胃癌患者外周血树突状细胞的体外诱导和扩增

目的探讨从胃癌患者外周血中体外诱导、扩增树突状细胞（DCs）的方法。方法从胃癌患者外周血中分离单个核细胞（PBMNCs）,在GM-CSF、IL-4和TNF-α的诱导下培养扩增DCs。用相差显微镜观察所得DCs细胞形态学特征,用扫描电镜和透射电镜观察细胞结构。用混合淋巴细胞反应法检测所得DCs刺激同种异体T细胞增殖的能力。结果经过GM-CSF、IL-4和TNF-α诱导培养12 d后,所得细胞具有DCs的典型形态学特征和结构,且具有极强的激发刺激同种异体T细胞增殖的能力。结论用GM-CSF、IL-4和TNF-α可以在体外诱导、扩增胃癌患者外周血中的DCs。     

CpG ODN对食管癌细胞RNA转染的脐血树突状细胞诱导CTL抗肿瘤功能的影响

目的探讨食管癌细胞RNA转染脐血树突状细胞（DCs）对细胞毒性T淋巴细胞（CTL）特异性抗肿瘤作用的影响,以及CpG寡脱氧核糖核苷酸（CpG ODN）的免疫佐剂作用。方法分离脐血单个核细胞,经SCF、GM-CSF和IL-4诱导和食管癌RNA转染形成成熟DCs,加入CpG ODN,检测DCs表面标志及DCs诱导的T细胞增殖、CTL杀伤活性。结果食管癌RNA转染后DCs高表达抗原提呈分子、协同刺激分子和黏附分子,对T细胞的促增殖作用和CTL杀伤活性显著增强（P〈0.01）,CpG ODN能显著促进DCs功能。结论CpG ODN具有增强食管癌细胞RNA转染的DCs对T细胞的促增殖作用和促CTL杀伤活性的作用。     

K562细胞可溶性抗原负载树突状细胞体外诱导CTL作用的研究

目的探讨经K562细胞裂解物冲击致敏的外周血单个核细胞衍生的树突状细胞（DC）的生物特性及体外诱导抗原特异性CTL应答的能力。方法采集健康人抗凝外周血分离单个核细胞，贴壁细胞用含rhGM—CSF、rhIL-4、TNF—α的RPM1640＋10％FBS培养基体外诱导培养产生DC，5天收获细胞并将细胞分组：A组：未负载抗原DC；B组：加入K562细胞裂解液脉冲DC。7天后用流式细胞仪检测成熟DC免疫表型，并将非贴壁细胞（淋巴细胞）作为效应细胞与各组DE共育，以产生细胞毒性T淋巴细胞（CTL）。12天用LDH释放试验测定对K562细胞的杀伤活性。并用ELISA方法测定细胞上清液中IL-12的含量。结果（1）经细胞因子联合体外诱导的各组DC较培养前在数量，形态及免疫表型上差异有统计学意义，CD86、CD83、CD40、CD1a表达增加，其中经K562细胞裂解液冲击的DC的CD83CD86表达率明显升高。（2）效应细胞与K562细胞混合培养时，负载K562细胞裂解液的DC刺激后的T细胞比单独DC刺激后的T细胞对K562细胞的杀伤作用更明显。（3）负载K562细胞裂解液的DC细胞培养上清液中产生IL-12含量较未负载抗原的DC明显增加。结论用GM—CSF、IL-4以及TNF—α诱导培养健康人外周血单个核细胞可以得到成熟的DC，且经K562细胞裂解液致敏可以进一步促进DC的成熟并体外诱导特异性杀伤靶细胞的CTL。     

