NDV-ATV与树突状细胞联合诱导抗胃癌作用研究

目的研究负载新城鸡瘟病毒修饰的自体肿瘤细胞瘤苗(NDV ATV)抗原的树突状细胞(DCs)诱导的抗胃癌效应。方法新城鸡瘟病毒感染MNK45细胞并灭活,粒巨细胞集落刺激因子(GM CSF)、IL4和TNFα体外诱导扩增DCs并负载NDV修饰后的胃癌抗原,制备胃癌抗原特异性细胞毒性淋巴细胞(CTL);用CytoTox96TM检测其对MNK45细胞体外杀伤效应,并以冻融胃癌细胞抗原为对照。结果负载新城鸡瘟病毒胃癌抗原DCs诱导的特异性CTL对MNK45细胞的杀伤率达(90.15±9.82)%,显著高于冻融抗原的杀伤率〔(60.57±5.74)%〕,P<0.05,且其对同种不同分化类型的胃癌细胞株MGC803及SGC7901也有较高的杀伤效应〔(52.23±6.45)%,(61.75±8.84)%〕,而对LOVO及HepG2肿瘤细胞无显著杀伤作用〔(9.11±3.42)%,(8.30±3.12)%〕,P<0.05。结论新城鸡瘟病毒修饰的胃癌细胞,联合树突状细胞可诱导产生高效和特异的抗胃癌效应。提示NDV ATV联合DCs可作为胃癌免疫治疗的一种有效的新方法。     

负载NDV-ATV的树突状细胞诱导的抗胃癌效应研究

目的研究负载新城鸡瘟病毒修饰的自体肿瘤细胞瘤苗(NDV-ATV)抗原的树突状细胞(DCs)诱导的抗胃癌效应。方法新城鸡瘟病毒感染MNK45细胞并灭活,GMCSF、IL-4和TNF-α体外诱导扩增DCs并负载NDV修饰后的胃癌抗原,制备胃癌抗原特异性CTL;用CytoTox96TM检测其对MNK45体外杀伤效应。并以冻融胃癌细胞抗原为对照。结果负载新城鸡瘟病毒胃癌抗原DCs诱导的特异性CTL对MNK45的杀伤率达90.15%,显著高于冻融抗原的杀伤率(P<0.05)。且其对同种不同分化类型的胃癌细胞株MGC803、SGC7901也有较高的杀伤效应,而对LOVO及HepG2肿瘤细胞无显著杀伤作用(P<0.01)。结论新城鸡瘟病毒修饰的胃癌细胞,联合树突状细胞可诱导产生高效和特异的抗胃癌效应。提示NDV-ATV联合DCs可作为胃癌免疫治疗的一种有效的新方法。     

负载膀胱癌酸洗抗原肽对树突状细胞分泌IL-12的影响及意义

目的：研究负载膀胱癌酸洗抗原肽对树突状细胞（DCs）分泌IL-12的影响和意义.方法：用弱酸洗脱法获得膀胱癌细胞株BIU-87肿瘤抗原肽;联合应用重组人GM-CSF、IL-4和TNF-α体外诱导健康志愿者DCs并负载膀胱癌肿瘤抗原肽;DC诱导膀胱癌抗原特异性CTLs;用MTT法检测其对BIU-87体外杀伤效应;用ELISA法检测第3、6、9天培养细胞的上清液,评价DCs分泌IL-12 p70的能力.结果：联合应用重组人GMCSF、IL-4和TNF-α可在体外诱导出DCs;负载膀胱癌抗原肽的DC诱导的特异性CTLs可杀伤高达（78.5±7.0）%的BIU-87细胞,显著高于各对照组（P〈0.01）;经ELISA方法检测不同时期的DCs分泌IL-12的量不同,而且负载抗原肽DCs较未负载抗原DCs有更强的分泌能力（P〈0.05）.结论：IL-12 p70在DCs刺激特异性CTLs的过程中发挥重要作用,其分泌量受DCs的成熟状态及某些刺激信号的影响,膀胱癌酸洗抗原肽是其刺激信号之一.     

