雷公藤内酯醇对浆细胞样树突状细胞功能及成熟的影响

目的 通过体外试验研究雷公藤内酯醇(triptolide)对浆细胞样树突状细胞(plasmacytoid dendritic cell,pDC)功能及成熟的影响,为进一步阐明雷公藤内酯醇的免疫学活性提供依据.方法 从健康志愿者外周血分离单个核细胞,流式细胞仪分选pDC,加入0、5、10、30 μg/L的雷公藤内酯醇共孵育,24 h后收集上清液,ELISA检测pDC分泌的IFN-α、IL-6、TNF-α量,5 d后收集细胞,流式细胞仪检测树突状细胞表型CD11c、CD80、CD86阳性率,光镜观察DC的形态,扫描电镜观察DC的超微结构.结果 雷公藤内酯醇显著降低pDC分泌的IFN-α、IL-6、TNF-α,并呈雷公藤内酯醇浓度依赖性(P＜0.05);雷公藤内酯醇可抑制pDC向DC的分化和成熟,并呈雷公藤内酯醇浓度依赖性(P＜0.05).结论 雷公藤内酯醇能够降低pDC的功能,并抑制其向DC的分化和成熟.

Abstract：

Objective To explore the mechanism of immunomodulatory activity of triptolide on healthy volunteers peripheral blood mononuclear cells (PBMC)-derived plasmacytoid dendritic cells (pDCs). Methods Healthy volunteers-derived pDCs were sorted by flow cytometry, then incubated with triptolide (0, 5, 10, 30 μg/L). After 24 hours, we detected the concentration of IFN-α, IL-6, TNF-α using ELISA. After 5 days, the cultrural cells were collected and analyzed by flow cytometry, light microscope and electron microscope scanning. Results Triptolide-treated pDCs secreted lower level of IFN-α,IL-6 ,TNF-α, triptolide could inhibit pDCs differentiation to DCs which displayed more immature morphology and immunophenotypes than untreated-pDCs. Conclusion Triptolide could decrease the immune function of pDCs, inhibit differentiation and maturation of pDCs.     

母亲不同HBV感染状态所生新生儿树突状细胞亚群及功能研究

目的 探讨新生儿mDC、pDC频数及表面分子表达,以及母亲不同HBV感染状态对新生儿树突状细胞生物学特性的影响.方法 采集HBsAg阳性/HBeAg阳性HBV感染母亲、HBsAg阳性/HBeAg阴性HBV感染母亲以及HBV感染标志物阴性母亲所生新生儿脐带血、健康成人外周血,采用流式细胞仪检测mDC的频数及其CD86的表达、pDC的频数及其CD80、CD83、CD86表达、并采用FlowJo软件进行分析,比较各组间上述指标的差异.结果 分别采集HBsAg阳性/HBeAg阳性、HBsAg阳性/HBeAg阴性和HBV感染标志物阴性母亲所生新生儿脐带血14、12和13例,健康成人外周血7例.新生儿mDC频数(0.29±0.16)及其CD86阳性率(10.72±10.01)显著低于成年人(分别是0.81±0.17和32.13±7.46),(t=-7.86,P=0.00和t=-5.36,P=0.00)；新生儿pDC频数(0.15±0.07)以及pDC表面CD86/CD83阳性率(31.61±12.81,42.66±20.83)显著低于成年人(0.30±0.07;74.96±9.78;82.00±6.94),(t=-5.43,P =0.00;t=-8.49,P=0.00;t=-4.90,P=0.00).结论 新生儿脐带血mDC、pDC频数以及表面功能分子表达低于成人外周血,HBeAg可能降低生新生儿mDC表面CD86的表达.     

