hKDR融合基因致敏树突状细胞对小鼠肝癌细胞杀伤活性研究

目的:探讨hKDR融合基因致敏树突状细胞(DC)激发的细胞毒性T淋巴细胞(CTL)对小鼠肝癌细胞的杀伤活性.方法:自小鼠骨髓中获取DC,以粒细胞-巨噬细胞集落刺激因子(GM-CSF)和hKDR融合基因的mRNA致敏DC;用致敏的DC免疫小鼠取其脾脏获得CTL,即效应细胞,用脂质体将pCDNA hKDR转染至小鼠肝癌Hepal-6细胞株中,作为靶细胞;将效应细胞与靶细胞按不同比例混合,用乳酸脱氢酶(LDH)释放法检测细胞毒作用.结果:未经修饰的DC激活的CTL与靶细胞比例为100:1、50:1、25:1情况下,其杀伤率分别为19.2%、12.3%、6.9%;经hKDR修饰的DC激活的CTL与靶细胞比例为100:1、50:1、25:1情况下,其杀伤率分别为71.6%、55.8%、22.7%,将两组结果在相同的比例下两两比较,均有显著性差异(P＜0.05).结论:hKDR融合基因致敏DC激活的CTL对小鼠肝癌细胞具有较强的杀伤作用,且杀伤能力随效应细胞与靶细胞比例增高而增强.     

hKDR融合基因致敏树突状细胞对小鼠肝癌细胞杀伤活性研究

目的:探讨hKDR融合基因致敏树突状细胞(DC)激发的细胞毒性T淋巴细胞(CTL)对小鼠肝癌细胞的杀伤活性.方法:自小鼠骨髓中获取DC,以粒细胞-巨噬细胞集落刺激因子(GM-CSF)和hKDR融合基因的mRNA致敏DC;用致敏的DC免疫小鼠取其脾脏获得CTL,即效应细胞,用脂质体将pCDNA hKDR转染至小鼠肝癌Hepal-6细胞株中,作为靶细胞;将效应细胞与靶细胞按不同比例混合,用乳酸脱氢酶(LDH)释放法检测细胞毒作用.结果:未经修饰的DC激活的CTL与靶细胞比例为100:1、50:1、25:1情况下,其杀伤率分别为19.2%、12.3%、6.9%;经hKDR修饰的DC激活的CTL与靶细胞比例为100:1、50:1、25:1情况下,其杀伤率分别为71.6%、55.8%、22.7%,将两组结果在相同的比例下两两比较,均有显著性差异(P＜0.05).结论:hKDR融合基因致敏DC激活的CTL对小鼠肝癌细胞具有较强的杀伤作用,且杀伤能力随效应细胞与靶细胞比例增高而增强.     

前列腺特异性抗原细胞毒性T淋巴细胞表位多抗原肽的抗肿瘤免疫效应研究

目的:研究前列腺特异性抗原(PSA)来源的细胞毒性T淋巴细胞(CTL)表位多抗原肽对前列腺癌的抗肿瘤免疫效应。方法:体外分离培养来源于人白细胞抗原(HLA)-A2.1阳性的健康志愿者外周血单个核细胞的成熟树突状细胞(DC),按单抗原肽组(PSA146-154)、多抗原肽组(PSA146-154-MAP4)、阴性对照组(人类免疫缺陷病毒表位肽HIVpol476-484)培养制备相应的效应细胞,以前列腺癌细胞株LNCaP、DU-145和结肠癌SW480细胞为靶细胞,采用标准4 h51Cr释放试验检测不同效应细胞/靶细胞细胞个数比(效/靶比,10∶1、20∶1、40∶1、80∶1)的特异性杀伤效应(以特异性杀伤效率为指标),酶联免疫斑点(ELISPOT)法检测各组效应细胞分泌γ干扰素(IFN-γ)的CD8+效应细胞数量。结果:各组效应细胞对DU-145、SW480细胞均无特异性杀伤效应,单抗原肽组和多抗原肽组效应细胞对LNCaP细胞具有明显的特异性杀伤效应,且多抗原肽组强于单抗原肽组,与效/靶比呈正相关。与阴性对照组比较,单抗原肽组和多抗原肽组分泌IFN-γ的CD8+效应细胞数量明显增加;与单抗原肽组比较,多抗原肽组分泌CD8+细胞数量明显增加(P<0.05)。结论:PSA多抗原肽能诱导机体产生强于单抗原肽的PSA特异性抗肿瘤免疫效应,并可以在一定程度上增强非特异性的抗肿瘤效果。     

