融合基因mGM-CSF/hIL-2在肝癌细胞瘤苗特异性免疫治疗中的作用

背景与目的:手术切除是治疗肝癌的主要方法,但对其术后的复发转移却无更好的办法。近年来,免疫学的发展使肝癌的治疗有了更好的治疗方法。本研究旨在通过制备人白细胞介素2(hIL-2)与鼠粒-单核细胞集落刺激因子(mGM-CSF)融合基因修饰的H22肝癌瘤苗,观察其特异性抗肿瘤免疫作用。方法:用含hIL-2与mGM-CSF融合基因的真核表达载体,在体外转染H22细胞,制成疫苗,皮下接种小鼠,同时建立荷瘤小鼠模型。用51Cr释放法测定瘤苗免疫组、空载组、未转染组小鼠脾细胞对亲本H22细胞的杀伤活性。取血检测血清中IL-10、IFN-γ水平,观察小鼠存活期。结果:成功制备了含有hIL-2与mGM-CSF融合基因的H22肝癌瘤苗。免疫小鼠脾细胞体外对H22细胞的杀伤率为38.3%,显著高于对S180细胞的9.1%,以及空载组和未转染组的13.6%和7.5%(P<0.05)。转基因瘤苗免疫组血清IFN-γ为(12.83±0.75)pg/mL,较空载瘤苗组的(7.83±0.65)pg/mL明显升高(P<0.01),血清IL-10[(4.58±0.34)pg/mL]较空载瘤苗组的(8.15±0.28)pg/mL明显降低(P<0.01)。同时,转基因瘤苗免疫组小鼠存活期为(40±6)d,较对照组[空载瘤苗组(30±3)d,未转染组(19±4)d]明显延长。结论:转染hIL-2与mGM-CSF融合基因的同系肿瘤细胞瘤苗可激发特异性细胞介导的免疫反应,改善抗肿瘤免疫反应,延长荷瘤小鼠存活期。     

磷酸钙纳米颗粒修饰的甲胎蛋白和树突状细胞疫苗对小鼠皮下移植瘤的抑制作用

目的 探讨转染小鼠带有信号肽的AFP_1 cDNA和去掉信号肽的AFP_2 cDNA树突状细胞(DC)在体外的免疫活性,以及其对Balb/c小鼠皮下移植瘤的抑制作用.方法 应用磷酸钙纳米颗粒将带有信号肽的AFP_1 cDNA和去掉信号肽的AFP_2 cDNA 真核表达载体pcDNA3.1转染DC,将DC疫苗与同源小鼠脾淋巴细胞混合培养,采用酶联免疫吸附(ELISA)法,检测上清液中干扰素γ(IFN-γ)的表达情况.采用四甲基偶氮唑蓝(MTT)法,检测AFP_1/DC和AFP_2/DC刺激同基因小鼠脾淋巴细胞增殖能力及诱导特异性细胞毒性T淋巴细胞(CTL)的杀伤能力.观察AFP_1/DC和AFP_2/DC对Balb/c小鼠皮下移植瘤生长的抑制作用.结果 AFP_2/DC能明显促进脾淋巴细胞增殖并提高CTL的特异性杀伤作用.AFP_1/DC和AFP_2/DC瘤内注射均可显著抑制肝癌移植瘤的生长,但AFP_2/DC的抑制作用更明显,治疗2周后,AFP_2/DC组小鼠肿瘤体积为(726.7±298.2)mm3,明显小于AFP_1/DC组[(1486.2±457.2)mm~3]和空质粒对照组[(2137.2±547.2)mm~3,P<0.05].AFP_2/DC组和AFP_1/DC组的抑瘤率分别达79.2%和39.7%,而空质粒对照组和空白对照组的抑瘤率为0.AFP_2/DC组和AFP_1/DC 组小鼠的生存时间分别为(58.5±4.2)d和(45.2±4.8)d,较空质粒对照组[(30.6±6.2)d]显著延长(P<0.05).结论 AFP_2/DC 疫苗在体外能够诱导出高效而特异的抗肿瘤免疫效应,在体内具有抑制Balb/c小鼠皮下移植瘤生长的作用.     

