树突状细胞和巨噬细胞提呈日本血吸虫抗原作用

目的　探讨树突状细胞和巨噬细胞提呈日本血吸虫抗原的作用。方法　利用日本血吸虫可溶性虫卵抗原 (SEA)致敏树突状细胞和巨噬细胞 ,将致敏的细胞免疫小鼠 3次 ,检测血清抗体水平。结果　抗原致敏树突状细胞免疫组小鼠血清平均抗体水平 ( 0 10 6 8± 0 0 16 )高于抗原致敏巨噬细胞免疫组小鼠 ( 0 0 880± 0 0 17) ,且高于未处理树突状细胞免疫组小鼠 ( 0 0 82 5± 0 0 12 )。结论　树突状细胞提呈日本血吸虫抗原的作用强于巨噬细胞 ,是日本血吸虫感染中的主要抗原提呈细胞     

树突状细胞在免疫应答中的作用及其临床意义

树突状细胞（dendritic cell，DC）是指呈典型树突状或伪足状突起、膜表面高表达MHCⅡ类分子、能移行至次级淋巴器官和刺激初始型T细胞（naive T cells）增殖活化，并具有一些相对特异性表面标志的专职抗原提呈细胞（professional antigen presenting cell, pAPC）^[1]。DC是目前已知的功能最强抗原递呈     

宫颈癌细胞冻融抗原负载树突状细胞激发细胞毒性T淋巴细胞杀伤宫颈癌细胞的体外研究

目的研究HeLa、SiHa两种宫颈癌细胞株冻融抗原负载的树突状细胞(dendritic cells,DC)诱导细胞毒性T淋巴细胞(CTL)体外抗原特异性杀伤官颈癌细胞的能力.方法利用免疫磁珠分离法(MACS)分离纯化脐血CD34 细胞并在体外诱导分化为DC,用反复冻融法分别从两种宫颈癌细胞株(HeLa利SiHa)中提取可溶性冻融抗原并负载DC.流式细胞学检测负载两种抗原后DC表面分子的表达,ELISA法检测DC上清中IL-12的表达,MLR测定DC刺激T细胞增殖的能力,MTT法检测DC经抗原负载后激活的CTL分别对HeLa和SiHa细胞的杀伤作用.结果与未经抗原负载的DC相比,经HeLa和SiHa冻融抗原负载的DC能更好的表达各种DC分化相关抗原(CD1a、CD83、CD80、HLA-DR以及CD54),刺激T淋巴细胞增殖和IL-12分泌的能力也显著加强(P＜0.05).与未经抗原负载相比,两种宫颈癌细胞冻融抗原负载DC后激发的CTL在体外对官颈癌细胞的杀伤能力也明显加强.此外,HeLa细胞抗原负载DC诱导的CTL在体外对HeLa细胞的杀伤率为72.2%,显著高于它对SiHa细胞的杀伤率(22.3%);而SiHa细胞抗原负载DC诱导的CTL对SiHa细胞的杀伤率为69.5%,也明显高于它对HeLa细胞的杀伤率(28.1%),具有显著的抗原特异性.结论经宫颈癌细胞冻融抗原负载DC激活的CTL在体外具有更强的增殖能力,并具有抗原特异性杀伤宫颈癌细胞的作用.     

碘硒对大鼠腹腔巨噬细胞抗原提呈作用的影响

目的 通过观察碘硒对大鼠腹腔巨噬细胞抗原提呈作用的影响,初步探讨两者对自身免疫性甲状腺疾病发病的影响及其免疫学机制.方法 选用雌性Lewis大鼠20只,根据随机抽样的原则,分为4组:低硒适碘组(LSeN1),低硒高碘组(LSeH1),适硒适碘组(NSeN1),适硒高碘组(NSeH1).各组用人工合成的低硒低碘饲料和饮用含不同浓度硒或碘的去离子水(用K103和Na,SeO3配置)喂养3个月.制备各组大鼠腹腔巨噬细胞及卵白蛋白(OVA)致敏的T细胞,将两者共同培养,进行抗原提呈实验,采用ELISA方法测定上清中IL-2水平;采用RT-PCR方法检测各组脾细胞共刺激分子CD86 mRNA的表达水平.结果 NSeH1组培养上清中IL-2水平为(43.22±3.27)pg/ml明显高于NSeN1组IL-2水平(25.74±2.45)pg/ml.LSeN1组IL-2水平为(15.79±2.13)pg/ml明显低于NSeN1组.NSeH1组大鼠脾细胞CD86mRNA表达水平(CD86/βp-actin:0.52±0.10)明显高于NSeN1组(CDB6/13.actin:0.35±0.04).结论 高碘使巨噬细胞的抗原提呈作用呈现高于正常状态,成为诱发甲状腺自身免疫发生的一个重要的因素.低硒可使大鼠腹腔巨噬细胞对OVA抗原识别和提呈作用减弱,维持免疫自稳机制失调,可能也是构成诱发自身免疫病的一个因素.     