热休克蛋白抗原肽致敏的脐血树突状细胞治疗肺癌的实验研究

目的研究热休克蛋白抗原肽致敏的脐血树突状细胞体外对肺癌细胞的杀伤作用。方法用体外构建的热休克蛋白-抗原肽复合物刺激经组合细胞因子诱导的脐血树突状细胞,MTT法检测其对肿瘤细胞的杀伤作用。结果所得蛋白经电泳及Western blot进行蛋白分子量及性质鉴定为热休克蛋70。热休克蛋白-抗原肽负载可以促进脐血树突状细胞对T细胞的激发作用,使其对靶细胞有了更强的生长抑制作用。各组T细胞杀伤活性分别为:负载抗原组(85.77±1.03)%(正常T细胞)、(45.01±1.66)%(肺癌患者T细胞);未负载抗原组(41.92±1.38)%(正常T细胞)、(13.99±3.07)%(肺癌患者T细胞)。组间比较差异有统计学意义(P<0.01)。结论热休克蛋白抗原肽负载脐血树突状细胞能有效激发外周血T淋巴细胞,使其对靶细胞有了更强的生长抑制作用。本研究为解决树突状细胞的来源及开发特异性CTL的治疗开创了条件。     

负载肝癌排斥抗原肽的树突状细胞瘤苗活化T细胞的应用

目的　探讨负载肝癌排斥抗原肽SLIVHLNEV(C met突变肽 ) [1 ] 的树突状细胞瘤苗活化的T细胞在体外及裸鼠肝癌模型体内诱导的特异性抗肿瘤免疫。方法　用肝癌排斥抗原肽SLIVHLNEV致敏从脾脏中分离培养的树突状细胞 ,再与同源T淋巴细胞混合培养。乳酸脱氢酶 (LDH)释放法检测细胞毒作用。同时建立荷人肝癌细胞系HHCC的裸鼠移植瘤模型 ,观察DC瘤苗活化的T细胞预防移植瘤发生和抑制移植瘤生长的作用。结果　在体外实验中 ,DC瘤苗诱导的CTLs能够特异性杀伤HHCC肝癌细胞。在裸鼠模型中 ,CTLs不但能预防裸鼠移植瘤的发生 ,而且抑制裸鼠移植瘤生长。结论　负载肝癌排斥抗原肽SLIVHLNEV的树突状细胞瘤苗活化的T细胞在体外和裸鼠模型中均可诱导较强的抗肿瘤免疫     

人树突状细胞融合肝癌细胞体外诱导特异性抗肿瘤免疫

目的 探讨人树突状细胞(DC)融合肝癌细胞(HCC)体外诱导T淋巴细胞产生特异性抗肝癌免疫的作用.方法 应用人重组粒细胞/巨噬细胞集落刺激因子(rhGM-CSF)和重组人白细胞介素4(rhIL-4)对人外周血单个核细胞进行体外诱导产生树突状细胞,流式细胞仪检测DC表面标志物表达水平,聚乙二醇融合DC与肝癌细胞HerG2,MTT法测定融合细胞(HerG2/DC)刺激T淋巴细胞增生、分化能力,细胞毒性实验检测HerG2/DC诱导的细胞毒T淋巴细胞(CTL)对HerG2的特异性杀伤作用.结果 融合细胞HerG2/DC刺激T淋巴细胞增值能力明显提高,HerG2/DC活化的CTL对HerG2具有明显的特异性杀伤作用.结论 人树突状细胞融合肝癌细胞可有效诱导T淋巴细胞产生特异性的抗肝癌肿瘤免疫.     