HEC-1-B细胞培养上清液对子宫内膜癌树突状细胞成熟的影响

目的探讨子宫内膜癌细胞株HEC-1-B培养上清液对子宫内膜癌患者外周血单核细胞源性树突状细胞（DCs）成熟的影响。方法梯度离心法分离子宫内膜癌患者外周血单核细胞,在含重组人粒细胞一巨噬细胞集落刺激因子和重组人自介素4的DCs培养液中培养,使其分化为DCs。实验组将收集的子宫内膜癌细胞株HEC-1-B培养上清液过滤后与DCs共同孵育48h,对照组不给予任何干预措施。镜下观察DCs形态变化,采用流式细胞术检测DCs表型（CD1α、CD83、CD86）表达,ELISA法检测细胞上清液中自细胞介素10（IL-10）、IL-12含量。结果实验组细胞生长状态较好,具有典型的树枝状突起,数量不等、形态不一;实验组DCs表型CD1α、CD83、CD86分子的表达分别较对照组升高（P〈0．01）;实验组OCs分泌的细胞因子IL-10水平较对照组降低（P〈0．01）,IL-12水平较对照组升高（P〈0．01）。结论子宫内膜癌细胞株HEC-1-B培养上清液能够在体外诱导子宫内膜癌患者外周血单核细胞源性DCs成熟。     

贴壁生长与悬浮生长树突细胞的免疫学功能比较

目的 探索树突细胞(DCs)的可能生长状态,比较分析不同生长状态DCs的免疫学功能.方法 从正常人外周血分离获得单个核细胞,用不同培养介质常规培养2 h,取贴壁细胞,用含细胞因子的培养液进行培养,在培养过程中光镜下对其进行形态鉴别,然后检测两种DCs促淋巴细胞增殖和诱导免疫反应的功能.结果 7 d后,培养瓶中DCs基本处于悬浮状态,而培养板中DCs基本处于贴壁状态;培养瓶中DCs与培养板中DCs形态与理论相符,但培养板中DCs分化形态优于培养瓶中DCs.而在体外促进淋巴细胞增殖及体外诱导免疫反应方面,培养瓶中DCs的能力优于培养板中DCs.结论 在DCs体外培养至发育成熟的过程中,DCs先贴壁后悬浮的生长性质可能对其免疫学功能有着重要意义.     

树突状细胞负载甲胎蛋白、细胞毒性T细胞表位肽后的免疫应答

目的 用负载hAFP 218-226位LLNQHACAV九肽的树突状细胞（dendritic cells,DC)诱导自身淋巴细胞，使之活化为肝细胞癌（HCC）特异性细胞毒性T细胞（cytotoxic T lymphocyte,CTL)，并特异性杀伤HCC细胞。方法 在体外用GM-CSF和IL-4诱导HLA-A2^ 健康志愿者外周血单核细胞，使其分化为高纯度DC。用负载hAFP 218-226位LLNQHACAV九肽的DC诱导自身淋巴细胞，使之成为HCC特异性CTL。结果 诱导的DC分泌较高水平的IL-12（17.6-21.5pg/ml)，其中以第7天为最高（21.5pg/ml)，经DC和DC负载AFP表位肽后活化的淋巴细胞培养上清液中TNF水平均比在IL-2条件下培养的未活化淋巴细胞高。L929细胞死亡百分率分别为80.65和11.6%；经DC和DC负载AFP表位肽后活化的淋巴细胞培养上清液中IL-12水平分别为20.5pg/ml和46.9pg/ml，经DC活化后淋巴细胞增殖指数大于3。活化后的淋巴细胞不但特异性杀伤HLA-A2^ 的HCC细胞HepG2和负载AFP表位肽的T2细胞，而且还不同程度杀伤HLA-A3^ HCC细胞Alexander和其它表型HCC细胞，对NK细胞敏感的靶细胞慢性髓原白血病细胞K562也有较强的杀伤作用。结论 负载hAFPHLA-A2限制性CTL九肽的DC对表达AFP HCC的研究和治疗有潜在应用价值。     

负载酸洗脱食管癌抗原肽的脐血树突状细胞对CTL杀伤活性的影响

分离脐血中的单个核细胞,以酸洗脱食管癌抗原肽负载获得分化成熟树突状细胞（DCs）,检测其对特异性细胞毒T淋巴细胞（CTL）的诱导和体外杀伤效应的影响。结果发现,负载食管癌抗原肽的脐血DCs诱导的CTL对酸洗前食管癌细胞株T.Tn的杀伤率显著高于酸洗后组和各对照组（P均〈0．05）,而对无关肿瘤细胞株Hep2无显著杀伤作用。认为负载食管癌抗原肽的脐血DCs体外可诱导CTL产生特异性杀伤效应。     

