巨细胞病毒感染与细胞凋亡的研究进展

CMV病毒和细胞凋亡的关系相当复杂，有时表现为诱导凋亡作用，有时表现为抗凋亡作用。从病毒的角度看，病毒诱导小胶质细胞、巨噬细胞、造血干细胞、中性粒细胞及淋巴细胞等发生凋亡，可干扰机体抗病毒免疫反应，使病毒不至于被机体免疫系统消除，对于病毒感染的有些宿主细胞，病毒则利用自身抗凋亡作用以利于在感染细胞内完成病毒的复制和生活周期。从宿主的角度出发，机体会调动全身和局部抗病毒免疫反应、宿主细胞会启动凋亡机制来清除病毒。     

树突状细胞递呈抗原的分子机制

树突状细胞(DCS)是重要的抗原递呈细胞,其参与免疫应答的机制十分复杂,首先要形成“三力子复合体”,加上 DCs表达的其他刺激因子和粘附分子等信号的传递才能完成。DCs尚有一些其他功能,如表达IgE受体或作为感觉受体等,具有其他非职业性APC有明显不同     

慢性乙肝患者树突状细胞抗原递呈功能状态的初步研究

目的:研究慢性乙肝患者树突状细胞(DC)抗原递呈功能的改变。方法:从慢乙肝患者和健康人外周血分离单个核细胞,诱导培养出DC,显微镜下观察DC形态并计数,流式细胞仪检测DC表面协同刺激分子(CD80,CD86),MTT法检测DC刺激同种异体淋巴细胞增殖的能力,ELISA法测定培养上清液中IL-12、r-IFN水平。结果:慢乙肝患者外周血单个核细胞诱导培养所获得的DC数量及表面协同刺激分子(CD80,CD86)表达水平、刺激同种异体淋巴细胞增殖能力和细胞因子产生水平均明显低于健康者(P<0.05)。结论:慢乙肝患者DC的数量及抗原递呈、免疫刺激功能状态均处于低下水平。     

囊泡运输相关蛋白VAP-33在小鼠树突状细胞肉瘤细胞株DCS细胞中的表达及其功能

目的 探讨囊泡运输相关蛋白VAP-33在小鼠树突状细胞肉瘤细胞株DCS细胞中的表达及其功能.方法 用1%TritonX-114提取DCS细胞膜蛋白,用已制备的DCS细胞多克隆抗体(pAb)及蛋白A+G琼脂糖进行免疫沉淀,所得样品利用质谱技术进行分析,检测到VAP-33蛋白在DCS细胞中有表达,继而DCS细胞经不同量抗原(150、850、1500μl)刺激24、48、72 h后观察细胞形态及吞噬能力的变化,同时通过间接免疫荧光、共聚焦显微镜、Western blot等方法检测了VAP-33的分布及表达变化.0.5 mol/L胰岛素刺激DCS细胞20 min后,采用Western blot检测DCS细胞全蛋白、胞质蛋白、膜蛋白中VAP-33、葡萄糖转运蛋白4(GLUT-4)的表达变化,共聚焦方法观察胰岛素刺激前后VAP-33和GLUT-4在DCS细胞中的表达及定位变化.实验中均以常规培养的DCS细胞作为对照.结果 VAP-33蛋白主要表达在DCS细胞膜和胞质中;在外来抗原刺激下,随抗原量的增加及作用时间的延长DCS细胞趋向成熟树突状细胞,VAP-33表达量降低,对辣根过氧化物酶的吞噬能力增强.胰岛素刺激后,VAP-33与GLUT-4有共定位.结论 VAP-33在树突状细胞来源的肿瘤细胞中表达,与树突状细胞的抗原加工有关,在葡萄糖的转运中起一定作用.     

增强树突状细胞抗原呈递功能的新进展

随着分子和细胞生物学技术的不断发展，基于树突状细胞(DC)的肿瘤疫苗在恶性肿瘤的临床治疗中已获得越来越广泛的应用。DC是功能最强的专职抗原呈递细胞(APC)，具有独特的刺激T细胞增殖的能力，是机体免疫应答的始动者，一种天然的“免疫佐剂”通过DC可诱导特异性抗肿瘤免疫应答，调动患者的免疫系统清除肿瘤或预防微小残留病灶的     

