK细胞的活化,同种异体NK细胞的输注,体外扩增NK细胞的输注以及NK细胞系的过继转输等。由于体外NK细胞的纯化和大规模培养仍具有较高的技术难度,NK细胞系以其易于体外扩增和大规模培养、具有较强的细胞毒作用等优点,克服了自体以及同种异体NK细胞治疗的局限性,具有广阔的应用前景。NK细胞系在体外生长状态良好,能够通过基因修饰等手段增强抗肿瘤活性。
在众多活化NK细胞的细胞因子中,IFN-α占有非常重要的地位。IFN-α属于I型干扰素家族,广泛应用于造血系统肿瘤和某些实体肿瘤的治疗中,并且能够通过下调癌基因的表达,诱导肿瘤抑制基因进而影响肿瘤细胞的功能,还能够促进免疫细胞,比如T细胞,DC,NK细胞的分化和活化,介导IFN诱导的抗肿瘤免疫活性。IFN-α现已被广泛应用于DC的基因修饰,使其适应肿瘤过继免疫治疗的要求
白细胞介素-15(IL-15)是由Grabstein等于1994年在猴肾表皮细胞系cv21/EBVA的培养上清中发现,早期被定义为T细胞生长因子,在自体内环境的稳定,调节天然免疫和获得性免疫方面发挥多重作用。IL-15与IL-2在功能上有许多相似之处,均可以诱导CD34+的细胞分化为NK细胞,但IL-15在NK细胞的分化和功能上发挥了更为重要的作用,实验证实,在IL-15Rα-1-和IL-15-/-小鼠中,NK细胞无法正常发育、成熟并发挥功能。
本研究选用了两种对NK细胞发育、分化、功能等起关键作用的细胞因子IL-15和IFN-α,对NK细胞系NKL进行基因修饰。建立了hIFN-a基因修饰的NKL细胞,证实修饰后的细胞能够有效抑制肝癌的生长,并探讨了作用机制。前期实验中建立了hIL-15基因修饰的NKL细胞系,并证实了其在体外对肝癌细胞的杀伤效果,本研究进一步确证了hIL-15修饰后NKL细胞在体内对肝癌的治疗效果,并初步探讨了其作用机制。同时研究了hIL-15基因修饰后的NKL细胞对白血病的抑制效果和作用机制。结果证实,修饰后的NKL细胞具有更为显著地抗肿瘤作用,更加符合肿瘤过继免疫治疗的要求。
研究方法
首先,我们建立了hIFN-a基因修饰的NKL细胞,并研究其对肝癌的抑制作用和杀伤机制。通过PT-PCR方法从外周血PBMC中扩增hIFN-α1基因全长,插入pSecTagB真核表达载体中；通过电穿孔技术将hIFN-a基因转染入NKL细胞中；通过RT-PCR方法检测hIFN-a基因转染后,NKL细胞IFN-a的mRNA水平,和肝癌细胞上凋亡相关分子Fas、Mcl-1、Bcl-xl的表达水平；通过RNAi技术,下调HepG2细胞上Fas的表达水平；通过Real-time PCR检测NK细胞杀伤相关分子的mRNA水平；通过流式细胞术,检测NK细胞杀伤相关分子和HepG2细胞Fas的蛋白水平；通过MTT法检测NK细胞对肝癌细胞系以及原代肝癌细胞的杀伤；通过抗体阻断实验,检测IFN-γ和TNF-α在NKL-IFNa细胞对HepG2细胞杀伤过程中的作用；利用皮下注射和腹腔注射两种不同荷瘤方式建立裸鼠荷瘤模型,通过检测荷瘤小鼠瘤体积和小鼠生存期来验证NKL-IFNα细胞的治疗效果。
其次,我们验证了hIL-15基因修饰的NKL细胞对肝癌的治疗作用,并探讨其作用机制。利用皮下注射和腹腔注射两种不同荷瘤方式建立裸鼠荷瘤模型,通过检测荷瘤小鼠脾重、肝重、瘤重、脾脏淋巴细胞数和小鼠生存期来验证NKL-IL15细胞的治疗效果；通过ELISA检测NK细胞上清中IFN-y和TNF-a的分泌水平；通过流式细胞术检测NK细胞杀伤相关分子NKp80、颗粒酶B、Perforin、TRAIL和HepG2细胞上NKG2D配体的表达水平；通过细胞计数法检测与NKL-IL15细胞共培养后,HepG2细胞的增殖能力；通过MTT法检测NK细胞对肝癌细胞的杀伤活性；通过抗体阻断实验,检测NKG2D、IFN-y和TNF-a在NKL-IL15细胞对HepG2细胞杀伤过程中的作用。