慢性乙型肝炎干扰素治疗前后树突状细胞的变化及其与疗效相关性研究

目的 探讨慢乙肝干扰素α(IFNα)治疗前后髓样树突状细胞(mDC)和浆样树突状细胞(pDC)的变化及其与病毒学、生化学指标的关系.方法 采集30例HBeAg阳性慢乙肝患者IFNα治疗前和治疗12周时的外周血,用流式细胞仪检测mDC和pDC的频数及CD86表达水平.根据IFNα治疗后生化学指标和病毒学指标变化,把患者分为ALT复常组、ALT未复常组及病毒学应答组、病毒学无应答组,分析IFNα治疗前后不同组DC变化.结果 (1)IFNα治疗12周时ALT复常组pDC频率(0.25％±0.14％)较基线(0.18％±0.09％)升高(P =0.023),ALT未复常组mDC频率(0.58％±0.34％)及表面CD86阳性率(61.80％±22.52％)则较基线(分别为0.88％±0.51％,79.92％±25.94％)下降(P =0.025;P=0.036).(2)IFNα治疗12周时病毒学应答组pDC表面CD86阳性率(46.86±12.22％)较基线(29.42±15.16％)升高(P =0.002),病毒学无应答组的mDC频率(0.51％±0.22％)及表面CD86阳性率(59.63％±22.94％)则较基线(分别为0.94％±0.58％,80.11％±29.34％)下降(P =0.006;P =0.049).结论 慢乙肝干扰素α治疗后,生化和病毒应答与pDC频率和功能增强相关,而mDC频率及功能的降低与IFN治疗后无生化和病毒应答相关.     

慢性HIV感染者抗逆转录病毒治疗过程中树突状细胞亚群的变化特点

目的 了解慢性人免疫缺陷病毒(HIV)感染者抗逆转录病毒治疗(ART)过程中树突状细胞(DC)亚群的变化特点.方法 选取ART治疗的慢性HIV感染者17例,分别于治疗0,4,8,12,24,48,60周采集静脉血,同时选取健康者、长期不进展者(LTNPs)各15例为对照.常规进行CD4+/CD8+T细胞计数和病毒载量测定;用流式细胞术测定DC亚群,ELISA测定血浆IFN-α水平;采用SPSS16.0软件分析数据特点.结果 (1) ART治疗前HIV感染者髓样树突状细胞(mDC)百分比及绝对计数明显低于健康组和LTNP组(P＜0.001).ART治疗60周后,HIV感染者mDC明显增加,与健康组、LTNP组之间差异无统计学意义.(2) ART治疗过程中浆细胞样树突状细胞(pDC)数量和血浆IFN-α水平保持相对稳定,且接近健康组、LTNP组水平.(3) ART治疗前DC亚群细胞计数与CD4+T细胞计数正相关.ART治疗12、24、60周,mDC细胞计数与CD4+T细胞计数正相关,与病毒载量负相关.ART治疗8周mDC细胞计数增加值与治疗60周CD4+T细胞计数增加值正相关,与病毒载量下降值负相关.结论 HIV感染者mDC细胞数量明显减少,ART治疗后明显上升,与CD4+T细胞计数正相关,提示mDC在控制HIV感染方面可能具有重要作用.治疗早期mDC细胞数量可能是ART治疗后免疫重建的早期预测指标.     

黄芪多糖对浆细胞样树突状细胞功能及成熟的影响

目的:通过体外实验研究黄芪多糖(Astragalus polysaccharide,APS)对浆细胞样树突状细胞(Plasmacytoid dendritic cell,pDC)功能及成熟的影响,为进一步阐明黄芪多糖的免疫学活性提供依据。方法:从健康志愿者外周血分离单个核细胞,流式细胞仪分选pDC,加入0、50、100、200mg/L的黄芪多糖共孵育,24小时后收集上清液,应用酶联免疫吸附试验检测pDC分泌的IFN-α、TNF-α、IL-6量,5天后收集细胞,应用流式细胞仪检测树突状细胞(Dendritic cell,DC)表型CD11c、CD80、CD86阳性率、光镜观察DC的形态、扫描电镜观察DC的超微结构。结果:黄芪多糖显著提高pDC分泌的IFN-α、TNF-α、IL-6量,并且呈黄芪多糖浓度依赖性(P0.05);黄芪多糖可促进pDC向DC的分化和成熟,并呈黄芪多糖浓度依赖性(P0.05)。结论:黄芪多糖能够增强pDC的功能,并促进其向DC的分化和成熟。     

