树突状细胞在肝癌免疫治疗中的应用研究
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摘要
目的
肝细胞癌(hepatocellular carcinoma,HCC)是世界上主要的恶性肿瘤之一。大多数肝癌患者诊断时已经不能再进行手术。尽管有一些姑息治疗方法可供选择,但复发和肝内转移率都非常高。因此,非常有必要开发新的治疗方法如免疫基因治疗来降低肿瘤的复发率。
树突状细胞(dendritic cell,DC)是一类抗原提呈细胞并始动大多数免疫应答。其能够非常有效的捕获,运输并提呈抗原,以及吸引和活化特异性T淋巴细胞,这使得成熟的树突状细胞成为已知最强大的抗原提呈细胞(antigen presenting cell,APC)。一个很重要的事实是,树突状细胞能够提呈肿瘤相关抗原,以主要组织相容性复合体Ⅰ类和Ⅱ类分子为基础,它们不仅能刺激和活化细胞毒性T淋巴细胞(cytotoxic T cell,CTL),也能刺激和活化辅助T细胞。
最近,一些研究表明:在IFN-α和GM-CSF共同刺激下体外培养单核细胞3天就可诱导生成树突状细胞。然而,目前对IFN-γ在树突状细胞发育和功能方面作用的相关研究不多。IFN-γ是一种具有多种生物活性的免疫调节因子,在细胞及体液免疫中起着重要作用,包括上调靶细胞表面MHC-Ⅰ类分子,增强CTL细胞对肿瘤和病毒感染的靶细胞的杀伤作用;促进CD4+初始T细胞向Th1细胞分化,增强细胞免疫功能;对IL-4具有拮抗作用,可抑制Th2细胞的生成,降低体液免疫功能等。我们在常规培养条件中加入IFN-γ,以观察IFN-γ对培养的树突状细胞形态学、细胞表型、吞噬功能和迁移能力以及刺激成熟后IL-12分泌的影响,并通过MTT法检测成熟树突状细胞对T淋巴细胞增殖能力的作用。
glypican-3与AFP相似,是一种癌胚胎肝脏蛋白,在胎儿肝脏中高表达,但是在正常成人肝脏中无表达。glypican-3在肝癌细胞中过表达,而在成人正常组织,癌旁组织,肝炎和肝硬化组织中仅低表达或不表达。本研究中,我们使树突状细胞提呈完整的磷脂酰肌醇蛋白聚糖-3分子给T细胞用以产生抗肝癌的细胞毒性T细胞,带有更多的表位包括主要和次要组织相容性抗原。
很多体外实验研究表明,通过负载肝癌抗原的树突状细胞疫苗,可诱导出特异性较强的免疫应答,然而,如何有效地筛选出最为有效的致敏树突状细胞的途径仍无定论。本研究将GPC3作为肝癌免疫治疗的切入点,体外应用GPC3基因转染的方法致敏树突状细胞,并与肝癌裂解物负载树突状细胞的方法相比较。
方法
人外周血单个核细胞的获取及体外诱导生成树突状细胞:取健康成人外周血,肝素抗凝后,用Ficoll密度梯度离心法分离PBMC,分离步骤简述如下:将肝素化的新鲜外周全血用生理盐水稀释1倍,在15mL离心管内加入3mL淋巴细胞分离液,再缓慢注入稀释血液6mL,20℃250×g离心20min,吸取界面层单个核细胞,洗涤2次,备用诱导DC。将Ficoll法分离出的PBMC用RPMI 1640培养液重悬,并添加2mM L-谷氨酰胺,100U/mL青霉素,100mg/L链霉素和10%胎牛血清,接种到6孔培养板进行培养。37℃2h后,将悬浮细胞转移至另一培养板进行培养,备用作为T细胞;贴壁的细胞继续培养并添加100ng/mL GM-CSF和50ng/mL IL-4两种刺激因子,进行DC培养。
致敏树突状细胞:主要为树突状细胞培养第5天,对细胞进行转染pEF-hGPC3和空载体质粒。24小时后,除去质粒并用含有细胞因子的普通树突状细胞培养基重悬细胞。之后再过2天,获得大量的未贴壁细胞作为刺激细胞;主要为将1×10~7个HepG2细胞用生理盐水洗涤3次,加入100μL生理盐水,置液氮中,10min后迅速置入37℃水浴中,待其完全融化后,再次放入液氮中,反复3次,0.2μm微孔滤膜过滤,上清液进行蛋白定量,调整浓度至100mg/L,然后加入树突状细胞培养液中,进行负载肿瘤抗原。
电镜分析:收集两组树突状细胞悬液(1×10~6/mL),1000 r/min离心10 min,弃去上清液,加入4℃预冷的2.5%戊二醛,4℃固定2h,漂洗;用PBS缓冲液浸洗2次,每次10min,再用4℃预冷的1%锇酸进行后固定2h,PBS缓冲液浸洗2次,梯度酒精脱水;醋酸异戊酯置换15min,冷冻干燥,真空喷金,扫描电镜观察摄片。
细胞免疫化学检测转染后GPC3在DCs中的表达细胞膜或细胞质中出现棕黄色颗粒视为GPC3阳性表达细胞。
Western blot分析GPC3基因的表达:用适量体积的裂解缓冲液含有150mMNaCl,50mM Tris pH7.4,1%NP-40,1mM Na3VO4,1mM EDTA和1mM PMSF。裂解后的上清进行10%SDS-PAGE凝胶电泳,之后转移至PVDF膜上。用含有5%脱脂奶粉和0.2%Tween-20的TBS封闭,再分别用鼠单克隆抗-GPC3和抗-β-actin抗体4℃孵育过夜,用含有0.2%Tween-20的TBS充分漂洗后,再用山羊抗鼠IgG进行检测,最后DAB显色。
树突状细胞表面表型的检测:分别在两组培养的第6d、8d收集树突状细胞并制成单细胞悬液(轻轻吹打并用细胞刮刀轻轻刮取贴壁部分的细胞),计数1×10~6/mL,用PBS悬浮并离心(250g,5min)洗涤2次去除上清,调整细胞密度至1×106/mL,各测量管加入细胞悬液500μL,再分别加入FITC标记的CD83、CD80和CD86荧光单克隆抗体,置4℃避光反应标记40min,PBS洗2次,免疫染色后的细胞悬于含有0.5%多聚甲醛的PBS细胞固定液中。24h内上机流式细胞仪进行树突状细胞表型分析。
树突状细胞摄取FITC-dextran能力的检测:简述如下:将两组培养的树突状细胞分别用PBS缓冲液洗涤、悬浮计数后,调整细胞浓度为1×10~6/mL。然后,分别取150μL细胞悬液,加入150μL FITC-dextran(浓度为1mg/mL);同时另取150μL细胞悬液,加入150μL PBS缓冲液置于37℃,5%CO2条件下孵育1h,用PBS液洗涤4次,并在流式细胞仪上进行分析。每个标本检测10~4个细胞以确定FITC-dextran阳性的细胞比率和相对荧光密度(relative fluorescence intensity,RFI)。阳性细胞比率和相对荧光密度经Cell Quest软件计算所得。
树突状细胞迁移能力测定及上清液中IL-12检测:采用Transwell小室法,经TNF-α刺激生成成熟后,取2组树突状细胞各1×10~5个,分别加于Transwell小室的上室中,100μL;下室加入含50ng/mL CCL21的RPMI-1640培养基600μL,置于37℃,5%CO2条件下培养120min,结束前30min,向下室加入5mM EDTA,然后收集下室的培养液中迁移的细胞通过镜下计数,从而计算出迁移率,即迁移率=下室细胞数/总细胞数;并留取培养第8d的树突状细胞上清,用ELISA法检测IL-12的浓度。
体外混合淋巴细胞反应(MLR):调整上述效应T细胞的浓度为10~5个细胞/孔,进行所有分析。由50ng/mL TNF-α和1μg/mL PG-E2作用48小时,促进树突状细胞成熟,之后再用25μg/mL丝裂霉素C(Sigma-Aldrich,Los Angeles,USA)37℃处理30分钟,用培养基漂洗细胞3次,再接种在培养板里。树突状细胞以不同比例与效应T细胞混合。不与效应T细胞混合的树突状细胞作为对照。应用96孔板设置3个复孔培养混合细胞2天,检测前4小时加入20μL WST-1。测定检测波长(450nm)和参考波长(650nm)处的吸光度值。每组细胞均以本底吸光度值作为参考。刺激指数(SI)的计算公式为:SI=实验组OD值/(效应T细胞组OD值+刺激细胞组OD值)。
乳酸脱氢酶法测定细胞毒性:采用LDH细胞毒作用检测试剂盒,刺激后的效应T细胞作为效应细胞,而HepG2及SW620细胞作为靶细胞。简单来说,将靶细胞和效应细胞重悬于分析培养基中(含1%牛血清白蛋白的RPMI 1640培养基),在96-孔板中靶细胞(10~4个/孔)与效应细胞37℃条件下以不同比例混合培养。孵育5小时后,对培养板离心后将上清(100μL/孔)转移至另一个ELISA板里。100μL/孔加入LDH检测混合物,于暗室内室温孵育30分钟。每孔加入50μl中止缓冲液后,于490nm波长处测定样品的吸光度值,以650nm作为参考波长。靶细胞或效应细胞的LDH自发释放吸光度值由无效应细胞条件下靶细胞的吸光度值确定,反之亦然。LDH的最大释放由在含1%Triton X-100的分析培养基中孵育的靶细胞的吸光度值确定。特异细胞介导的细胞毒作用百分比的计算公式为:特异细胞毒作用(%)=[(效应细胞与靶细胞混合-效应细胞自发-靶细胞自发)/(最大-靶细胞自发)]×100%。
统计学分析:应用SPSS11.0软件包进行数据分析及处理,p＜0.05时具有统计学意义,p＜0.01具有显著性差异。
结果
树突状细胞体外培养形态:分离人外周血单个核细胞,加入细胞因子培养后第1d及6d,两组在高倍倒置显微镜下观察,形态大致相同,可见贴壁细胞,体积较小,绝大多数为圆形,仅有少量的半悬浮细胞,随后悬浮细胞开始逐渐增加,体积也逐渐变大,形态变得不规则;继续培养至第8d,刺激成熟后进行电镜观察,可见IFN-γ组树突状细胞表面较常规组细胞更为粗糙,形态呈不规则状,并有大量的皱折和不规则突起,明显区别于常规组,由此形态可推断出IFN-γ可增加树突状细胞接触抗原的有效面积,从而增加其摄取抗原的机会。培养第6d,IFN-γ组树突状细胞表面CD80、CD86的表达率分别为45.8%±7.5%、83.5%±6.2%明显高于常规组(15.3%±5.7%、59.8%±7.6%),p＜0.05,而CD83表达率为9.5%±3.6%与常规组(7.8%±5.3%)近似;TNF-α刺激生成成熟后,两组的CD80、CD86、CD83表达均有所升高,但仍以IFN-γ组(96.8%±2.8%、95.3%±3.9%、87.3%±6.5%)明显,p＜0.05,而常规组分别为88.6%±5.6%、91.6±4.7%及75.6%±8.1%。