最后,我们探讨了hIL-15基因修饰的NKL细胞对白血病的治疗作用。通过MTT法检测NK细胞对白血病细胞系以及原代白血病细胞的杀伤活性；通过ELISA检测NK细胞上清中IFN-y和TNF-a的分泌水平；通过流式细胞术检测NK细胞杀伤相关分子NKp80、颗粒酶B、Perforin和K562细胞上NKG2D配体的表达水平；通过通过细胞计数法检测与NKL-IL15细胞共培养后,K562细胞的增殖能力；通过抗体阻断实验,检测NKG2D、IFN-y和TNF-a在NKL-IL15细胞对K562细胞杀伤过程中的作用；利用皮下注射和腹腔注射两种不同荷瘤方式建立NOD/SCID鼠荷瘤模型,通过检测荷瘤小鼠瘤体积、瘤重和小鼠生存期来验证NKL-IL15细胞的治疗效果。
结果
结果一：hIFN-a基因修饰NKL细胞系的建立及其抗肝癌功能与机制研究
1.构建hIFN-a基因修饰的NKL细胞系我们已经成功建立了hIFN-a基因修饰的NKL细胞系,NKL-IFNa细胞能够分泌大量的IFN-α。
2IFN-a基因修饰能够增强NKL细胞对HCC的杀伤活性NKL-IFNa细胞对肝癌细胞系HepG2、H7402细胞和原代肝癌细胞的杀伤活性显著增强。特别是,IFN-α基因修饰亦能增强NKL细胞对HBV+肝癌肿瘤细胞HepG2.2.15细胞的杀伤效果。
3.IFN-α基因修饰增强NKL细胞的活性hIFN-α基因修饰能够上调NKL细胞中杀伤相关分子TNF-α、IFN-γ、perforin、Granzyme B和FasL的表达,从而增强NKL细胞对HCC的细胞毒性。
4.NKL-IFNa细胞诱导的Fas表达能够增强对HCC的杀伤敏感性HCC表面Fas的表达在NKL-IFNa细胞介导的细胞毒性反应中起到主导作用。阻断HepG2细胞表面Fas的表达,会使NKL-IFNa细胞所介导的杀伤效果显著下降。
5NKL-IFNa细胞通过分泌TNF-a和IFN-y诱导HCC细胞上Fas的表达阻断TNF-α和IFN-γ能够降低HepG2表面Fas的表达,进而抑制了NK细胞介导的细胞毒性作用。说明,NKL-IFNa细胞对HCC的杀伤是Fas依赖的,TNF-α和IFN-γ介导的。
6.IFN-α基因修饰能够增强NKL细胞在小鼠体内抑制肝癌的效果NKL-IFNα细胞能够在腹腔荷瘤和皮下荷瘤小鼠模型中,抑制小鼠体内肝癌细胞的生长,延长小鼠的生存期。
结果二：hIL-15基因修饰的NKL细胞系在小鼠体内抗HCC作用及机制研究
1.在小鼠荷瘤模型中,IL-15基因修饰的NKL细胞具有显著抗HCC作用NKL-IL15细胞治疗能够有效抑制腹腔和皮下荷瘤两种荷瘤小鼠模型中肝癌细胞在荷瘤小鼠体内的生长,延长小鼠的生存期,发挥良好的治疗效果。
2.IL-15基因修饰能够增强NKL细胞对HCC细胞的应答IL-15基因修饰后的NKL细胞,对HepG2细胞的刺激更加敏感,能够上调杀伤相关分子的表达,从而增强对HCC的杀伤效果。
3.IL-15基因修饰的NKL细胞能够抑制HCC细胞的增殖IL-15基因修饰的NKL细胞能够通过分泌细胞因子,抑制HCC的增殖,增强抗肿瘤功效。
4.IL-15基因修饰的NKL细胞通过分泌TNF-α和IFN-γ上调HCC细胞上NKG2D配体的表达NKL-IL15细胞通过分泌TNF-a和IFN-y上调HCC细胞上NKG2D配体的表达,进而增强HCC对NK细胞的杀伤敏感性。
5.NKL-IL15细胞杀伤功能的增强依赖于NKG2D的识别和TNF-α的分泌NKG2D的识别和TNF-α的分泌,在NKL-IL15介导的HCC杀伤过程中起到非常重要的作用。
结果三：hIL-15基因修饰NKL细胞的抗白血病功能及机制研究
