米非司酮对人外周血来源树突状细胞成熟和功能的影响

目的观察米非司酮对人外周血来源树突状细胞(DCs)成熟及生物学功能的影响,探讨米非司酮作为紧急避孕药的免疫学机制。方法人外周血CD14+单核细胞在体外经GM-CSF、IL-4培养6d诱导分化为未成熟DCs,加入1800 nmol/L米非司酮继续培养48h,流式细胞仪检测细胞表型,ELSIA检测DCs培养上清液中IL-12p70水平,混合淋巴细胞反应检测DCs刺激同种异体T细胞增殖的能力。结果与阴性对照组相比,米非司酮处理后的DCs,其细胞表面HLA-DR及CD83分子表达上调(t分别=15.23、7.63,P均<0.05),IL-12p70分泌增加(t=12.62,P<0.05),且刺激同种异体T细胞增殖的能力明显增强,差异均有统计学意义(t分别=9.12、13.24、6.78,P均<0.05)。结论米非司酮能诱导人外周血来源DCs成熟,促进DCs诱导的免疫应答启动。     

人外周血单核细胞来源的树突状细胞的体外诱导

目的:探讨在体外从人外周血单核细胞诱导培养成熟的树突状细胞(dendritic cell,DC)的方法.方法:培养过程分两阶段,第一阶段:采用密度梯度离心法分离健康成人外周血中的单个核细胞,再以黏附法分离出单核细胞,经重组人粒细胞-巨噬细胞集落刺激因子(rhGM-CSF)100 ng/mL、重组人白介素-4(rhIL-4)100 ng/mL体外诱导.第二阶段:第5天加入重组人肿瘤坏死因α-(rhTNF-α)100 ng/mL,继续培养2 d,刺激DC成熟.倒置显微镜下观察DC形态,流式细胞仪检测DC表面标志物CD83、CD1a、CD86、CD40、CD14表达水平,用MTT法测定DC刺激同种异体T细胞增殖的能力.结果:人外周血单核细胞经rhGM-CSF及rhIL-4诱导培养5 d后,多数细胞呈集落生长,细胞表型CD83、CD1a、CD86、CD40及CD14分别是14.3%、12.8%、20.1%、19.9%及16.2%.加入rhTNF-α诱导后,即培养第7天,细胞表型CD83、CD1a、CD86、CD40及CD14分别是29.8%、18.2%、33.6%、28.1%及8.0%(与第5天比较,均P<0.05).成熟后的DC具备较强刺激T细胞增殖的能力.结论:rhGM-CSF联合rhlL-4诱导人外周血单核细胞,可获得大量不成熟的DC,该体系有利于Dc扩增,加入rhTNF-α,继续培养2 d,可诱导出成熟的DC,成熟的DC具备较强刺激T细胞增殖的能力.     

Efficient transduction of human monocyte-derived dendritic cells by chimpanzee-derived adenoviral vector

Using recombinant adenoviruses (Ads) to target host dendritic cells (DCs) presents an attractive prospect for immunization. The efficacy of commonly used human Ad-derived gene transfer vectors for antigen delivery in humans is often compromised by preexisting anti-Ad immunity, acquired by the majority of human population as a result of frequent naturally occurring virus infections. As an alternative vector we propose chimpanzee-derived recombinant adenoviruses, which are poorly neutralized by human sera. In the present study we examine the ability of one such vector, AdC68, to transduce and activate human monocyte-derived DCs in culture. We found that AdC68 could efficiently transduce both immature and mature DCs at levels similar to those by the human serotype 5 Ad recombinant. Exposure of immature DCs to AdC68 did not alter the expression of activation and maturation marker molecules on the cell surface. Nevertheless, the transduction induced DCs to secrete interferon alpha and interleukin (IL)-6, but not IL-12 or tumor necrosis factor alpha. In addition, AdC68-transduced immature DCs could stimulate proliferation of autologous T lymphocytes. This is the first report describing a chimpanzee-derived recombinant Ad as a vector for transduction of human DCs.     

