Phenotype and function of human dendritic cells derived from M-DC8(+) monocytes

Dendritic cells (DC) generated from peripheral blood monocytes have been used with promising results as a new approach for the immunotherapy of cancer. However, at least four different subpopulations of peripheral blood monocytes have been recognized and their contribution to the generation of functional DC is not known. Recently, the monoclonal antibody M-DC8 has been shown to react with 0.2 - 1 % of blood leukocytes. We have identified M-DC8(+) cells as monocytes which represent about 40 % of CD14(low)CD16(+) monocytes. Similar to M-DC8(-) monocytes, they develop in the presence of GM-CSF and IL-4 into a very homogenous population of cells with DC phenotype and function. M-DC8(+) DC show on average a twofold higher expression of HLA class I and class II molecules than M-DC8(-) DC. These DC produce IL-12p75 both in response to LPS and to CD40 ligation. M-DC8(+) DC induced a strong Th1 immune response and were two to four old more potent than M-DC8(-) DC for the priming of cord blood T cells. M-DC8(+) monocytes can be used as a source of very potent dendritic cells with the potential to significantly improve the efficacy of DC-based immunotherapies.     

Generation and characterization of ovine dendritic cells derived from peripheral blood monocytes

Dendritic cells (DCs) are professional antigen‐presenting cells with a highly immunostimulatory function and the capacity to activate naïve T cells. In recent years the rapid progress in mouse and human DC research can be mainly attributed to the generation of DCs from precursor cells in vitro, although a lack of reagents has hampered DC research in many large animal models. Here we describe the generation and characterization of ovine monocyte‐derived DCs in vitro. In addition to the characteristic morphology and non‐adherence of DCs, peripheral blood mononuclear cell monocytes cultured with ovine granulocyte–macrophage colony‐stimulating factor (GM–CSF) and interleukin‐4 (IL‐4) expressed CD11c and major histocompatibility complex (MHC) class II, but did not express CD14. High levels of endocytosis and an ability to stimulate antigen‐specific proliferation of CD4 T lymphocytes were also demonstrated.     

慢性乙型肝炎患者树突状细胞表型的研究

目的探讨慢性乙型肝炎患者外周血来源树突状细胞（Dendritic cell，DC）数量及表型的改变，并对其与肝功能、乙肝病毒复制水平的关系进行研究。方法检测37例慢性乙肝患者和21例健康人肝功能及血清HBV DNA水平，并提取外周血单个核细胞（Peripheral blood mononuclear cell，PBMC）进行体外诱导培养，促使其发育成DC，计数其数量并检测膜表面分子的变化。分析DC数量及表型与肝功能、乙肝病毒复制水平的关系。结果与正常对照组比较，慢性乙肝患者的树突状细胞数量明显减少（P〈0．05），且其膜表面分子CD83、CD86的表达均明显降低（P〈0．05）。在慢性乙肝患者中，DC数量、DC膜表面分子CD83和CD86与血清HBV DNA之间呈负相关关系，而与肝功能之间无明显相关关系。结论慢性乙肝患者体内存在DC数量减少及成熟障碍，这种改变与肝内炎症反应程度不相关，但与乙肝病毒（Hepatitis Bvirus，HBV）的复制水平呈负相关，提示DC参与慢性乙肝患者体内HBV的清除。     

Functional and phenotypic characterization of distinct porcine dendritic cells derived from peripheral blood monocytes

Dendritic cells (DCs) are bone marrow-derived antigen-presenting cells that have an exquisite capacity to interact with T cells and modulate their responses. Little is known about porcine DCs despite the fact that they represent an important target in strategies that are aimed at modulating resistance to infection in pigs and may be of major importance in transplantation biology. We generated immature monocyte-derived porcine dendritic cells (MoDCs) directly from adherent peripheral blood cells treated with porcine granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4). The cells were observed via electron microscopy and their phenotype was characterized using monoclonal antibodies. The functionality of the porcine MoDCs was demonstrated showing that the cells were capable of different specialized functions relevant to antigen capture and were potent stimulators in a primary allo-mixed leucocyte reaction. Treatment of the MoDCs with porcine cell line-derived necrotic factors resulted in the phenotypic and functional maturation of MoDCs. We confirmed also that monocyte-derived DCs were differentially regulated by cytokines, showing that transforming growth factor-beta1 (TGF-beta1) is able to redirect monocytic precursors into the differentiation pathway of Langerhans' cells presenting typical Birbeck granules. Interestingly, and in contrast to the human and murine model, we showed that the monocyte-derived porcine Langerhans'-type cells (MoLCs) were much more potent activators of allogeneic T cells than MoDCs obtained without TGF-beta1.     

