H22细胞全细胞抗原负载的树突状细胞激活肿瘤浸润性淋巴细胞抗小鼠肝癌的实验

目的探讨H22小鼠肝癌细胞（H22细胞）全细胞抗原致敏的树突状细胞激活肿瘤浸润淋巴细胞抗小鼠肝癌细胞活性。方法取得小鼠骨髓细胞并诱导生成树突状细胞,由冻融法制备的H22细胞全细胞抗原致敏,然后用已致敏的树突状细胞激活肿瘤浸润性淋巴细胞,测定致敏前后的DC表面抗原CD11c、CD80、CD86、CD40、MHCⅡ,并评估激活前后的TIL对H22细胞的杀伤活性,同时脾淋巴细胞作为杀伤对照。结果CD11c阳性细胞中CD80、CD86、CD40、MHCⅡ阳性细胞所占比例在致敏后的DC表现为明显上调。经致敏后成熟DC激活的TIL对H22细胞杀伤活性明显高于未激活的TIL,并高于激活或未激活的小鼠脾脏淋巴细胞。结论在H22细胞全抗原致敏后,小鼠成熟DC中CD80、CD86、CD40、MHCⅡ的表达率明显高于未成熟DC。经H22细胞全细胞抗原致敏的DC能诱导活化TIL,明显提高其在体外对H22细胞的杀伤活性。     

抗原致敏树突状细胞诱导CIK对胃癌细胞杀伤作用的研究

[目的]探讨抗原致敏的树突状细胞（DC）诱导CIK（cytokine induced killer）的杀伤作用。[方法]联合应用粒/巨细胞集落刺激因子（GM-CSF）及白介素-4（IL-4）从外周血单个核细胞中培养DC，用人胃癌细胞SGC提取肿瘤抗原致敏DC，流式细胞仪检测致敏前后DC表型的变化，用人胃癌细胞SGC提取肿瘤抗原致敏DC诱导CIK，用MTT法检测淋巴细胞的增殖及原致敏DC诱导CIK对SGC的杀伤效应。[结果]①利用GM-CSF及IL-4可从外周血单个核细胞中获取DC，肿瘤抗原可促进DC的成熟，肿瘤抗原致敏可促进DC成熟，细胞表面分子HLA-DR、CD86、CD86升高，CD14下降。②混合淋巴细胞反应提示成熟的DC可促进CIK细胞增殖。③SGC肿瘤抗原致敏DC诱导CIK对胃癌细胞SGC有特异性的杀伤作用，随着效靶比的升高，杀伤效应随之增强。[结论]抗原致敏的DC可通过诱导特异性CIK细胞及促进CIK细胞增殖两方面显著提高CIK细胞的杀瘤效应。     

肿瘤抗原致敏树突状细胞治疗胶质瘤的实验研究

 目的　研究肿瘤冻融抗原致敏树突状细胞 (DC)治疗胶质瘤的疗效。方法　从大鼠骨髓中分离出单核细胞后 ,加入添加rGM CSF和rIL 4的IMDM培养基中培养后鉴定。制备大鼠C6胶质瘤冻融抗原以及DC疫苗 ,同时肿瘤冻融抗原致敏DC体外细胞毒试验 ;建立动物模型 ,体内应用DC疫苗并观察疗效。结果　单核细胞培养 8天后可获得大量的树突状细胞 ;体外细胞毒试验发现对相应的C6肿瘤细胞有明显的杀伤作用 ;动物实验发现治疗组肿瘤生长明显抑制 ,而对照组肿瘤明显生长 ,两组肿瘤体积有显著性差异P <0 .0 1。结论　建立了体外扩增大鼠骨髓DC以及制备DC疫苗的方法 ,采用冻融抗原致敏DC疫苗治疗荷瘤动物生存时间明显延长 ,肿瘤生长明显抑制 ,为研究其在脑胶质瘤临床免疫治疗打下基础     