Interleukin-10 increases mannose receptor expression and endocytic activity in monocyte-derived dendritic cells

Human monocyte-derived dendritic cells were differentiated in vitro for 7 days with granulocyte macrophage-colony stimulating factor and interleukin-13. These cultured dendritic cells are at an immature stage of differentiation and exhert high endocytic activity via surface mannose receptor and via fluid-phase macropinocytosis. We have investigated the modulation of endocytosis by interleukin-10 in these cells. When added during the last 24 h of the 7-day culture, interleukin-10 significantly stimulated the uptake of fluorescein-labelled dextran (39 ± 16% increase, mean ± SD of 6 experiments), a sugar binding to the mannose receptor. This effect was dose dependent and correlated with the length of exposure to interleukin-10, with a maximal effect (more than seven-fold increase) when the cytokine was added at the beginning of the culture (day 0). The interleukin-10-increased fluorescein-labelled-dextran endocytosis was mostly mediated via the mannose receptor, as unlabelled mannose and specific antimannose receptor monoclonal antibody inhibited most of the uptake. Moreover, interleukin-10-treated cells expressed increased levels (up to four-fold) of mannose receptor. Interleukin-10 also increased, although to a lesser extent, the fluid-phase endocytosis (macropinocytosis) of fluorescein-labelled albumin. Interleukin-10 had the opposite effect on the differentiation and functional activity of monocyte-derived dendritic cells; cells having a very low stimulatory capacity and reduced expression of MHC class II and CD1a after a 7-day exposure. Thus interleukin-10 had a strong immunosuppressive effect on the differentiation and functional activity of monocyte-derived dendritic cells and yet strongly stimulated endocytosis in these cells. We speculate that an increased endocytic activity would eventually result in a decreased availability of antigens in the external milieu, thus contributing to the immunosuppressive and tolerogenic activity of interleukin-10.     

Recombinant adenovirus vector activates and protects human monocyte-derived dendritic cells from apoptosis

The aim of this study was to examine the effect of two of the most commonly used viral vectors, that is, retrovirus and adenovirus, on the antigen presentation of dendritic cells (DCs). DCs were generated from CD34(+) hematopoietic precursors and CD14(+) monocytes of the same prostate cancer patients. Adenoviral transduction of monocyte-derived DCs (MO-DCs) resulted in upregulation of CD80, CD86, and CD83 expression. Adenovirus-transduced MO-DCs were also more potent stimulators of allogeneic lymphocytes, produced increased amounts of the cytokines tumor necrosis factor alpha and interleukin 12 p70, and exhibited increased expression of NF-kappaB and antiapoptotic molecules Bcl-X(L) and Bcl-2. Enhanced expression of the antiapoptotic molecules correlated with increased resistance of adenovirus-transduced MO-DCs to spontaneous as well as Fas-mediated cell death. In contrast to the adenoviral construct, no significant transduction of MO-DCs with the retrovirus could be obtained. Transduction of CD34(+) cell-derived DCs with the retrovirus or the adenovirus did not significantly alter expression of the costimulatory molecules or cytokines studied. At lower stimulation ratios, CD34(+) cell-derived DCs transduced with retrovirus were less potent in their ability to stimulate allogeneic lymphocytes in comparison with nontransduced DCs. Our results indicate that adenoviral vectors may be more suitable for gene delivery to DCs for immunotherapy.     

Enhanced lentiviral transduction of monocyte-derived dendritic cells in the presence of conditioned medium from dying monocytes

Lentiviral vectors (LVs) are attractive vehicles for the transduction of human dendritic cells (DCs) in order to mobilize their endogenous antigen presentation pathways. We analyzed here how to improve the efficiency of LV transduction, which we performed at the initial stages of the differentiation of purified monocytes into dendritic cells (Mo-DCs). Using LVs pseudotyped with the vesicular stomatitis virus envelope G glycoprotein (VSV-G), we found that a conditioned medium derived from dying monocytes (MCM) improved by 2- to 10- fold the proportion of transduced Mo-DCs. This enhanced transduction efficiency requires the presence of MCM during the initial stage of LV transduction and does not affect the phenotype and antigen presentation function of terminally differentiated Mo-DCs. Importantly, we found that MCM derived from a human acute monocytic leukemia cell line, THP-1, was equally effective. The MCM activity was heat stable (56 degrees C) and was present in the soluble fraction after high-speed centrifugation. Altogether our results show that a soluble factor present in dying monocyte cultures can replace advantageously facilitating agents such as Polybrene, to achieve high LV transductions levels. This protocol can be performed with autologous monocytes and is therefore applicable in clinical settings.     