Ad介导人AFP和IFN-у协同诱发抗小鼠肝癌免疫效应

背景与目的:原发性肝癌(hepatocellular carcinoma,HCC)是常见的恶性肿瘤之一,目前对HCC的治疗尚无行之有效的手段。本研究探讨腺病毒(Adenovirus,Ad)载体介导异种甲胎蛋白(Alpha-fetoprotein,AFP)和酌干扰素(Interferon-gamma,IFN-γ酌)的协同抗肝癌效应。方法:用RT-PCR(reverse transcri-ptase-polymerase chain reaction)方法克隆小鼠IFN-γ酌基因并构建复制缺损型腺病毒编码人AFP和小鼠IFN-γ酌联合表达载体(Ad-hAFP/IFN-γ酌)。皮内免疫C57BL/6小鼠7d后,取脾细胞行51Cr释放实验检测特异性细胞毒T淋巴细胞(Cytotoxic Tlymphocytes,CTLs)杀伤活性;或给免疫小鼠皮下接种Hepa1-6肝癌细胞,观察荷瘤小鼠成活情况。结果:51Cr释放实验显示,Ad-hAFP/IFN-γ酌免疫小鼠1周后其诱导产生的特异性CTL杀伤活性明显强于Ad-hAFP或Ad-IFN-γ酌单独免疫,在效∶靶比(E∶T)为10∶1时,Ad-hAFP/IFN-γ酌、Ad-hAFP和Ad-IFN-γ酌诱发的CTL杀伤率分别(43.8±5.5)%、(28.2±3.2)%和(12.8±1.9)%;30∶1时,为(79.6±6.4)%、(51.9±4.3)%和(15.6±2.3)%以及90∶1时(88.2±6.3)%、(62.5±4.8)%和(26.5±2.4)%。荷瘤试验表明,Ad-hAFP或Ad-IFN-γ酌单独免疫小鼠后1周接种5×106Hepa1-6肝瘤细胞,观察2个月,Ad-hAFP免疫组80%的小鼠荷瘤,Ad-IFN-γ酌免疫组小鼠则100%荷瘤;而Ad-hAFP/IFN-γ酌免疫小鼠在接种同样数量的Hepa1-6细胞,2个月无小鼠荷瘤,小鼠100%存活。结论:腺病毒载体介导异种AFP能有效地诱发针对小鼠AFP的特异性细胞免疫反应,IFN-γ酌能明显增强这种效应。     

树突状细胞与细胞因子诱导的杀伤细胞共培养对多药耐药肿瘤细胞系的杀伤活性

目的探讨在细胞因子诱导的杀伤细胞(CIK)表达特异性抗原的肿瘤细胞过程中,是否存在抗原特异性杀伤。方法分离健康人骨髓获得单个核细胞,分别诱导为树突状细胞(DC)和CIK细胞,将人类乳腺癌耐药细胞株MCF-7/ADR细胞的冻融物抗原冲击或未冲击DC与CIK细胞共培养(pulsed-DC CIK、DC CIK),CIK细胞单独培养作对照。用流式细胞仪分析细胞表型,用酶联免疫吸附法(ELISA)检测IL-12、和IFN-γ分泌水平,用二苯基溴化四氮唑蓝(MTT)法测定细胞毒效应。结果DC与CIK共育后,两组DC成熟表型较共育前明显提高(P=0.003、P=0.001);pulsed- DC CIK组与DC CIK组、CIK组比较,细胞表型(CD3、CD8、CD56)明显提高(P=0.003、P= 0.011),CD3 CD56 细胞明显增多(P=0.001,P<0.001),CD3 CD8 细胞亦明显增多(P=0.002, P=0.002);CD45RA表型则明显降低(P<0.001,P=0.004)。IL-12和IFN-γ水平在pulsed-DC CIK组表达最高,分别为(254±14.5)pg/ml和(3100±286)pg/ml。对有耐药抗原表达的MCF-7/ADR细胞,pulsed-DC CIK组杀伤效应最强,pulsed-DC CIK组、DC CIK组和CIK组比较,差异均有统计学意义(pulsed-DC CIK组与DC CIK组比较,P=0.039;pulsed-DC CIK组与CIK组比较,P= 0.002;DC CIK组与CIK组比较,P=0.049);而对于无P-gp抗原表达的MCF-7细胞的杀伤效应,pulsed- DC CIK组和DC CIK组之间无明显差异,但均高于CIK组,差异有统计学意义(pulsed-DC CIK组与CIK组比较,P=0.007;DC CIK组与CIK组比较,P=0.048)。结论从人骨髓培养得到DC和CIK细胞共培养后,能促进各自特征性表面标志的表达上调,并分泌大量相关细胞因子。细胞杀伤效应的明显提高及可能的特异性细胞杀伤效应,为多药耐药肿瘤的临床生物免疫治疗提供实验基础。     