巨细胞病毒对骨髓基质细胞粘附功能的影响

目的:探讨巨细胞病毒(CMV)对骨髓基质细胞表面粘附分子ICAM-1 和VCAM-1的表达及骨髓基质细胞对正常造血细胞的粘附率的影响。方法:用流式细胞仪检测骨髓基质细胞表达粘附分子ICAM-1和VCAM-1阳性率,用MTT方法检测骨髓基质细胞对正常骨髓造血细胞的粘附率。结果:本实验所用的CMV可以感染骨髓基质成纤维细胞;100TCID₅₀剂量以下CMV对骨髓基质细胞无明显破坏作用;活性CMV 感染骨髓基质细胞早期(18 h)粘附分子ICAM-1表达明显较高,晚期(120 h)ICAM-1表达明显较低;灭活的CMV也能使骨髓基质细胞ICAM-1表达增高,作用与有活性的CMV相似;骨髓基质细胞在CMV感染早期,对造血细胞的粘附率增高。CMV对VCAM-1的表达无明显影响。结论:CMV 感染骨髓基质细胞早期,骨髓基质细胞对造血细胞粘附能力增高,CMV感染晚期骨髓基质细胞对造血细胞粘附能力降低。     

人巨细胞病毒感染诱导宿主细胞凋亡的实验研究

人巨细胞病毒 (ＨＣＭＶ)活动性感染可以通过胎盘垂直传播 ,导致流产、畸形、死胎、新生儿神经系统及其他脏器的损害 ,已成为影响胎儿及新生儿生长发育的常见病原。现已证实 ,许多病毒性疾病 (如病毒性肝炎和艾滋病等 )的发生与凋亡失常有关〔1〕 。但目前有关ＨＣＭＶ与细胞凋亡的关系报道尚少。我们采用流式细胞技术对ＨＣＭＶ感染细胞进行细胞周期和凋亡分析 ,以探讨ＨＣＭＶ对宿主细胞的影响及机理。1　材料和方法1 1　病毒和细胞　ＨＣＭＶＡＤ16 9毒株由湖北省病毒研究所提供 ,按常规方法传代 ,滴定 ,其滴度为 10 3 ＴＣＩＤ5 0 0 .1…     

人巨细胞病毒感染诱导宿主细胞凋亡的实验研究

人巨细胞病毒 (ＨＣＭＶ)活动性感染可以通过胎盘垂直传播 ,导致流产、畸形、死胎、新生儿神经系统及其他脏器的损害 ,已成为影响胎儿及新生儿生长发育的常见病原。现已证实 ,许多病毒性疾病的发生与凋亡失常有关〔1〕。我们采用流式细胞技术对ＨＣＭＶ感染细胞进行细胞周期和凋亡分析 ,以探讨ＨＣＭＶ对宿主细胞的影响及机理。1　材料和方法1 1　病毒和细胞　ＨＣＭＶＡＤ16 9毒株由湖北省病毒研究所提供 ,按常规方法传代 ,滴定 ,其滴度为 10 3ＴＣＩＤ5 0 0 .1ｍｌ;人胚肺成纤维细胞 (ＨＥＬ)由同济医科大学附属同济医院儿科病毒室提供 ,…     

巨细胞病毒感染时免疫功能的变化及其意义

目的　探讨巨细胞病毒感染时免疫变化及其意义。方法　以 2 0名CMV IgM(Cytomegalovirus immunoglobulinM)阳性的病儿为观察对象 ,另设对照组 30例。ELISA法检测柯萨奇B组病毒抗原 (CVB Ag)、IgM抗体 (CVB IgM)、巨细胞病毒IgM抗体 (CMV IgM)、EB病毒IgM抗体 (EBV IgM) ,单克隆抗体标记间接ABC免疫法检测外周血T细胞亚群。结果　CMV感染组的LAK细胞、NK细胞活性均明显降低 (P <0 .0 1)。合并其它感染原的CMV混合感染组CD3含量减少 ,CD8含量增加 (P <0 .0 5 ) ,CD4 CD8数值明显降低 (P <0 .0 1)。病毒混合感染并合并支原体感染组CD3含量减少 (P <0 .0 5 )、CD4 CD8数值、IgA含量明显降低 (P <0 .0 1)。结论　巨细胞病毒感染影响了T细胞亚群的平衡 ,在合并其他感染时天然免疫细胞功能下降和T细胞亚群紊乱更明显     