树突状细胞与1型糖尿病的关系

1型糖尿病是一种T淋巴细胞介导的器官特异性自身免疫性疾病,表现为胰岛β细胞的选择性破坏.树突状细胞(DCs)作为体内最重要的专职性抗原递呈细胞,在1型糖尿病的发病机制中具有极其重要的作用.DCs具有功能上的可塑性,不同类型DCs、不同成熟状态的DCs以及DCs所处微环境的不同,都将诱导其产生不同的功能状态.通过调节DCs的不同功能状态促进中枢或外周免疫耐受、扩展胰岛特异性调节性T细胞(CD4~+ CD25~+ Treg cell)、以及介导辅助性T(Th)细胞发生Th1/Tk2细胞的免疫偏离,可诱导免疫耐受,预防1型糖尿病的发生.     

阿维A对正常人外周血树突细胞的影响

目的研究阿维A对正常人外周血树突细胞(DCs)功能的影响。方法抽取健康人外周血,分离单个核细胞,体外诱导培养DCs。不同浓度阿维A(1 nmol/L和10 nmol/L)作用DCs 12 h,对照组只加培养基,流式细胞术(FCM)检测DCs中CD83及CD86表达水平,酶联免疫吸附试验(ELISA)检测细胞培养上清液中白介素(IL)-12表达水平。结果体外成功培养出DCs,FCM检测到1、10 nmol/L阿维A作用DCs 12 h后,CD83表达率分别为(56.65±7.09)%、(63.82±5.81)%,显著高于阴性对照组〔(43.55±4.32)%〕(P<0.05);CD86表达率分别为(65.43±1.79)%、(73.73±2.10)%,显著高于阴性对照组〔(52.96±1.10)%〕(P<0.05)。ELISA检测到1、10 nmol/L阿维A作用DCs 12 h后,DCs分泌到细胞培养液中的IL-12表达水平分别为(51.377±4.527)、(60.659±4.973)pg/ml,显著高于对照组〔(39.927±2.809)pg/ml〕(P<0.05)。结论阿维A可提高正常人外周血DCs表面标志CD83、CD86的表达率及细胞因子IL-12的分泌量,增强正常人外周血DCs的抗原提呈能力并可促进DCs向成熟方向分化,从而起到调控机体免疫功能的作用。     

华支睾吸虫成虫抗原致敏树突状细胞诱导免疫应答的研究

目的研究华支睾吸虫成虫虫体抗原（CA）致敏树突状细胞（DCs）诱导免疫应答的类型。方法利用rm-GMCSF诱导分化BALB/c小鼠骨髓细胞获得DCs,倒置显微镜和透射电镜观察DCs形态;CA致敏DCs,检测IL-12p70水平;将致敏后的DCs和淋巴细胞共培养,用CCK-8试剂盒检测淋巴细胞增殖,用ELISA试剂盒检测IFN-γ和IL-4水平。结果倒置显微镜和透射电镜下见典型的DCs,CA致敏的DCs分泌高水平IL-12p70;共培养后,淋巴细胞明显增殖,分泌的IFN-γ水平显著升高,IL-4水平与其他各组比较差异无统计学意义（P〉0.05）。结论CA刺激后DCs被活化,诱导的免疫应答向Th1方向极化,该应答与华支睾吸虫感染早期的保护性免疫密切相关。     