树突状细胞疫苗对人肺腺癌GLC-82细胞体外杀伤作用的实验观察

目的 通过建立负载人肺腺癌细胞株GLC-82可溶性抗原的树突状细胞(DC)疫苗,探讨应用DC疫苗的致敏特异性杀伤性T细胞(CTL)体外杀瘤细胞的可行性和实验条件,为后期l临床应用提供实验依据.方法 通过用定量摩尔氯化钾提取法获得人肺腺癌细胞GLC-82的可溶性抗原多肽(TSA),从人外周血单核细胞(PBMC)中用GM-CSF、白细胞介素-4和肿瘤坏死因子-α体外诱导扩增并鉴定获取DC,构建DC疫苗;利用DC疫苗刺激同种异体外周血T淋巴细胞活化增殖,诱导产生具有识别肺癌细胞抗原的特异性CTL的可行性及MTT法检测该CTL对GLC-82、肺癌CALU-6和人红白血病K 562细胞的体外杀伤效应.结果 人PBMC体外经7d诱导出的DC,经形态学、免疫组化证实具有典型的树突状细胞特性;负载GLC-82抗原的DC疫苗能有效诱导同种异体T淋巴细胞活化增殖产生CTL,最适浓度为1:10;诱导活化的CTL对靶细胞的杀伤活性明显高于未经肿瘤抗原致敏的组.结论 诱导培养人外周血PBMC中的Mo可获取大量DC,诱导出的DC功能较强,适宜临床应用;DC疫苗能强烈刺激初始型同种异体T淋巴细胞增殖产生CD 8+表达增加的CTL;激活的CTL对肺癌靶细胞发挥高效而特异的细胞毒效应,对非肺组织瘤靶细胞也具有非特异性杀伤效应.     

人树突状细胞融合肝癌细胞体外诱导特异性抗肿瘤免疫

目的探讨人树突状细胞（DC）融合肝癌细胞（HCC）体外诱导T淋巴细胞产生特异性抗肝癌免疫的作用。方法应用人重组粒细胞／巨噬细胞集落刺激因子（rhGM—CSF）和重组人白细胞介素4（rhIL-4）对人外周血单个核细胞进行体外诱导产生树突状细胞,流式细胞仪检测DC表面标志物表达水平,聚乙二醇融合DC与肝癌细胞HerG2,MTT法测定融合细胞（HerG2／DC）刺激T淋巴细胞增生、分化能力,细胞毒性实验检测HerG2／DC诱导的细胞毒T淋巴细胞（CTL）对HerG2的特异性杀伤作用。结果融合细胞HerG2／DC刺激T淋巴细胞增值能力明显提高,HerG2／DC活化的CTL对HerG2具有明显的特异性杀伤作用。结论人树突状细胞融合肝癌细胞可有效诱导T淋巴细胞产生特异性的抗肝癌肿瘤免疫。     

不同细胞因子诱导树突状细胞体外抗肿瘤效应研究

目的研究不同细胞因子诱导的树突状细胞(DC)体外抗肿瘤效应。方法常规分离健康人外周血单核细胞,分别采用粒巨噬集落刺激因子(GM-CSF),白细胞介素(IL)-4,肿瘤坏死因子(TNF),GM-CSF、IL-4、α干扰素(IFN-α)细胞因子组合将其诱导为DC,并分别负载SMMC7721细胞冻融抗原;用流式细胞仪检测第9天时细胞表面HLA-DR、CD1α、CD80、CD83的表达;采用四甲基偶氮唑盐比色(MTT)法检测各组刺激自体淋巴细胞增殖能力,并检测各组DC诱导的细胞毒性T淋巴细胞(CTL)对SMMC7721细胞株的杀伤率。结果第9天显微镜下观察各组细胞,其细胞形态无明显差异,但流式细胞仪显示IFN-α组DC其膜表面标志CD80、CD83、CD1a表达上调,在刺激同种T细胞增殖能力方面,较TNF-α组DC效果明显。且IL-12分泌明显增加。在肝癌细胞株SMMC7221杀伤实验中,培养液中加入IFN-α后杀伤效应明显。结论与TNF-α相比,IFN-α诱导DC一定程度上能增强其特异性杀伤能力。     