IFN-γ对树突状细胞摄取抗原能力的影响:由于未成熟的树突状细胞转变为成熟的树突状细胞,不仅成熟的标志和共刺激分子表达上调,而且抗原捕获能力也会下降,所以本研究主要为未成熟的树突状细胞的吞噬能力。IFN-γ组树突状细胞中FITC-dextran阳性细胞比率为85.5%±4.5%,相对荧光密度为839.9±46.8;常规组的阳性细胞比率及相对荧光密度分别为68.9%±8.6%、592.3±41.7,两组间存在显著性差异。
IFN-γ对树突状细胞迁移能力及分泌IL-12的影响:尽管一些炎症介质如IFN-α、TNF-α等可上调抗原提呈细胞MHC-Ⅱ类分子的表达水平,提高其抗原提呈能力并能促进其分泌IL-12,但是,IFN-γ对树突状细胞的迁移能力及分泌IL-12的影响仍未可知。Mailliard RB曾报道:常规方法体外培养树突状细胞体内及体外均有较强的迁移能力,但是并不分泌IL-12。由此,为了使以树突状细胞为基础的肿瘤免疫治疗活在体内能诱导出有效的Th1抗肿瘤的免疫应答,必须增强其分泌IL-12的能力。流式细胞仪结果表明:IFN-γ组细胞的迁移率为47.0%±5.5%,而常规组为50.4%±3.8%,两组无明显差别(p＞0.05);而ELISA法检测IL-12的结果显示:IFN-γ组的上清液中的IL-12量为1103.6pg/mL±147.0 pg/mL,而常规组则为708.0pg/mL±170.7pg/mL,两组间存在显著性差异(p＜0.05),提示IFN-γ可明显提高树突状细胞对IL-12的分泌。
在相差显微镜下观察,我们发现转染了pEF-hGPC3质粒的DCs具有相似的形态学特征,我们进行了免疫细胞化学染色方法来证实转染了pEF-hGPC3质粒的DCs成功表达GPC3靶抗原。Western blot结果显示转染成功而且转染效率较高。转染了pEF-hGPC3质粒的DCs中GPC3的表达由单克隆抗体检测到。贴壁的外周血单核细胞经GM-CSF和IL-4培养6天后,用pEF-hGPC3质粒转染不成熟DCs48小时。抗GPC3抗体对细胞进行免疫细胞化学染色。在pEF-hGPC3质粒转染的不成熟DCs中检测到GPC3胞浆或胞膜着色。而在空载体转染的不成熟DCs中未发现GPC3表达。
效应T细胞的增殖由WST-1方法检测,对不同组间的SI值进行了统计学分析比较。GPC3修饰的DCs刺激的效应T细胞的增殖情况在不同组间没有显著差异,1:10(DC:R)时:3.25±0.33、3.11±0.19、3.09±0.20;1:50(DC:R)时:2.08±0.13、2.09±0.43、1.94±0.78;1:100(DC:R)时:1.20±0.20、1.12±0.20、1.37±0.50,p＞0.05。IFN-γ是一种Th1相关细胞因子,主要参与细胞介导免疫应答的发生发展,因此我们也对DCs刺激T细胞分泌IFN-γ的情况进行了分析。IFN-γ的分泌由ELISA方法检测,并比较不同组间上清液中IFN-γ浓度的差异(p＞0.05)。GPC3修饰的DCs中IFN-γ的浓度在不同组间没有显著差异,不论DC/R比例多少,1:10(DC:R)时,516.78 pg/mL±25.86 pg/mL、484.31 pg/mL±22.84 pg/mL、505.22pg/mL±27.08 pg/mL;1:50(DC:R)时:259.04 pg/mL±52.33 pg/mL、227.12pg/mL±26.87 pg/mL、238.24 pg/mL±8.01 pg/mL;1:100(DC:R)时:183.62pg/mL±40.27 pg/mL、197.69 pg/mL±35.84 pg/mL、190.69 pg/mL±12.04 pg/mL。
我们选择了HCC细胞系HepG2作为GPC3特异性CTLs的靶细胞,因为其表达GPC3。效应细胞抗HepG2的细胞毒作用由LDH方法检测,HepG2裂解物负载和GPC3转染的DCs诱导的特异性细胞毒作用。结果证实转染了GPC3的DCs刺激的效应细胞能够有效裂解表达GPC3的HepG2细胞(p＜0.01)。10.1(E:T)时杀伤率:20.02%±4.00%、8.45%±2.25%、7.1 0%±3.24%,p＜0.01;50:1(E:T)时杀伤率:35.36%±5.34%、12.12%±3.44%、13.84%±2.84%,p＜0.01;100:1(E:T)时杀伤率:37.51%±1.77%、11.72%±2.15%、14.71%±4.38%,p＜0.01。
致敏树突状细胞后,GPC3基因表达蛋白的测定:通过Western blot检测各组肝癌相关抗原蛋白一磷脂酰肌醇蛋白聚糖-3的表达,其结果显示GPC3转染组和HepG2裂解物负载组均有表达磷脂酰肌醇蛋白聚糖-3蛋白,但是尤以前者更为明显,条带明显强于后者,而其他两组未见磷脂酰肌醇蛋白聚糖-3条带。
树突状细胞表面CD80、CD86和CD83的表达:培养第6d,TNF-α刺激成熟前,肿瘤裂解物负载组树突状细胞表面CD80、CD86及CD83的表达率分别为55.8%±7.5%、83.5%±6.2%和19.5%±5.2%明显高于GPC3转染组(20.3%±6.7%、59.8%±6.6%及9.8%±3.6%)及其他两对照组,p＜0.05;TNF-α和PGE2刺激生成成熟后,四组的CD80、CD86、CD83表达均有所升高,最终三种表面分子的表达,各组间未见显著性差异。
我们选择了HCC细胞系HepG2和结肠癌细胞系SW620作为GPC3特异性CTLs的靶细胞,因为其表达GPC3。效应细胞抗HepG2的细胞毒作用由LDH方法检测,HepG2裂解物负载和GPC3转染的树突状细胞诱导的特异性细胞毒作用。LDH方法分析各组效应细胞对HepG2细胞毒作用:对照组、裂解物负载组、空质粒组和GPC3转染组杀伤率分别为:10:1(E:T),4.10%±1.24%、16.52%±3.60%、3.45%±1.25%、20.02%±4.00%;50:1(E:T),5.84%±2.84%、38.36%±4.34%、4.12%±1.44%、35.73%±5.34%;100:1(E:T),9.71%±4.38%、40.51%±3.77%、8.72%±2.15%、38.65%±5.57%。各组效应细胞对SW620细胞毒作用:10:1(E:T),5.68%±2.56%、15.57%±5.17%、4.51%±1.95%、6.02%±2.68%;50:1(E:T),5.84%±1.83%、23.16%±5.69%、5.53%±2.68%、7.35%±3.52%;100:1(E:T),10.71%±3.37%、31.58%±4.28%、8.32%±3.54%、9.68%±3.31%。结果证实转染了GPC3的树突状细胞刺激的效应细胞能够有效裂解表达GPC3的HepG2细胞。
结论
1、IFN-γ明显增强正常人DC的功能,可为体外制作以DC为基础的肿瘤疫苗提供更为有效的培养方法,提高瘤苗的免疫活性。
2、GPC3基因修饰DC,能够有效地诱导针对HepG2肝癌细胞的细胞免疫反应,GPC3为基础免疫治疗开启了一条新的肝癌治疗途径。
3、GPC3基因转染DC产生的对肝癌细胞杀伤作用较裂解物负载的方法更具有特异性,理论上证明前者更适合临床应用,减少因此产生自身免疫反应的不良后果的可能。
Objective
Hepatocellular carcinoma(HCC)is one of the major malignancies worldwide. Most of the HCC patients are inoperable at the time of diagnosis.Despite several palliative therapeutic options there is a high rate of recurrence or of intra-hepatic metastases.Therefore,novel treatment strategies such as immuno-gene therapy are necessary to be developed to lower the frequency of tumor recurrence.
Dendritic cells(DCs)represent the class of antigen presenting cells(APCs)that initiate most immune responses.And their unique efficiency in capturing,transporting, and presenting antigen,as well as attracting and activating specific T cells,make mature DCs the most potent APCs known.Critical to the antigen-presenting function of DCs is the fact that they can present tumor-associated antigens and stimulate cytotoxicity T cells to induce specific immune responses in the context of both MHC classⅠandⅡ; thus,they can stimulate both cytotoxicity and helper T cells.Much attention has been directed to the problem of how and what antigens should be pulsed to DCs.At present, DCs pulsed with tumor-associated antigens in various forms,including whole cell lysate,peptides,proteins,RNA or DNA,have been studied for anti-tumor effects in experimental tumor models.In these