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。     

致敏树突细胞治疗膀胱肿瘤血液细胞毒性T淋巴细胞的变化

目的观察致敏的树突细胞（DC）治疗膀胱肿瘤（BT）后血液细胞毒性T淋巴细胞（CTL）的变化并探讨其机制。方法选取F344大鼠44只，称重后随机分为4组；均采用膀胱灌注N-甲基亚硝基脲（MNU）制成BT；第11周4组分别经皮下注射磷酸缓冲液（PBS）、未致敏DC、肿瘤抗原及致敏DC，每周1次，共4次；实验第15周，经显微镜和流式细胞仪（FCM）检测。结果致敏DC组膀胱重量比其他三组轻（P〈0．05）。致敏DC组BT病理分期低于PBS组和未致敏DC组（P〈0．05）；CD3^＋ T细胞在致敏DC组低于其余三组（P〈0．05）；CTL在致敏DC组高于其余三组（P〈0．001）。结论应用致敏DC经皮下注射治疗大鼠BT可降低肿瘤的病理分期，未致敏DC皮下注射对膀胱肿瘤治疗无效，肿瘤抗原皮下注射对膀胱肿瘤治疗效果欠佳。致敏DC可通过呈递抗原来激活CTL增生而发挥其免疫杀伤作用。     

亚胺培南经验性治疗肿瘤患者中性粒细胞缺乏伴发热的回顾性分析

目的评价碳青酶烯类抗生素亚胺培南经验性治疗肿瘤患者中性粒细胞缺乏伴发热的疗效和安全性。方法回顾性分析51例肿瘤患者化疗后出现中性粒细胞缺乏伴发热应用亚胺培南进行经验性治疗的临床资料。结果接受亚胺培南/西司他丁进行初始经验性治疗的51例患者中,痊愈29例,显效8例,进步7例,无效7例,有效率72.5%。2例患者出现了可耐受的不良反应。结论亚胺培南作为肿瘤患者中性粒细胞缺乏伴发热的初始经验性抗菌治疗,具有较高的疗效和良好的耐受性。     

宫颈癌患者外周血树突状细胞抗宫颈癌免疫反应

[目的]研究自宫颈癌患者外周血分离、培养而来的树突状细胞体外对宫颈癌Hela细胞的抑制作用.[方法]应用细胞因子诱导培养从宫颈癌患者外周血分离的单核细胞,获得成熟DC.宫颈癌组织来源抗原致敏DC,激活T细胞增殖分化为细胞毒性T淋巴细胞(CTL).MTT法检测比较负载抗原DC与未负载抗原DC诱导的CTL对Hela细胞和SKOV3细胞不同的杀伤效应.[结果]宫颈癌细胞抗原致敏的DC,激活肿瘤抗原特异性CTL,对宫颈癌Hela细胞产生高效而特异的杀伤作用(56.410%),对卵巢癌SKOV3细胞仅有一定的杀伤作用(24.901%);而未致敏DC激活的CTL对以上两种肿瘤细胞只有很低的杀伤作用,分别为10.256%和20.151%.单纯肿瘤抗原刺激下的T细胞,对Hela和SKOV3细胞产生非特异性杀伤作用,其效率仅为1.865%及15.613%.[结论]宫颈癌患者外周血DC在体外能诱导高效而特异的抗宫颈癌细胞免疫反应,提示肿瘤抗原激活的DC作为疫苗在宫颈癌的免疫治疗中具有重要意义.     

肝癌细胞对重组人HSP70联合肝癌组织冻融抗原修饰树突状细胞的免疫应答

目的:研究重组人热休克蛋白70(rhHSP70)联合肝癌组织冻融抗原修饰的树突状细胞(dendritic cell,DC)诱导对肝癌细胞的免疫杀伤效应.方法:外周血单个核细胞经粒-巨噬细胞集落刺激因子(GM-CSF)、白细胞介素4 (IL-4)诱导生成DC,负载冻融抗原的同时加入rhHSP70,不同分组致敏的DC激活淋巴细胞生成肿瘤抗原特异性细胞毒性T淋巴细胞(cytotoxic T cells,CTL),四甲基偶氮唑蓝(MTT)法及3H-TdR法检测DC刺激淋巴细胞增殖能力, MTT法检测CTL对肝癌细胞的体外杀伤活性,酶联免疫吸附试验(ELISA)测定细胞因子的分泌,流式细胞术(FCM)检测DC表型变化.结果:冻融抗原致敏的DC可明显促进淋巴细胞增殖,能有效呈递肝癌冻融抗原,诱导产生抗原特异性CTL,联合rhHSP70能进一步增强CTL对肝癌细胞的杀伤作用.结论:肝癌冻融抗原联合rhHSP70修饰的DC诱导CTL对肝癌细胞能产生高效杀伤作用.     