胎儿来源的树突状细胞体外诱导抗前列腺癌效应的实验研究

目的研究胎儿来源树突状细胞(DC)体外诱导抗前列腺癌的特异性细胞免疫效果。方法从胎儿骨髓、肝脏获得单个核细胞,经重组人粒细胞-巨噬细胞集落刺激因子(rhGM-CSF)、重组人白细胞介素-4(rhIL-4)和重组人肿瘤坏死因子-α(rhTNF-α)诱导产生DC。50%~70%硫酸铵饱和沉淀法获取前列腺癌细胞DU145含热休克蛋白(HSP)成分的细胞溶解物,以该抗原负载DC,激活胎脾细胞产生肿瘤特异性细胞毒性T淋巴细胞(CTL)。MTT法检测CTL对DU145、PC3和EJ细胞的杀伤效应。结果胎儿骨髓、肝脏可诱导出功能成熟的DC,高表达CD1 a、CD86、HLA-DR和CD83。负载DU145抗原的DC可诱导产生CD8+CTL。CD8+T细胞表型由转化前的(14.09±2.46)%变为转化后的(62.76±2.64)%。对DU145细胞有明显细胞毒作用,显著高于对EJ细胞杀伤效应(P<0.01)。结论含HSP成分的肿瘤细胞溶解物负载胎儿来源DC,体外可诱导出特异性抗肿瘤免疫应答。     

肾癌树突状细胞疫苗的体外活性研究

目的 探讨负载人肾癌细胞抗原肽制备树突状细胞(DC)疫苗体外杀伤肾癌细胞的作用.方法 利用细胞膜酸洗脱法获得人肾透明细胞癌细胞株786-0细胞表面抗原肽.外周血单个核细胞在体外经人粒细胞一巨噬细胞集落刺激因子,白细胞介素4和脂多糖诱导获得成熟的DC,并负载分离到的抗原肽制备DC疫苗.利用疫苗体外诱导出特异性细胞毒性T淋巴细胞(CTL)作为实验组.同时设置4个对照组,对照组1用未负载抗原肽的DC和单个核细胞混合培养,对照组2用单个核细胞进行培养,对照组3用抗原肽与单个核细胞混合培养,对照组4用未负载抗原肽的DC单独培养.4个对照组也加入同实验组相同的各种因子进行培养.51Cr释放法检测特异性CTL的杀伤活性.结果 疫苗诱导的特异性CTL对肾癌细胞的杀伤活性为(31.93±5.05)%,与对照组(5.88±2.26)%、(8.03±6.70)%、(9.70±2.09)%、(9.35±3.58)%相比差异有统计学意义(P<0.05).结论 负载抗原肽的DC疫苗体外试验有高效的抗肾癌细胞活性.     

树突状细胞激活的肿瘤浸润性淋巴细胞体外特异性抗小鼠肝癌研究

目的树突状细胞(DC)是目前已知的功能最强的抗原提呈细胞(APC),可以向包括肿瘤浸润性淋巴细胞(TIL)在内的T淋巴细胞提呈抗原,并诱发细胞毒T淋巴细胞(CTL)反应。本文旨在探讨H22细胞和B16细胞全细胞性抗原致敏的树突状细胞激活的肿瘤浸润性淋巴细胞体外抗小鼠肝癌活性。方法从小鼠四肢长骨骨髓中获取DC,应用粒/巨噬细胞集落刺激因子(GM-CSF)、白介素-4(IL-4)和肿瘤全细胞性抗原致敏DC,然后用DC激活TIL,观察TIL在体外对H22细胞、Hepal-6细胞和B16细胞的杀伤活性。结果经H22细胞全细胞性抗原致敏的DC激活的TIL具有很高的对H22细胞杀伤活性,杀伤率为(71·31±3·11)%,明显高于其对Hepal-6和B16细胞的杀伤活性[杀伤率分别为(50·11±3·03)%,(30·31±2·89)%];也明显高于未经H22细胞全细胞性抗原致敏的DC激活的TIL、DC激活的脾淋巴细胞和未经DC激活的脾淋巴细胞对H22细胞杀伤活性[杀伤率分别为(49·80±3·21)%,(48·76±3·60)%和(19·23±2·71)%]和对Hepal-6细胞杀伤活性[杀伤率分别为(39·4±3·21)%,(38·62±2·87)%和(18·73±2·40)%]以及对B16细胞杀伤活性[杀伤率分别为(26·38±2·51)%,(25·82±2·70)%和(18·34±3·01)%],同时经B16细胞全细胞性抗原致敏的DC激活的TIL(来源于H22瘤体)也可诱导相对较低的对B16细胞的特异性细胞杀伤活性。结论来源于H22瘤体的TIL经H22细胞全细胞性抗原致敏的DC激活后可产生很强的针对H22细胞的特异性杀伤活性,明显高于其他各组,说明DC能诱导TIL产生高效而特异的体外抗小鼠肝癌免疫。     