models,immunization with tumor antigens presented by DCs has been shown much promise in effectively priming the cellular immune response.Some clinical applications using DC-based tumor vaccines have been reported.Although anti-tumor cellular immune responses could be induced by DCs vaccination in most patients,clinical objective responses were limited in tumor models.Thus,a new strategy for DC-based tumor vaccines is expected to improve the clinical effectiveness of treatment.In this regard,we exerted GPC-3 modification of DCs for presenting tumor antigen to T cells so as to produce specific cytotoxicity against HCC with minimal side effects.
Glypican-3(GPC-3)potentially behaves as an oncofetal liver protein. Overexpression of GPC-3 is detected in fetal liver,whereas it is absent in normal adult liver and benign hepatocellular nodules,including cases of focal nodular hyperplasia and adenoma.And some researches have reported that GPC-3 was overexpressed in most HCC.By comparing AFP and GPC-3 immunostaining in different types of HCC, Wang et al.confirmed that GPC-3 is more sensitive,especially in HCC developed from cirrhosis.In general,oncofetal proteins do not seem to play a critical role in tumor progression,but may be used as tumor markers or as targets for immunotherapy. Hiroyuki Komori et al.reported that they had identified the HLA-A2 restricted cytotoxicity epitopes possibly useful for GPC-3 specific immunotherapy of HCC and raised the possibility that some GPC-3 peptides may be applicable to cancer therapy.In this study,human peripheral blood monocytes derived DCs were obtained,and HepG2 lysate pulsed DCs had more power in promoting effector cells proliferation and IFN-γproduction than GPC-3 transfected DCs only at the ratio of 1:10.However,DCs with GPC-3 modified showed intensive ability to induce highly specific cytotoxicity against HepG2 cells.Thus,genetic modification of human monocyte-derived DCs with cDNA sequences encoding GPC-3 might be a promising strategy for effective HCC immunotherapy.
Methods
Generation of DCs from a healthy donor.DCs were generated from peripheral blood mononuclear cells(PBMCs),as described previously,with few modifications. Briefly,PBMCs were isolated from 50ml peripheral blood by using a Ficoll-Pague gradient(Haoyang,China).The cells were resuspended in complete medium(CM) using RPMI1640(Gibco,USA)supplemented with 10%FCS,100U/ml penicillin, 100mg/ml streptomycin,and allowed to adhere into a six-well plate.After 2h at 37℃, non-adherent cells were transferred to another plate as responder cells and adherent cells were cultured in CM supplemented with 100ng/ml recombinant human granulocyte-macrophage colony-stimulating factor(rhGM-CSF)and 50ng/ml interleukin-4(rhIL-4)(both from PeproTech,USA).Fresh medium containing cytokines was substituted every other day.
Cell lines culture.HCC cell line HepG2 and colon cancer cell line SW620 were selected in this study for their identical phenotype of HLA-A2+.The liver cell line THLE-3 was used as a normal control.The cells were all maintained in DMEM supplemented with 10%FCS,100U/ml penicillin and 100mg/ml streptomycin.Previous studies have demonstrated that GPC-3 was expressed specific for HepG2 cells,while SW620 cells and THLE-3 cells were GPC-3 negative,which was demonstrated preliminarily.
Preparations of HepG2 lysate.HepG2 cells were allowed to grow until confluent and washed three times in phosphate-buffered saline(PBS).Cells were collected and underwent 4 times of freeze(liquid nitrogen)and thaw(37℃)cycles,after which particles were removed by centrifugation.The supernatant was passed through a 0.2μm filter,and protein concentrations were determinated by BCA assay(Pierce,USA).
Generation of mature DCs with GPC-3 transfected or HepG2 lysate pulsed.The pEF-hGPC-3 plasmid containing full-length GPC-3 was generous gift from Dr.Jorge Filmus(Toronto University,Canada).5d after GM-CSF plus IL-4 incubation, transfections were performed according to the manufacturer's instructions.Briefly,cells were reuspended in FCS-free CM supplemented with 4μg plasmids plus 10μl Lipofectamine2000(Invitrogen,USA)and 100ng/ml TNF-α.Simultaneously,cells were reuspended in CM supplemented with 200μg HepG2 lysate and 100ng/ml TNF-α. 48h later,the non-adherent cells were harvested and regarded as mature DCs.