IL-2基因转染的CIK细胞联合DC对肝癌细胞的杀伤作用

目的 探讨IL-2基因转染的细胞因子诱导的杀伤细胞(CIK)联合树突状细胞(DC)对肝癌细胞的杀伤作用.方法 采用密度梯度离心法从健康人外周血中分离单个核细胞(PMBC),分别诱导CIK细胞和DC.用携带IL-2基因的重组腺病毒AdBM5-GFP-IL-2转染CIK细胞,HepG2肝癌冻融抗原冲击致敏DC,然后将转染IL-2基因的CIK细胞(IL2-CIK)与肝癌抗原致敏DC(Lysate-DC)共培养,制备肝癌抗原致敏DC激活的IL-2基因修饰的CIK细胞(Lysate-DC-IL-2-CIK).采用MTT法检测单纯CIK、DC联合CIK(DC-CIK)、肿瘤抗原致敏DC激活的CIK(Lysate-DC-CIK)、Dc激活的IL2基因转染的CIK(DC-IL2-CIK)和肿瘤抗原致敏Dc激活的IL-2基因转染的CIK(Lysate-DC-IL2-CIK)对肝癌HepG2细胞的杀伤作用,效靶比分别为10:1、20:1、40:1,同时以肝癌细胞H7404、白血病细胞K562作为靶细胞进行杀伤对照实验.结果 研究结果表明,不同效靶比的Lysate-DC-IL-2-CIK效应细胞对HepG2的杀伤活性均显著高于对照组(P<0.05或0.01),且其杀瘤作用随着效靶比的增高而增强,在效靶比为40:1时达到最佳抗肿瘤活性.结论 经肝癌抗原致敏DC激活的IL-2基因转染的CIK细胞对AFP分泌功能的肝癌细胞具有更强的杀伤作用.     

负载肝癌抗原的树突状细胞瘤苗诱导的抗肿瘤作用

目的 探讨原发性肝癌患者外周血树突状细胞（DC）体外经自体肝癌细胞抗原致敏后诱导的抗肿瘤作用。方法肝癌患者外周血经梯度密度离心法分离，获得DC前体细胞，用重组人粒细胞-巨噬细胞集落刺激因子（rhGM—CSP）和重组人白细胞介素-4（rhIL-4）联合培养，诱导扩增DC。制备自体肝癌细胞抗原，体外脉冲DC，检测DC诱导自体T细胞增殖能力及细胞毒性T细胞（CTL）在体外对自体肝癌细胞的杀伤活性，并检测肿瘤抗原致敏DC分泌的IL-12水平。结果经自体肝癌细胞抗原致敏的DC能分泌IL-12和诱导较强的自体T细胞增殖，且能诱导特异性CTL，该CTL对自体肝癌细胞具有很强的杀伤活性，杀伤率明显高于DC、未经肝癌细胞抗原致敏的DC激活的CTL及T淋巴细胞的杀伤率，而对3LLLEWIS肺癌细胞、H22肝癌细胞则无明显的．杀伤作用。结论肝癌患者外周血DC经自体肝癌细胞抗原致敏后能诱导高效而特异的抗肝癌免疫，其机制可能与增强T细胞应答和诱导机体产生肿瘤特异CTL而发挥特异性的抗肿瘤作用有关。     

树突状细胞与肿瘤疫苗治疗

树突状细胞(dendriticcells,DC)是人体内功能最强大的专职抗原递呈细胞,是肿瘤免疫反应中不可缺少的辅助细胞。未成熟DC可摄取抗原,当有危险信号刺激时可迁徙至淋巴器官,并递呈抗原给T细胞进而激发免疫应答。目前普遍采用的获取DC的方法是对外周血单核细胞(PBMC)进行体外诱导和培养。证据显示DC浸润与许多恶性肿瘤的预后有关。DC数量减少和功能缺陷,也是肿瘤细胞逃避机体免疫监视的机制之一。用于装载DC的抗原包括肿瘤细胞或肿瘤细胞提取物、肿瘤抗原蛋白、肿瘤抗原肽、肿瘤抗原DNA/RNA等。肿瘤抗原经脂质体介导在体内转染DC,也可激发特异性免疫应答。     

