双歧杆菌完整肽聚糖对脐血来源树突状细胞分化成熟的影响

目的研究两歧双歧杆菌完整肽聚糖（WPG）对脐血来源树突状细胞（DC）形态及分泌细胞因子的影响,了解双歧杆菌WPG对DC分化、成熟及免疫调节功能的作用;并为益生菌及其生物活性成分的进一步开发提供依据。方法分离正常孕妇脐血单个核细胞诱导生成未成熟树突状细胞（Dendritic cells,DCs）,实验组在培养的第7天分别加入两歧双歧杆菌WPG（5μg／ml）、两歧双歧杆菌全菌（100μg/ml）,阳性对照组加入脂多糖（LPS）,阴性对照组仅加入培养基。倒置显微镜在培养各期形态学观察,流式细胞术检测表面标志物CD83及CD1a的表达,NLR检测DCs刺激同种异体T淋巴细胞能力,ELISA法测定DCs培养上清中IL-12p70、IL-10的分泌。结果脐血单核细胞在双歧杆菌WPG与GM-CSF、IL-4协同诱导作用下,能成为形态上具有典型树突状突起的DCs;诱导后的CB-MDDCs刺激同种异体T细胞的增殖能力及分泌IL-12：p70、IL-10的水平显著高于阴性对照组（P〈0．01）且细胞表面标志物CD83及CD1a的表达增加。结论（1）双歧杆菌WPG能影响CB-MDDCs的成熟状态。（2）双歧杆菌WPG对CB-MDDCs成熟程度及分泌细胞因子水平的影响强于双歧杆菌全菌,说明WPG是双歧杆菌主要免疫活性成分。     

甘草甜素对树突状细胞表型及生物学功能的影响

培养小鼠髓系DC2．4细胞,加入LPS（阳性对照组）或甘草甜素,用扫描电镜观察DC的超微结构、流式细胞仪检测DC表面分子MHCII、CD86及CD40的表达、4-氨基安替比林（4-AAP）比色检测DC内酸性磷酸酶活性、ELISA方法检测DC培养上清中IL—12的浓度,体外刺激淋巴细胞增殖实验检测DC对同种异体T淋巴细胞的刺激能力。结果表明,与对照组相比,甘草甜素刺激后,DC表面树突状突起增多,表面分子MHCⅡ、CD86及CD40表达增加,酸性磷酸酶活性下降,培养上清中IL-12浓度升高,刺激同种异体T淋巴细胞的能力也明显增强。结果表明,甘草甜素能够促进小鼠髓系DC2.4表型及功能的成熟。     

小鼠骨髓源性树突状细胞体外诱导培养方法的比较研究

目的探讨最佳体外诱导培养小鼠成熟树突状细胞（dendritic cells,DC）的方法。方法分离、纯化6周龄C57BL／6小鼠骨髓单核细胞,以含10％胎牛血清、20ng／ml重组小鼠粒细胞-巨噬细胞集落刺激因子（GM—CSF）和10ng／ml重组小鼠白细胞介素-4（IL-4）的RPMI-1640培养基培养7d,然后将细胞分成对照未刺激组、肿瘤坏死因子-α（TNF-α）刺激组和TNF-α＋脂多糖（lipopolysaccharides,LPS）刺激组。继续培养48h后,观察各组细胞形态,检测IL-12、IL-6浓度及细胞表面标志CD11c、CD80、CD86和MHC II。结果培养9d后,两刺激组培养的细胞经相差显微镜观察有DC生长。TNF—α刺激组细胞培养上清液中IL-6、IL-12含量显著高于对照组（P〈0．01）,但显著低于TNF—α＋LPS刺激组（P〈0．05）。3组均高表达CD11c,各组间无显著差异;而CD80、CD86和MHC II表达阳性率TNF-α刺激组显著高于对照组（P〈0．01）,TNF-α＋LPS刺激组显著高于单纯TNF—α刺激组（P〈0．05）。结论联合使用TNF-α与LPS刺激可使DC成熟度提高,分泌IL-6、IL-12增加。     