An unbalanced PD-L1/CD86 ratio in CD14^＋＋CD16^＋ monocytes is correlated with HCV viremia during chronic HCV infection

Circulating monocyte subsets with distinct functions play important roles in hepatitis C virus （HCV） infection. However, the mechanisms have not been well studied. In this study, we analyzed the distributions and phenotypic characteristics of three circulating monocyte subsets--CD14^＋＋CD16^-, CD14^＋＋CD16^＋ and CD14^＋＋mCD16^——in chronic HCV-infected patients, HCV spontaneous resolvers and healthy controls, and we evaluated the possible link between HCV viremia and disease progression. Our results indicated that the frequency of the CD 14^＋＋CD 16^＋ monocyte subset was decreased, and negatively correlated with HCV RNA and core antigen levels during chronic HCV infection. PD-L1 expression and the PD-L1/CD86 ratio in CD14^＋＋CD16^＋ monocytes were higher during chronic HCV infection than in spontaneous HCV resolvers and healthy controls. The PD-L1/CD86 ratio positively correlated with HCV viral load and core antigen levels. Finally, PD-L1 was significantly increased, while cytokine secretions were dramatically decreased upon Toll-like receptor （TLR） ligand binding and HCV JFH-lstimulation. These findings indicates the compromised immune status of the CD14^＋＋CD16^＋ monocytes during chronic HCV infection and provides new insights into the specific role of the CD14^＋＋CD16^＋ monocytes and their significance in chronic HCV infection.     

呼吸道合胞病毒感染细支气管炎患儿外周血树突状细胞的数量及其与临床症状的相关性

目的 检测呼吸道合胞病毒(RSV)感染细支气管炎患儿外周血髓样树突状细胞(mDC)及浆细胞样树突状细胞(pDC)的数量,并分析它们与疾病严重程度之间的关系.方法 采用PCR方法检测鼻咽部分泌物的呼吸道合胞病毒.通过流式细胞仪检测71例RSV感染(轻度、中度和重度)细支气管炎患儿及48例正常婴幼儿外周血mDC及pDC的数量.结果 RSV感染细支气管炎患儿外周血mDC数量明显高于正常婴幼儿(P＜0.01),pDC数量明显低于正常婴幼儿(P＜0.01).重度RSV感染细支气管炎患儿入院时外周血mDC、pDC数量均明显低于轻度组,差异有统计学意义(P＜0.05).结论 RSV感染细支气管炎患儿早期外周血mDC明显升高、pDC降低.mDC数量越高,提示其病情越轻;pDC数量越低,提示喘息越重.     

子痫前期患者外周血树突状细胞与T细胞亚群的变化

目的 观察子痫前期患者外周血中的树突状细胞亚群与T细胞亚群相关细胞因子的变化.方法 实验组为子痫前期患者32例,对照组为未孕妇女20例,正常妊娠妇女20例.采集研究对象外周血细胞,流式细胞术检测全血细胞中髓系树突状细胞(mDC)和浆细胞样树突状细胞(pDC);分离外周血单个核细胞,经胞内细胞染色检测Th1、Th2、Th17细胞数量及Th1/Th2比值.结果 子痫前期组mDC百分比(0.33±0.12)％和mDC/pDC比值(2.96±1.65)均高于正常妊娠组,二组数据有明显差异(P＜0.05);pDC百分比(0.16±0.13)％较正常妊娠组(0.21 ±0.12)％有所下降,二组差异有统计学意义(P＜0.05).子痫前期组IFN-γ、IL-4和IL-17的百分比分别为(18.67 ±1.96)％、(1.88±0.51)％和(1.36±0.59)％,与正常妊娠组相比均有显著性差异(P＜0.01).子痫前期组mDC/pDC比率和Th1/Th2之间呈显著正相关(r=0.637,P＜0.01);Th17表达率与pDC表达率之间呈负相关(r=-0.670,P＜0.05),与mDC/pDC比率之间呈显著正相关(r=0.772,P＜0.01).结论 子痫前期患者外周血中树突状细胞亚群和Th1、Th2、Th17型细胞因子异常表达,可能是患者发生免疫失衡的重要原因.     