DC疫苗对荷人前列腺癌免疫重建NOD/SCID小鼠的抑瘤作用

目的 探讨PC-3细胞冻融抗原致敏的树突状细胞(dendritic cells,DC)疫苗(PC-3-DC)对荷人前列腺癌免疫重建NOD/SCID小鼠(hu-PBL-NOD/SCID)的抑瘤作用.方法 采用人外周血淋巴细胞(peripheral blood lymphocytes,PBL)腹腔注射法建立hu-PBL-NOD/SCID小鼠模型,随机分为实验组(PC-3-DC组)和对照组(DC组、PBS组),腹腔分别注射PC-3-DC疫苗、未致敏的DC和PBS.每周1次,共2次,然后接种1×107 PC-3细胞,观察鼠成瘤率、成瘤潜伏期、肿瘤体积以及测定特异性CTL活性.结果 ELISA法可检测到小鼠血清中人IgG水平,hu-PBL-NOD/SCID嵌合模型重建成功,各组小鼠间成瘤率无明显差异,但PC-3-DC组成瘤潜伏期延长,肿瘤生长缓慢,2周后肿瘤体积明显小于DC组和PBS组,差异有统计学意义(P＜0.05),实验组脾淋巴细胞对PC-3细胞有特异性杀伤效应,而对K562细胞则无杀伤活性.结论 负载PC-3冻融抗原的DC疫苗可诱导人T淋巴细胞活化增殖,能有效抑制hu-PBL-NOD/SCID小鼠肿瘤的生长.     

负载冻融抗原的树突状细胞诱导特异性抗膀胱癌作用的研究

目的研究负载膀胱癌冻融抗原的树突状细胞（DC）诱导的对膀胱癌细胞的特异性杀伤效应。方法反复冻融法获得BIU-87细胞抗原;人单个核细胞在含重组人GM—CSF、IL-4和TNF-a的RPMI1640培养基中体外诱导出人DC并负载肿瘤抗原;9d后成熟DC与自体T细胞共孵育诱导膀胱癌抗原特异性CTLs; 用MTT法检测其对BIU-87体外杀伤效应;用ELISA法检测DC分泌IL-12的能力。结果负载膀胱癌抗原的DC诱导的特异性CTLs可明显杀伤BIU-87细胞,在效靶比为40：1时杀伤率为（65．5±6．4）％,显著高于各对照组（P〈0．01）;负载抗原DCs较未负载抗原DCs有更强的分泌IL-12能力（P〈0．05）,而且不同时期的DC分泌的量不同。结论负载膀胱癌抗原的DC在体外可诱导出高效而特异的抗膀胱癌效应,提示以DC为中心的肿瘤生物治疗有望提高膀胱癌综合治疗水平。     

树突状细胞肿瘤疫苗诱导抗胃癌作用的实验研究

目的探讨树突状细胞(dendriticcells,DCs)肿瘤疫苗诱导的抗胃癌效应对荷瘤裸小鼠的作用。方法使用胃癌细胞冻融抗原,体外致敏从小鼠骨髓诱导分化来源的DCs成为肿瘤疫苗,用其来刺激脾脏淋巴细胞,得到肿瘤抗原特异的细胞毒性T淋巴细胞(CTL),观察其对荷瘤裸小鼠肿瘤生长的影响以及早期凋亡诱导作用。结果经重组小鼠粒细胞巨噬细胞集落刺激因子(rmGM-CSF)和重组小鼠白细胞介素4(rmIL-4)体外诱导小鼠骨髓细胞,得到大量形态典型、具备强烈刺激增殖能力、高表达CD1a、CD11c、CD40和CD80的DCs。肿瘤抗原致敏DCs刺激脾脏淋巴细胞成为CTL后,可显著抑制裸小鼠皮下移植瘤生长并致瘤细胞早期凋亡增加。结论DCs肿瘤疫苗可通过CTL的作用,抑制荷瘤裸小鼠的胃癌细胞生长及促进胃癌细胞早期凋亡。     