树突状细胞瘤苗诱导的抗神经胶质瘤作用体外实验研究

目的 :研究负载自体神经胶质瘤抗原的树突状细胞 (DCs)瘤苗在体外诱导的特异性细胞毒性淋巴细胞 (CTL)对神经胶质瘤细胞的杀伤效应。方法 :以组合酶消化法从新鲜神经胶质瘤手术标本中获取神经胶质瘤细胞 ,冻融制备神经胶质瘤抗原。GM -CSF、IL -4体外诱导外周血单个核细胞 (PBMC)获得DCs并负载神经胶质瘤抗原 ,继而以其刺激自体T淋巴细胞制备神经胶质瘤抗原特异性CTL ;用CytoTox96TM检测CTL对患自身神经胶质瘤细胞体外杀伤效应。结果 :负载神经胶质瘤抗原DCs诱导的特异性CTL对患者自身神经胶质瘤细胞的杀伤率达88 17 % ,显著高于LAK细胞的杀伤率 (P<0 05)。且其对同种不同分化类型的神经胶质瘤细胞株 (P<0 01)。结论 :负载神经胶质瘤抗原的DCs体外可诱导出高效而特异的抗神经胶质瘤效应 ,提示以DCs为中心的肿瘤生物治疗作用可望提高神经胶质瘤综合治疗水平     

Methadone therapy modulate the dendritic cells of heroin addicts

Evidence from various studies suggests that narcotics abuse may exert adverse immunomodulatory effects on immune responses. The aim of this research was to understand the effects of detoxification with methadone on the percentage of dendritic cells (DCs) and expression of its markers in heroin addicts. In this study, myeloid DCs (CD11c⁺) and plasmacytoid DCs (CD123⁺) were examined in two groups. These groups comprised of 20 healthy volunteers and 20 chronic heroin addicts, before and after detoxification with methadone. The percentages of myeloid DCs and plasmacytoid DCs were lower in addict subjects than in the control. The HLA-DR expression on DCs was significantly lower in addict subjects than in the control, whereas CD11c and CD123 expression in DCs subsets were increased in them. Most of these changes were modified after the methadone therapy. Dendritic cells are essential to the initiation of primary immune responses, therefore the disruption of their function can be one of the reasons for the increased prevalence of infections in heroin addicts. The methadone therapy can improve the imposed changes by heroin.     

慢病毒介导的人MUC1核心肽串联重复片段修饰的树突状细胞疫苗的制备

目的探讨制备慢病毒介导的人mucin糖粘蛋白1(MUC1)核心肽串联重复序列(TR)修饰的树突状细胞(DC)疫苗的可行性。方法人工合成编码MUC1 5个TR片段的基因序列,将其克隆入慢病毒载体pLV-GFP中,经酶切、基因测序鉴定。构建成功的慢病毒载体转染293T细胞,制备病毒。病毒液感染DC细胞。流式细胞术检测感染效率及DC表型,RT-PCR及Westernblot检测MUC1目的基因的表达。结果酶切及测序证实慢病毒载体构建成功。制备的慢病毒能有效感染DC细胞,感染效率达32.54%;流式检测证实慢病毒感染后DC成熟表型未受影响;RT-PCR及Western blot检测证实转染后的DC可有效表达MUC1。结论成功制备慢病毒介导的MUC1基因TR片段修饰的DC疫苗。     

Auranofin,an immunosuppressive drug,inhibits MHC class I and MHC class II pathways of antigen presentation in dendritic cells

Auranofin (AF), an orally administered, gold-based, anti-arthritic agent, has emerged as a clinically useful therapeutic drug for the treatment of rheumatoid arthritis. In the present study, we examined the effects of AF on major histocompatibility complex (MHC)-restricted antigen presentation in dendritic cells (DCs), which are the most important accessory cells for the induction of T cell responses. A mouse dendritic cell line, DC2.4 cells, and DCs that were generated from mouse bone marrow cells by culturing with granulocyte macrophage-colony stimulating factor (GM-CSF) and interleukin (IL)-4 were each pretreated with AF for 2 hr, and then incubated with ovalbumin (OVA). After the 2-hr incubation, the DCs were fixed, and the amounts of OVA peptide-H-2K^b complexes were assessed using OVa-specific CD8+ T cells. AF inhibited MHC class I-restricted presentation of exogenous OVA. This inhibitory activity of AF appeared to be due not only to the inhibition of the phagocytic activity of DCs, but also to the suppression of MHC molecule expression on DCs. AF also inhibited MHC class II-restricted presentation of exogenous OVA. These results show that AF exerts immunosuppressive activity at least in part by inhibiting MHC-restricted antigen presentation in professional antigen-presenting cells.     