树突状细胞与马尔尼菲青霉酵母体外相互作用的研究

本文探讨了树突状细胞(DCs)在抗马尔尼菲青霉感染免疫中的作用。用细胞因子 rhGM-CSF 和rhIL-4诱导人外周血单核细胞分化为树突状细胞, 观察DCs的形态, 并用流式细胞仪进行DCs的表型测定, ELISA方法检测培养上清液IL-12p70的浓度, 混合淋巴细胞反应检测DCs刺激T淋巴细胞的增殖能力, 实时荧光定量PCR检测趋化因子受体CCR7、CXCR4的mRNA的表达。倒置显微镜下可见诱导获得的DCs细胞形态不规则, 表面伸展出大量树突。与马尔尼菲青霉酵母共同培养24 h后DCs的胞内含有大量的酵母细胞; 细胞表型CD86、CD83、HLA-DR和CD40的表达明显增高; 刺激T淋巴细胞增殖的能力增强; 趋化因子受体CCR7和CXCR4的mRNA表达量增加且能够产生IL-12p70但产生的量低于LPS刺激组。DCs能吞噬加热灭活的马尔尼菲青霉酵母, 并趋于成熟, 抗原呈递能力增加, 但是产生IL-12p70的量较低, 可能造成宿主抗马尔尼菲青霉酵母的细胞免疫功能的不足。     

CpG-ODN2216致敏的单核细胞培养上清对HBV相关性肝癌病人的树突状细胞表型和功能的影响

目的:研究CpG-ODN2216致敏的外周血单核细胞(PBMC)培养上清液对HBV相关性肝癌病人的树突状细胞(DC)成熟与功能的影响,寻求一种增强DC疫苗效果的方法。方法:从9例HBV相关性肝癌患者PBMC中诱导出未成熟的单核细胞来源的DC(MoDC),经HBV核心抗原(HBcAg)负载后,用CpG-ODN2216刺激的PBMC上清液、“细胞因子鸡尾酒(IL-1β、IL-6、TNF-α和PGE2)”以及两者的联合作用促进MoDC的进一步成熟,检测MoDC表型和功能;选择其中5例HLA-A2 病人,用成熟MoDC诱导自身T细胞产生HBV特异性CD8 的细胞毒性T淋巴细胞(CTL)。结果:用细胞因子鸡尾酒和CpG-ODN2216刺激的PBMC上清液联合作用可明显增强MoDC表面的CD80、CD83和CD1a表达,其对HBcAg负载的MoDC促成熟作用大于单独用细胞因子鸡尾酒或单独用CpG-ODN2216刺激PBMC的上清液。联合作用促进MoDC分泌IL-12和IL-10的能力明显强于单独应用PBMC上清液或细胞因子鸡尾酒,其刺激自体T细胞分泌IFN-γ、TNF-α、IL-6的能力也明显增高。联合作用促成熟的MoDC诱导HLA-A2 病人的自体T细胞产生HBVcore18-27特异性CD8 CTL的频率明显高于细胞因子鸡尾酒单独促成熟的MoDC。结论:CpG-ODN2216刺激PBMC的上清液和细胞因子鸡尾酒联合作用可以明显促进HBcAg负载的HBV相关性肝癌病人的MoDC成熟,增强MoDC分泌细胞因子、刺激自体特异性T淋巴细胞应答、诱导HBV特异性细胞毒性T细胞的能力。为提高HBV特异性树突状细胞疫苗的效果提供了一种可行方案。     