Antitumor Activity of Lentivirus-mediated Interleukin -12 Gene Modified Dendritic Cells in Human Lung Cancer in Vitro

Objectives: Dendritic cell (DC)-based tumor immunotherapy needs an immunogenic tumor associatedantigen (TAA) and an effective approach for its presentation to lymphocytes. In this study we explored whethertransduction of DCs with lentiviruses (LVs) expressing the human interleukin-12 gene could stimulate antigenspecificcytotoxic T cells (CTLs) against human lung cancer cells in vitro. Methods: Peripheral blood monocytederivedDCs were transduced with a lentiviral vector encoding human IL-12 gene (LV-12). The anticipated targetof the human IL-12 gene was detected by RT-PCR. The concentration of IL-12 in the culture supernatant of DCswas measured by ELISA.Transduction efficiencies and CD83 phenotypes of DCs were assessed by flow cytometry.DCs were pulsed with tumor antigen of lung cancer cells (DC+Ag) and transduced with LV-12 (DC-LV-12+Ag).Stimulation of T lymphocyte proliferation by DCs and activation of cytotoxic T-lymphocytes (CTL) stimulatedby LV-12 transduced DCs pulsed with tumor antigen against A549 lung cancer cells were assessed with methylthiazolyltetrazolium (MTT). Results: A recombinant lentivirus expressing the IL-12 gene was successfullyconstructed. DC transduced with LV-12 produced higher levels of IL-12 and expressed higher levels of CD83than non-transduced. The DC modified by interleukin -12 gene and pulsed with tumor antigen demonstratedgood stimulation of lymphocyte proliferation, induction of antigen-specific cytotoxic T lymphocytes and antitumoreffects. Conclusions: Dendritic cells transduced with a lentivirus-mediated interleukin-12 gene have anenhanced ability to kill lung cancer cells through promoting T lymphocyte proliferation and cytotoxicity.     

Apoptotic pancreatic tumor cells are superior to cell lysates in promoting cross-priming of cytotoxic T cells and activate NK and gammadelta T cells

Tumor vaccines using dendritic cells (DCs) have been shown to induce antitumor CTL responses. The choice of the tumor antigen preparation used for DC loading is still an unresolved issue. We compared DCs pulsed with cell lysates, whole apoptotic tumor cells or their supernatants of the HLA-A2(+) human pancreatic carcinoma cell line Panc-1 for their capacity to activate T cells. Monocyte-derived DCs from HLA-A2(+) donors were pulsed with tumor antigen, matured subsequently, and cocultured with autologeous peripheral blood mononuclear cells. After three weekly restimulations with DCs, T-cell activation was assessed by intracellular IFN-gamma staining and cytotoxicity assays. Compared with lysate, pulsing DCs with the supernatant of apoptotic tumor cells induced a higher frequency of activated CTLs and T-helper cells, as well as an enhanced MHC class I-restricted tumor cell lysis. No activation of natural killer (NK) or gammadelta T cells was detected. Pulsing DCs with whole apoptotic tumor cells induced an even more pronounced lytic effect. However, in this case, MHC class-I blocking was only partially effective, and unrelated cell lines were also killed. IFN-gamma staining revealed activation of CTLs and T-helper cells, as well as NK and gammadelta T cells. Trans-well cultures of NK cells, apoptotic tumor cells, and DCs showed that NK cell activation was dependent on direct cell-to-cell contact with tumor cells and the presence of interleukin-12 produced by DCs. These results indicate that the choice of antigen preparation is a critical determinant in the induction of antitumor immunity. Tumor vaccines consisting of DCs and apoptotic tumor cells may be able to activate CTLs, as well as effector cells of the innate immune system.     