IL-24基因修饰的树突状细胞促进CIK细胞对肺癌细胞的杀伤作用

Objective To study the antitumor effect and mechanism of co-cultured cytokine-induced killer(CIK) cells and autologous DC modified with IL-24 gene on A549 cells in vitro. Methods DC and CIK cells were prepared routinely from human peripheral blood mononuclear cells(PBMC). Recombinant adenovirus vector pAdEasy-1-pTrack-CMV-IL-24 was extracted from DH5α, it was lineared with Pac I and transfected into A293 cells, and then the IL-24 recombined adenovirus(Ad-IL-24) was obtained. Ad-IL-24 was used to infect DC. The cells obtained were named DC-IL-24. RT-PCR and ELISA were used to evaluate the expression of IL-24 gene in transfected DC. The phenotypes change of DC were identified by flow cytometry analysis, the concen-tration of IL-12 and TNF-α in supernatant of DC were determined by EIJSA. The ability of CIK producing per-forin was measured by homolysis method. FCM was used to determine the cytotoxicity of cocultured CIK cells and autologous DC modified with IL-24 gene to A549 cells. Results We obtained the high titre of Ad-IL-24.IL-24 gene was transfered into DC successfully via Ad-IL-24. The green fluorescence was observed on DC by fluorescence microscope. The expression rate of CD80, CD83, HI.A-DR, CD40, CXCR4 on DC-IL-24 was sig-nificantly increased compared with that of the control group. DC-IL-24 produced markedly higher levels of IL-12 and TNF-α as compared with DC. DC-IL-24 can enhance the ability of CIK cells producing perforin. On com-parison with non-transfected DC co-cultured with CIK cells, transfected DC co-cultured with CIK cells had a sig-nificantly higher lytic activity against A549 cells. Conclusion IL-24 gene modification can enhance the anti-tu-moral immunity of DC. The mechanism of which might be related to the increased secretion of IL-12 and TNF-α, up-regulation expression of co-stimulatory molecules and MHC Ⅱ class molecules on DC, promoting the acti-vation and maturation of DC, and then enhancing CIK cells to generate specific anti-tumoral immunity.     

慢性乙型肝炎患者树突状细胞形态、表型和功能的变化

目的 观察慢性乙型肝炎病毒（hepatitis B virus,HBV)感染者树突状细胞（dendritic cells,DC)形态、表型和功能的改变。方法 从13例慢性乙肝患者和11例健康人外周血中分离和培养DC,观察DC的形态。用流式细胞仪检测DC的表面标志,HLA－DR、CD1a,CD80和CD86的表达。用^3H-TdR掺入法,检测DC诱导混合性淋巴细胞反应（mixed leukocytes reaction,MLR)的能力。结果 正常人的DC较慢乙肝患者的DC在形态上更为典型。前者的DC不规则,细胞可表达较多的HLA－DR、CD80和CD86分子（P＜0．05）,诱导MLR的能力也较强（P＜0．05）。结论 慢乙肝患者外周血DC处于不完全成熟状态,其免疫刺激能力较低。     

Increased PD‐L1 expression and PD‐L1/CD86 ratio on dendritic cells were associated with impaired dendritic cells function in HCV infection

Impaired hepatitis C virus (HCV)‐specific T cell immunity was associated with the persistence of HCV infection. Dysfunction of dentritic cells (DCs) was believed to be involved in T cell exhaustion, but the mechanisms were rarely understood. In this study, surface costimulatory marker (CD83, CD86, and CD40), coinhibitory marker (PD‐L1) expression and allostimulatory capacity of plasmacytoid DCs (pDCs) and myeloid DCs (mDCs) were evaluated in HCV‐infected patients. Results showed that the expression of both costimulatory and coinhibitory markers was increased in HCV‐infected patients compared with healthy controls. PD‐L1/CD86 ratio was increased and positively correlated with PD‐L1 expression on DCs in HCV‐infected patients. Allostimulatory capacity of DCs was impaired and inversely correlated with PD‐L1 expression and PD‐L1/CD86 ratio. These findings suggested that the effect of inhibitory marker PD‐L1 overwhelmed the effect of costimulatory markers and down regulated DC‐T activation in HCV‐infected patients. The results will be helpful to understand the mechanism of dysfunction of DCs in HCV infection and shed light on the DC‐based immunotherapeutic strategy. J. Med. Virol. 82: 1152–1159, 2010. © 2010 Wiley‐Liss, Inc.     