IL-12 production by human monocyte-derived dendritic cells: looking at the single cell

Dendritic cells (DCs) are under investigation as immunotherapeutic agents in the treatment of cancer and infectious diseases. One of the important factors in skewing the immune response toward clinically beneficial TH1-type immunity is interleukin-12p70. IL-12p70 is synthesized and secreted in response to inflammatory cytokines, bacterial/viral components, and CD40 ligation. This study investigated the production of IL-12 by DCs at the single-cell level using a sensitive intracellular cytokine flow cytometry-based assay system. The authors observed that immature DCs could be stimulated with several compounds to produce IL-12, but that IL-12 production was a feature of a minority of activated DCs. IL-12+ DCs were characterized as being partially matured (ie, absent or low CD83 expression, with variable expression of CD1a and CD64). Interestingly, activated DCs lacked expression of the CD16 and CD64 Fc gammaR, which may have important implications for exogenous antigen-loading strategies.     

Potent maturation of monocyte-derived dendritic cells after CD40L lentiviral gene delivery

Dendritic cells (DCs) are being evaluated in immunization protocols to enhance immunity against infectious diseases and cancer. Interaction of T-helper cells expressing CD40 ligand (CD40L) with its cognate CD40 receptor on DCs leads to a mature DC phenotype, characterized by increased capacity of antigen presentation to cytotoxic T cells. The authors examined the ability of third-generation self-inactivating lentiviral vectors expressing CD40L to induce autonomous maturation of ex vivo expanded human monocyte-derived dendritic cells. Transduction with lentiviral vectors achieved a highly efficient gene transfer of CD40L to DCs, which correlated with phenotypic maturation as shown by the expression of immunologic relevant markers (CD83, CD80, MHCI) and secretion of IL-12, whereas DC phenotype was not affected by a control vector expressing only the green fluorescent protein marker. Addition of recombinant IFN-gamma to DCs at the time of CD40L transduction further enhanced IL-12 production, and when co-cultured with allogeneic and autologous CD8+ and CD4+ T cells, a potent activation was observed. Autologous responses against an HLA-A2-restricted influenza peptide (Flu-M1) and a tumor-associated antigenic peptide (gp100 210M) were significantly enhanced when CD40L transduced DCs were used as antigen-presenting cells for in vitro stimulation of CD8+ cytotoxic T lymphocytes. These results demonstrate that endogenous expression of CD40L by lentivirally transduced DCs induced their autonomous maturation to a phenotype comparable to that induced by optimal concentrations of soluble CD40L, providing a novel tool for genetic manipulation of DCs.     

Differential effects of autologous serum on CD34(+) or monocyte-derived dendritic cells

Dendritic cells (DC) with potentially important clinical applications have been generated from human peripheral blood monocytes and CD34(+) cells in the presence of recombinant cytokines granulocyte-macrophage colony-stimulating factor (GM-CSF) + interleukin-4 (IL-4) and GM-CSF + tumor necrosis factor-alpha (TNF-alpha), respectively. Many of the studies generating DC have included fetal calf serum, which is not desirable due to the risk of immune reactions and infectious disease transmission. Additionally, low DC yields have been reported using serum-free media. In this study, we investigate supplementing serum-free media with autologous serum and plasma for DC generation from monocytes and CD34(+) cells. Our results show that functional DC can be reproducibly obtained in the presence of autologous serum using monocytes and CD34(+) cells as the starting populations. However, with the addition of autologous serum, a differential effect is observed in the phenotypic characterization of these culture-derived DC. Monocytes cultured for 7 days in X-VIVO 15 serum-free media in the presence of GM-CSF + IL-4 showed down-regulation of CD14 with increased expression of HLA-DR, mannose receptor, CD80, and CD86, along with highly up-regulated CD1a(+) expression. The addition of autologous serum to serum-free media in monocyte cultures resulted in a dose-dependent decrease in the CD1a(+) expression generating a distinct subset of CD1a(+/-) cells expressing HLA-DR, mannose receptor, CD80, and CD86. Upon stimulation with CD40L cells, both monocyte-derived DC subsets CD1a(+/-) and CD1a(++) were capable of maturation measured by CD83 and CD86 up-regulation. Data suggest the differences in the monocyte-derived DC in serum-free (CD1a(++)) or autologous serum (CD1a(+/-)) supplemented cultures is of a qualitative nature, rather than quantitative. CD1a(+) and CD14(+) cells expressing HLA-DR, mannose receptor, CD80, and CD86 were generated in 7 days from CD34(+) cells in serum-free media. A quantitative effect was obtained when cultures were supplemented with autologous serum, resulting in a significant enhancement of CD34-derived DC generated. These results demonstrate generation of DC from two different starting populations using serum-free media that can be enhanced with the addition of autologous serum. Interestingly, a differential effect was observed in the phenotypic characterization of these culture-derived DC.     