Flow cytometric analysis.The cell surface expression of DCs markers was assessed by flow cytometric analysis.Antibodies used to evaluate the phenotype of DCs were anti-CD80-FITC,anti-CD86-FITC,and anti-CD83-FITC(Biolegend,USA).DCs were stained for 40 min at 4℃and analyzed by the FACScan using CellQuest software. About ten thousand cells were examined for each determination.
Immunocytochemical staining.Cytospin preparations of GPC-3 transfected or HepG2 lysate pulsed DCs were fixed in 4%paraformaldehyde and incubated overnight with mouse monoclonal antibody detecting GPC-3(Santa Cruz,USA),following 5% rabbit serum treatment at 37℃for 1h.After which they were incubated with rabbit anti-mouse IgG and SP complex for 30min(both from Maixin,China),followed by DAB development.HCC cells were used as positive controls for GPC-3,and negative controls were prepared by non immune rabbit serum at the same dilution as for the primary antibody.Cells with brown particles appearing in membrane or cytoplasm were as regarded as positive cells.
Western blot.Cells were lysed in appropriate amounts of lysing buffer containing 150mM NaCl,50mM Tris,pH 7.4,1%NP-40,1mM Na3VO4,1mM EDTA and 1mM PMSF.The supernatants were electrophoresed on 10%SDS-PAGE and transferred to a PVDF membrane.After blocking with 5%BSA and 0.05%Tween-20 in Tris-buffered saline,the membrane was incubated with mouse monoclonal anti-GPC-3 and anti-β-actin antibodies(both from Santa Cruz,USA),finally subjected to chemiluminescence detection using goat anti-mouse IgG(Zhongshan,China),using a DAB kit(Maixin,China).
Proliferation of responder cells.The responder cells described above were adjusted at the concentration of 1×103 cells per well for all experiments.Matured DCs with various treatments were incubated with 25μg/ml mitomycin C(Sigma,USA)at 37℃for 30 min and subsequently mixed with responder cells at various DC-to-responder ratios of 1:10,1:50 and 1:100.DCs cultured in the absence of responder cells served as controls for background proliferation.Cells in triplicate wells of a 96-well plate were cultured for 48h and subsequently pulsed with 20μl WST-1 (Roche,Switzerland)during the last 4h of culture.The absorbance for measuring wavelength(450nm)and the reference wavelength(650nm)were measured.Stimulator index(SI)represented proliferation of responder cells and was calculated as follows: SI=A experiment/(A responder cells+A DCs).
IFN-γsecretion.Supernatants of the cultures were harvested and assayed in triplicate by ELISA using commercially available reagents(BD,USA),which were performed according to the manufacturer's instructions.The concentrations of IFN-γin the supernatants were determined by regression analysis.
Cytotoxicity assay.The responder cells stimulated by DCs with various treatments mentioned above were used as effector cells in the cytotoxicity assay using LDH cytotoxicity detection kit(Roche,USA).HepG2,THLE-3 and SW620 were used as target cells in this assay.Briefly,target cells and effector cells were resuspended in assay medium(RPMI 1640 supplemented with 1%BSA),and then target cells(103 cells per well)were cocultured with effector cells at different effector-to-target ratios of 10:1,50:1 and 100:1 in 96-well plate at 37℃.After 24h of incubation,the plate was centrifuged and the supernatant was removed.100μl per well LDH detection mixture was then added and incubated in the dark for 30 min at room temperature.After adding 50ul stop solution per well,the absorbance of each sample was measured at 490 nm with 650 nm as reference.The spontaneous release of LDH by target cells or effector cells was assessed by incubation of target cells in the absence of effector cells and vice versa.The maximum release of LDH was determined by incubation of target cells in 1%TritonX-100 in assay medium.The percentage of specific cytotoxicity was determined as follows:cytotoxicity(%)=[(effector&target mixture-effector spontaneous-target spontaneous)/(maximum-target spontaneous)]×100.
Statistical analysis.The SPSS 13.0 software was applied to complete data processing.Independent-samples t-test was used to evaluate the differences between DCs with various treatments in responder cells proliferation and IFN-γsecretion,as well as cytotoxicity against target cells.All data were represented as mean±SD of three independent experiments.Results were considered statistically significant when the p-value was less than 0.05.
Results
These cells exhibited irregular shapes,and many fine processes at their edges.The expression of GPC-3 in GPC-3 transfected or HepG2 lysate pulsed immuture DCs were examined by immunocytochemistry.Positive cells were defined as cytoplasm or membrane staining.Compared with empty vector,the intense expression of GPC-3 was observed in GPC-3 transfected DCs,which suggested an effective transfection.In HepG2 lysate pulsed DCs,GPC-3 immunoreactivity was occasionally detected and poorly distributed.
Western blot was also performed to evaluate the expression of GPC-3 in GPC-3 transfected or HepG2 lysate pulsed immature DCs.GPC-3 transfected DCs expressed GPC-3,which was detected at a much lower level in HepG2 lysate pulsed DCs, demonstrating a successful uptake of GPC-3 antigen by DCs.These date indicated that GPC-3 transfected DCs might have the ability to effectively present the GPC-3 antigen to responder cells.
To evaluate the effects of exogenous GPC-3 or HepG2 lysate on the phenotypes of immature DCs,we analyzed the cell surface markers of DCs transfected with GPC-3 or pulsed with HepG2 lysate by flow cytometry.The intensity of expression of surface markers remained stable in GPC-3 transfected or HepG2 lysate pulsed DCs,compared with that in control immature DCs.These results suggest that both GPC-3 and HepG2 lysate had no apparent influence on DC phenotypes.
We also investigated whether GPC-3 or HepG2 lysate treated DCs could promote the activation of effector cells,which is represented by proliferation and IFN-γsecretion.Compared with the GPC-3 transfected DCs,HepG2 lysate pulsed DCs had more power to induce the proliferation of effector cells at the ratio of 1:10(4.05±0.08 vs 3.46±0.11,p=0.002).However,the proliferations of effector cells promoted by either GPC-3 transfected or HepG2 lysate pulsed DCs were no significant difference at the ratio of 1:10 and 1:50,respectively(p＞0.05).
After a culture period of another 48h,the levels of IFN-γin the supernatants of effector cells sitmulated by GPC-3 transfected and HepG2 lysate pulsed mature DCs,as well as the control cells were measured by ELISA.The effector cells stimulated by HepG2 lysate pulsed DCs producted more IFN-γthan GPC-3 transfected DCs at the ratio of 1:10(708.45±11.81 pg/ml vs 643.43±22.11 pg/ml,p=0.011),indicating that HepG2 lysate was efficient in promoting IFN-γproduction of effector cells to some extent.However,the IFN-γsecreted by effector cells in GPC-3 transfected DCs was observed nearly the same as that in HepG2 lysate pulsed DCs at the ratio of either 1:50 or 1:100(p＞0.05).
To identify the specificity and validity of GPC-3 modified DCs involved in the process of HepG2 cells being lysed,we selected another HLA-A2+human tumor cell line SW620,except for HepG2 cells,as target cells for cytotoxicity assay.HepG2 cells exhibit overexpression of GPC3,SW620 cells are GPC3 negative.The cytotoxicity against the two tumor cell lines were assessed by a standard LDH release assay.The effector cells stimulated by HepG2 lysate pulsed DCs showed intensive lytic activity of HepG2 and SW620 cells with no statistical significance(p＞0.05).However,effector cells stimulated with GPC-3 transfected DCs effectively lyse HepG2 cells,at the ratios of both 50:1 and 100:1(16.6%and 34.1%,respectively).On the other hand,the GPC3-null SW620 cells were only observed minimally lysed at the ratio of 100:1 (12.0%),yet the lytic activity of effector cells stimulated with HepG2 lysate pulsed DCs was significantly elevated at different ratios(p＜0.05).
Conclusions
1、IFN-γcan enhance the properties of DCs,and provide a better method of culturing DCs in vitro to increase the immune activity.
2、DCs modified with full-length GPC-3 could generate specific cytotoxicity against HepG2 cells,which may provide a new method of treating HCC.
3、Compared with lysate pulse,GPC3 genetically modified DCs induced specific cytotoxity against HCC cell in vitro,which have the potential to serve as novel vaccine for HCC with less nonspecific cytotoxicities.