丝裂霉素诱导凋亡癌细胞致敏树突状细胞后的免疫应答

目的 观察树突状细胞(DC)从丝裂霉素诱导的凋亡胆管癌细胞获取抗原后,抗肿瘤免疫应答及对胆管癌细胞的特异性免疫杀伤效果。 方法 用粒细胞-巨噬细胞集落刺激因子(GM-CSF)加IL-4从人外周血分化、诱导DC,丝裂霉素在体外诱导培养的人胆管癌细胞凋亡,将DC、T淋巴细胞和凋亡胆管癌细胞共培养,同时设计不同类型肿瘤细胞(坏死胆管癌细胞及培养胆管癌细胞)作对照,7d后,分离、富集DC、T淋巴细胞进行免疫应答及肿瘤细胞杀伤试验。 结果 与凋亡胆管癌细胞共培养的DC可以有效提呈胆管癌细胞抗原,有强烈的免疫应答,刺激的细胞毒T淋巴细胞特异性地杀伤胆管癌细胞。 结论 丝裂霉素诱导凋亡癌细胞可以致敏rhGM-CSF加rhIL-4从人外周血单个核细胞诱导、扩增出的DC,并产生显著的杀伤胆管癌细胞的免疫反应,可望成为特异性免疫治疗肿瘤的一条新途径。     

Functional changes of dendritic cells derived from allogeneic partial liver graft undergoing acute rejection in rats

AIM: To investigate functional change of dendritic cells (DCs) derived from allogeneic partial liver graft undergoing acute rejection in rats. METHODS: Allogeneic (SD rat to LEW rat) whole and 50 % partial liver transplantation were performed. DCs from liver grafts 0 hr and 4 days after transplantation were isolated and propagated in the presence of GM-CSF in vitro. Morphological characteristics of DCs propagated for 4 days and 10 days were observed by electron microscopy. Phenotypical features of DCs propagated for 10 days were analyzed by flow cytometry. Expression of IL-12 protein and IL-12 receptor mRNA in DCs propagated for 10 days was also measured by Western blotting and semiquantitative RT-PCR, respectively. Histological grading of rejection were determined. RESULTS: Allogeneic whole liver grafts showed no features of rejection at day 4 after transplantation. In contrast, allogeneic partial liver grafts demonstrated moderate to severe rejection at day 4 after transplantation. DCs derived from allogeneic partial liver graft 4 days after transplantation exhibited typical morphological characteristics of DC after 4 days' culture in the presence of GM-CSF. DCs from allogeneic whole liver graft 0 hr and 4 days after transplantation did not exhibit typical morphological characteristics of DC until after 10 days' culture in the presence of GM-CSF. After 10 days' propagation in vitro, DCs derived from allogeneic whole liver graft exhibited features of immature DC, with absence of CD40, CD80 and CD86 surface expression, and low levels of IL-12 proteins (IL-12 p35 and IL-12 p40) and IL-12 receptor (IL-12Rbeta(1) and IL-12Rbeta(2)) mRNA, whereas DCs from allogeneic partial liver graft 4 days after transplantation displayed features of mature DC, with high levels of CD40, CD80 and CD86 surface expression, and as a consequence, higher expression of IL-12 proteins (IL-12 p35 and IL-12 p40) and IL-12 receptors (IL-12Rbeta1 and IL-12Rbeta2) mRNA than those of DCs both from partial liver graft 0 hr and whole liver graft 4 days after transplantation (P<0.001) was observed. CONCLUSION: DCs derived from allogeneic partial liver graft undergoing acute rejection display features of mature DC.     

Comparative analysis of murine marrow-derived dendritic cells generated by Flt3L or GM-CSF/IL-4 and matured with immune stimulatory agents on the in vivo induction of antileukemia responses