负载滋养层细胞抗原对小鼠树突状细胞表型及功能的影响

目的 研究负载滋养层细胞抗原对小鼠髓源性树突状细胞(DC)分化成熟过程的影响,获得致耐受性DC.方法 体外使用粒细胞巨细胞集落刺激因子(GM-CSF)诱导小鼠骨髓细胞定向分化、经LPS刺激获得成熟DC;通过外胎盘锥组织块培养法获得滋养层细胞,制备可溶性抗原,加入DC培养体系.流式细胞术检测DC表面共刺激分子及MHC-Ⅱ的表达,ELISA法检测DC分泌IL-10和IL-12的浓度,混合淋巴细胞培养评估 DC刺激同种T细胞增殖、活化的功能.结果 成熟DC表型为CD40high CD80highCD86highMHC-Ⅱhigh,分泌大量的IL-12和极少量的IL-10 ,体外能有效刺激T细胞的增殖;负载滋养层细胞抗原的DC表型为CD40midCD80lo wCD86lowMHC-Ⅱlow,在分泌大量IL-12的同时IL-10也明显升高,不能有效刺激T细胞增殖,并使T细胞分泌细胞因子呈现明显Th2偏倚.结论 负载滋养层细胞抗原后的DC表面共刺激分子及MHC-Ⅱ表达降低,刺激T细胞增殖能力下降;其自分泌和促使T细胞旁分泌的细胞因子呈现Th2偏倚,是一种耐受性DC.     

Generation of feline dendritic cells derived from peripheral blood monocytes for in vivo use

Dendritic cells (DCs) are professional antigen-presenting cells that can prime T cells and polarize the cellular immune response. Because Th1-type immune responses have been connected to success in combating viral infection, a promising therapeutic application of DCs would be their differentiation in vitro and injection back into the host to boost an immune response in infected animals. This study was aimed both at developing a protocol to cultivate feline DCs in the absence of exogenous proteins for their use in vivo and at investigating what might be the most appropriate stimulus to induce their maturation in vitro and finding correlates of maturation. We generated DCs from peripheral blood monocytes in the presence of feline interleukin-4 and granulocyte-macrophage colony stimulating factor, and after 5 days their maturation was induced with either lipopolysaccharide, human recombinant tumor necrosis factor alpha, poly(I:C), or activated feline platelets. After 48 h, their CD14, CD1a, major histocompatibility complex class II, and B7.1 surface expression was analyzed in parallel with their ability to uptake antigen or prime a mixed leukocyte reaction. The results presented show that feline DCs cultured in autologous plasma differentiate and are able to mature in the presence of stimuli similar to the ones currently used for other species. The present work sets the grounds for future use of DCs obtained by the protocol described for in vivo vaccination and immunotherapy of feline immunodeficiency virus-infected cats.     

恒河猴骨髓来源未成熟树突状细胞的培养及鉴定

目的建立一种体外诱导恒河猴骨髓CD34+细胞分化为树突状细胞(DC)的方法,并对其细胞形态及表面标志进行鉴定。方法用免疫磁珠分选系统(MACS)分选恒河猴骨髓细胞,收集高纯度的CD34+造血干细胞;加入重组粒细胞-巨噬细胞集落刺激因子(GM-CSF)和白细胞介素4(IL-4)及肿瘤坏死因子-α(TNF-α),诱导其分化为骨髓来源的树突状细胞,并采用电镜观察细胞形态。用树突状细胞表面标志CD1a及成熟树突状细胞(mDC)表面标志CD83流式抗体染色后分析。结果细胞因子诱导6d后成功培养出表面呈绒毛状和树枝状突起的典型DC样细胞,流式细胞术分析结果显示92.35%的细胞为DC,27.61%的细胞为m DC。结论用此方法可在体外培养出高纯度且典型的恒河猴骨髓源性未成熟树突状细胞(im DC),为进一步研究其生物学特性及功能奠定了基础。     