Tet-off系统对小鼠骨髓基质细胞中人IFN-γ基因表达的调控作用

背景与目的:本实验室已成功构建了质粒pTre-IFN-γ,并证实四环素基因调控系统(Tet-off system)能在体外调控人γ-干扰素(interferon-gamma,IFN-γ)基因在小鼠骨髓基质细胞(marrow stromal cells,MSCs)中的表达。本实验进一步研究Tet-off系统体外调控的可逆性,以及其对小鼠MSCs中人IFN-γ基因表达的调控作用。方法:脂质体介导pTet-off和pTre-IFN-γ质粒共转染小鼠MSCs。用ELISA法检测MSCs培养液中人IFN-γ蛋白的分泌量。把共转染后的MSCs回输给BALB/c裸小鼠,用实时荧光定量RT-PCR方法检测各组BALB/c裸小鼠脾组织中的IFN-γmRNA含量。结果:用ELISA法可检测到共转染后的MSCs培养液中有IFN-γ蛋白分泌,分泌高峰在前72h内;共转染后84h加入含300ng/mL盐酸四环素的培养液培养12h,每1×107个细胞的分泌量为(67.11±22.14)pg,无四环素处理组为(319.96±29.04)pg,两组相比差异有统计学意义(P<0.001);去除四环素后,IFN-γ蛋白分泌显著增加(P=0.032)。用实时荧光定量RT-PCR可检测到回输共转染MSCs的各组BALB/c裸小鼠脾组织中均有IFN-γmRNA转录,不用四环素处理组最高,达(1.5±0.7)×105copy·(100mg)-1,定期四环素处理组为(6.9±5.3)×102copy·(100mg)-1(P<0.001),接受1次四环素处理组表达量介于前两者之间,差异均有统计学意义(P<0.01)。结论:Tet-off系统在体外和体内都可以迅速、有效地调控人IFN-γ基因在小鼠MSCs中的表达,而且调控是可逆的。     

小鼠3LL肿瘤局部高表达GM-CSF增强抗肿瘤免疫的实验研究

背景与目的:粒-单核细胞集落刺激因子(grannulocyte/monocyte-colony stimulating factor,GM-CSF)具有促进未成熟树突状细胞(dendritic cell,DC)成熟、上调其MHC分子和协同刺激分子表达以及增强DC对肿瘤抗原的提呈等作用.本文研究GM-CSF在肿瘤局部的高表达可以促进肿瘤局部DC成熟及亚群改变,从而诱导增强的抗肿瘤免疫效应.方法:以GM-CSF转染小鼠Lewis肺癌细胞株3LL获得3LL-GM,分别以3LL、3LL-vec(空载体对照)和3LL-GM细胞接种C57BL/6小鼠,分离肿瘤局部免疫细胞,流式细胞术检测DC成熟度、DC亚群比例、CD8 效应T细胞的活化及其功能.结果:3LL-GM肿瘤局部GM-CSF浓度高达441.22 ng/g肿瘤组织,显著高于母本3LL组的0.53 ng/g肿瘤组织(P<0.05)和空载体对照3LL-vec组的0.42 ng/g肿瘤组织(P<0.05).在3LL-GM、3LL和3LL-vec组小鼠肿瘤内浸润的CD11c DC细胞中,I-Ab 细胞的比例分别为60.62%、19.98%和23.12%(P<0.05),CD80 细胞的比例分别为60.93%、37.43%和47.03%(P<0.05),表明肿瘤局部GM-CSF促进DC成熟;同时,三组小鼠肿瘤内浸润的CD11c CD8α CD4-DC亚群比例分别为60.82%、40.00%和29.27%(P<0.05),显示肿瘤局部GM-CSF促使DC分化为CD11c CD8α CD4-亚群.3LL-GM组小鼠肿瘤内浸润淋巴细胞(tumor infiltrating lymphocyte,TIL)中,CD3 CD62Llow细胞比例高达20.84%,显著高于3LL组的6.34%(P<0.05)和3LL-vec组的15.18%(P<0.05);并且分泌IFN-γ的CD8 T淋巴细胞比例为2.77%,与对照组相比差异具有显著性(P<0.05).结论:肿瘤局部高表达GM-CSF,可通过提高DC成熟度及上调CD11c CD8α CD4-DC亚群在肿瘤局部的比例,诱导增强的抗肿瘤免疫效应,最终导致肿瘤消退.     