肝细胞癌(hepatocellular carcinoma,HCC)是世界上主要的恶性肿瘤之一。大多数肝癌患者诊断时已经不能再进行手术。尽管有一些姑息治疗方法可供选择,但复发和肝内转移率都非常高。因此,非常有必要开发新的治疗方法如免疫基因治疗来降低肿瘤的复发率。
树突状细胞(dendritic cell,DC)是一类抗原提呈细胞并始动大多数免疫应答。其能够非常有效的捕获,运输并提呈抗原,以及吸引和活化特异性T淋巴细胞,这使得成熟的树突状细胞成为已知最强大的抗原提呈细胞(antigen presenting cell,APC)。一个很重要的事实是,树突状细胞能够提呈肿瘤相关抗原,以主要组织相容性复合体Ⅰ类和Ⅱ类分子为基础,它们不仅能刺激和活化细胞毒性T淋巴细胞(cytotoxic T cell,CTL),也能刺激和活化辅助T细胞。
最近,一些研究表明:在IFN-α和GM-CSF共同刺激下体外培养单核细胞3天就可诱导生成树突状细胞。然而,目前对IFN-γ在树突状细胞发育和功能方面作用的相关研究不多。IFN-γ是一种具有多种生物活性的免疫调节因子,在细胞及体液免疫中起着重要作用,包括上调靶细胞表面MHC-Ⅰ类分子,增强CTL细胞对肿瘤和病毒感染的靶细胞的杀伤作用;促进CD4+初始T细胞向Th1细胞分化,增强细胞免疫功能;对IL-4具有拮抗作用,可抑制Th2细胞的生成,降低体液免疫功能等。我们在常规培养条件中加入IFN-γ,以观察IFN-γ对培养的树突状细胞形态学、细胞表型、吞噬功能和迁移能力以及刺激成熟后IL-12分泌的影响,并通过MTT法检测成熟树突状细胞对T淋巴细胞增殖能力的作用。
glypican-3与AFP相似,是一种癌胚胎肝脏蛋白,在胎儿肝脏中高表达,但是在正常成人肝脏中无表达。glypican-3在肝癌细胞中过表达,而在成人正常组织,癌旁组织,肝炎和肝硬化组织中仅低表达或不表达。本研究中,我们使树突状细胞提呈完整的磷脂酰肌醇蛋白聚糖-3分子给T细胞用以产生抗肝癌的细胞毒性T细胞,带有更多的表位包括主要和次要组织相容性抗原。
很多体外实验研究表明,通过负载肝癌抗原的树突状细胞疫苗,可诱导出特异性较强的免疫应答,然而,如何有效地筛选出最为有效的致敏树突状细胞的途径仍无定论。本研究将GPC3作为肝癌免疫治疗的切入点,体外应用GPC3基因转染的方法致敏树突状细胞,并与肝癌裂解物负载树突状细胞的方法相比较。
方法
人外周血单个核细胞的获取及体外诱导生成树突状细胞:取健康成人外周血,肝素抗凝后,用Ficoll密度梯度离心法分离PBMC,分离步骤简述如下:将肝素化的新鲜外周全血用生理盐水稀释1倍,在15mL离心管内加入3mL淋巴细胞分离液,再缓慢注入稀释血液6mL,20℃250×g离心20min,吸取界面层单个核细胞,洗涤2次,备用诱导DC。将Ficoll法分离出的PBMC用RPMI 1640培养液重悬,并添加2mM L-谷氨酰胺,100U/mL青霉素,100mg/L链霉素和10%胎牛血清,接种到6孔培养板进行培养。37℃2h后,将悬浮细胞转移至另一培养板进行培养,备用作为T细胞;贴壁的细胞继续培养并添加100ng/mL GM-CSF和50ng/mL IL-4两种刺激因子,进行DC培养。
致敏树突状细胞:主要为树突状细胞培养第5天,对细胞进行转染pEF-hGPC3和空载体质粒。24小时后,除去质粒并用含有细胞因子的普通树突状细胞培养基重悬细胞。之后再过2天,获得大量的未贴壁细胞作为刺激细胞;主要为将1×10~7个HepG2细胞用生理盐水洗涤3次,加入100μL生理盐水,置液氮中,10min后迅速置入37℃水浴中,待其完全融化后,再次放入液氮中,反复3次,0.2μm微孔滤膜过滤,上清液进行蛋白定量,调整浓度至100mg/L,然后加入树突状细胞培养液中,进行负载肿瘤抗原。
电镜分析:收集两组树突状细胞悬液(1×10~6/mL),1000 r/min离心10 min,弃去上清液,加入4℃预冷的2.5%戊二醛,4℃固定2h,漂洗;用PBS缓冲液浸洗2次,每次10min,再用4℃预冷的1%锇酸进行后固定2h,PBS缓冲液浸洗2次,梯度酒精脱水;醋酸异戊酯置换15min,冷冻干燥,真空喷金,扫描电镜观察摄片。
细胞免疫化学检测转染后GPC3在DCs中的表达细胞膜或细胞质中出现棕黄色颗粒视为GPC3阳性表达细胞。
Western blot分析GPC3基因的表达:用适量体积的裂解缓冲液含有150mMNaCl,50mM Tris pH7.4,1%NP-40,1mM Na3VO4,1mM EDTA和1mM PMSF。裂解后的上清进行10%SDS-PAGE凝胶电泳,之后转移至PVDF膜上。用含有5%脱脂奶粉和0.2%Tween-20的TBS封闭,再分别用鼠单克隆抗-GPC3和抗-β-actin抗体4℃孵育过夜,用含有0.2%Tween-20的TBS充分漂洗后,再用山羊抗鼠IgG进行检测,最后DAB显色。
树突状细胞表面表型的检测:分别在两组培养的第6d、8d收集树突状细胞并制成单细胞悬液(轻轻吹打并用细胞刮刀轻轻刮取贴壁部分的细胞),计数1×10~6/mL,用PBS悬浮并离心(250g,5min)洗涤2次去除上清,调整细胞密度至1×106/mL,各测量管加入细胞悬液500μL,再分别加入FITC标记的CD83、CD80和CD86荧光单克隆抗体,置4℃避光反应标记40min,PBS洗2次,免疫染色后的细胞悬于含有0.5%多聚甲醛的PBS细胞固定液中。24h内上机流式细胞仪进行树突状细胞表型分析。
树突状细胞摄取FITC-dextran能力的检测:简述如下:将两组培养的树突状细胞分别用PBS缓冲液洗涤、悬浮计数后,调整细胞浓度为1×10~6/mL。然后,分别取150μL细胞悬液,加入150μL FITC-dextran(浓度为1mg/mL);同时另取150μL细胞悬液,加入150μL PBS缓冲液置于37℃,5%CO2条件下孵育1h,用PBS液洗涤4次,并在流式细胞仪上进行分析。每个标本检测10~4个细胞以确定FITC-dextran阳性的细胞比率和相对荧光密度(relative fluorescence intensity,RFI)。阳性细胞比率和相对荧光密度经Cell Quest软件计算所得。
树突状细胞迁移能力测定及上清液中IL-12检测:采用Transwell小室法,经TNF-α刺激生成成熟后,取2组树突状细胞各1×10~5个,分别加于Transwell小室的上室中,100μL;下室加入含50ng/mL CCL21的RPMI-1640培养基600μL,置于37℃,5%CO2条件下培养120min,结束前30min,向下室加入5mM EDTA,然后收集下室的培养液中迁移的细胞通过镜下计数,从而计算出迁移率,即迁移率=下室细胞数/总细胞数;并留取培养第8d的树突状细胞上清,用ELISA法检测IL-12的浓度。
体外混合淋巴细胞反应(MLR):调整上述效应T细胞的浓度为10~5个细胞/孔,进行所有分析。由50ng/mL TNF-α和1μg/mL PG-E2作用48小时,促进树突状细胞成熟,之后再用25μg/mL丝裂霉素C(Sigma-Aldrich,Los Angeles,USA)37℃处理30分钟,用培养基漂洗细胞3次,再接种在培养板里。树突状细胞以不同比例与效应T细胞混合。不与效应T细胞混合的树突状细胞作为对照。应用96孔板设置3个复孔培养混合细胞2天,检测前4小时加入20μL WST-1。测定检测波长(450nm)和参考波长(650nm)处的吸光度值。每组细胞均以本底吸光度值作为参考。刺激指数(SI)的计算公式为:SI=实验组OD值/(效应T细胞组OD值+刺激细胞组OD值)。
乳酸脱氢酶法测定细胞毒性:采用LDH细胞毒作用检测试剂盒,刺激后的效应T细胞作为效应细胞,而HepG2及SW620细胞作为靶细胞。简单来说,将靶细胞和效应细胞重悬于分析培养基中(含1%牛血清白蛋白的RPMI 1640培养基),在96-孔板中靶细胞(10~4个/孔)与效应细胞37℃条件下以不同比例混合培养。孵育5小时后,对培养板离心后将上清(100μL/孔)转移至另一个ELISA板里。100μL/孔加入LDH检测混合物,于暗室内室温孵育30分钟。每孔加入50μl中止缓冲液后,于490nm波长处测定样品的吸光度值,以650nm作为参考波长。靶细胞或效应细胞的LDH自发释放吸光度值由无效应细胞条件下靶细胞的吸光度值确定,反之亦然。LDH的最大释放由在含1%Triton X-100的分析培养基中孵育的靶细胞的吸光度值确定。特异细胞介导的细胞毒作用百分比的计算公式为:特异细胞毒作用(%)=[(效应细胞与靶细胞混合-效应细胞自发-靶细胞自发)/(最大-靶细胞自发)]×100%。
统计学分析:应用SPSS11.0软件包进行数据分析及处理,p＜0.05时具有统计学意义,p＜0.01具有显著性差异。
结果
树突状细胞体外培养形态:分离人外周血单个核细胞,加入细胞因子培养后第1d及6d,两组在高倍倒置显微镜下观察,形态大致相同,可见贴壁细胞,体积较小,绝大多数为圆形,仅有少量的半悬浮细胞,随后悬浮细胞开始逐渐增加,体积也逐渐变大,形态变得不规则;继续培养至第8d,刺激成熟后进行电镜观察,可见IFN-γ组树突状细胞表面较常规组细胞更为粗糙,形态呈不规则状,并有大量的皱折和不规则突起,明显区别于常规组,由此形态可推断出IFN-γ可增加树突状细胞接触抗原的有效面积,从而增加其摄取抗原的机会。培养第6d,IFN-γ组树突状细胞表面CD80、CD86的表达率分别为45.8%±7.5%、83.5%±6.2%明显高于常规组(15.3%±5.7%、59.8%±7.6%),p＜0.05,而CD83表达率为9.5%±3.6%与常规组(7.8%±5.3%)近似;TNF-α刺激生成成熟后,两组的CD80、CD86、CD83表达均有所升高,但仍以IFN-γ组(96.8%±2.8%、95.3%±3.9%、87.3%±6.5%)明显,p＜0.05,而常规组分别为88.6%±5.6%、91.6±4.7%及75.6%±8.1%。