肺癌抗原负载的树突细胞体外诱导细胞毒性T淋巴细胞的杀瘤作用

目的探讨不同方式肺癌抗原负载的树突状细胞(DC)在抗肿瘤免疫中的作用。方法取肺癌患者外周静脉血体外诱导和培养DC,分别以肺癌细胞总RNA转染DC(转染组)、肺癌细胞融合DC(融合组)和肺癌细胞冻融抗原负载DC(冻融组),以未负载抗原的DC(未负载组)和T细胞组作为对照,比较各组(每组n=6)DC诱导的细胞毒性T淋巴细胞(CTL)对肺癌细胞A_(549)的杀伤率(%)。结果转染组、融合组、冻融组、未负载组和T细胞组的杀伤率分别为(73．2±5．9)%、(61．6±6．2)%、(55．3±6．9)%、(22．3±6．1)%和(19．8±6．3)%(P＜0．05)；其中转染组、融合组和冻融组高于未负载组和T细胞组(P＜0．05)；转染组和融合组之间差异无统计学意义(P＞0．05),但两者均大于冻融组(P＜0．05)。RNA转染DC所需的肿瘤细胞数仅是冻融及融合方式的1/5和1/6。结论3种肺癌抗原负载DC方式均有诱导效应细胞杀伤靶细胞的增效作用,而以肺癌细胞总RNA转染方式为佳。     

Induction of tumor-specific cytotoxic T lymphocytes in cancer patients by autologous tumor RNA-transfected dendritic cells

OBJECTIVE: To demonstrate the feasibility of inducing tumor antigen-specific immune responses in patients with metastatic cancer using total tumor RNA-loaded dendritic cells (DCs). SUMMARY BACKGROUND DATA: The authors have shown that DCs transfected with mRNA encoding defined tumor antigens induce tumor antigen-specific T-cell responses in vitro and in vivo. There may be significant advantages to inducing immune responses against the entire repertoire of antigens expressed by a patient's autologous tumor. METHODS: RNA was extracted from a metastatic colon cancer and used to load autologous DCs. The DCs were coincubated with autologous T cells and the cytolytic activity of the T cells was assessed by the ability to lyse the autologous tumor cells. RNA was then extracted from a metastatic lung cancer and used to load autologous DCs, followed by four injections of the DC vaccine given every 4 weeks. Tumor antigen-specific cytotoxic T lymphocyte activity was then evaluated by testing peripheral blood mononuclear cells for their ability to lyse an antigen-expressing target. RESULTS: DCs transfected with the total RNA content of autologous tumor cells stimulated antigen-specific T-cell responses that are capable of recognizing and lysing autologous, primary tumor cells in vitro. Tumor-specific immune responses were induced in a patient with a carcinoembryonic antigen-expressing adenocarcinoma after immunization with autologous DCs transfected with total tumor RNA. CONCLUSIONS: DCs transfected with total tumor RNA may represent a method for inducing immune responses against the entire repertoire of tumor antigens of surgically resected malignancies.     