In vitro maturation potential of monocyte-derived dendritic cells is impaired in patients with spinal cord injury: a case-control study

OBJECTIVE: To determine whether the T-cell reactivity and maturation potential of dendritic cells are impaired in subjects with spinal cord injury (SCI). DESIGN: Cross-sectional, case-control study. SETTING: University hospital in Taiwan. PARTICIPANTS: Thirty male SCI subjects, including 14 with paraplegia and 16 with tetraplegia, and 30 age- and sex-matched healthy controls. INTERVENTIONS: Not applicable. MAIN OUTCOME MEASURES: CD4+ and CD8+ T-cell reactivity was assessed by lymphoproliferative response (LPR) and CD69 expression in response to stimulation with antigens, anti-CD3 monoclonal antibody, and mitogen. Dendritic cell maturation potential was assessed by phenotypic (CD80 and CD83 expression in stimulated monocyte-derived dendritic cells) and functional (the LPR of alloreactive T cells in mixed leukocyte reactions) analysis. RESULTS: The potential of phenotypic and functional maturation of dendritic cells of subjects with SCI was significantly impaired compared with healthy controls, and that potential was worse for tetraplegic patients than for paraplegic patients. However, no significant difference was found in T-cell responses of CD4+ and CD8+ subsets between subjects with SCI and healthy controls. CONCLUSIONS: Impaired maturation potential of dendritic cells is a novel defect in innate immunity in people with SCI.     

Activation of immature monocyte-derived dendritic cells after transduction with high doses of lentiviral vectors

Dendritic cells (DCs) are an attractive tool for immunomodulation, targeting mature DCs (mDCs) for immunization or immature/semimature DCs (iDCs) for tolerization. Therefore, introducing antigens into DCs has become a prime topic in various immunological disciplines. Numerous studies have shown that lentiviruses are an efficient vehicle for this purpose. This study evaluates the effects of lentiviral transduction on iDC activation. Immature DCs are efficiently transduced with increasing doses of lentivirus without affecting cell viability. Transduction at low multiplicities of infection (MOIs) did not result in phenotypical or functional maturation. Higher doses of lentivirus, however, resulted in upregulation of adhesion, costimulatory, and HLA molecules, as well as in increased allostimulatory capacity and secretion of interleukin (IL)-6, IL-8, and tumor necrosis factor-alpha. Production of IL-12 p70, IL-10, and interferon-alpha was observed only at extremely high doses. Protein kinase R phosphorylation on transduction at an MOI of 150 was demonstrated by Western blotting. A Toll-like receptor (TLR)-driven luciferase reporter assay showed dose-dependent activation of TLR2, TLR3, and TLR8, which was independent of the pseudotype, production, or transduction protocol and was abrogated on heat inactivation. These data show that lentiviral vectors provide not only the antigen but also appropriate activation signals to iDCs, favoring their use for immunotherapy and vaccine development.     