Effect of PSK on the maturation of dendritic cells derived from human peripheral blood monocytes

Dendritic cells (DCs) are powerful antigen-presenting cells (APCs) that have attracted attention in recent years from the viewpoint of DC vaccine therapy against cancer. However, the existence of a strongly immunosuppressed state in cancer-bearing individuals inhibits DC maturation, which is one of the problems facing anti-cancer DC vaccine therapy. Protein-bound polysaccharide K (PSK), which is extracted from the cultured mycelium of Coriolus versicolor (Fr.) Quél, is used as an anti-cancer agent in Japan. PSK is reported to improve the immunosuppressed state and might be associated with DC maturation directly. We examined the effect of PSK on the maturation of DC derived from CD14-positive cells obtained from human peripheral blood monocytes using a negative selection method. CD14-positive cells cultured in the presence of PSK significantly increased the expression of HLA class II antigen and CD40; significantly increased the number and expression of CD80-, CD86- and CD83-positive cells; decreased Fluorescein isothiocyanate (FITC)-dextran uptake, augmented IL-12 production; augmented the allogeneic mixed lymphocyte reaction; and induced antigen-specific cytotoxicity. These results indicate that PSK promotes both the phenotypic and functional maturation of DC derived from human CD14-positive mononuclear cells. The clinical significance of the combined use of PSK in DC vaccine therapy remains for study.     

Phenotype and function of intestinal dendritic cells

It is now appreciated that dendritic cells (DCs) play a primary role in oral tolerance and defense against mucosal pathogens. Specific DC subpopulations are localized to discrete regions within primary inductive tissues, like the Peyer's patch and mesenteric lymph node, and effector sites, like the lamina propria, and may have unique roles in driving regulatory, effector and memory T cell responses. Certain DC subpopulations may also help maintain T cell responses at sites of abnormal intestinal inflammation. While early in our understanding, knowledge about the involvement of DC subpopulations in the regulation of mucosal immunity may well provide a basis for the development of novel vaccines and therapeutics.     

Lymphotropic papovaviruses isolated from African green monkey and human cells

A lymphotropic papovavirus was isolated from a lymphoblastoid cell line of African green monkey (AGM) cells which also contained a herpesvirus and a paramyxovirus-like agent. The papovavirus was analyzed by restriction endonuclease cleavage; its biochemical and serological crossreactivity with SV40 and host range have been determined. Thus far, only B-lymphoblasts of primate and human origin have been found to be susceptible to infection. Although more than 50% of the tested monkey sera were reactive with antigens of this virus, all human sera tested failed to react. Cleavage patterns and hybridization studies with the viral DNA indicate that the virus represents a novel member of the papovavirus group that is characterized by its lymphotropic host range. Papovavirus particles were also demonstrated in a human lymphoblastoid cell line (CCRF-SB) originally derived from a leukemic child. These cells revealed nuclear fluorescence when tested with human sera, but failed to react with AGM sera. Although characterization of this agent has not yet been completed, available evidence suggests that it represents another lymphotropic papovavirus which seems to be spread within the human population.     