自体肿瘤瘤苗对荷瘤鼠Th1、Th2型细胞因子及细胞毒作用的影响

目的研究经处理的H22肝癌细胞肿瘤瘤苗作为全细胞瘤苗对H22荷瘤小鼠体内Th1/Th2细胞比例和细胞因子的影响以及CTL的杀伤活性.方法用加重组白细胞介素2、重组粒细胞单核细胞集落刺激因子及福氏不完全佐剂制成疫苗,建立荷瘤小鼠模型,用51Cr释放法测定瘤苗免疫组、荷瘤组、正常组小鼠脾细胞对亲本H22肝癌细胞的杀伤活性;流式细胞仪检测单个核细胞中的Th1和Th2细胞,并取血检测血清中IL-10、IFN-γ水平.结果效靶比为200:1时,免疫小鼠脾细胞体外杀伤亲本H22肝癌细胞的杀伤率为38.3%,显著高于荷瘤组的13.6%,正常组的7.5%,以及对S180细胞的9.1%(P均＜0.05).瘤苗免疫组Th1细胞及Th1/Th2细胞的比值显著升高(P＜0.01),血清IFN-γ较对照组明显升高(P＜0.01);血清IL-10较对照组明显降低(P＜0.01).结论肿瘤细胞加小剂量IL-2和GM-CSF及佐剂组成的肿瘤细胞瘤苗可激发特异性细胞介导的免疫反应,改善抗肿瘤免疫反应.     

人乳腺癌OX40L转基因细胞株的构建及其对T细胞的共刺激作用

背景与目的:共刺激分子OX40(CD134)和OX40配体(OX40L、CD134L)分属TNFR、TNF家族,OX40-OX40L信号能促进细胞因子的产生及提高抗原特异性记忆T细胞的数量,发挥重要的免疫调节作用。本实验旨在构建稳定表达OX40配体(OX40L)的人乳腺癌转基因细胞株,进而研究OX40-OX40L信号对T细胞体外活化和功能的调节作用。方法:运用逆转录聚合酶链反应(RT-PCR)技术从人成熟树突细胞(dendriticcell,DC)获得全长人OX40L基因,构建真核表达载体pcDNA3.1-OX40L转染人乳腺癌细胞株MDA-MB-435;经过G418抗性筛选和亚克隆,获得稳定表达OX40L的转基因细胞,并经RT-PCR和免疫荧光标记确证靶分子的表达;采用MTT、酶联免疫吸附试验(ELISA)和免疫荧光标记等方法分析转基因细胞对T细胞的增殖、IL-2、IL-4、IFN-γ的分泌和T细胞上Fas分子表达的影响。结果:成功地获得稳定表达人OX40L分子的人乳腺癌MDA-MB-435转基因细胞株,该细胞株可有效地促进T细胞增殖,促进T细胞分泌IL-2和IFN-γ,在第7天,T细胞、T细胞 M435、T细胞 M435-mock、T细胞 M435-OX40L各组IL-2的浓度依次为:315ng/ml、322ng/ml、586ng/ml、973.4ng/ml;IFN-γ浓度依次为:(2518±117.6)pg/ml、(2490±124.2)pg/ml、(2695±134.5)pg/ml、(3755±187.75)pg/ml,该基因转染细胞同时还能下调T细胞上Fas分子的表达[(68.3±5.6)%,P<0.05]。结论:稳定表达人OX40L的乳腺癌转基因细胞在体外能有效地活化T细胞,介导其增殖、分泌细胞因子及抑制T细胞活化诱导的细胞死亡。     

人结肠癌主动特异性免疫治疗的初步研究

目的:探讨自体肿瘤疫苗的作用机制及临床意义。方法:50例进展期结肠癌病人术后,以自体肿瘤细胞疫苗辅助主动免疫治疗。术后第4周开始免疫接种(共4次,每次间隔7~10天);接种前3天及第4次接种后1周,采集外周血。采集血清监测血清IFN-γ、IL-10水平;用PPD及自体肿瘤抗原做皮肤迟发型过敏试验(DTH),48小时后测量红斑、硬结大小(mm);对DTH反应部位皮肤活检,用免疫组化染色,了解局部免疫活性细胞浸润;临床随访。结果:自体肿瘤细胞疫苗治疗后:血清IFN-γ水平升高,由(6.01±2.30)pg/ml升至(12.98±4.65)pg/ml;而IL-10水平由(19.80±10.15)pg/ml降至(8.92±4.60)pg/ml,差异有非常显著性(P<0.05);治疗前后病人对自体肿瘤抗原的特异性DTH反应明显增强(P<0.01);DTH反应部位,CD8 T细胞、CD4 T细胞及DC细胞浸润明显增多;病人耐受性良好,无溃疡等严重副作用发生;随访结果显示:术后辅助自体肿瘤疫苗主动特异性免疫治疗,可延长结肠癌病人的无瘤生存时间,降低复发率及死亡率。结论:自体肿瘤疫苗可激发病人特异性细胞介导的免疫反应;可改善肿瘤病人的抗瘤免疫反应;自体肿瘤疫苗主动特异性免疫治疗,对于杀灭残留癌细胞、抗转移及复发有重要作用。     