1.hIL-15基因修饰能够增强NKL细胞对白血病细胞的杀伤功能hIL-15基因修饰能够增强NKL细胞对白血病细胞系和原代白血病细胞的杀伤功能。
2.IL-15基因修饰的NKL细胞能够抑制白血病细胞的增殖,诱导白血病细胞上NKG2D配体的表达IL-15基因修饰的NKL细胞能够通过分泌细胞因子抑制白血病细胞的增殖,同时诱导白血病细胞表面NKG2D配体的表达,促进了NKL细胞对白血病细胞的杀伤。
3.IL-15基因修饰促进了NKL细胞的活化IL-15基因修饰能够增强NKL细胞杀伤相关受体和分子的表达,进而增强NKL细胞对白血病细胞的杀伤功能。
4.IL-15基因修饰NKL细胞杀伤功能的增强依赖于NKG2D和IFN-y NKG2D的识别和IFN-y的分泌,在NKL-IL15介导的白血病细胞杀伤过程中起到非常重要的作用。
5.IL-15基因修饰能够增强NKL细胞在小鼠体内的抗肿瘤效果NKL-IL15细胞能够抑制小鼠体内白血病细胞的生长,延长小鼠的生存期。
6.NKL-IL15细胞能够活化hPBMC,增强其对白血病细胞的杀伤NKL-IL15细胞具有对hPBMC的活化作用。
结论
1.构建hIFN-α基因修饰NKL细胞,在体内和体外实验中均证实其对肝癌的抑制作用,且NKL-IFNa细胞通过分泌TNF-α和IFN-y诱导HCC细胞上Fas的表达,依赖Fas/FasL途径发挥细胞毒作用。
2.验证前期构建的hIL-15基因修饰的NKL细胞,在小鼠体内对肝癌的抑制作用,其杀伤作用依赖于NKG2D的识别和’TNF-α的分泌。
3.探讨hIL-15基因修饰的NKL细胞对白血病的治疗作用,初步研究其杀伤机制是依赖于NKG2D和IFN-γ。
基因修饰的NK细胞系,可以通过细胞因子IL-15和IFN-α的修饰,增强细胞毒作用,使其适用于肿瘤的过继免疫治疗。基因修饰NK细胞系的应用,能够为疾病治疗带来新的思路。
Object
As a frequently occurring disease, malignant tumors have a great threat to human health. The death of malignant tumors accounts for about1/4of total deaths in the world, and shows an increasing trend in death rate. There are many treatment methods for malignant tumors, including surgery, chemotherapy, radiotherapy, endocrine therapy and radiofrequencyablation therapy, but most of them are lack of specificity and targeting, as they could not only kill tumor cells but also destroy normal cells, leading to a serious decline in immune function of tumor patients. In recent years, immunotherapy based the cutting-edge science of 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, immunotherapy is attempting to stimulate the immune system to reject and destroy tumors.