人DC体外分化成熟特性及抗P-选择素功能域单抗对其干预调节

结合树突状细胞(DC)生物学特性, 探讨抗P-选择素lectin-EGF功能域单抗(PsL-EGFmAb)对体外培养人DC成熟和功能干预调节的作用. 通过SCF, GM-CSF, TGF-β₁, Flt-3L及TNF-α体外培养体系, 从脐血CD34⁺造血干细胞中诱导扩增获得DC, 并于细胞成熟过程中用PsL-EGFmAb及辅以IL-10作为对照进行干预. 分别观察和检测DC形态学及细胞活力, 细胞表面分子HLA-DR, CD1a, CD11c, CD54, CD83, CD80, CD86, CD209(DC-SIGN)及CD62P, E, L(P-、E-、L-选择素)表达, 细胞内活性氧(ROS)水平, 及IL-12p35, p40 mRNA与NF-κBP50, P65 mRNA表达, 培养上清液中IL-12p70分泌含量, 以及DC体外对T淋巴细胞刺激能力, 以此分析PsL-EGFmAb 对DC成熟与功能的干预状况. 结果显示, 未成熟DC高表达属模式识别受体的C型凝集素DC-SIGN外, 且胞内蓄积适量ROS, 具备了细胞吞噬能力. 成熟DC除仍高表达DC-SIGN, 伴随细胞内NF-κB基因明显表达, 其表面黏附共刺激分子CD11c, CD83, CD80, CD86表达上调, 且细胞因子IL-12合成分泌增加, 并具明显的体外刺激T淋巴细胞增殖能力, 符合于抗原提呈细胞特征. 此外, 未成熟和成熟DC基本不表达P-, E-选择素, 而分别高表达和低表达L-选择素. 进一步发现, PsL-EGFmAb较对照IL-10对DC表面DC-SIGN表达有抑制作用; 也能抑制细胞内NF-κB基因表达, 并相应抑制或下调DC黏附共刺激分子CD11c, CD83, CD80, CD86及HLA-DR表达, 抑制IL-12基因转录及其合成分泌, 以及抑制DC体外刺激T细胞增殖的能力. 上述结果表明, PsL-EGFmAb对DC分化成熟及功能具有抑制作用, 提示此作用与其抑制作为DC模式识别受体及功能分子DC-SIGN有关, 并可能是通过影响NF-κB信号途径起作用.     

过敏性哮喘小鼠树突状细胞负荷Der p2后改变及与T细胞增殖分化的关系研究

目的:探讨哮喘小鼠与正常小鼠骨髓源性树突状细胞(DC)负荷Der p2抗原后表达表面分子(CD11c、CD86)和细胞因子(IL-10、IL-12p70)的差异及其对Th1和Th2型细胞因子平衡的影响,进一步研究过敏性哮喘发生中DC的可能作用。方法:分别从哮喘组和对照组提取骨髓培养DC,第五天负荷Der p2,24小时后吹打收集细胞,观察DC形态,用流式细胞仪检测孵育后细胞表面CD11c、CD86表达。并留取负荷Der f2前后培养上清,ELISA法检测IL-10及IL-12p70含量。同时以DC:反应细胞比例为1:10混合培养,72 h后ELISA法检测混合培养上清中IL-4、IL-5、IFN-γ的水平。结果:1负荷Der p2后,哮喘组CD86、CD11c表达比对照组高,分别为(t=11,P0.05)(t=4.9,P0.05),差异有统计学意义;2在细胞因子分泌方面,Der p2负荷前,两组DC均能分泌IL-10与IL-12p70,IL-10水平哮喘组高(t=9.5,P0.05),而IL-12p70水平对照组高(P0.05);负荷Der p2后,对照组IL-10、IL-12p7分泌量比负荷前明显增加(P0.05),哮喘组无明显差异(P0.05);3在DC刺激同种T细胞因子分泌方面,负荷Der p2后哮喘组DC刺激T细胞分泌IL-4、IL-5分泌能力明显增强(P0.05),而刺激INF-γ能力降低(P0.05)。结论:DC在过敏性哮喘中起着重要作用,异常DC通过增加CD86、CD11c的表达和减少IL-10及IL-12的合成,致使T细胞向Th2细胞优势分化。     