小鼠肝癌全细胞抗原致敏骨髓DC激活TIL抗癌机制的研究

目的:通过分析小鼠肝癌H22细胞全细胞性抗原致敏小鼠骨髓树突状细胞(DC)前后DC表型变化及其分泌细胞因子的变化,探讨肝癌细胞全细胞性抗原致敏DC激活肿瘤浸润性淋巴细胞(TIL)的抗癌机制。方法:取得小鼠骨髓细胞并诱导生成DC,用冻融法制备的小鼠肝癌H22细胞全细胞抗原致敏,然后用已致敏的DC激活TILs,测定DC致敏前后DC表面抗原CD80、CD86、CD40和MHCⅡ表达变化及DC分泌细胞因子IL-12、IL-2、IFN-γ和TNF-α的水平,评估激活前后TIL对H22细胞的杀伤活性,同时以小鼠脾淋巴细胞作为杀伤对照。结果:致敏后小鼠骨髓DC表面抗原CD80、CD86、CD40和MHCⅡ表达率分别为(57.55±7.32)%、(54.49±14.20)%、(46.79±8.25)%和(53.94±13.94)%,明显高于未致敏DC的(20.01±5.22)%、(24.56±9.08)%、(18.06±5.13)%和(30.24±8.39)%,P值均<0.05;DC上清液中IL-12、IL-2、IFN-γ和TNF-α浓度分别为(80.40±1.33)、(94.67±3.36)、(29.83±1.20)和(75.01±4.10)ρg/mL,明显高于未致敏DC的(19.35±0.99)、(11.25±0.50)、(1.05±0.09)和(2.02±0.27)ρg/mL,P值均为0.000。经致敏后成熟DC激活的TIL对H22细胞杀伤率为(81.80±2.90)%,明显高于未激活TIL、激活或未激活小鼠脾淋巴细胞的(62.64±3.94)%、(47.35±3.40)%和(28.45±2.56)%。结论:H22细胞全细胞抗原致敏DC后,其表型变化及细胞因子分泌增多是其诱导活化TIL抗癌活性的可能机制。     

IL-21对CTL抗非霍奇金淋巴瘤影响的研究

目的：探讨白介素21（IL-21）对细胞毒T淋巴细胞（CTL）杀伤非霍奇金淋巴瘤（NHL）的影响,阐述该影响是通过IFN-γ和TNF-α的高表达而实现。方法：分选树突状细胞（DCs）,用NHL细胞株（Raji）冲击,诱导产生CTL;将此培养液分为对照组和实验组,实验组加入100ng/ml的IL-21。ELISA、RT-PCR检测IFN-γ和TNF-α水平。结果：IL-21作用组,IFN-γ水平：469.32±2.83pg/ml,TNF-α：82.52±4.72pg/ml,对照组,IFN-γ水平：27.63±2.76pg/ml,TNF-α：11.63±1.54pg/ml,两组差异显著（P〈0.05）。结论：IL-21是通过IFN-γ和TNF-α的高表达而实现增强CTL杀伤NHL的作用,有重要的临床意义。     

树突状细胞与肿瘤细胞融合后诱导T细胞介导的抗肿瘤效应

目的：研究树突状细胞（DC）与肿瘤细胞（SP2／0）融合作为肿瘤疫苗免疫小鼠后抑制肿瘤生长的作用及其机理。方法：BALB／C小鼠DC与SP2／0细胞体外融合,形成的杂交瘤分2次免疫接种同基因小鼠皮下,然后用SP2／0细胞攻击免疫的小鼠,观察肿瘤的生长情况及免疫小鼠脾细胞特异性CTL功能。结果：DC－SP2／0杂交瘤接种小鼠能产生明显的抗肿瘤效应,其拮抗SP2／0细胞攻击的能力明显强于用灭活的死SP2／0细胞与DC混合,和单用死SP2／0细胞免疫的小鼠及用生理盐水的对照组小鼠。DC－SP2／0杂交瘤免疫接种小鼠的脾细胞特异性CTL杀伤活性强于其它各组小鼠。结论：DC与肿瘤细胞融合后作为肿瘤疫苗接种可在体内产生明显的抗肿瘤免疫,其机理主要是特异性CTL的作用。     

Dendritic cells transduced with tumor-associated antigen gene elicit potent therapeutic antitumor immunity: comparison with immunodominant peptide-pulsed DCs