In vitro induction of tumor-specific HLA class I-restricted CD8+ cytotoxic T lymphocytes from patients with locally advanced breast cancer by tumor antigen-pulsed autologous dendritic cells

BACKGROUND: Early dissemination of treatment-resistant tumor cells remains the major cause of metastatic recurrence and death in breast cancer patients. Dendritic cells (DCs) are the most powerful antigen-presenting cells, and recently DC-based vaccination has shown great promise for the treatment of human malignancies by immunological intervention. MATERIALS AND METHODS: CD8+ T lymphocytes derived from peripheral blood mononuclear cells stimulated in vitro with autologous breast tumor antigen-pulsed DCs were tested for their ability to induce a HLA class I restricted cytotoxic T lymphocyte (CTL) response against autologous tumor cells. To correlate cytotoxic activity by CTL with T cell phenotype, two-color flow cytometric analysis of surface markers and intracellular cytokine expression was performed. RESULTS: DC pulsed with breast tumor extracts consistently elicited a tumor-specific HLA class I restricted CTL response in vitro in three consecutive patients harboring locally advanced breast cancer. CTL expressed strong cytolytic activity against autologous tumor cells but did not lyse autologous Epstein Barr virus-transformed lymphoblastoid cell lines and showed variable cytotoxicity against the natural killer-sensitive cell line K-562. In all patients, two color flow cytometric analysis of surface markers and intracellular cytokine expression demonstrated that tumor-specific CTL exhibited an heterogeneous CD8+/CD56+ expression and a striking Th1 cytokine bias (IFNgamma(high)/IL-4 (low)). CONCLUSIONS: Tumor lysate-pulsed DCs can consistently stimulate specific CD8+ CTLs able to kill autologous tumor cells in patients with locally advanced breast cancer in vitro. Tumor antigen-pulsed DC-based vaccinations may be appropriate for the treatment of residual and/or chemotherapy-resistant breast cancer refractory to standard salvage treatment modalities.     

Advances in specific immunotherapy for prostate cancer

OBJECTIVES: The absence of effective therapies for advanced prostate cancer has entailed an intensive search for novel treatments. This review presents an overview of specific immunotherapeutic strategies for prostate cancer. METHODS: Current literature was reviewed regarding the identification of tumor antigens and the design of T-cell- and antibody-based immunotherapy for prostate cancer. The PubMed database was searched using the key words antibodies, clinical trials, dendritic cells, immunotherapy, prostate cancer, and T cells. RESULTS: T cells and antibodies are powerful components of the specific antitumor immune response. CD8+ cytotoxic T lymphocytes (CTLs) efficiently destroy tumor cells. CD4+ T cells improve the antigen-presenting capacity of dendritic cells (DCs) and support the stimulation of tumor-reactive CTLs. Monoclonal antibodies exhibit their antitumor effects via antibody-dependent cellular cytotoxicity and complement activation. Consequently, much attention has been given to the identification of tumor antigens that represent attractive targets for specific immunotherapy. Several prostate cancer-related antigens were described and used in clinical trials. Such studies were based on the administration of peptides, proteins, or DNA. Furthermore, men with prostate cancer were vaccinated with peptide-, protein-, or RNA-loaded DCs, which display an extraordinary capacity to induce tumor-reactive T cells. Monoclonal antibodies directed against surface antigens were also used. Clinical trials revealed that immunotherapeutic strategies represent safe and feasible concepts for the induction of immunologic and clinical responses in men with prostate cancer. CONCLUSIONS: Specific immunotherapy represents a promising treatment modality for prostate cancer. Further improvement of the current approaches is required and may be achieved by combining T-cell- and antibody-based vaccination strategies with radio-, hormone-, chemo-, or antiangiogenic therapy.     

Tumor specific cytotoxicity and telomerase down-regulation in prostate cancer by autologous dendritic cells loaded with whole tumor cell antigens