Toll样受体增强树突状细胞中腺苷受体A2B亚型的表达及功能

树突状细胞(dendritic cell,DC)表面所表达的腺苷受体A2B亚型(ADOR-A2B)可促进DC对辅助性T淋巴细胞(T helper cell,Th)的激活,导致自身免疫性疾病的发生或加重.本文旨在研究作为免疫反应的诱导分子Toll样受体(Toll-like receptors,TLRs)是否可调节ADOR-A2B在DC中的表达并籍此影响其功能.体外诱导小鼠骨髓细胞分化为树突状细胞(BM-DC),以多种TLRs的配体,Pam3csk4、polyIC、LPS及CpG进行干预.提取细胞总RNA,real-time PCR测定ador-a2a、ador-a2b的表达;放射性配体结合实验测定BM-DC对3H-腺苷结合能力的变化.以LPS及选择性ADOR-A2B激动剂BAY 60-6583协同干预BM-DC,ELISA测定培养基中IL-1、IL-6及IL-12的含量.以干预后的BM-DC刺激naive CD4细胞,ELISA测定培养基中IL-17A、IFNγ的含量,荧光抗体染色及流式细胞仪分析检测CD4细胞的分化.结果显示,TLR-4的配体LPS可显著提高BM-DC中ador-a2b的表达及对腺苷的结合能力.BAY 60-6583与LPS相协同可刺激BM-DC分泌多种致炎因子,并增加其诱导CD4细胞向Th1及Th17分化的能力.由此可见,Toll样受体可上调adora2b在DC中的表达,并可籍此增加DC分泌促炎因子的能力及对CD4细胞的刺激作用.     

树突状细胞与马尔尼菲青霉酵母体外相互作用的研究

本文探讨了树突状细胞(DCs)在抗马尔尼菲青霉感染免疫中的作用.用细胞因子rhGM-CSF和rhIL-4诱导人外周血单核细胞分化为树突状细胞,观察DCs的形态,并用流式细胞仪进行DCs的表型测定,ELISA方法检测培养上清液IL-12p70的浓度,混合淋巴细胞反应检测DCs刺激T淋巴细胞的增殖能力.实时荧光定量PCR检测趋化因子受体CCR7、CXCR4的mRNA 的表达.倒置显微镜下可见诱导获得的DCs细胞形态不规则,表面伸展出大量树突.与马尔尼菲青霉酵母共同培养24 h后DCs的胞内含有大量的酵母细胞;细胞表型CD86、CD83、HLA-DR和CD40的表达明显增高;刺激T淋巴细胞增殖的能力增强;趋化因子受体CCR7和CXCR4的mRNA表达量增加且能够产生IL-12p70但产生的量低于LPS刺激组.DCs能吞噬加热灭活的马尔尼菲青霉酵母,并趋于成熟,抗原呈递能力增加,但是产生IL-12p70的量较低,可能造成宿主抗马尔尼菲青霉酵母的细胞免疫功能的不足.     

未成熟树突状细胞防治移植物抗宿主病的实验研究

目的：观察供者未成熟树突状细胞（imDC）刺激自体T细胞增殖的能力,探讨利用imDC防治移植物抗宿主病（GVHD）临床应用的可行性。方法：Ak健康供者外周血分离单核细胞,采用重组人粒细胞巨噬细胞集落刺激因子（rhGM-CSF）和白细胞介素（IL）4联合培养4d,诱导其分化成imDC;培养7d,分化成mDC。并通过倒置显微镜和HE染色观察细胞形态、流式细胞仪检测细胞表型。采用MLR方法,构建GVHD发生机制的模型,比较供者imDC和mDC刺激自体T细胞增殖的能力。结果：（1）培养4天后细胞具有典型的imDC特征,CDla、CD83和双抗分别表达为55．79％、64、67％和46、67％,成熟标志CD83表达较低;培养7天后具有典型mDC特征,CDla、CD83和双抗表达分别为61．56％、82．40％和64．12％,成熟标志CD83表达较高。（2）MLR法共孵育72小时后,加入CCK-8检测OD值,imDC组与对照组比较无统计学意义（P〉0．05）,不能刺激自体T细胞增殖（SI〈1．00）;mDC组与对照组、imDC组比较均有显著统计学意义（P〈0．01）,能够刺激自体T细胞增殖（SI〉2．00）。结论：供者imDC能够诱导自体T细胞低反应,有望用来防治GVHD。     