Several studies have shown that vaccine therapy using dendritic cells (DCs) pulsed with specific tumor antigen peptides can effectively induce antitumor immunity. Peptide-pulsed DC therapy is reported to be effective against melanoma, while it is still not sufficient to show the antitumor therapeutic effect against epithelial solid tumors such as gastrointestinal malignancies. Recently, it has been reported that vaccine therapy using DCs transduced with a surrogate tumor antigen gene can elicit a potent therapeutic antitumor immunity. In this study, we investigated the efficacy of vaccine therapy using DCs transduced with the natural tumor antigen in comparison with peptide-pulsed DCs. DCs derived from murine bone marrow were adenovirally transduced with murine endogenous tumor antigen gp70 gene, which is expressed in CT26 cells, or DCs were pulsed with the immunodominant peptide AH-1 derived from gp70. We compared these two cancer vaccines in terms of induction of antigen-specific cytotoxic T lymphocyte (CTL) responses, CD4+ T cell response against tumor cells, migratory capacity of DCs and therapeutic immunity in vivo. The cytotoxic activity of splenocytes against CT26 and Meth-A pulsed with AH-1 in mice immunized with gp70 gene-transduced DCs was higher than that with AH-1-pulsed DCs. CD4+ T cells induced from mice immunized with gp70 gene-transduced DCs produced higher levels of IFN-gamma by stimulation with CT26 than those from mice immunized with AH-1-pulsed DCs (p < 0.0001), and it was suggested that DCs transduced with tumor-associated antigen (TAA) gene induced tumor-specific CD4+ T cells, and those CD4+ T cells played a critical role in the priming phase of the CD8+ T cell response for the induction of CD8+ CTL. Furthermore, DCs adenovirally transduced with TAA gene showed an enhancement of expression of CC chemokine receptor 7 and improved the migratory capacity to draining lymph nodes. In subcutaneous models, the vaccination using gp70 gene-transduced DCs provided a remarkably higher therapeutic efficacy than that using AH-1-pulsed DCs. These results suggested that vaccine therapy using DCs adenovirally transduced with TAA gene can elicit potent antitumor immunity, and may be useful for clinical application.     

人AFP腺病毒载体感染的树突状细胞诱导小鼠抗肝癌免疫

目的探讨复制缺损型腺病毒载体(Ad)介导异种AFP修饰的树突状细胞(DCs)诱发抗肝癌免疫、打破肿瘤免疫耐受的效果。方法从HepG2和Hepa16细胞中克隆人和小鼠AFP,插入Ad中构建AdhAFP和AdmAFP。用AdhAFP或AdmAFP感染小鼠骨髓来源的DC后,在无或有删除CD4或CD8情况下免疫C57BL/6小鼠,7d后取脾细胞行51Cr释放实验,检测特异性CTL杀伤活性;或给免疫小鼠接种Hepa16肝癌细胞,观察荷瘤小鼠成活情况。结果AdhAFP/DCs免疫小鼠1周后其细胞毒性T淋巴细胞杀伤活性明显强于AdmAFP/DCs。AdhAFP/DCs免疫小鼠后1周接种5×106Hepa16肝瘤细胞,2个月后仍然有80%的小鼠无瘤生长;而接种1×106Hepa16细胞至AdmAFP/DCs免疫小鼠,2个月后小鼠成活率为20%。删除小鼠CD4或CD8T细胞均使AdhAFP/DCs诱发的抗肿瘤免疫反应消失。结论Ad介导异种AFP修饰的DCs能有效地打破肿瘤的免疫耐受,诱发强烈的抗原特异性细胞免疫反应,这种特异性细胞免疫反应是CD4和CD8依赖性的。     

Tumor cell lysate-pulsed dendritic cells induce a T cell response against colon cancer in vitro and in vivo

To investigate whether tumor cell lysate-pulsed (TP) dendritic cells (DCs) induce cytotoxic T lymphocyte (CTL) activity against colon cancer in vitro and in vivo. Hematopoietic progenitor cells were magnetically isolated from BALB/c mice bone marrow cells. These cells were cultured with cytokines GM-CSF, IL-4, and TNFα to induce their maturation. They were analyzed by morphological observation and phenotype analysis. DCs were pulsed with tumor cell lysate obtained by rapid freezing and thawing at a 1:3 DC:tumor cell ratio. CTL activity and interferon gamma (IFNγ) secretion was evaluated ex vivo. In order to determine whether or not vaccination with CT26 TP DCs induce the therapeutic potential in the established colon tumor model, CT26 colon tumor cells were implanted subcutaneously (s.c.) in the midflank of naïve BALB/c mice. Tumor-bearing mice were injected with vaccination with CT26 TP DCs on days 3 and 10. Tumor growth was assessed every 2–3 days. Finally, CTL activity and IFNγ secretion were evaluated in immunized mice. Hematopoietic progenitor cells from mice bone marrow cells cultured with cytokines for 8 days showed the character of typical mature DCs. Morphologically, these cells were large with oval or irregularly shaped nuclei and with many small dendrites. Phenotypically, FACS analysis showed that they expressed high levels of MHC II, CD11b, CD80, and CD86 antigen, and were negative for CD8α. However, immature DCs cultured with cytokines for 5 days did not have typical DCs phenotypic markers. Ex vivo primed T cells with CT26 TP DCs were able to induce effective CTL activity against CT26 tumor cells, but not B16 tumor cells (E:T = 100:1, 60.36 ± 7.11% specific lysis in CT26 group vs. 17.36 ± 4.10% specific lysis in B16 group), and produced higher levels of IFNγ when stimulated with CT26 tumor cells but not when stimulated with B16 tumor cells (1210.33 ± 72.15 pg/ml in CT26 group vs. 182.25 ± 25.51 pg/ml in B16 group, P < 0.01). Vaccination with CT26 TP DCs could induce anti-tumor immunity against CT26 colon tumor in murine therapeutic models (tumor volume on day 19: CT26 TP DCs 342 ± 55 mm³ vs. the other control groups, P < 0.05). In addition, all splenic CD3⁺ T cells obtained from mice vaccinated with CT26 TP DCs produced high levels of IFNγ and shown specific cytotoxic activity against CT26 tumor cells, but no cytotoxic activity when stimulated with B16 tumor cells. Tumor cell lysate-pulsed DCs can induce tumor-specific CTL activity against colon cancer in vitro and in vivo.     