IFN-γ对树突状细胞摄取抗原能力的影响:由于未成熟的树突状细胞转变为成熟的树突状细胞,不仅成熟的标志和共刺激分子表达上调,而且抗原捕获能力也会下降,所以本研究主要为未成熟的树突状细胞的吞噬能力。IFN-γ组树突状细胞中FITC-dextran阳性细胞比率为85.5%±4.5%,相对荧光密度为839.9±46.8;常规组的阳性细胞比率及相对荧光密度分别为68.9%±8.6%、592.3±41.7,两组间存在显著性差异。
IFN-γ对树突状细胞迁移能力及分泌IL-12的影响:尽管一些炎症介质如IFN-α、TNF-α等可上调抗原提呈细胞MHC-Ⅱ类分子的表达水平,提高其抗原提呈能力并能促进其分泌IL-12,但是,IFN-γ对树突状细胞的迁移能力及分泌IL-12的影响仍未可知。Mailliard RB曾报道:常规方法体外培养树突状细胞体内及体外均有较强的迁移能力,但是并不分泌IL-12。由此,为了使以树突状细胞为基础的肿瘤免疫治疗活在体内能诱导出有效的Th1抗肿瘤的免疫应答,必须增强其分泌IL-12的能力。流式细胞仪结果表明:IFN-γ组细胞的迁移率为47.0%±5.5%,而常规组为50.4%±3.8%,两组无明显差别(p＞0.05);而ELISA法检测IL-12的结果显示:IFN-γ组的上清液中的IL-12量为1103.6pg/mL±147.0 pg/mL,而常规组则为708.0pg/mL±170.7pg/mL,两组间存在显著性差异(p＜0.05),提示IFN-γ可明显提高树突状细胞对IL-12的分泌。
在相差显微镜下观察,我们发现转染了pEF-hGPC3质粒的DCs具有相似的形态学特征,我们进行了免疫细胞化学染色方法来证实转染了pEF-hGPC3质粒的DCs成功表达GPC3靶抗原。Western blot结果显示转染成功而且转染效率较高。转染了pEF-hGPC3质粒的DCs中GPC3的表达由单克隆抗体检测到。贴壁的外周血单核细胞经GM-CSF和IL-4培养6天后,用pEF-hGPC3质粒转染不成熟DCs48小时。抗GPC3抗体对细胞进行免疫细胞化学染色。在pEF-hGPC3质粒转染的不成熟DCs中检测到GPC3胞浆或胞膜着色。而在空载体转染的不成熟DCs中未发现GPC3表达。
效应T细胞的增殖由WST-1方法检测,对不同组间的SI值进行了统计学分析比较。GPC3修饰的DCs刺激的效应T细胞的增殖情况在不同组间没有显著差异,1:10(DC:R)时:3.25±0.33、3.11±0.19、3.09±0.20;1:50(DC:R)时:2.08±0.13、2.09±0.43、1.94±0.78;1:100(DC:R)时:1.20±0.20、1.12±0.20、1.37±0.50,p＞0.05。IFN-γ是一种Th1相关细胞因子,主要参与细胞介导免疫应答的发生发展,因此我们也对DCs刺激T细胞分泌IFN-γ的情况进行了分析。IFN-γ的分泌由ELISA方法检测,并比较不同组间上清液中IFN-γ浓度的差异(p＞0.05)。GPC3修饰的DCs中IFN-γ的浓度在不同组间没有显著差异,不论DC/R比例多少,1:10(DC:R)时,516.78 pg/mL±25.86 pg/mL、484.31 pg/mL±22.84 pg/mL、505.22pg/mL±27.08 pg/mL;1:50(DC:R)时:259.04 pg/mL±52.33 pg/mL、227.12pg/mL±26.87 pg/mL、238.24 pg/mL±8.01 pg/mL;1:100(DC:R)时:183.62pg/mL±40.27 pg/mL、197.69 pg/mL±35.84 pg/mL、190.69 pg/mL±12.04 pg/mL。
我们选择了HCC细胞系HepG2作为GPC3特异性CTLs的靶细胞,因为其表达GPC3。效应细胞抗HepG2的细胞毒作用由LDH方法检测,HepG2裂解物负载和GPC3转染的DCs诱导的特异性细胞毒作用。结果证实转染了GPC3的DCs刺激的效应细胞能够有效裂解表达GPC3的HepG2细胞(p＜0.01)。10.1(E:T)时杀伤率:20.02%±4.00%、8.45%±2.25%、7.1 0%±3.24%,p＜0.01;50:1(E:T)时杀伤率:35.36%±5.34%、12.12%±3.44%、13.84%±2.84%,p＜0.01;100:1(E:T)时杀伤率:37.51%±1.77%、11.72%±2.15%、14.71%±4.38%,p＜0.01。
致敏树突状细胞后,GPC3基因表达蛋白的测定:通过Western blot检测各组肝癌相关抗原蛋白一磷脂酰肌醇蛋白聚糖-3的表达,其结果显示GPC3转染组和HepG2裂解物负载组均有表达磷脂酰肌醇蛋白聚糖-3蛋白,但是尤以前者更为明显,条带明显强于后者,而其他两组未见磷脂酰肌醇蛋白聚糖-3条带。
树突状细胞表面CD80、CD86和CD83的表达:培养第6d,TNF-α刺激成熟前,肿瘤裂解物负载组树突状细胞表面CD80、CD86及CD83的表达率分别为55.8%±7.5%、83.5%±6.2%和19.5%±5.2%明显高于GPC3转染组(20.3%±6.7%、59.8%±6.6%及9.8%±3.6%)及其他两对照组,p＜0.05;TNF-α和PGE2刺激生成成熟后,四组的CD80、CD86、CD83表达均有所升高,最终三种表面分子的表达,各组间未见显著性差异。
我们选择了HCC细胞系HepG2和结肠癌细胞系SW620作为GPC3特异性CTLs的靶细胞,因为其表达GPC3。效应细胞抗HepG2的细胞毒作用由LDH方法检测,HepG2裂解物负载和GPC3转染的树突状细胞诱导的特异性细胞毒作用。LDH方法分析各组效应细胞对HepG2细胞毒作用:对照组、裂解物负载组、空质粒组和GPC3转染组杀伤率分别为:10:1(E:T),4.10%±1.24%、16.52%±3.60%、3.45%±1.25%、20.02%±4.00%;50:1(E:T),5.84%±2.84%、38.36%±4.34%、4.12%±1.44%、35.73%±5.34%;100:1(E:T),9.71%±4.38%、40.51%±3.77%、8.72%±2.15%、38.65%±5.57%。各组效应细胞对SW620细胞毒作用:10:1(E:T),5.68%±2.56%、15.57%±5.17%、4.51%±1.95%、6.02%±2.68%;50:1(E:T),5.84%±1.83%、23.16%±5.69%、5.53%±2.68%、7.35%±3.52%;100:1(E:T),10.71%±3.37%、31.58%±4.28%、8.32%±3.54%、9.68%±3.31%。结果证实转染了GPC3的树突状细胞刺激的效应细胞能够有效裂解表达GPC3的HepG2细胞。
结论
1、IFN-γ明显增强正常人DC的功能,可为体外制作以DC为基础的肿瘤疫苗提供更为有效的培养方法,提高瘤苗的免疫活性。
2、GPC3基因修饰DC,能够有效地诱导针对HepG2肝癌细胞的细胞免疫反应,GPC3为基础免疫治疗开启了一条新的肝癌治疗途径。
3、GPC3基因转染DC产生的对肝癌细胞杀伤作用较裂解物负载的方法更具有特异性,理论上证明前者更适合临床应用,减少因此产生自身免疫反应的不良后果的可能。
Objective
Hepatocellular carcinoma(HCC)is one of the major malignancies worldwide. Most of the HCC patients are inoperable at the time of diagnosis.Despite several palliative therapeutic options there is a high rate of recurrence or of intra-hepatic metastases.Therefore,novel treatment strategies such as immuno-gene therapy are necessary to be developed to lower the frequency of tumor recurrence.
Dendritic cells(DCs)represent the class of antigen presenting cells(APCs)that initiate most immune responses.And their unique efficiency in capturing,transporting, and presenting antigen,as well as attracting and activating specific T cells,make mature DCs the most potent APCs known.Critical to the antigen-presenting function of DCs is the fact that they can present tumor-associated antigens and stimulate cytotoxicity T cells to induce specific immune responses in the context of both MHC classⅠandⅡ; thus,they can stimulate both cytotoxicity and helper T cells.Much attention has been directed to the problem of how and what antigens should be pulsed to DCs.At present, DCs pulsed with tumor-associated antigens in various forms,including whole cell lysate,peptides,proteins,RNA or DNA,have been studied for anti-tumor effects in experimental tumor models.In these models,immunization with tumor antigens presented by DCs has been shown much promise in effectively priming the cellular immune response.Some clinical applications using DC-based tumor vaccines have been reported.Although anti-tumor cellular immune responses could be induced by DCs vaccination in most patients,clinical objective responses were limited in tumor models.Thus,a new strategy for DC-based tumor vaccines is expected to improve the clinical effectiveness of treatment.In this regard,we exerted GPC-3 modification of DCs for presenting tumor antigen to T cells so as to produce specific cytotoxicity against HCC with minimal side effects.