树突状细胞在人巨细胞病毒感染中的作用

人巨细胞病毒(human cytomegalovirus,HCMV)在人群中感染率极高,通过多种免疫逃避机制,实现在宿主体内的长期潜伏感染。树突状细胞(dendritic cells,DC)是重要的抗原提呈细胞,在诱导和维持特异性免疫应答中发挥重要的作用。人体内的DC根据来源、表型分为两群:髓系DC(myeloid DC,mDC)和浆细胞样DC(plasmacytoid DC,pDC),大量研究证实HCMV介导的多种免疫逃避机制中,部分是通过影响DC功能实现的。HCMV不仅可以感染mDC,影响mDC表型、迁移、分泌细胞因子、激活T细胞功能,而且还可以抑制pDC分泌干扰素水平及激活T细胞能力,并且激活过度的B细胞反应,导致机体抗病毒细胞免疫反应的抑制和体液免疫紊乱,实现病毒长期潜伏感染。本文主要讨论HCMV是如何改变两种DC亚型的功能以实现免疫逃避目的。     

Rat cytomegalovirus infection depletes MHC II in bone marrow derived dendritic cells

While cytomegalovirus (CMV) infects and replicates in a multitude of cell types, the ability of the virus to replicate in antigen presenting cells (APCs) is believed to play a critical role in the viral dissemination and latency. CMV infection of APCs and manipulation of their function are important areas of investigation. CMV down regulation of MHC II is reportedly mediated by the HCMV proteins US2, US3, UL83, UL111a (vIL10) or through the induction of cellular IL10. In this study, we demonstrate that rat CMV (RCMV) significantly reduces MHC II expression neither by mechanisms that do not involve orthologues of the known HCMV genes nor by an increase in cellular IL10. Rat bone marrow derived dendritic cells (BMDC) were highly susceptible to infection with RCMV and a recombinant RCMV expressing eGFP. RCMV infection of BMDCs depleted both surface and intracellular MHC II to nearly undetectable levels as well as reduced surface expression of MHC I. The effect on MHC II only occurred in the infected GFP positive cells and is mediated by an immediate early or early viral gene product. Furthermore, treatment of uninfected immature DCs with virus-free conditioned supernatants from infected cells failed to down regulate MHC II. RCMV depletion of MHC II was sensitive to treatment with lysosomal inhibitors but not proteasomal inhibitors suggesting that the mechanism of RCMV-mediated down regulation of MHC II occurs through endocytic degradation. Since RCMV does not encode homologues of US2, US3, UL83 or UL111a, these data indicate a novel mechanism for RCMV depletion of MHC II.     

Reprogramming dendritic cells through the immunological synapse: A two-way street

Dendritic cells (DCs) bridge innate and adaptive immunity. Their main function is to present antigens to prime T cells and initiate and shape adaptive responses. Antigen presentation takes place through intimate contacts between the two cells, termed immune synapses (IS). During the formation of IS, information travels towards the T-cell side to induce and tune its activation; but it also travels in reverse via engagement of membrane receptors and within extracellular vesicles transferred to the DC. Such reverse information transfer and its consequences on DC fate have been largely neglected. Here, we review the events and effects of IS-mediated antigen presentation on DCs. In addition, we discuss novel technological advancements that enable monitoring DCs interactions with T lymphocytes, the main effects of DCs undergoing productive IS (postsynaptic DCs, or psDCs), and how reverse information transfer could be harnessed to modulate immune responses for therapeutic intervention.     

Induction of immunosuppressive functions of dendritic cells in vivo by CD4+CD25+ regulatory T cells: role of B7-H3 expression and antigen presentation

Suppressive functions of CD4+CD25+ regulatory T cells (Treg) are mainly studied by their interaction with conventional T cells. However, there is evidence that Treg also interact with antigen-presenting cells (APC), leading to suppression of APC function in in vitro coculture systems. Studying the in vivo distribution of Treg after injection, we found that Treg are located in direct proximity to dendritic cells (DC) and affect their functional maturation status. After contact to Treg, DC up-regulate the inhibitory B7-H3 molecule and display reduced numbers of MHC-peptide complexes, leading to impaired T cell stimulatory function. When Treg-exposed DC were used to immunize animals against antigens, the DC failed to produce a robust immune response as compared to control DC. Thus, these data indicate that Treg are able to inhibit DC activation and produce an inhibitory phenotype of DC. Accordingly, Treg may recruit DC for the amplification of immunosuppression by restraining their maturation in vivo and inducing an immunosuppressive phenotype of DC.     