树突状细胞和脑胶质瘤自体免疫治疗的实验和临床研究

目的 对树突状细胞(DC)肿瘤疫苗进行实验和临床研究并探讨其应用前景。方法 从脑胶质瘤患者外周血提取DC进行培养并对其进行形态学和免疫学鉴定。采用乳酸脱氢酶(LDH)释放法检测负载不同肿瘤抗原的DC诱导细胞毒性T细胞(CTL)体外杀伤活性。在实验研究的基础上拟定临床治疗方案:将56例脑胶质瘤术后患者分为3组,实验组19人接受冻融法制备的自体肿瘤抗原DC疫苗和卡介苗(BCG)治疗,阳性对照组17人接受全肿瘤组织匀浆和BCG治疗,阴性对照组20人,术后仅接受放疗。结果 采用粒细胞、巨噬细胞集落刺激因子(GM-CSF)及白细胞介素-4(IL-4)培养可获得高纯度的DC。DC在负载肿瘤抗原后诱导CTL杀伤活性大大提高而DC与肿瘤细胞共培养对CTL杀伤活性无显著影响。临床治疗中实验组中位数生存时间大于38个月,阳性对照组为24个月,二者均高于阴性对照组(13个月)。生存分析示3组间的生存曲线分布差异有显著性意义。结论 采用冻融法制备的自体脑胶质瘤抗原DC疫苗可抑制脑胶质瘤的复发和进展,从而有效地延长脑胶质瘤患者的生存时间,有良好的临床应用前景。     

树突状细胞和脑胶质瘤自体免疫治疗的实验和临床研究(英文)

目的对树突状细胞(DC)肿瘤疫苗进行实验和临床研究并探讨其应用前景。方法从脑胶质瘤患者外周血提取 DC 进行培养并对其进行形态学和免疫学鉴定。采用乳酸脱氢酶(LDH)释放法检测负载不同肿瘤抗原的 DC 诱导细咆毒性 T 细胞(CTL)体外杀伤活性。在实验研究的基础上拟定临床治疗方案:将56例脑胶质瘤术后患者分为3组,实验组19人接受冻融法制备的自体肿瘤抗原 DC 疫苗和卡介苗(BCG)治疗,阳性对照组17人接受全肿瘤组织匀浆和 BCG治疗,阴性对照组20人,术后仪接受放疗。结果采用粒细胞、巨噬细胞集落刺激因子(GM-CSF)及白细胞介素-4(IL-4)培养可获得高纯度的 DC。DC 在负载肿瘤抗原后诱导CTL 杀伤活性大大提高而 DC 与肿瘤细胞共培养对 CTL 杀伤活性无显著影响。临床治疗中实验组中位数生存时间大于38个月,阳性对照组为24个月,二者均高于阴性对照组(13个月)。生存分析示3组间的生存曲线分布差异有显著性意义。结论采用冻融法制备的自体脑胶质瘤抗原 DC 疫苗可抑制脑胶质瘤的复发和进展,从而有效地延长脑胶质瘤患者的生存时间,有良好的临床应用前景。     

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。     

腺病毒介导的HBsAg基因修饰树突状细胞的体外生物学特性

目的： 〖HT5"SS〗探讨腺病毒介导乙型肝炎病毒表面抗原（AdVHBsAg）基因修饰树突状细胞（dendritic cell,DC）瘤苗体外生物学活性。〖HT5W〗方法〖HT5"SS〗：将腺病毒表达载体AdVHBsAg转染人单个核细胞来源的DC,构建AdVHBsAgDC肝癌瘤苗,采用Western blotting法鉴定转染基因表达,FACS检测表面分子和内吞功能,3HTdR法检测T细胞增殖反应的能力,MTT法检测CTL活性。〖HT5W〗结果〖HT5"SS〗：HBsAg基因转染后,Western blotting法检测结果示HBsAg基因表达于转染的DC,表明腺病毒介导的HBsAg基因转染的有效性。AdVHBsAgDC可高表达CD1a、CD11c、 CD83、CD86和HLADR,但内吞功能较DC组降低(P<0.05)。AdVHBsAg DC刺激自体T细胞增殖功能均明显高于DC对照组和AdVLacZDC组(P<0.05)。AdVHBsAg DC体外诱导CTL对HepG2215肿瘤细胞的杀伤作用具有特异性。〖HT5W〗结论〖HT5"SS〗：肝癌相关基因HBsAg可作为乙型肝炎病毒相关性肝癌的切入点,该研究为HBV相关肝癌DC体内免疫治疗提供了实验依据。     

Dendritic cells fused with allogeneic breast cancer cell line induce tumor antigen-specific CTL responses against autologous breast cancer cells