Natural killer (NK) cells are a key component of the innate immune system, and characterized by a strong cytolytic activity against various types of tumor cells and virus-infected cells via non-major histocompatibility complex (MHC)-restricted and non-T-cell receptor (TCR)-restricted mechanisms. NK cell-based immunotherapy has been in development, and can potentially be implemented through the administration of cytokines or immunomodulatory drugs to activate endogenous NK cells, or the adoptive transfer of induced alloreactive NK cells developed from allogeneic stem cells, ex vivo expanded autologous NK cells or donor-derived allogenic NK cells. By comparison, there are some limitations as to the efficacy of the reinfusion of ex vivo expanded autologous or allogeneic NK cells.Clinical use of human permanent NK cell lines would overcome some of the limitations, which are more cytotoxic and can be easily expanded and maintained invitro without contamination by other lymphocytes.
IFN-a, a pleiotropic type I IFN (Interferon), is one of the most important cytokines which activates NK cells. IFN-a is extensively used in the treatment of patients with hematological malignancies or certain solid tumors, such as melanoma and renal carcinoma. IFN-a not only affects tumor cell function, for example by downregulating oncogene expression and inducing tumor suppressor genes, but can also promote the differentiation and activity of host immune cells, including T cells, dendritic cells and NK cells, resulting in IFN-a-induced antitumor immunity. IFN-a has also been widely used in the gene-modification of DCs (Dendritic cells) for cancer immunotherapy.
IL-15is the major physiologic growth factor responsible for NK cell ontogeny; additionally, it is a potent regulator for NK cell proliferation, survival and cytolytic activity, and there are no functional NK cells in IL-15Rα-/-and IL-15-/-mice.
On this basis, we used electroporation technology to genetically modify NKL cells with the human IFN-a gene and human IL-15gene, and explored the potential of these gene-modified NKL cells for adaptive immunotherapy.
Methods
Firstly, we established human IFN-a gene-modified NKL cells and explored the potential of NKL-IFNa cells for HCC adaptive immunotherapy. Electroporation, RT-PCR, Flow cytometry, ELISA, MTT, RNAi methods were performed for gene-modification, molecular expression, cytokines secretion, cytotoxicity assays and mechanism discussion. Different therapeutic schedules were investigated in a xenograft model of HCC in nude mice, to assess the application of gene-modified NKL cells in adoptive cellular immunotherapy.
Secondly, to further assess the applicability of NKL-IL15cells in adoptive cellular immunotherapy, in present study, we further investigated their natural cytotoxicity against HCC in vivo. HCC xenograft nude mice were established by intraperitoneal or subcutaneous injection with HepG2cells, and then treated with irradiated NKL cells. Flow cytometry, ELISA, cell counting and MTT methods were performed for molecular expression, cell proliferation and cytotoxicity assays.
At last, to further confirm the applicability of NKL-IL15cells in leukemia, we evaluated the efficiency of NKL-IL15cells against leukemia. Leukemia xenograft NOD/SCID mice were established by intraperitoneal or subcutaneous injection with K562cells, and then treated with irradiated NK cells. Flow cytometry, ELISA, cell counting and MTT methods were performed for molecular expression, cell proliferation and cytotoxicity assays.
Results
Part1:hIFN-alpha gene modification augments human natural killer cell line anti-human hepatocellular carcinoma function
1.Establishment of the hIFN-a gene-modified NKL cell line We had successfully established a hIFN-α gene-modified NKL cell line.
2.IFN-a gene-modification enhances NKL cell-mediated natural cytotoxicity against human hepatocarcinoma cells The enhanced natural cytotoxicity of NKL-INFa cells against human HCC cells is mediated directly by IFN-a acting on the NKL cells, rather than IFN-a acting on the tumor cells.
3.IFN-a gene-modification increases the activation of NKL cells hlFN-a gene modification altered the expression of cytotoxicity-associated genes, which in turn enhanced the natural cytotoxicity of NKL cells against HCC cells.