Analysis of maturation states of rat bone marrow-derived dendritic cells using an improved culture technique

In this study, we examined in more detail the development of rat bone marrow-derived dendritic cells (BMDC). A two-stage culture system was used to propagate BMDC from rat bone marrow precursors. BMDC developed within clusters of proliferating cells after repetitive addition of rat granulocyte/macrophage colony-stimulating factor and rat interleukin (IL)-4 at a concentration of 5 ng/ml to the cultures. Fluorescence-activated cell sorter analysis performed at an early stage of development (day 6) revealed an immature phenotype with intermediate levels of major histocompatibility complex (MHC) class II expression and low levels of the costimulator molecules CD80 and CD86. Upon further culture, a strong upregulation of MHC class II, costimulatory and adhesion molecules could be observed, whereas macrophage marker antigens were downregulated. Late-stage BMDC (day 10) showed a high expression of MHC class I and II, ICAM-1, Ox62 and CD11c, and revealed a split pattern of B7-1 and B7-2. The cell yield was about 40% of the initially plated bone marrow cells with 80% MHC class II-high and less than 20% MHC class II-low positive cells. Full maturation of rat BMDC (day 12) with an almost uniform expression of B7 was achieved by subsequent subculture and further stimulation with rat tumour necrosis factor alpha (TNF-alpha), lipopolysaccharide (LPS) or soluble CD40 ligand (CD40L). Analysis of the cell supernatant revealed a strong IL-12 production after LPS or CD40L, and to a lesser extent after TNF-alpha stimulation. Additionally, LPS-treated, but not CD40L-treated BMDC secreted TNF-alpha into the supernatant. Early-stage BMDC sufficiently triggered a T cell receptor (TCR) downregulation, but did not stimulate naive T cells in an allogeneic mixed leukocyte reaction (MLR) and revealed a low stimulatory capacity in an antigen-specific T cell assay. In contrast, late-stage BMDC and especially fully mature BMDC strongly induced TCR internalisation, elicited high T cell responses in the allogeneic MLR similar to those obtained by mature rat spleen dendritic cells and efficiently activated antigen-specific T cells. In conclusion, this protocol allows easy access to large numbers of rat BMDC at defined maturation stages and selective studies for the manipulation of immune responses in rat models.     

Dendritic cells process and present antigens across a range of maturation states

We isolated dendritic cells (DC) from lymphoid organs of mice bearing a transgene for a membrane-bound form of the model protein hen egg white lysozyme (HEL). DC from the spleen had a lower representation of costimulatory molecules and class II MHC molecules than those isolated from lymph nodes and thymi. Splenic DC were capable of further maturation by in vivo treatment of mice with LPS. The immature DC from spleen processed HEL and displayed the chemically dominant epitope as evidenced by FACS analysis. These immature DC also presented this epitope to CD4(+) T cells. Splenic DC from another transgenic mouse (ML-5) containing serum HEL also showed the ability to process and present Ag despite low levels of circulating HEL. In vitro-derived DC from the bone marrow (bone marrow-derived DC) of mHEL mice also displayed immature to mature features and in both cases displayed HEL peptides as well as SDS-stable MHC class II molecules. Immature bone marrow-derived DC also processed exogenous HEL. We conclude that the DC sets normally found in tissue show a scale of maturation features but even the most immature process and present peptides by MHC class II molecules.     