The basal activation state of DC subsets correlates with anti-HCV treatment outcome in HCV/HIV co-infected patients

In this work we evaluated plasmacytoid (pDC) and myeloid dendritic (mDC) cells activation before and during anti-HCV treatment in HCV+/HIV+ individuals. HCV+/HIV+ patients received Peg-IFN-α2b subcutaneously for 28 days, followed by oral weight-based ribavirin. DCs activation was evaluated by flow cytometry. Baseline pDC CD80 and CD86 expression was correlated with HIV, but not with HCV viral load. A transient decrease of HIV RNA was found not associated with DC activation. When patients were grouped according to early/sustained virological response (EVR/SVR) to anti-HCV treatment, baseline pDC CD80 and CD86 expression was higher in non-EVR and non-SVR compared to EVR and SVR. Moreover, in responder patients CD80 and CD86 were upregulated by IFN-α. Our data suggest a correlation between DCs activation and response to therapy. These findings could be helpful to better understand the mediators of IFN-α action in HCV+/HIV+ patients and to explore possible exploitation of this knowledge to improve therapeutic response.     

Decreased interferon-alpha production in HIV-infected patients correlates with numerical and functional deficiencies in circulating type 2 dendritic cell precursors.

Peripheral blood mononuclear cells from patients with human immunodeficiency virus (HIV) infection exhibit a progressively marked decrease in the production of virus-induced interferon (IFN)-alpha, a finding that correlates with and is highly predictive of disease progression and opportunistic infections. The major IFN-alpha producing population has recently been defined as the precursor to type 2 dendritic cells (pDC2) or plasmacytoid DC (pDC). Using four-color flow cytometry, we have enumerated the pDC2 vs non-IFN-alpha producing myeloid DC1 in peripheral blood from HIV-infected patients and healthy controls and related these values to CD4 cell numbers, viral load, and functional activity. The patients had reductions in the numbers of both pDC2 (lin-/HLA-DR+/CD123(bright)) and DC1 (lin1-/HLA-DR+/CD123(dim)/CD11c+), both at an absolute level and as a percentage of cells. The decreases were most evident in patients with decreased CD4 levels. Viral load correlated with the functional frequency of the IFN producing cells but not with absolute pDC2 levels. Using intracellular flow cytometric analysis for IFN-alpha, the patients were demonstrated to have fewer pDC2, as well as a lower percentage of responding cells among those remaining. We conclude that deficient production of IFN-alpha by pDC2 from HIV-infected patients results from both selective loss of these cells and their qualitative dysfunction. Given the central role of DC, and in particular, DC2, in linking innate and adaptive immune responses, these qualitative and quantitative changes in pDC2 are likely to be key contributors to HIV pathogenesis.     

Preferential depletion of CD2(low) plasmacytoid dendritic cells in HIV-infected subjects

Plasmacytoid dendritic cells (pDCs) are decreased in number and are functionally impaired in HIV act reasons for pDCs depletion are still unknown. It was recently reported that pDCs can be divided into two functionally distinct populations based on their CD2 expression level. To determine how the CD2(high) and CD2(low) populations are affected by HIV infection, we analyzed their frequencies in the peripheral blood of HIV-infected subjects and healthy controls. We found that the CD2(low) pDC subset was preferentially depleted in infected individuals. The frequency of CD2(low) pDCs correlated with the CD4(+) T-cell count but not with the plasma viral load. This finding furthers our understanding of the causes and consequences of pDC depletion during HIV infection.     