4.Elevated Fas on HCC cells contributes to the augmented sensitivity of HCC cells to NKL-IFNa-mediated cytolysis Fas expressed on HCC cells plays a critical role in NKL-IFNa cell-mediated cytolysis.
5.TNF-a and IFN-y secreted by NKL-IFNa cells induce the expression of Fas on HCC cells Cytokines produced by IFN-a gene-modified NKL cells, such as TNF-a and IFN-y, are also involved in NKL-IFNa cell-mediated cytolysis, possibly by inducing increased expression of Fas on the target cells.
6.IFN-a gene-modified NKL cells exert an augmented anti-tumor effect in a xenograft model of HCC in nude mice Treatment with NKL-IFNa cells is effective in suppressing the tumor growth and prolonging the survival time of mice bearing HCC xenograft tumors.
Part2. hIL-15gene modified human natural killer cells exerted augmented anti-human hepatocellular carcinoma effect in vivo
1. Immunotherapy of IL-15gene-modified NKL cells in HCC xenograft mouse models The treatment of NKL-IL15cells were effective in suppressing tumor growth and prolonging the survival time of HCC bearing mice in different tumor-bearing models.
2. IL-15gene-modification promoted the activation of NKL cells IL-15gene-modified NKL cells showed more sensitive to HepG2cells stimulation, companied with the up-regulation of cytolysis-related molecules, thereby augmenting NKL cell cytotoxicity activity against HCC cells.
3. The products of IL-15gene-modified NKL cells decreased the proliferation of HCC cells IL-15gene-modified NKL cells could decrease the proliferation of HCC cells, and some cytokines produced by NKL-IL15might be involved in this process.
4. The expression of NKG2D ligands in HCC cells were up-regulated by TNF-a and IFN-y produced by IL-15gene-modified NKL cells TNF-a and IFN-y secreted by NKL-IL15cells promoted the expression of NKG2D ligands, which would increase the sensitivity of HCC to NK cell cytolysis.
5. The enhanced cytotoxicity of NKL-IL15cells was mainly dependent on NKG2D recognition and TNF-a secretion NKG2D recognition and TNF-a production were important for NKL-IL15cells-mediated cytolysis against HepG2cells. Part3. hIL-15gene modified human natural killer cells (NKL-IL15) anti-human leukemia function1. hIL-15gene-modification enhanced NKL cell-mediated natural cytotoxicity against human leukemia cells hIL-15gene modification of NKL cells could enhance the natural cytotoxicity against human leukemia cells.2. The products of IL-15gene-modified NKL cells decreased the proliferation of leukemia cells and induced the expression of NKG2D ligands in leukemia cells IL-15gene-modified NKL cells decreased the proliferation and up-regulated the expression of NKG2D ligands in leukemia cells by secreting some certain factors, which promoted the natural cytotoxicity of NKL cells against human leukemia cells.
3. IL-15gene-modification mediated the enhancement of NKL cell cytotoxicity was dependent on NKG2D and IFN-γ The high cytotoxicity mediated by NKL-IL15cells against K562cells depended on NKG2D and IFN-γ.
4. IL-15gene-modification improved the antitumor effects of NK cells in vivo The treatment of NKL-IL15cells was effective in suppressing tumor growth and prolong the survival time of leukemia-bearing mice.
5. NKL-IL15cells activated hPBMCs and enhanced the natural cytotoxicity against leukemia IL-15gene-modified NK cells could display active effects on hPBMCs cells.
Conclusion
Firstly, IFN-α gene-modified NKL cells could be suitable for the development of cell-based immunotherapeutic strategies for hepatocellular carcinoma.
Secondly, hIL-15gene-modified human natural killer cells exerted augmented anti-human hepatocellular carcinoma and anti-leukemia effect in vivo. The results suggested that hIL-15gene-modified NKL cells could be a promising new candidate for adoptive immunotherapy in the future.
We could gene-modified NK cell lines with cytokines which could induce the cytotoxicity effect to be suitable for the use of adoptive immunotherapy. The use of gene-modified NK cell lines may represent a novel and potential immunotherapeutic strategy in the future.
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