Generation of dendritic cells from acute myeloid leukaemia cells and monocytes: our local experience

Dendritic cells (DC) are efficient and potent antigen-presenting cells. Pilot clinical trials indicated that DC loaded with tumour antigen could induce tumour-specific immune responses in various cancers including B-cell lymphoma, melanoma and prostate cancer. Owing to extensively low number of DC in the blood circulation, a variety of sources have been used to generate DC including monocytes, CD34+ stem cells and even with leukaemic blast cells. We demonstrate here a simple method to generate DC from acute myeloid leukaemia (AML) cells and monocytes from healthy donor or remission samples. AML cells or monocytes were cultured in RPMI 1640 media supplemented with foetal bovine serum or autologous serum where possible and different combinations of cytokines GM-CSF, IL-4 and TNF-alpha. The generated DC were evaluated for their morphology by phase contrast microscopy and May Grunwald Giemsa staining. Viability of cells was determined by trypan blue dye exclusion. Percentage of yields and immunophenotypes were carried out by flow cytometry. We found that cultured AML cells and monocytes developed morphological and immuno-phenotypic characteristics of DC. Monocytes are better than AML blast in generating DC and serve as a ready source for dendritic cell vaccine development.     

树突状细胞诱导的CTL抗乙型肝炎病毒效应

目的　研究乙肝表面抗原 (HBsAg)负载的树突状细胞疫苗在抗乙肝病毒中的作用。方法　从健康人外周血中分离出单核细胞 ,在GM CSF和IL 4的作用下培养 7d诱导出DC ,然后以DC为刺激细胞在体外诱导出HBsAg特异性CTL ;将携有HBV S基因的pLXSN S重组质粒 ,电击导入肝癌细胞系 (HepG2 ) ,建立表达HBsAg的靶细胞模型HepG2 S ;用DC激活的HBsAg特异性CTL与HepG2 S细胞同时接种于BALB c裸鼠或用HBsAg特异性CTL治疗HepG2 S荷瘤裸鼠 ,观察肿瘤的发生和生长情况。结果　在接种后的 38d内 ,治疗组肿瘤明显比对照组小 ,差异有显著性 (P <0 .0 5 ) ,而且治疗组有 3只荷瘤鼠肿瘤消退 (6 0 % )。结论　DC激活的HBsAg特异性CTL细胞对HepG2 S移植瘤有治疗作用。     

Mechanism of NK cell activation induced by coculture with dendritic cells derived from peripheral blood monocytes.

Dendritic cells (DCs) have been regarded as one of the effective antigen-presenting cells, but the relationship between DCs and lymphocytes, in particular natural killer (NK) cells, remains unclear. In this study, we evaluated how DCs interact with both lymphocytes and NK cells using a coculture system. The number of lymphocytes increased significantly when cocultured with DCs (1.8-fold increase). In particular, the proliferation of NK cells was prominent. Furthermore, the coculture of DCs with lymphocytes induced a marked increase in IL-12 and IFN-gamma secretion. When contact between the DCs and lymphocytes was prevented, the secretion of both IL-12 and IFN-gamma was markedly reduced. IFN-gamma production was completely blocked by an anti-IL-12 antibody, indicating that IFN-gamma secretion was dependent on IL-12 secretion. The stimulating effect of the DCs on the proliferation of the lymphocytes was partially suppressed by anti-IL-12 antibodies, and was completely attenuated when cellular contact was prevented. Furthermore, the NK cell proliferation induced by coculture with DCs was significantly blocked by the inhibition of the interaction of either CD40-CD40L or CD28-B7 molecule. The coculture with DCs enhanced NK activity by 40%, and this was partially suppressed by anti-IL-12 antibodies and was completely blocked by the inhibition of cell-to-cell contact. These results indicate that the activation of NK cells by DCs is partially mediated by IL-12 secretion, and that direct contact between DCs and NK cells play a major role in this response.     