Bone marrow (BM)-derived dendritic cells (DCs) cultured in granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin 4 (IL-4) have been used to generate antitumor immune responses. The cytokine Flt3 ligand (Flt3L) also has been shown to generate BM DCs. We sought to determine if DCs generated by using Flt3L then matured with lipopolysaccharide (LPS) could lead to DCs with in vivo anti-acute myelogenous leukemia (anti-AML) activity. LPS and tumor necrosis factor alpha (TNF-alpha) are effective agents for maturing DCs; however, they have potential in vivo toxicities. Cytosine-phosphorothioate-guanine oligodeoxynucleotides (CpGs) are considered relatively nontoxic, potent activators of DC function and maturation in vitro and in vivo. We investigated whether CpGs would be comparable to TNF-alpha or LPS for the maturation of GM-CSF/IL-4-generated DCs. DCs cultured with GM-CSF/IL-4 and matured with TNF-alpha, LPS, or CpG produced a greater allogeneic T-cell response compared with Flt3L/LPS-generated DCs. All 4 distinct DC types were pulsed with AML-lysate and administered before tumor challenge produced an increase in the total number of splenic anti-AML-specific cytotoxic T-lymphocyte precursors and led to significantly (P < or =.0001) improved survival compared with nonvaccinated controls. GM-CSF/IL-4/LPS was superior to Flt3L/LPS for generating anti-AML effects in vivo. Whereas TNF-alpha was comparable to LPS in conferring on GM-CSF/IL-4 DCs anti-AML effects in vivo, CpGs were superior to LPS. These data have important clinical implications and are the first to show that Flt3L-generated DCs can provide antitumor protection and that nontoxic agents such as CpGs and Flt3L may be useful in the clinical development of DC vaccines.     

Monocyte-derived dendritic cells from chronic HCV patients are not infected but show an immature phenotype and aberrant cytokine profile.

BACKGROUND: Chronic hepatitis C virus (HCV) infection is characterized by an insufficient immune response, possibly owing to impaired function of antigen-presenting cells such as myeloid dendritic cells (DCs). Therapeutic vaccination with in vitro generated DCs may enhance the immune response. Subsets of DCs can originate from monocytes, but the presence of HCV in monocytes that develop into DCs in vitro may impair DC function. Therefore, we studied the presence of HCV RNA in monocytes and monocyte-derived DCs from chronic HCV patients. METHODS: Monocytes were cultured with granulocyte macrophage colony-stimulating factor (GM-CSF) and interleukin 4 (IL-4) for 6 days, and then with GM-CSF, IL-4, tumour necrosis factor-alpha (TNF-alpha), prostaglandin E2, IL-1beta and IL-6 for 2 days to generate mature DCs. HCV RNA was assessed by polymerase chain reaction. Surface molecules were assessed by flow cytometry. Cytokine production was assessed by cytokine bead array. RESULTS: HCV RNA was present in monocytes in 11 of 13 patients, but undetectable in mature DCs in 13 of 13 patients. The morphology of patient DCs was comparable with DCs from healthy controls, but the percentage of cells expressing surface molecules CD83 (P=0.001), CD86 (P=0.023) and human leucocyte antigen-DR (P=0.028) was lower in HCV patients. Compared with control DCs, patient DCs produced enhanced levels of IL-10 (P=0.0079) and IL-8 (P=0.0079), and lower levels of TNF-alpha (P=0.032), IL-6 (P=NS) and IL-1beta (P=0.0079). Patient and control DCs did not produce IL-12. CONCLUSIONS: Monocyte-derived DCs from chronic HCV patients are not infected but show an immature phenotype and aberrant cytokine profile.     

4种甲胎蛋白抗原表位肽段致敏树突状细胞诱导细胞毒性T淋巴细胞对肝癌细胞的杀伤效应比较

目的 通过体外杀伤试验,比较4种人肝癌特异性甲胎蛋白抗原表位肽段PLFQVEPV[hAFP(137～145)],FMNKFIYEI[hAFP(158～166)]、GLSPNLNRFL[hAFP(325-334)]和GVAL,QTMKQ[hAFP(542～550)]修饰树突状细胞(DC)后诱导的细胞毒性T淋巴细胞(CTL)对人肝癌细胞的特异性杀伤效应. 方法 选取健康人外周血单个核细胞,体外诱导成熟DC;合成4种hAFP抗原表位肽段分别修饰和诱导DC,体外刺激CTL,通过流式细胞仪法和细胞毒性检测法检测该修饰后的DC所诱导的CTL在体外对肝癌细胞株SMMC-7721细胞的杀伤作用.组间比较应用t检验进行统计学分析. 结果 4种人肝癌特异性甲胎蛋白片段均能在体外修饰和诱导DC细胞的增殖和成熟;这些成熟DC在体外均能诱导特异性CTL,CTL对SMMC-7721细胞均可产生杀伤效应.其中FMNKFIYEI[hAFP(158～166)]肽段诱导的CTL在效靶比分别为80:1、40:1及10:1时对肿瘤细胞的杀伤效应分别达到了78.1%±9.8%、43.9%±5.9%和28.2%±4.9%,比其他3种肽段的杀伤效应高(P<0.05). 结论 短肽段具有单独体外诱导特异CTL的作用,其诱导效果和杀伤效果均优于完整hAFP蛋白,其诱导的CTL有特异地杀伤SMM C-7721细胞的作用.     