不同培养基对人外周血单核细胞来源树突状细胞发育的影响

本研究旨在探讨RPM I1640和IMDM两种培养液对诱导人外周血单核细胞向树突状细胞(DC)发育分化的影响。利用GM-CSF+IL-4细胞因子组合,在培养条件一致的前提下,改变培养液种类,通过对成熟、未成熟DC形态观察,用流式细胞术检测细胞表型和吞噬能力、应用混合淋巴细胞反应(MLR)检测其刺激T细胞的增殖能力,用悬浮芯片技术检测DC与同种异体T细胞共培养后上清中所含细胞因子的变化,分析不同培养液对DC功能的影响。研究结果表明,两种培养液培养所得的DC在形态上无显著差异,DC的吞噬能力以及CD14和CD83的表达亦无显著差异,但应用IMDM培养的DC的CD1a表达明显降低,且对T细胞增殖的刺激能力也显著降低;IMDM培养的DC高表达IL-6、IL-8和IL-10,而IL-12的表达则显著降低。结论:不同的培养体系可获得功能不同的DC,IMDM培养的DC可能与免疫耐受有关,本研究结果为DC在临床上的应用提供了新思路。     

α-干扰素诱导外周血单核细胞向树突状细胞分化

背景：研究表明，树突状细胞接受刺激的性质和微环境及其起源是决定免疫反应中初始CD4^＋T细胞发生Th1或Th2应答的关键因素，树突状细胞是否成熟对其功能的发挥影响很大。 目的：观察广泛应用的细胞因子α-干扰素对人外周血单核细胞向树突状细胞分化成熟的影响。 设计、时间及地点：细胞水平的观察对照实验，于2007-05／12在深圳市人民医院临床医学研究中心实验室完成。 材料：取10名健康成人志愿者的外周血。 方法：采集外周血10mL，肝素抗凝，常规淋巴细胞分离液分离单核细胞，然后将单核细胞与粒-巨噬细胞集落刺激因子、白细胞介素4体外培养7d。分3组进行树突状细胞培养，对照组不加α-干扰素；100U／L α-干扰素组于培养第5天加入100U／L的α-干扰素；300U／Lα-干扰素组于培养第5天加入300U／L的α-干扰素。主要观察指标：培养第7天用流式细胞术检测树突状细胞膜表面CD83和MHC—DR表达水平；应用四氮唑盐法测定树突状细胞刺激同种异体T细胞增殖的能力：采用酶联免疫吸附法检测树突状细胞培养上清中白细胞介素12p40＋p70含量。 结果：100，300U／Lα-干扰素组中CD83^＋和MHC—DR^＋树突状细胞的数量、刺激同种异体T细胞增殖的能力及培养上清液中白细胞介素12p40＋p70水平均显著高于对照组（P〈0．01），并以300U／Lα-干扰素组作用更强。 结论：α-干扰素可以有效促进单核细胞源树突状细胞的功能成熟，并呈剂量依赖效应。     

Rapid induction of tumor-specific type 1 T helper cells in metastatic melanoma patients by vaccination with mature,cryopreserved, peptide-loaded monocyte-derived dendritic cells

There is consensus that an optimized cancer vaccine will have to induce not only CD8+ cytotoxic but also CD4+ T helper (Th) cells, particularly interferon (IFN)-gamma-producing, type 1 Th cells. The induction of strong, ex vivo detectable type 1 Th cell responses has not been reported to date. We demonstrate now that the subcutaneous injection of cryopreserved, mature, antigen-loaded, monocyte-derived dendritic cells (DCs) rapidly induces unequivocal Th1 responses (ex vivo detectable IFN-gamma-producing effectors as well as proliferating precursors) both to the control antigen KLH and to major histocompatibility complex (MHC) class II-restricted tumor peptides (melanoma-antigen [Mage]-3.DP4 and Mage-3.DR13) in the majority of 16 evaluable patients with metastatic melanoma. These Th1 cells recognized not only peptides, but also DCs loaded with Mage-3 protein, and in case of Mage-3DP4-specific Th1 cells IFN-gamma was released even after direct recognition of viable, Mage-3-expressing HLA-DP4+ melanoma cells. The capacity of DCs to rapidly induce Th1 cells should be valuable to evaluate whether Th1 cells are instrumental in targeting human cancer and chronic infections.     