Glypican-3(GPC-3)potentially behaves as an oncofetal liver protein. Overexpression of GPC-3 is detected in fetal liver,whereas it is absent in normal adult liver and benign hepatocellular nodules,including cases of focal nodular hyperplasia and adenoma.And some researches have reported that GPC-3 was overexpressed in most HCC.By comparing AFP and GPC-3 immunostaining in different types of HCC, Wang et al.confirmed that GPC-3 is more sensitive,especially in HCC developed from cirrhosis.In general,oncofetal proteins do not seem to play a critical role in tumor progression,but may be used as tumor markers or as targets for immunotherapy. Hiroyuki Komori et al.reported that they had identified the HLA-A2 restricted cytotoxicity epitopes possibly useful for GPC-3 specific immunotherapy of HCC and raised the possibility that some GPC-3 peptides may be applicable to cancer therapy.In this study,human peripheral blood monocytes derived DCs were obtained,and HepG2 lysate pulsed DCs had more power in promoting effector cells proliferation and IFN-γproduction than GPC-3 transfected DCs only at the ratio of 1:10.However,DCs with GPC-3 modified showed intensive ability to induce highly specific cytotoxicity against HepG2 cells.Thus,genetic modification of human monocyte-derived DCs with cDNA sequences encoding GPC-3 might be a promising strategy for effective HCC immunotherapy.
Methods
Generation of DCs from a healthy donor.DCs were generated from peripheral blood mononuclear cells(PBMCs),as described previously,with few modifications. Briefly,PBMCs were isolated from 50ml peripheral blood by using a Ficoll-Pague gradient(Haoyang,China).The cells were resuspended in complete medium(CM) using RPMI1640(Gibco,USA)supplemented with 10%FCS,100U/ml penicillin, 100mg/ml streptomycin,and allowed to adhere into a six-well plate.After 2h at 37℃, non-adherent cells were transferred to another plate as responder cells and adherent cells were cultured in CM supplemented with 100ng/ml recombinant human granulocyte-macrophage colony-stimulating factor(rhGM-CSF)and 50ng/ml interleukin-4(rhIL-4)(both from PeproTech,USA).Fresh medium containing cytokines was substituted every other day.
Cell lines culture.HCC cell line HepG2 and colon cancer cell line SW620 were selected in this study for their identical phenotype of HLA-A2+.The liver cell line THLE-3 was used as a normal control.The cells were all maintained in DMEM supplemented with 10%FCS,100U/ml penicillin and 100mg/ml streptomycin.Previous studies have demonstrated that GPC-3 was expressed specific for HepG2 cells,while SW620 cells and THLE-3 cells were GPC-3 negative,which was demonstrated preliminarily.
Preparations of HepG2 lysate.HepG2 cells were allowed to grow until confluent and washed three times in phosphate-buffered saline(PBS).Cells were collected and underwent 4 times of freeze(liquid nitrogen)and thaw(37℃)cycles,after which particles were removed by centrifugation.The supernatant was passed through a 0.2μm filter,and protein concentrations were determinated by BCA assay(Pierce,USA).
Generation of mature DCs with GPC-3 transfected or HepG2 lysate pulsed.The pEF-hGPC-3 plasmid containing full-length GPC-3 was generous gift from Dr.Jorge Filmus(Toronto University,Canada).5d after GM-CSF plus IL-4 incubation, transfections were performed according to the manufacturer's instructions.Briefly,cells were reuspended in FCS-free CM supplemented with 4μg plasmids plus 10μl Lipofectamine2000(Invitrogen,USA)and 100ng/ml TNF-α.Simultaneously,cells were reuspended in CM supplemented with 200μg HepG2 lysate and 100ng/ml TNF-α. 48h later,the non-adherent cells were harvested and regarded as mature DCs.