Effects of mizoribine on MHC-restricted exogenous antigen presentation in dendritic cells

Mizoribine (MZR) has been shown to possess immunosuppressive activity that selectively inhibits the proliferation of lymphocytes by interfering with inosine monophosphate dehydrogenase. The efficacy of MZR is not only in patients who have had renal transplantation, but also in patients with rheumatoid arthritis (RA), lupus nephritis, and primary nephritic syndrome. Because the exact mechanism of its immunosuppressive action is not clear, the object of this study was to examine the ability of MZR to regulate the antigen presenting cells (APCs), dendritic cells (DCs). In this work, we tested whether MZR (1-10 microg/mL) could inhibit the cross-presentation of DCs. DC2.4 cells (H-2K(b)) or bone marrow-derived DCs (BM-DCs) generated from BM cells of C57BL/6 mouse (H-2K(b)) were cultured in the presence of MZR with OVA-microspheres, and the amount of OVA peptide-class I MHC complexes was measured by a T cell hybridoma, B3Z, that recognizes OVA (257-264 : SIINFEKL)-H-2Kb complex and expresses-galactosidase. MZR profoundly inhibited the expression of SIINFEKL-H-2K(b) complexes. This inhibitory activity of MZR appeared to affect the phagocytic activity of DCs. MZR also decreased IL-2 production when we examined the effects of MZR on CD4+ T cells. These results provide an understanding of the mechanism of immunosuppressive activity of MZR on the inhibition of MHC-restricted antigen presentation and phagocytic activity in relation to their actions on APCs.     

Preparation and targeted delivery of immunoliposomes bearing poly(ethylene glycol)-coupled humanized anti-hepatoma disulfide-stabilized Fv (hdsFv25) in vitro

Peptides in bee venom (PBV) have attracted considerable attention for anti-cancer therapy. In this study, sterically stabilized liposomal PBV (PBV-SL) was prepared using Soybean phosphatidylcholine, cholesterol, and the cholesterol derivatives of PEG with terminal COOH groups. The humanized anti-hepatoma disulfide-stabilized Fv (hdsFv25) was coupled to sterically stabilized liposomes. The hdsFv25-immunoliposome has strong affinity and specificity to SMMC-7721 cells in vitro. PBV-loaded sterically stabilized liposomes modified with the hdsFv25 can kill SMMC-7721 cells in vitro with higher efficiency than non-targeted liposomes. These results demonstrate that this strategy should also be applicable to immunotherapy for other cancers.     

Immunization of retrovirus-transfected dendritic cells induces specific cytotoxic T lymphocytes for two distinct malarial peptides presented by Kd molecule

Plasmodium yoelli sporozoite surface protein 2 (pySSP2) is considered as an important antigen for protection studies in malaria vaccine development. For the liver stage protection, anti-pySSP2 cytotoxic T lymphocyte (CTL) activity in BALB/c mice was investigated by immunization of genetically engineered bone marrow-derived dendritic cells (DCs) expressing pySSP2 peptides. Retrovirus-transfected bone marrow cells cultured with GMCSF and IL-4 for 7 days demonstrated 70-80% of DCs with high CD11c, CD80, CD86, and MHC class I (I-Kd) expression. Dividing bone marrow cells were infected with retrovirus expressing SSP2 on fifth, sixth, and seventh days of culture by prolonged centrifugation for 1 h at 32 degrees C. Transfection efficacy of DCs was assessed using retrovirus-shuttled green fluorescence vector (pMSCV-EGFP neo). A total of 64% of CD11c positive transfected DCs showed green fluorescence. The degree of SSP2 expression in transfected DCs was assessed by immunoprecipitation with SSP2 antibody. Both SSP2 and EGFP transfected DCs had prolonged expression of the engineered gene until day 6 since the transfection. Antigen presentation to nai;ve CTLs was assessed by immunization of retrovirus-infected DCs into BALB/c mice. Kd restricted, antigen-specific two new MHC class I (I-Kd) binding motifs were identified (A and C) in pySSP2 protein. Both A and C induced peptide-specific, IFN-gamma-secreting cytolytic CTLs upon antigen recognition on target cells. Taken together, these data indicate that genetically modified DCs by prolonged centrifugation is effective in enhanced antigen presentation. Immunization of DCs encoding SSP2 gene resulted in identification of two K(d) restricted CTL epitopes and induction of IFN-gamma-secreting cytolytic CD8+ T cells.     