Dendritic cells post-maturation are reprogrammed with heightened IFN-gamma and IL-10

Mature dendritic cells (mDCs) undergo "exhaustion" in producing cytokines. Nevertheless, whether this "exhaustion" of mDCs is selective to certain cytokines, or whether mDCs have specific cytokine-producing profiles has yet to be defined. Herein, we investigated the cytokine production in vitro by immature DCs (iDCs) and LPS-induced mDCs. Compared to iDCs, mDCs produced comparable levels of IL-6 and TNF-alpha. Strikingly, mDCs produced significantly higher IFN-gamma and IL-10. IL-12 production of mDCs was suppressed. Kinetic studies of the responses of iDCs and mDCs to LPS or CD40L showed that mDCs acquired progressively heightened activity in producing IFN-gamma and IL-10. TNF-alpha-, IL-6-producing capability of mDCs was maintained. Nevertheless, IL-12 production by mDCs was not recovered at any time point. Mature DCs were potent in priming both Th1 and Th2 cells. In conclusion, upon maturation, DCs are reprogrammed with a distinct cytokine-secreting profile, which may play an important role in regulating T cell functions.     

Production of IL-12 by human monocyte-derived dendritic cells is optimal when the stimulus is given at the onset of maturation, and is further enhanced by IL-4

Dendritic cells produce IL-12 both in response to microbial stimuli and to T cells, and can thus skew T cell reactivity toward a Th1 pattern. We investigated the capacity of dendritic cells to elaborate IL-12 with special regard to their state of maturation, different maturation stimuli, and its regulation by Th1/Th2-influencing cytokines. Monocyte-derived dendritic cells were generated with GM-CSF and IL-4 for 7 days, followed by another 3 days +/- monocyte-conditioned media, yielding mature (CD83(+)/dendritic cell-lysosome-associated membrane glycoprotein(+)) and immature (CD83(-)/dendritic cell-lysosome-associated membrane glycoprotein(-)) dendritic cells. These dendritic cells were stimulated for another 48 h, and IL-12 p70 was measured by ELISA. We found the following: 1) Immature dendritic cells stimulated with CD154/CD40 ligand or bacteria (both of which concurrently also induced maturation) secreted always more IL-12 than already mature dendritic cells. Mature CD154-stimulated dendritic cells still made significant levels (up to 4 ng/ml). 2) Terminally mature skin-derived dendritic cells did not make any IL-12 in response to these stimuli. 3) Appropriate maturation stimuli are required for IL-12 production: CD40 ligation and bacteria are sufficient; monocyte-conditioned media are not. 4) Unexpectedly, IL-4 markedly increased the amount of IL-12 produced by both immature and mature dendritic cells, when present during stimulation. 5) IL-10 inhibited the production of IL-12. Our results, employing a cell culture system that is now being widely used in immunotherapy, extend prior data that IL-12 is produced most abundantly by dendritic cells that are beginning to respond to maturation stimuli. Surprisingly, IL-12 is only elicited by select maturation stimuli, but can be markedly enhanced by the addition of the Th2 cytokine, IL-4.     

Dendritic cells trigger tumor cell death by a nitric oxide-dependent mechanism

Dendritic cells (DCs) are well known for their capacity to induce adaptive antitumor immune response through Ag presentation and tumor-specific T cell activation. Recent findings reveal that besides this role, DCs may display additional antitumor effects. In this study, we provide evidence that LPS- or IFN-gamma-activated rat bone marrow-derived dendritic cells (BMDCs) display killing properties against tumor cells. These cytotoxic BMDCs exhibit a mature DC phenotype, produce high amounts of IL-12, IL-6, and TNF-alpha, and retain their phagocytic properties. BMDC-mediated tumor cell killing requires cell-cell contact and depends on NO production, but not on perforin/granzyme or on death receptors. Furthermore, dead tumor cells do not exhibit characteristics of apoptosis. Thus, intratumoral LPS injections induce an increase of inducible NO synthase expression in tumor-infiltrating DCs associated with a significant arrest of tumor growth. Altogether, these results suggest that LPS-activated BMDCs represent powerful tumoricidal cells which enforce their potential as anticancer cellular vaccines.     