4种甲胎蛋白抗原表位肽段致敏树突状细胞诱导细胞毒性T淋巴细胞对肝癌细胞的杀伤效应比较

目的 通过体外杀伤试验,比较4种人肝癌特异性甲胎蛋白抗原表位肽段PLFQVEPV[hAFP(137～145)],FMNKFIYEI[hAFP(158～166)]、GLSPNLNRFL[hAFP(325-334)]和GVAL,QTMKQ[hAFP(542～550)]修饰树突状细胞(DC)后诱导的细胞毒性T淋巴细胞(CTL)对人肝癌细胞的特异性杀伤效应. 方法 选取健康人外周血单个核细胞,体外诱导成熟DC;合成4种hAFP抗原表位肽段分别修饰和诱导DC,体外刺激CTL,通过流式细胞仪法和细胞毒性检测法检测该修饰后的DC所诱导的CTL在体外对肝癌细胞株SMMC-7721细胞的杀伤作用.组间比较应用t检验进行统计学分析. 结果 4种人肝癌特异性甲胎蛋白片段均能在体外修饰和诱导DC细胞的增殖和成熟;这些成熟DC在体外均能诱导特异性CTL,CTL对SMMC-7721细胞均可产生杀伤效应.其中FMNKFIYEI[hAFP(158～166)]肽段诱导的CTL在效靶比分别为80:1、40:1及10:1时对肿瘤细胞的杀伤效应分别达到了78.1%±9.8%、43.9%±5.9%和28.2%±4.9%,比其他3种肽段的杀伤效应高(P<0.05). 结论 短肽段具有单独体外诱导特异CTL的作用,其诱导效果和杀伤效果均优于完整hAFP蛋白,其诱导的CTL有特异地杀伤SMM C-7721细胞的作用.     

4种甲胎蛋白抗原表位肽段致敏树突状细胞诱导细胞毒性T淋巴细胞对肝癌细胞的杀伤效应比较

目的 通过体外杀伤试验,比较4种人肝癌特异性甲胎蛋白抗原表位肽段PLFQVEPV[hAFP(137～145)],FMNKFIYEI[hAFP(158～166)]、GLSPNLNRFL[hAFP(325-334)]和GVAL,QTMKQ[hAFP(542～550)]修饰树突状细胞(DC)后诱导的细胞毒性T淋巴细胞(CTL)对人肝癌细胞的特异性杀伤效应. 方法 选取健康人外周血单个核细胞,体外诱导成熟DC;合成4种hAFP抗原表位肽段分别修饰和诱导DC,体外刺激CTL,通过流式细胞仪法和细胞毒性检测法检测该修饰后的DC所诱导的CTL在体外对肝癌细胞株SMMC-7721细胞的杀伤作用.组间比较应用t检验进行统计学分析. 结果 4种人肝癌特异性甲胎蛋白片段均能在体外修饰和诱导DC细胞的增殖和成熟;这些成熟DC在体外均能诱导特异性CTL,CTL对SMMC-7721细胞均可产生杀伤效应.其中FMNKFIYEI[hAFP(158～166)]肽段诱导的CTL在效靶比分别为80:1、40:1及10:1时对肿瘤细胞的杀伤效应分别达到了78.1%±9.8%、43.9%±5.9%和28.2%±4.9%,比其他3种肽段的杀伤效应高(P<0.05). 结论 短肽段具有单独体外诱导特异CTL的作用,其诱导效果和杀伤效果均优于完整hAFP蛋白,其诱导的CTL有特异地杀伤SMM C-7721细胞的作用.     