Flow cytometric analysis.The cell surface expression of DCs markers was assessed by flow cytometric analysis.Antibodies used to evaluate the phenotype of DCs were anti-CD80-FITC,anti-CD86-FITC,and anti-CD83-FITC(Biolegend,USA).DCs were stained for 40 min at 4℃and analyzed by the FACScan using CellQuest software. About ten thousand cells were examined for each determination.
Immunocytochemical staining.Cytospin preparations of GPC-3 transfected or HepG2 lysate pulsed DCs were fixed in 4%paraformaldehyde and incubated overnight with mouse monoclonal antibody detecting GPC-3(Santa Cruz,USA),following 5% rabbit serum treatment at 37℃for 1h.After which they were incubated with rabbit anti-mouse IgG and SP complex for 30min(both from Maixin,China),followed by DAB development.HCC cells were used as positive controls for GPC-3,and negative controls were prepared by non immune rabbit serum at the same dilution as for the primary antibody.Cells with brown particles appearing in membrane or cytoplasm were as regarded as positive cells.
Western blot.Cells were lysed in appropriate amounts of lysing buffer containing 150mM NaCl,50mM Tris,pH 7.4,1%NP-40,1mM Na3VO4,1mM EDTA and 1mM PMSF.The supernatants were electrophoresed on 10%SDS-PAGE and transferred to a PVDF membrane.After blocking with 5%BSA and 0.05%Tween-20 in Tris-buffered saline,the membrane was incubated with mouse monoclonal anti-GPC-3 and anti-β-actin antibodies(both from Santa Cruz,USA),finally subjected to chemiluminescence detection using goat anti-mouse IgG(Zhongshan,China),using a DAB kit(Maixin,China).
Proliferation of responder cells.The responder cells described above were adjusted at the concentration of 1×103 cells per well for all experiments.Matured DCs with various treatments were incubated with 25μg/ml mitomycin C(Sigma,USA)at 37℃for 30 min and subsequently mixed with responder cells at various DC-to-responder ratios of 1:10,1:50 and 1:100.DCs cultured in the absence of responder cells served as controls for background proliferation.Cells in triplicate wells of a 96-well plate were cultured for 48h and subsequently pulsed with 20μl WST-1 (Roche,Switzerland)during the last 4h of culture.The absorbance for measuring wavelength(450nm)and the reference wavelength(650nm)were measured.Stimulator index(SI)represented proliferation of responder cells and was calculated as follows: SI=A experiment/(A responder cells+A DCs).
IFN-γsecretion.Supernatants of the cultures were harvested and assayed in triplicate by ELISA using commercially available reagents(BD,USA),which were performed according to the manufacturer's instructions.The concentrations of IFN-γin the supernatants were determined by regression analysis.
Cytotoxicity assay.The responder cells stimulated by DCs with various treatments mentioned above were used as effector cells in the cytotoxicity assay using LDH cytotoxicity detection kit(Roche,USA).HepG2,THLE-3 and SW620 were used as target cells in this assay.Briefly,target cells and effector cells were resuspended in assay medium(RPMI 1640 supplemented with 1%BSA),and then target cells(103 cells per well)were cocultured with effector cells at different effector-to-target ratios of 10:1,50:1 and 100:1 in 96-well plate at 37℃.After 24h of incubation,the plate was centrifuged and the supernatant was removed.100μl per well LDH detection mixture was then added and incubated in the dark for 30 min at room temperature.After adding 50ul stop solution per well,the absorbance of each sample was measured at 490 nm with 650 nm as reference.The spontaneous release of LDH by target cells or effector cells was assessed by incubation of target cells in the absence of effector cells and vice versa.The maximum release of LDH was determined by incubation of target cells in 1%TritonX-100 in assay medium.The percentage of specific cytotoxicity was determined as follows:cytotoxicity(%)=[(effector&target mixture-effector spontaneous-target spontaneous)/(maximum-target spontaneous)]×100.
Statistical analysis.The SPSS 13.0 software was applied to complete data processing.Independent-samples t-test was used to evaluate the differences between DCs with various treatments in responder cells proliferation and IFN-γsecretion,as well as cytotoxicity against target cells.All data were represented as mean±SD of three independent experiments.Results were considered statistically significant when the p-value was less than 0.05.
Results
These cells exhibited irregular shapes,and many fine processes at their edges.The expression of GPC-3 in GPC-3 transfected or HepG2 lysate pulsed immuture DCs were examined by immunocytochemistry.Positive cells were defined as cytoplasm or membrane staining.Compared with empty vector,the intense expression of GPC-3 was observed in GPC-3 transfected DCs,which suggested an effective transfection.In HepG2 lysate pulsed DCs,GPC-3 immunoreactivity was occasionally detected and poorly distributed.
Western blot was also performed to evaluate the expression of GPC-3 in GPC-3 transfected or HepG2 lysate pulsed immature DCs.GPC-3 transfected DCs expressed GPC-3,which was detected at a much lower level in HepG2 lysate pulsed DCs, demonstrating a successful uptake of GPC-3 antigen by DCs.These date indicated that GPC-3 transfected DCs might have the ability to effectively present the GPC-3 antigen to responder cells.
To evaluate the effects of exogenous GPC-3 or HepG2 lysate on the phenotypes of immature DCs,we analyzed the cell surface markers of DCs transfected with GPC-3 or pulsed with HepG2 lysate by flow cytometry.The intensity of expression of surface markers remained stable in GPC-3 transfected or HepG2 lysate pulsed DCs,compared with that in control immature DCs.These results suggest that both GPC-3 and HepG2 lysate had no apparent influence on DC phenotypes.
We also investigated whether GPC-3 or HepG2 lysate treated DCs could promote the activation of effector cells,which is represented by proliferation and IFN-γsecretion.Compared with the GPC-3 transfected DCs,HepG2 lysate pulsed DCs had more power to induce the proliferation of effector cells at the ratio of 1:10(4.05±0.08 vs 3.46±0.11,p=0.002).However,the proliferations of effector cells promoted by either GPC-3 transfected or HepG2 lysate pulsed DCs were no significant difference at the ratio of 1:10 and 1:50,respectively(p＞0.05).
After a culture period of another 48h,the levels of IFN-γin the supernatants of effector cells sitmulated by GPC-3 transfected and HepG2 lysate pulsed mature DCs,as well as the control cells were measured by ELISA.The effector cells stimulated by HepG2 lysate pulsed DCs producted more IFN-γthan GPC-3 transfected DCs at the ratio of 1:10(708.45±11.81 pg/ml vs 643.43±22.11 pg/ml,p=0.011),indicating that HepG2 lysate was efficient in promoting IFN-γproduction of effector cells to some extent.However,the IFN-γsecreted by effector cells in GPC-3 transfected DCs was observed nearly the same as that in HepG2 lysate pulsed DCs at the ratio of either 1:50 or 1:100(p＞0.05).
To identify the specificity and validity of GPC-3 modified DCs involved in the process of HepG2 cells being lysed,we selected another HLA-A2+human tumor cell line SW620,except for HepG2 cells,as target cells for cytotoxicity assay.HepG2 cells exhibit overexpression of GPC3,SW620 cells are GPC3 negative.The cytotoxicity against the two tumor cell lines were assessed by a standard LDH release assay.The effector cells stimulated by HepG2 lysate pulsed DCs showed intensive lytic activity of HepG2 and SW620 cells with no statistical significance(p＞0.05).However,effector cells stimulated with GPC-3 transfected DCs effectively lyse HepG2 cells,at the ratios of both 50:1 and 100:1(16.6%and 34.1%,respectively).On the other hand,the GPC3-null SW620 cells were only observed minimally lysed at the ratio of 100:1 (12.0%),yet the lytic activity of effector cells stimulated with HepG2 lysate pulsed DCs was significantly elevated at different ratios(p＜0.05).
Conclusions
1、IFN-γcan enhance the properties of DCs,and provide a better method of culturing DCs in vitro to increase the immune activity.
2、DCs modified with full-length GPC-3 could generate specific cytotoxicity against HepG2 cells,which may provide a new method of treating HCC.
3、Compared with lysate pulse,GPC3 genetically modified DCs induced specific cytotoxity against HCC cell in vitro,which have the potential to serve as novel vaccine for HCC with less nonspecific cytotoxicities.
引文
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