ObjectivesWe investigated the efficacy of cytotoxic activity of whole tumor cell-antigen loaded dendritic cells in the treatment of hormone refractory prostate cancer.Materials and methodsFrom 10 patients with HRPC, peripheral blood samples were obtained and cultured with GM-CSF and IL-4 to provide differentiation of peripheral blood mononuclear cells (PBMs) into dendritic cells (DCs). DC phenotype was confirmed by flow cytometry (MHC class II HLA-DR, CD80, CD86, CD83, CD14 expression analysis). Subsequently, whole tumor cell lysates of LNCaP, DU-145, and PC-3 lines were incubated with DCs. Direct antitumoral activity of induced DCs and activation of PBM cells by these DCs was assessed by lactate dehydrogenase (LDH) cytotoxicity assay. Post-treatment changes in the telomerase gene expression of tumor cells were investigated by real time RT-PCR analysis.ResultsLDH activity was highest in the PC-3 cell line (9.5%) and lowest in the DU-145 line (3.2%). Co-incubation of PBMs with activated DCs resulted in a significant increase at the levels of cytotoxicity in all cell lines. Likewise, incubation of tumor cells with activated DCs caused significant down-regulation of telomerase gene expression in all cell lines. Most pronounced suppression was in the LNCaP cell line (decrease by 97.1%). The decrease in the level of telomerase gene expression in DU-145 and PC-3 cell lines was 80% and 70%, respectively.ConclusionsCytotoxic immune response to prostate cancer-associated antigens can be elicited in vitro in patients with HRPC using an allogeneic tumor cell-based strategy. DC-based active immunotherapy appears as an effective treatment method in the pre-clinical setting and further phase I/II trials are warranted.     

Identification of an HLA-A*0201-restricted T-cell epitope derived from the prostate cancer-associated protein trp-p8

BACKGROUND: New concepts for the immunotherapy of prostate carcinoma (PCa) largely depend on the identification of suitable target antigens that are present in a high percentage of prostate tumors. Their expression in normal tissues should be restricted to the prostate and they should be immunogenic in vivo. The number of antigens displaying these properties is still limited. Here, we identify for the first time an immunogenic peptide derived from the prostate-specific protein transient receptor potential-p8 (trp-p8) that is recognized by cytotoxic T lymphocytes (CTLs) from PCa patients. METHODS: To determine the abundance of trp-p8 in prostate tumors, the expression level of trp-p8 mRNA was quantitatively analyzed in a panel of prostate cancer tissues. Trp-p8-derived human leukocyte antigen (HLA)-A*0201-restricted peptides were selected and tested for the in vitro activation of CTLs when loaded on autologous dendritic cells (DCs). RESULTS: Trp-p8 mRNA was found to be expressed in all prostate tumors and in the corresponding normal prostate tissue. Of five selected trp-p8-derived peptides, only peptide GLMKYIGEV was shown to activate specific CTLs, which effectively lysed PCa cells confirming the endogenous generation and presentation of this peptide by tumor cells. CONCLUSIONS: Our results suggest this antigen as a suitable target for the T-cell-based immunotherapy of PCa.     

Human tumor-rejection antigens and peptides from genes to clinical research

One of the most significant advances in the field of modern tumor immunology is the identification of genes encoding tumor-rejection antigens that are recognized by human leukocyte antigen (HLA) class I-restricted and tumor-specific cytotoxic T lymphocytes (CTLs). Several peptides encoded by these genes are now under clinical trial as cancer vaccines, and major tumor regression has been observed in some melanoma patients. These results indicate that identification of the peptides capable of inducing CTLs may provide a new modality of cancer therapy. We investigated tumor-rejection antigens from epithelial cancers, and reported 7 genes encoding tumor-rejection antigens and peptides available for specific immunotherapy of HLA-A26 or -A24 patients with epithelial cancers. Furthermore, we identified more than 10 genes encoding tumor-rejection antigens and peptides available for specific immunotherapy of HLA-A2 patients with epithelial cancers. Therefore these new antigens and peptides could be applicable to the treatment of numerous epithelial cancer patients worldwide. Phase I clinical trials of cancer vaccine with these peptides for epithelial cancer patients are in progress at our university. Basic and clinical research will provide new insights for a better understanding of the molecular basis of T cell-mediated recognition of cancer cells and be important for the development of cancer vaccines.     

人卵巢癌细胞光动力学细胞裂解物负载树突状细胞对体外免疫应答的影响

目的:研究人卵巢癌细胞光动力学细胞裂解物对体外免疫应答反应的影响.方法:体外诱导培养人脐血树突状细胞(DC).人卵巢癌细胞SKOV3分别用光动力学方法(激光照射)及冻融的方法制备细胞裂解物,与DC共培养48 h,RPMI1640与DC培养作为阴性对照,采用流式细胞仪检测DC免疫表型CD1a、CD80、CD86、HLA-...