Ciglitazone inhibits oxidized-low density lipoprotein induced immune maturation of dendritic cells

BACKGROUND: The peroxisome proliferator-activated receptor (PPAR) activation has generally been shown to have anti-inflammatory effects and dendritic cells (DCs) are the most efficient antigen presenting cells that play an active role in the development of atherosclerosis. The effects of PPARgamma on DCs maturation and immune function remain unknown now and we, therefore, studied the influence of PPARgamma agonist ciglitazone on the maturation and immune function of DCs. METHODS: Human monocytes were purified and immature DCs derived; ciglitazone (25 micromol/L) was added to the medium for 24 hours; ox-LDL (50 microg/ml) was then added to the medium for another 24 hours. The immunophenotypic expressions (CD1a, CD40, CD86, and HLA-DR) were analyzed by FACS and endocytosis function by FITC-dextran and the cytokines secretions of culture supernatants (IL-12,IL-10,TNFalpha, and IL-2) were measured with ELISA. RESULTS: Ciglitazone reduced ox-LDL induced immunophenotypic expressions of DCs (CD40, CD1a, and HLA-DR). Ox-LDL inhibited the endocytosis of DCs, which was prevented by ciglitazone; ciglitazone attenuated ox-LDL induced cytokine secretions of DCs (IL-12, 116 +/- 29 versus 34 +/- 3 pg/ml*; IL-10, 49 +/- 1 versus 28 +/- 9 pg/ml*; TNFalpha, 46 +/- 16 versus 24 +/- 8 pg/ml*, *P < 0.05 compared with ox-LDL, respectively). CONCLUSION: Our study suggested that one of the anti-inflammatory mechanisms of PPAR-gamma agonist ciglitazone was mediated by inhibiting the ox-LDL induced maturation and immune function of DCs.     

The combination of immunosuppressive drugs with 8-methoxypsoralen and ultraviolet a light modulates the myeloid-derived dendritic cell function

The functional properties of myeloid dendritic cells (DCs) differ, depending on microenvironmental factors as well as on their stage of maturation. The main approaches for the selective enhancement of the tolerogenic properties of DCs include the induction of a pharmacological arrest of the DCs maturation and the genetical engineering of DCs expressing immunosuppressive molecules. Several immunosuppressive/anti-inflammatory agents have been discovered that potentially inhibit DC maturation and immunogenicity. Photopheresis (ECP) is an immunomodulatory therapy in which leucocytes are exposed to 8-methoxypsoralen (8-MOP) and ultraviolet (UV) A radiation (PUVA). The combination of ECP with immunosuppressive agents has demonstrated efficacy in the management of transplanted patients by reducing either the incidence of organ rejection or the pharmacological toxicity. In particular, we have observed in hepatitis C virus (HCV)-positive patients that the same combination has reduced the immunosuppressive burden and improved sustainability and efficacy of pre-emptive antiviral therapy after liver transplantation. Therefore, in our work we investigated the in vitro effects of PUVA, combined with immunosuppressive drugs (IDs), on both in vitro human DC generation and maturation, in order to contribute to understanding the immunological mechanisms underlying this pharmacological combination. Monocyte PUVA-treatment was performed by using an in vitro experimental protocol that we previously described. PUVA-treated or -untreated highly purified CD14+ cells were incubated with the association of the immunosuppressive drugs, used in the management of liver transplantation, at two different concentrations, in the presence of IL-4 and GM-CSF. The treatment with IDs at the highest concentration (corresponding to that used in clinical practice), alone or in association with PUVA, induced an immunosuppressive effect, by impairing both DC generation and maturation. Neither immunosuppressive drugs at the lowest concentration nor their combination with PUVA affected myeloid DC generation, but modified DC functions, strengthening the induction of a tolerogenic pattern. As this ID concentration was arbitrarily chosen, further experiments could highlight whether lower concentrations than those used in clinical practice would elicit the same effect on DCs and potentially improve their functional properties. This work describes an original experimental approach exploring the in vitro mechanism of action of the combined procedure of PUVA with immunosuppressive drugs, used in liver transplantation, on DCs generation and function. Our results contribute to the knowledge of the mechanisms of action of this combined procedure on DCs, suggesting useful therapeutic implications for the in vivo therapy.