Dendritic cell-derived TNF-α is responsible for development of IL-10-producing CD4 T cells

Immature dendritic cells (DCs) appear to be involved in peripheral immune tolerance via induction of IL-10-producing CD4⁺ T cells. We examined the role of TNF-α in generation of the IL-10-producing CD4⁺ T cells by immature DCs. Immature bone marrow-derived DCs from wild type (WT) or TNF-α^−/− mice were cocultured with CD4⁺ T cells from OVA specific TCR transgenic mice (OT-II) in the presence of OVA_323-339 peptide. The WT DCs efficiently induced the antigen-specific IL-10-producing CD4⁺ T cells, while the ability of the TNF-α^−/− DCs to induce these CD4⁺ T cells was considerably depressed. Addition of exogenous TNF-α recovered the impaired ability of the TNF-α^−/− DCs to induce IL-10-producing T cells. However, no difference in this ability was observed between TNF-α^−/− and WT DCs after their maturation by LPS. Thus, TNF-α appears to be critical for the generation of IL-10-producing CD4⁺ T cells during the antigen presentation by immature DCs.     

The synthetic analogue of mycoplasmal lipoprotein FSL-1 induces dendritic cell maturation through Toll-like receptor 2

Granulocyte-macrophage colony-stimulating factor-differentiated bone marrow-derived dendritic cells were stimulated with the synthetic lipopeptide S-(2,3-bispalmitoyloxypropyl)-CGDPKHSPKSF (FSL-1) or the Escherichia coli lipopolysaccharide. FSL-1 induced the production of TNF-alpha and IL-12 by C57BL/6-derived bone marrow-derived dendritic cells but not by bone marrow-derived dendritic cells from Toll-like receptor 2-deficient (TLR2(-/-)) mice. Lipopolysaccharide induced the production of TNF-alpha and IL-12 by bone marrow-derived dendritic cells derived from either type of mice. FSL-1 did not induce production of IL-10 by bone marrow-derived dendritic cells from either type of mice, whereas lipopolysaccharide induced small amounts of IL-10 by bone marrow-derived dendritic cells from both types of mice. The upregulation by FSL-1 of the expression of CD80, CD86 and the MHC class II molecule IA(b) was dose- and time-dependent on the surfaces of C57BL/6-derived bone marrow-derived dendritic cells but not on the surface of TLR2(-/-)-derived bone marrow-derived dendritic cells. Lipopolysaccharide upregulated the expression of these molecules on the surfaces of bone marrow-derived dendritic cells from both types of mice. The expression of CD11c on the surfaces of C57BL/6-derived bone marrow-derived dendritic cells was upregulated by stimulation with both FSL-1 and lipopolysaccharide up to 12 h; thereafter, the expression was downregulated. The results suggest that FSL-1 can accelerate maturation of bone marrow-derived dendritic cells and this FSL-1 activity is mediated by TLR2.     

Immature and Maturation-Resistant Human Dendritic Cells Generated from Bone Marrow Require Two Stimulations to Induce T Cell Anergy In Vitro

Immature dendritic cells (DC) represent potential clinical tools for tolerogenic cellular immunotherapy in both transplantation and autoimmunity. A major drawback in vivo is their potential to mature during infections or inflammation, which would convert their tolerogenicity into immunogenicity. The generation of immature DC from human bone marrow (BM) by low doses of GM-CSF (lowGM) in the absence of IL-4 under GMP conditions create DC resistant to maturation, detected by surface marker expression and primary stimulation by allogeneic T cells. This resistence could not be observed for BM-derived DC generated with high doses of GM-CSF plus IL-4 (highGM/4), although both DC types induced primary allogeneic T cell anergy in vitro. The estabishment of the anergic state requires two subsequent stimulations by immature DC. Anergy induction was more profound with lowGM-DC due to their maturation resistance. Together, we show the generation of immature, maturation-resistant lowGM-DC for potential clinical use in transplant rejection and propose a two-step-model of T cell anergy induction by immature DC.