Hepatitis B virus surface antigen impairs myeloid dendritic cell function: a possible immune escape mechanism of hepatitis B virus

Chronic hepatitis B virus (HBV) infection is the result of an inadequate immune response towards the virus. Myeloid dendritic cells (mDC) of patients with chronic HBV are impaired in their maturation and function, resulting in more tolerogenic rather than immunogenic responses, which may contribute to viral persistence. The mechanism responsible for altered mDC function remains unclear. The HBV-infected patients display large amounts of HBV particles and viral proteins in their circulation, especially the surface antigen HBsAg, which allows multiple interactions between the virus, its viral proteins and DC. To assess whether HBV directly influences mDC function, the effects of HBV and HBsAg on human mDC maturation and function were investigated in vitro. As already described for internalization of HBV by DC, the present study shows that peripheral blood-derived mDC of healthy controls also actively take up HBsAg in a time-dependent manner. Cytokine-induced maturation in the presence of HBV or HBsAg resulted in a significantly more tolerogenic mDC phenotype as demonstrated by a diminished up-regulation of costimulatory molecules and a decreased T-cell stimulatory capacity, as assessed by T-cell proliferation and interferon-gamma production. In addition, the presence of HBV significantly reduced interleukin-12 production by mDC. These results show that both HBV particles and purified HBsAg have an immune modulatory capacity and may directly contribute to the dysfunction of mDC in patients with chronic HBV. The direct immune regulatory effect of HBV and circulating HBsAg particles on the function of DC can be considered as part of the mechanism by which HBV escapes immunity.     

Aberrant Phenotype and Function of Dendritic Cells in Adult B Lineage Acute Lymphoblastic Leukemia

ABSTRACT

Dendritic cells (DCs) play a major role in regulating immune responses, but the aberrant phenotype and function of defective DCs in adult acute lymphoblastic leukemia (ALL) remain unclear. Here, B lineage ALL (B-ALL) patients were divided into groups according to different standards. By course of disease: newly diagnosed (ND), complete remission (CR), consolidation (CONS). By stratification: high risk (HR), standard risk (SR). By minimal residual disease (MRD): MRD positive(MRD+), MRD negative (MRD?). The proportion of plasmacytoid DC(pDC) and myeloid DC(mDC) were compared within these standards. The costimulatory molecule levels of pDC, mDC in ND and CR were measured and the function of peripheral blood monocyte-derived DC(MoDC)s were examined. We found proportions of pDC and mDC in ND were both lower compared to control group and gradually increased after CR. In HR and MRD+, the proportions were also lower compared to SR and MRD? at CR stage, respectively; but there were no difference between these comparisons when newly diagnosed. In ND, both CD80, CD86 levels in pDC, mDC were higher while the levels in activated MoDCs were lower when compared to control and CR group, respectively. The dextran uptake of MoDCs, T cell proliferation promoting ability, IL-12, BAFF, INF-α levels in supernatant and their mRNA relative expression in activated MoDCs in ND were also lower than those in control and CR group. So, DCs in B-ALL display suppressed status in phenotype and function,which would be gradually restored after effective chemotherapy. pDC and mDC could respond to patient condition, DCs proportion may be useful for monitoring disease progression.     

Effect of SIVmac infection on plasmacytoid and CD1c+ myeloid dendritic cells in cynomolgus macaques

Dendritic cells (DCs) are known to be essential for the induction and regulation of immune responses. Non-human primates are essential in biomedical research and contribute to our understanding of the involvement of DCs in human infectious diseases. However, no direct single-platform method for quantifying DC precursors has yet been optimized in macaques to give accurate absolute blood counts of these rare-event cell populations in the blood. We adapted a rapid whole-blood assay for the absolute quantification of DCs in cynomolgous macaques by four-colour flow cytometry, using a single-platform assay compatible with human blood. Cynomolgus macaque plasmacytoid DCs (pDCs) and CD1c⁺ myeloid DCs (CD1c⁺ mDCs) were quantified in the blood of 34 healthy macaques and the results obtained were compared with those for blood samples from 11 healthy humans. In addition, circulating absolute numbers of pDCs were quantified in cynomolgus macaques chronically infected with SIVmac. During infection, pDC counts decreased whereas circulating CD1c⁺ mDC counts increased. Information regarding absolute pDC and mDC counts in non-human primates may improve our understanding of the role of these cells in SIV/HIV infection and in other infectious diseases.