三拗汤对人外周血来源的树突状细胞成熟的影响

目的探讨三拗汤对人外周血单核细胞来源的树突状细胞分化成熟的影响。方法采用体外培养人外周血单核细胞来源的未成熟树突状细胞，将细胞随机分为模型组和用药组，用药组加入三拗汤水煎液＋LPS；模型组加入相应体积的无菌双蒸水＋INS，在倒置相差显微镜下观察细胞的形态改变并计数。结果模型组成熟树突状细胞数量较多，细胞表面有许多毛刺状突起；而用药组未成熟树突状细胞较多，多数细胞仍为圆形，表面少有突起。两者比较有差异（P〈0．05）。结论三拗汤可抑制树突状细胞的分化和成熟。     

Optimizing Dendritic Cell Preparation for Fusion with Melanoma Cells

Background: Fusion of dendritic cells (DCs) with melanoma cells could reinforce the antigenicity of tumors as a strategy for the treatment of malignant melanoma. However, the insufficient quantity of DCs and the low fusion efficiency limits the development of such approach. Objective: To define the dosage of the stimulating factors as well as the induction condition for the optimal DCs preparation and cell fusion. Methods: DCs were generated from murine bone marrow cells, and cultured with four different concentrations of the granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4). DCs were confirmed to be mature by detecting the expression of MHC-II, CD11c, CD80, and CD83 by flowcytometry. DCs-melanoma fusion cells were generated using polyethylene glycols (PEG) with different molecular weights and the fusion efficiency was detected by fluorescence-activated cell sorter (FACS). Results: The largest quantity of DCs was found when cells were cultured with 1000 U/ ml of GM-CSF and 500 U/ml of IL-4 (1.69 ± 0.04 ×10 6 ml-1, p<0.001 when compared with the other three groups). The expression levels of MHC-II and CD83 on day 7 after incubation were significantly lower than those on day 3 (MHC-II: p<0.001; CD83: p<0.001). The efficiency of cell fusion under induction of PEG-3000 was significantly higher than that of PEG-4000 (15.4 ± 0.56% vs. 11.1 ± 0.45%, p<0.001). Conclusions: The largest quantity for mature DCs was stimulated with 1000 U/ml of GM-CSF and 500 U/ml of IL-4 and the highest fusion efficiency was under induction of PEG-3000.     

Conditioned medium from HeLa cells enhances motility of human monocyte-derived dendritic cells but abrogates their maturation and endocytic activity

Progressive tumor proliferation may be associated with suppression of the immune response. Several different mechanisms can contribute to immune evasion. It is generally proposed that inhibition of dendritic cell functions would be a key mechanism by which tumors could escape immune surveillance. Therefore, the purpose of this study was to evaluate the capacity of HeLa cells conditioned medium (HeLa-CM) to modulate phenotypic and functional parameters of human peripheral blood monocyte-derived dendritic cells (DCs). Two types of reference DCs population were generated in vitro, the first cultured in the presence of IL-4 and GM-CSF which represented immature DCs (iDCs) and the second, representing mature DCs (mDCs), was raised from the iDCs by additional stimulation with a maturation cocktail - TNF-alpha, IL-1beta, IL-6, PGE2. In parallel, the iDCs were treated with HeLa-CM collected from the tumor cells. The analysis of DC populations demonstrated that the HeLa-CM prevented maturation of these cells and also impaired their capacity to uptake an antigen and stimulate proliferation of allogeneic T cells. In contrast, HeLa-CM modulated DCs exhibited a 3-fold increase mobility over iDCs. The latter functional capacity did not correlate with the levels of matrix metalloproteinase expression in the analysed cells. Altogether, our results provide evidence that HeLa cells produce soluble factors that might dramatically alter basic phenotypic and functional characteristics of DCs.