Enhanced T-cell immunity induced by dendritic cells with phagocytosis of heat shock protein 70 gene-transfected tumor cells in early phase of apoptosis

The dual role of heat shock protein 70 (HSP70), as antigenic peptide chaperone and danger signal, makes it especially important in dendritic cell (DC)-based vaccination. In this study, we investigated the impacts of apoptotic transgenic MCA/HSP tumor cells expressing HSP70 on DC maturation, T-cell stimulation and vaccine efficacy. We found that DCs with phagocytosis of MCA/HSP in early phase of apoptosis expressed more pMHC I complexes, stimulated stronger cytotoxic T lymphocyte (CTL) responses (40% specific killing at an E:T cell ratio of 50) and induced immune protection in 90% of mice against MCA tumor cell challenge, compared with 25% specific CTL killing activity and 60% immune protection seen in mice immunized with DC with phagocytosis of MCA/HSP in late phase of apoptosis (P<0.05). Similar results were confirmed in another EG7 tumor model also expressing HSP70. Taken together, our data demonstrate that HSP70 on apoptotic tumor cells stimulate DC maturation, and DC with phagocytosis of apoptotic tumor cells expressing HSP70 in early phase of apoptosis more efficiently induced tumor-specific CTL responses and immunity than DCs with phagocytosis of apoptotic tumor cells in late phase of apoptosis. These results may have an important impact in designing DC-based antitumor vaccines.     

Engineered fusion hybrid vaccine of IL-18 gene-modified tumor cells and dendritic cells induces enhanced antitumor immunity

Dendritic cell (DC)-tumor fusion hybrid vaccines that facilitate antigen presentation represent a novel powerful strategy in cancer immunotherapy. In our study, we investigated the antitumor immunity derived from the vaccination of fusion hybrids between engineered J558/IL-18 myeloma cells secreting Th1 cytokine IL-18 and DCs. DC/J558/IL-18 could secret a higher level of IL-18 than DCs, efficiently expressed J558 tumor antigen P1A, and enhanced ability of allogeneic T cell stimulation when compared to J558/IL-18. Our data showed that the immunization of BALB/c mice with DC/J558/IL-18 hybrids induced the most potent protective immunity against 1 x 10(6) cells with a J558 tumor challenge, compared to those immunized with the mixture of DCs and J558/IL-18, J558/IL-18, or J558. Furthermore, the immunization of mice with engineered DC/J558/IL-18 hybrids elicited stronger NK activity and J558 tumor-specific cytotoxic T lymphocyte (CTL) responses in vitro. In addition, DC/J558/IL-18 tumor cells into syngeneic mice induced a Th1 dominant immune response to J558 and resulted in tumor regression, which indicated that the antitumor effect mediated by DC/J558/IL-18 appeared to be dependent on TH1 cytokine production. These results demonstrate that the engineered fusion hybrid vaccines that combine Th1 gene-modified tumor with DCs may be an attractive strategy for cancer immunotherapy.     