人外周血单核细胞源性朗格汉斯细胞和表皮炎症样树突状细胞的诱导及表型分析

目的利用人外周血单核细胞体外诱导培养朗格罕氏细胞(Mo-LC)和炎症性树突状表皮细胞(Mo-IDEC)并观察其表型特点。方法联合应用LymphoPrepTM梯度离心试剂和CD14+磁珠分离和筛选外周血CD14+单核细胞;在重组人粒细胞-巨噬细胞集落刺激因子(GM-CSF)和重组人白介素4(IL-4)的基础上分别于第0、2、4天加入人天然TGF-β1或β-巯基乙醇(β-ME)。培养第6天时收集细胞,通过相差显微镜观察其形态,并利用单克隆抗体和流式细胞术对诱导细胞亚群检测细胞表面分子的表达水平。结果 CD14+单核细胞体外诱导培养6 d后可形成具有树突状外观的Mo-LC和Mo-IDEC,2种细胞均不同程度表达CD1a、FcεRI、FcεRII、TLR1、TLR2;Mo-LC表达CD207,Mo-IDEC表达CD206。特应性皮炎患者来源的Mo-IDEC表面CD1a表达高于Mo-LC,且CD23、TLR2、FcεRI的表达水平显著高于健康对照组。结论利用外周血单核细胞可在体外成功诱导Mo-LC和Mo-IDEC,这2种细胞表型特点与体内分离的细胞在形态和表型上类似,为体外进一步研究这2类细胞的功能打下基础。     

Vaccinia virus-related events and phenotypic changes after infection of dendritic cells derived from human monocytes

The in vitro interactions between vaccinia virus (VV) and monocyte-derived human dendritic cells (DC) have been studied to gain a better understanding of the mechanisms involved in the induction of an immune response by VV. This work showed that VV binds to DC less efficiently than to HeLa cells (HeLa). Capping of viral antigens on the DC surface and electron microscopic examinations suggested that VV enters into DC mainly by endocytosis instead of fusion as for HeLa. Early viral-encoded proteins were expressed in DC but late viral proteins and viral DNA synthesis did not occur. Nevertheless, when successfully infected, DC expressed a similar amount of a foreign, viral-encoded protein, as HeLa, if the early component of the p7.5 promoter was used. VV infection did not lead to DC maturation as determined by following the level of several cell surface markers associated with maturation, but an inhibition of the expression of the costimulatory molecule CD80 was noticed. The proliferation of allogeneic peripheral blood lymphocytes (PBL) was stimulated by VV-infected DC or inhibited depending on the particular donor lymphocytes employed. PBL from VV-vaccinated individuals with good memory responses to VV antigens proliferated in the presence of infected autologous DC. PBL from individuals with poor memory responses to VV and one unvaccinated individual also proliferated, albeit to a lower level, in the presence of infected autologous DC. These results suggest that VV-infected DC could both stimulate memory cells and prime naive cells in vitro.     

Recruitment of immature plasmacytoid dendritic cells (plasmacytoid monocytes) and myeloid dendritic cells in primary cutaneous melanomas

The present study has analysed the distribution and phenotype of dendritic cells (DCs) in primary cutaneous melanomas and sentinel lymph nodes by immunohistochemistry. In primary melanomas, an increase of DCs was found in the epidermis and the peritumoural area. Intraepidermal DCs were mostly CD1a⁺/Langerin⁺ Langerhans cells. Peritumoural DCs included a large population of DC‐SIGN⁺/mannose‐receptor⁺/CD1a^? DCs, a small subset of CD1a⁺ DCs, and, remarkably, plasmacytoid monocytes/plasmacytoid DCs (PM/PDCs). The PM/PDCs, most likely recruited by SDF‐1 secreted by melanoma cells, produced type I interferon (IFN‐I), but the expression of the IFN‐α inducible protein MxA was extremely variable and very limited in the majority of cases. All DC subsets were predominantly immature. The peritumoural area also contained a minor subset of mature CD1a⁺ DCs. However, the small amount of local interleukin (IL)‐12 p40 mRNA and the naïve phenotype of 20–50% of peritumoural T‐lymphocytes are consistent with poor T‐cell stimulation or erroneous recruitment. In sentinel lymph nodes, notable expansion of mature CD1a⁺/Langerin⁺ DCs was observed. The paucity of intratumoural DCs and the predominant immature phenotype of peritumoural dermal DCs indicate defective maturation of primary cutaneous melanoma‐associated DCs, resulting in lack of T‐cell priming. These results may explain why melanoma cells grow despite the presence of infiltrating immune cells. Copyright © 2003 John Wiley & Sons, Ltd.