IL-2基因修饰对树突状细胞的生物学特征和功能的影响

目的:观察白细胞介素2(IL-2)基因修饰对树突状细胞(DC)的生物学特征和功能的影响,探讨用IL-2基因修饰DC,增强DC介导特异性抗肿瘤免疫的机制。方法:IL－2基因修饰小鼠骨髓来源的DC后,用扫描电镜观察其表面形态的变化,FACS分析IL-2基因修饰对DC表面免疫分子表达的影响,RT-PCR方法检测DC中 IFN-γ mRNA表达。用3H-TdR掺入法检测IL-2基因修饰后,DC对同种异体T淋巴细胞的刺激作用和对肿瘤抗原的特异性提呈功能。结果:经IL-2基因修饰后,DC表面的伪足增多、变长;其表面与抗原提呈相关的免疫分子Ia、B7-1、B7-2和CD40的表达明显上调;il-2基因修饰的DC(DC-IL-2)中表达IFN-γ mRNA;CD-IL-2不但对同种异体T淋巴细胞有较强的促增殖作用,而且对肿瘤抗原的特异性提呈功能亦明显增强。结论:IL-2基因修饰DC,能促进DC的发育,上调DC表面与抗原提呈相关的免疫分子,增强了DC的生物活性。     

Characteristics of antigen-independent and antigen-dependent interaction of dendritic cells with CD4+ T cells

Dendritic cells (DC) are the main antigen-presenting cells for the initiation of primary T cell-mediated immune responses. In the first stage of activation, T cells bind to DC in an antigen-independent manner. We studied the adhesion characteristics of human CD4⁺ T cells to DC generated from CD34⁺ hematopoietic progenitors following 12 to 13 days of culture in the presence of granulo-cyte/macrophage colony-stimulating factor and tumor necrosis factor-α. A majority of these cells had the morphology, phenotype and functions of DC. CD4⁺ T/DC adhesion was measured by means of fluorescence microscopy and flow cytometry. Four independent receptor/ligand pathways, LFA-1/ICAM, ICAM/LFA-1, CD2/LFA-3 and CD28/CD80, were involved in the transient adhesion of DC to CD4⁺ T cells in antigen-independent and specific alloantigen-dependent situations, as shown by blocking experiments using monoclonal antibodies. The antibodies also blocked a primary mixed lymphocyte reaction (MLR) in which DC were used as stimulatory cells. Adhesion of alloreactive CD4⁺ T cells to antigen-presenting DC was stronger than that of resting CD4⁺ T cells, while peak adhesion occurred after 5 and 20 min, respectively. The LFA-1 ligands involved in adhesion of resting CD4 T cells to DC and alloreactive CD4⁺ T cells to specific DC differed in part, since ICAM-3 on resting T cells and ICAM-1 on alloreactive T lymphocytes preferentially bound LFA-1. Studies of interactions between DC and phorbol ester-activated T cells expressing the CD40 ligand revealed a fifth independent adhesion pathway, CD40/CD40 ligand. CD4-mediated regulation of CD4⁺ T/DC adhesion was suggested by the observation that preincubation of CD4⁺ T cells and DC individually with anti-CD4 antibodies inhibited adhesion. In addition, antibodies specific for HLA class II molecules inhibited adhesion when used to pretreat DC but not alloactivated CD4⁺ T cells.     

CD137 ligand reverse signaling has multiple functions in human dendritic cells during an adaptive immune response

T cell activation via dendritic cells (DC) is an important step in the adaptive immune response, which requires DC maturation, migration to lymph nodes and presentation of antigen to T cells. CD137 receptor expressed on activated T cells is a potent costimulatory molecule. Here, we investigated the functions of CD137 ligand (CD137L) in human monocyte-derived DC during an immune response. Cross-linking of CD137L on DC leads to cell maturation in an autocrine fashion, mostly via release of TNF-alpha. Reverse signaling of CD137L also mediates migration of DC via up-regulation of the CCR7 chemokine receptor, demonstrated by an in vivo MIP-3beta-dependent SCID mouse migration model. Finally, CD137L-activated DC induce differentiation of human T cells into potent Th1 effectors. Cocultivation of autologous T cells and CD137L-activated DC in an antigen-specific reaction leads to T cell proliferation and the release of IL-12p70 and IFN-gamma. These findings deliver new insights into the multiple effects of reverse signaling of CD137L in human DC during the initiation of an adaptive immune response, including the key features of DC maturation, migration and, ultimately, antigen-specific T cell differentiation.     