抗原冲击树突状细胞介导细胞毒性T淋巴细胞特异性抗白血病作用的体外研究

 目的　研究白血病来源树突状细胞体外诱导的有效方法;观察不同抗原诱导的特异性细胞毒性T淋巴细胞（CTL）的抗白血病效应。方法　分离白血病患者骨髓单个核细胞,经钙离子载体A23187诱导分化,将弱酸洗脱抗原、低渗抗原分别冲击树突状细胞,96 h后树突状细胞与T细胞共同培养,采用MTT法比较CTL细胞对白血病细胞的杀伤活性。结果　骨髓来源的单个核细胞经过100 ng／ml的GM-CSF、500 ng／ml钙离子载体A23187成功诱导为树突状细胞,倒置显微镜下具有树突状细胞的典型形态,流式细胞术测定其CD1a、CD83表达较诱导前明显升高,差异有统计学意义（P＜0.01）。弱酸洗脱法获得抗原冲击树突状细胞致敏T细胞后杀伤白血病的能力最高,未负载抗原的树突状细胞致敏T细胞杀伤白血病细胞的能力最低,两者差异具有统计学意义（P＜0.01）。结论　白血病来源的骨髓单个核细胞经GM-CSF、钙离子载体A23187能够成功诱导为树突状细胞;弱酸洗脱后获得的抗原冲击树突状细胞致敏T细胞能够获得更强的杀伤白血病细胞的能力。     

粒细胞-巨噬细胞集落刺激因子基因修饰的树突状细胞疫苗增强体外抗肿瘤免疫效应

目的 研究粒细胞-巨噬细胞集落刺激因子(GM-CSF)基因修饰树突状细胞(DC)后形态、表型及功能的变化,以及增强DC疫苗对肿瘤细胞的体外杀伤作用.方法 小鼠尾静脉注射趋化因子配体3(CCL3),分选得到B220- CDllc+细胞,经细胞因子培养诱导分化DC.在体外用含GM-CSF基因的重组腺病毒(AdGM-CSF)转染DC,酶联免疫吸附试验(ELJSA)检测转染后GM-CSF的水平.通过细胞形态学观察、表型分析及混合淋巴细胞反应(MLR),检测GM-CSF基因修饰前后DC的变化.反复冻融法制备胃癌可溶性抗原,将其与GM-CSF基因修饰的DC共同培养,制备DC疫苗,四甲基偶氮唑蓝(MTT)法检测活化的T淋巴细胞在体外对小鼠前胃癌细胞(MFC)的杀伤作用,ELISA法检测干扰素γ(INF-γ)的分泌情况.结果 CCL3注射后,外周血中B220- CD11c+细胞明显增加,48 h达到高峰[占外周血单个核细胞的(13.88±1.10)%].AdGM-CSF转染后,培养液上清中GM-CSF浓度升高,当感染复数(MOI)为1:100时达到高峰[(130.00±12.61)pg/m1].经GM-CSF.基因修饰的DC在形态上更趋成熟,MHCⅡ类分子、CD80、CD86等细胞表型明显上调,具有更强的刺激T细胞增殖的能力.荷载胃癌抗原的DC激活的T淋巴细胞对MFC细胞具有特异性杀伤作用,并产生高水平的INF-γ[(1245.00±13.75)pg/ml].结论 GM-CSF转染DC后,能大量表达GM-CSF,DC形态及细胞表型更趋成熟,刺激T细胞增殖能力明显增强.GM-CSF基因修饰的DC在体外可诱导出针对靶肿瘤细胞的特异性杀伤作用.     

Engineered fusion hybrid vaccine of IL-4 gene-modified myeloma and relative mature dendritic cells enhances antitumor immunity

Dendritic cell (DC)-tumor fusion hybrid vaccine which facilitates antigen presentation represents a new powerful strategy in cancer therapy. In the present study, we investigated the antitumor immunity derived from vaccination of fusion hybrids between wild-type J558 or engineered J558-IL-4 myeloma cells secreting cytokine interleukin-4 (IL-4) and immature DCs (DC(IMAT)) or relative mature DCs (DC(RMAT)). DC(RMAT) displayed an up-regulated expression of immune molecules (Ia(d), CD40, CD54, CD80 and CD86) and certain cytokines/chemokines, and enhanced ability of allogeneic T cell stimulation when compared to DC(IMAT). These DCs were fused with myeloma cells by polyethylene glycol (PEG). The fusion efficiency was approximately 20%. Our data showed that immunization of C57BL/6 mice with DC(RMAT)/J558 hybrids induced protective immunity against a high dose of J558 tumor challenge (1x10(6) cells) in 3 out of 10 immunized mice, compared with no protection seen in mice immunized with DC(IMAT)/J558 hybrids. Furthermore, immunization of mice with engineered DC(RMAT)/J558-IL-4 hybrids elicited stronger J558 tumor-specific cytotoxic T lymphocyte (CTL) responses in vitro and induced more efficient protective immunity (10/10 mice; tumor free) against J558 tumor challenge in vivo than DC(RMAT)/J558 hybrid vaccines. The results demonstrate the importance of DC maturation in DC-tumor hybrid vaccines and indicate that the engineered fusion hybrid vaccines which combine gene-modified tumor and DC vaccines may be an attractive strategy for cancer immunotherapy.