Dendritic cell (DC)/tumor cell fusion vaccine has been revealed as a promising tool for the antitumor immunotherapy. Previous research has shown that fusion hybrids of human DCs and autologous tumor cells can induce cytotoxic T lymphocyte (CTL) responses against autologous tumor cells in animal models and human clinical trials. However, a major restriction factor for the clinical use is the difficulty for preparation of sufficient amount of autologous tumor cells especially for the patients with metastasis cancer whose primary tumor lesion is not clear or has been resected. In this study, allogeneic breast cancer cell line MCF-7 cells were electrofused to autologous DCs from patient with breast cancer as a strategy to deliver shared breast cancer antigens to DCs. Fusion cells generated by autologous DCs and allogeneic MCF-7 were able to induce autologous T lymphocytes proliferation, high levels of IFN-gamma production and CTL responses. CTLs induced by DCs/allogeneic MCF-7 fusion cells were able to kill autologous breast cancer cells in an antigen specific and HLA restriction manner. Our study may provide the experiment basis for the use of allogeneic breast cancer cell line in the DC/tumor cell fusion cell vaccination strategy.     

Generation of antigen-presenting cells using cultured dendritic cells and amplified autologous tumor mRNA

Novel antigen-presenting cells (APCs) were generated using cultured dendritic cells (DCs) and amplified tumor mRNA, and the potential of tumor antigen-reactive T cell induction by the tumor RNA-introduced DCs (DC/tumor RNA) was analyzed in a patient with melanoma antigen-encoding gene (MAGE3)-positive malignant melanoma of the esophagus. DCs were generated from an adherent fraction of peripheral blood mononuclear cells in the presence of granulocyte macrophage colony-stimulating factor and interleukin-4. Tumor mRNA was purified from tumor tissue, amplified in vitro using a T7 RNA polymerase system, and then introduced into DCs by electroporation (150 V/150 microF or 100 V/200 microF). The gene introduction efficiency was 44-55% as measured by enhanced green fluorescent protein reporter gene expression, and the viability of RNA-introduced DCs was approximately 80%. DC/tumor RNA could induce tumor antigen-reactive cytotoxic T lymphocytes (CTLs) in an mRNA-specific manner, but had no effect on the self-antigen-reactive T cells. DC/tumor RNA could induce the polyspecific antigen-reactive CTL responses mediated by both human leukocyte antigen class I and class II molecules, whereas MAGE3 peptide-pulsed DCs induced only the monospecific MAGE3-reactive CTL responses mediated by human leukocyte antigen class I molecules, showing the superiority of the DC/tumor RNA over the DC/peptide. It is suggested that the use of DC/tumor RNA as antigen-presenting cells may be more effective, convenient and practical for the DC-based anti-cancer immunotherapy.     

Tumor cyclooxygenase 2-dependent suppression of dendritic cell function.

Dendritic cells (DCs) serve as professional antigen-presenting cells and are pivotal in the host immune response to tumor antigens. To define the pathways limiting DC function in the tumor microenvironment, we assessed the impact of tumor cyclooxygenase (COX)-2 expression on DC activities. Bone marrow-derived DCs were cultured in either tumor supernatant (TSN) or TSN from COX-2-inhibited tumors. After culture, DCs were pulsed with tumor-specific peptides, and their ability to generate antitumor immune responses was assessed following injection into established murine lung cancer. In vitro, DC phenotype, alloreactivity, antigen processing and presentation, and interleukin (IL)-10 and IL-12 secretion were evaluated. DCs cultured in TSN failed to generate antitumor immune responses and caused immunosuppressive effects that correlated with enhanced tumor growth. However, genetic or pharmacological inhibition of tumor COX-2 expression restored DC function and effective antitumor immune responses. Functional analyses indicated that TSN causes a decrement in DC capacity to (a) process and present antigens, (b) induce alloreactivity, and (c) secrete IL-12. Whereas TSN DCs showed a significant reduction in cell surface expression of CD11c, DEC-205, MHC class I antigen, MHC class II antigen, CD80, and CD86 as well as a reduction in the transporter-associated proteins, transporter associated with antigen processing 1 and 2, the changes in phenotype and function were not evident when DCs were cultured in supernatant from COX-2-inhibited tumors. We conclude that inhibition of tumor COX-2 expression or activity can prevent tumor-induced suppression of DC activities.