Role of CCR8 and other chemokine pathways in the migration of monocyte-derived dendritic cells to lymph nodes

Studying the influence of chemokine receptors (CCRs) on monocyte fate may reveal information about which subpopulations of monocytes convert to dendritic cells (DCs) and the migration pathways that they use. First, we examined whether prominent CCRs on different monocyte subsets, CCR2 or CX3CR1, mediated migration events upstream of the accumulation of monocyte-derived DCs in lymph nodes (LNs). Monocytes were labeled and traced by uptake of latex microspheres in skin. Unexpectedly, neither CCR2 nor CX3CR1 were required. However, absence of CCR2 led to an increased labeling of the minor Gr-1int monocyte population, and the number of latex+ DCs that emigrated to LNs was correspondingly increased. Characterization of Gr-1int monocytes revealed that they selectively expressed CCR7 and CCR8 mRNA in blood. CCR7 and CCR8 pathways were used by monocyte-derived DCs during mobilization from skin to LNs. The role of CCR8 in emigration from tissues also applied to human monocyte-derived cells in a model of transendothelial trafficking. Collectively, the data suggest that Gr-1int monocytes may be most disposed to become a lymphatic-migrating DCs. When these monocyte-derived DCs exit skin to emigrate to LNs, they use not only CCR7 but also CCR8, which was not previously recognized to participate in migration to LNs.     

Bacterial ghosts as novel efficient targeting vehicles for DNA delivery to the human monocyte-derived dendritic cells

Recombinant bacterial ghosts loaded with plasmids were tested as an antigen delivery system and as a potential mediator of maturation for human monocyte-derived dendritic cells (DCs). Bacterial ghosts are cell envelopes derived from Gram-negative bacteria; the intracellular content is released by the controlled expression of plasmid-encoded lysis gene E of PhiX174. All the cell surface structures of the native bacteria, including the outer membrane proteins, adhesins, LPS, lipid A, and peptidoglycans, are preserved. Co-incubation of immature DCs with ghosts resulted in decreased expression of CD1a, CD80, and CD83 molecules, while addition of maturation mix (TNF-alpha, IL-1 beta, IL-6, and PGE2) to the cultures enhanced expression of these molecules. No marked changes were observed in the expression of the CD11c, CD40, and CD86 surface molecules. The exposure of DCs to ghosts in combination with maturation mix resulted in a nonsignificant increase in their ability to activate T cells. DCs co-incubated with bacterial ghosts carrying plasmids encoding GFP in combination with maturation mix exhibited high expression levels of GFP (up to 85%). These results indicate that in addition to their well-established use as vaccines, bacterial ghosts can also be used as carriers of nucleic acid-encoded antigens.     

Induction of leukemic-cell-specific cytotoxic T lymphocytes by autologous monocyte-derived dendritic cells presenting leukemic cell antigens

Leukemic-dendritic cells (leukemic-DCs) have certain limitations, which include difficult generation in 30-40% of patients, and low levels of expression of several key molecules. Therefore, an alternative approach using monocyte-derived DCs pulsed with tumor antigens is required. We investigated the possibility of immunotherapy for AML using leukemic-cell-specific cytotoxic T lymphocytes that were stimulated in vitro by autologous DCs pulsed with tumor antigens. To generate DCs, CD14(+) cells were isolated from peripheral blood mononuclear cells using magnetic-activated cell sorting, and cultured in the presence of GM-CSF and IL-4. On day 6, maturation of DCs was induced by addition of cytokine cocktail (TNF-alpha, IL-1beta, IL-6, and prostaglandin E(2)) for 2 days, and then the mature DCs were pulsed with whole leukemic cell lysates or apoptotic leukemic cells. There were no differences in the phenotypic expressions of mature DCs generated by pulsing with or without leukemic antigens. The mature DCs pulsed with tumor cell lysates or apoptotic leukemic cells showed a higher allostimulatory capacity for allogeneic CD3(+) T cells as compared with mature non-pulsed DCs. Autologous CD3(+) T cells stimulated by the mature pulsed DCs showed more potent cytotoxic activities against autologous leukemic cells than those stimulated by mature non-pulsed DCs. These results suggest that use of DCs pulsed with leukemic cell lysates or apoptotic leukemic cells is a feasible alternative immunotherapeutic approach to overcome the limitations of leukemic-DCs for the treatment of AML patients.