Targeting dendritic cells to advance cross-presentation and vaccination outcomes

Dendritic cells (DCs) are a complex network of specialised antigen-presenting cells that are critical initiators of adaptive immunity. Targeting antigen directly to DCs in situ is a vaccination strategy that selectively delivers antigen to receptors expressed by DC subtypes. This approach exploits specific DC subset functions of antigen uptake and presentation. Here, we review DC-targeted vaccination strategies that are designed to elicit effective cross-presentation for CD8⁺ T cell immunity. In particular, we focus on approaches that exploit receptors highly expressed by mouse and human cDCs equipped with superior cross-presentation capacity. These receptors include DEC205, Clec9A and XCR1. Targeting DC receptors Clec12A, Clec4A4 and mannose receptor is also reviewed. Outcomes of DC-targeted vaccination in mouse models through to human clinical trials is discussed. This is a promising new vaccination approach capable of directly targeting the cross-presentation pathway for prevention and treatment of tumours and infectious diseases.     

Pathways utilized by dendritic cells for binding, uptake, processing and presentation of antigens derived from HIV-1

The outcome following HIV infection depends on the nature and durability of the HIV-specific T cell response induced initially. The activation of protective T cell responses depends upon dendritic cells (DC), antigen-presenting cells which have the capacity to process and present viral antigens. DC pulsed with aldrithiol-2-inactivated HIV and delivered in vivo were reported to induce immune responses and promote virologic control in chronically HIV-1-infected subjects. To gain an understanding of this phenomenon, we characterized the steps involved in the presentation of antigens derived from aldrithiol-2-treated vs. infectious HIV-1 by DC. Antigen presentation, on both MHC class I and II, was independent of DC-specific ICAM-3-grabbing integrin, DEC-205 and macrophage mannose receptor, C-type lectins expressed by the DC. Inhibitor studies showed that presentation on MHC class I was dependent on viral fusion in a CD4/coreceptor-dependent manner, both at the cell surface and within endosomes, and access to the classical endosomal processing pathway. MHC class II presentation of HIV-associated antigens was dependent on active endocytosis, probably receptor-mediated, and subsequent degradation of virions in acidified endosomes in the DC. Our study brings forth new facts regarding the binding, uptake, and processing of chemically inactivated virions leading to efficient antigen presentation and should aid in the design of more effective HIV vaccines.     

Targeting antigen to bone marrow stromal cell‐2 expressed by conventional and plasmacytoid dendritic cells elicits efficient antigen presentation

Bone marrow stromal cell‐2 (BST‐2) has major roles in viral tethering and modulation of interferon production. Here we investigate BST‐2 as a receptor for the delivery of antigen to dendritic cells (DCs). We show that BST‐2 is expressed by a panel of mouse and human DC subsets, particularly under inflammatory conditions. The outcome of delivering antigen to BST‐2 expressed by steady state and activated plasmacytoid DC (pDC) or conventional CD8⁺ and CD8^? DCs was determined. T‐cell responses were measured for both MHC class I (MHCI) and MHC class II (MHCII) antigen presentation pathways in vitro. Delivering antigen via BST‐2 was compared with that via receptors DEC205 or Siglec‐H. We show that despite a higher antigen load and faster receptor internalisation, when antigen is delivered to steady state or activated pDC via BST‐2, BST‐2‐targeted activated conventional DCs present antigen more efficiently. Relative to DEC205, BST‐2 was inferior in its capacity to deliver antigen to the MHCI cross‐presentation pathway. In contrast, BST‐2 was superior to Siglec‐H at initiating either MHCI or MHCII antigen presentation. In summary, BST‐2 is a useful receptor to target with antigen, given its broad expression pattern and ability to access both MHCI and MHCII presentation pathways with relative efficiency.     

Murine dendritic cells pulsed in vitro with tumor antigen induce tumor resistance in vivo

The aim of this work is to induce tumor resistance to a B cell lymphoma in BALB/c mice using elements of the immune system. It has indeed been shown by us and by others that antigen-presenting cells (APC) like dendritic cells can induce efficient immune responses and can even substitute for Freund's adjuvant. Here we show that mice immunized with syngeneic dendritic cells pulsed in vitro with tumor antigen (BCL1 idiotype expressed by lymphoma cells) are protected against a subsequent tumor inoculation. The in vivo resistance can be correlated with the induction of a humoral response specific for the idiotype expressed by the tumor. No such protection can be achieved when B cells are used as APC. These data show that effector cells in tumor-bearing animals can be recruited and activated using dendritic cells, providing long-lasting immune surveillance.