结核杆菌抗原对人γδT细胞体外增殖影响

目的 观察结核杆菌抗原(Mtb-Ag)对人γδT细胞体外增殖的影响,以解决医学研究中γδT细胞来源不足问题。方法 应用酸性改良罗氏培养基分离培养结核杆菌及丙酮酸钠液体培养基进行扩增,通过85℃水浴灭活,超声波细胞破碎制备Mtb-Ag。在体外以不同的剂量刺激人外周血单个核细胞(PBMC),采用四甲基偶氮噻唑蓝(MTT)法检测增殖程度,应用流式细胞仪检测γδT细胞增殖的百分率。结果 在一定范围内随着刺激剂量的增加PBMC增殖亦增加。流式细胞仪分析增殖结果显示,7μg/ml刺激组γδT细胞的增殖率最高(约为49.08%)。但当刺激剂量增加至9μg/ml时,γδT细胞增殖比率开始降低(约为32.62%),而αβT细胞增殖比率增加明显(约为36.76%)。结论 Mtb-Ag在合适刺激剂量时,可对人γδT细胞发挥优势扩增;当剂量过大时则对αβT细胞发挥优势扩增。     

调节性T细胞及其在感染性疾病中的作用

有关调节性T(Treg)细胞的研究是近年来免疫学领域的热点问题之一。大量研究表明,Treg细胞在免疫耐受、抗肿瘤、抗感染等重要免疫功能方面起中心作用。本文对Treg细胞的免疫功能、调节机制及其在感染性疾病中的作用进行综述。     

T细胞耗竭在结核分枝杆菌感染中的作用

机体感染结核分枝杆菌(MTB)后,由T细胞介导的细胞免疫在控制MTB感染中起关键作用。然而,在慢性MTB或病毒感染或肿瘤疾病中,T细胞持续大量接触抗原后,会出现T细胞耗竭的状态。T细胞耗竭表现为:T细胞增殖能力及细胞因子分泌水平呈渐进性下降,一些抑制性受体水平逐渐升高[如程序性死亡受体-1(PD-1)]。近期的文献研究表明,T细胞耗竭与机体对MTB感染的无效控制存在关联,在MTB的持续或潜伏感染的发展中可能起重要作用。因此,进一步探索T细胞耗竭在MTB持续感染中的作用及其机制,可能有助于找到潜在的治疗靶点。     

6种中药多糖对诱导树突状细胞成熟及刺激T细胞增殖作用的比较

[目的]比较6种中药多糖对树突状细胞诱导成熟及刺激T细胞增殖的能力。[方法]通过密度梯度离心法分离人外周血单核细胞,采用细胞因子体外培养7d后,分别加入6种中药多糖,观察细胞的形态变化及用免疫细胞化学法检测其表面标志的表达。通过混合淋巴细胞反应检测细胞提呈抗原的能力。[结果]经6种中药多糖诱导的DC分子表达有明显变化:MHCII+、CD86+、CD83+(P﹤0.05,P﹤0.01),而经RPMI1640培养的DC分子表达与培养前无明显变化。经6种中药多糖诱导的DC均能促进效应T细胞的增殖能力(P﹤0.01)。其中枸杞多糖对上述功能的作用最为明显(P﹤0.05)。[结论]上述6种中药多糖均可诱导DC分化、成熟和刺激T细胞增殖,枸杞多糖与其余5种多糖比较,对上述功能的作用最为明显。     

辅助性T细胞17及调节性T细胞调节初治肺结核患者免疫功能的作用

目的 探讨外周血辅助性T细胞17(Th17)和调节性T细胞(Treg)在调节初治肺结核患者免疫功能中的作用.方法 选取2012年1月至2013年6月在绍兴市第六人民医院就诊的初治肺结核患者169例,根据有无空洞分成空洞组(73例)和无空洞组(96例);根据空洞所占肺野将空洞组的73例患者分成1～2个肺野组、3～4个肺野组和5～6个肺野组.另外选取同期健康体格检查者51名作为健康对照组.流式细胞仪动态检测治疗前、治疗2个月时患者外周血CD4+T细胞上Th17和Treg细胞水平,并比较各组间Th17细胞和Treg细胞水平的差异.结果 肺结核组治疗前及治疗2个月后外周血Th17细胞表达水平分别为(1.63±0.38)％和(2.57±0.82)％,均低于健康对照组的(3.85±1.13)％,差异有统计学意义(t=4.18、2.69,P＜0.01);外周血Treg细胞表达水平分别为(7.53±1.77)％和(5.39±1.22)％,显著高于健康对照组的(3.99±0.84)％,差异有统计学意义(t=6.27、3.34,P＜0.01).空洞组Th17的细胞的表达水平为(1.49±0.25)％,低于无空洞组的(1.87±0.46)％;Treg细胞水平为(7.94±1.85)％,高于无空洞型组的(6.88±1.14)％,组间差异均有统计学意义(t=2.35、2.59,P＜0.05).1～2个肺野组、3～4个肺野组和5～6个肺野组间Th17细胞水平随着空洞数量增加而降低,但Treg细胞逐渐升高,组间差异有统计学意义(F=3.10、3.82,P＜ 0.05).结论 外周血Th17和Treg细胞水平与肺结核的严重程度密切相关,对结核免疫反应起重要调节作用.     

树突细胞与慢性乙型肝炎相关性研究进展

树突细胞作为目前已知的功能最强大的抗原提呈细胞,自发现以来始终受到人们的关注,并被认为将成为特异性免疫治疗的有效工具。近年来,以树突细胞为基础的慢性乙型肝炎免疫治疗研究也已开展。本文主要介绍树突细胞在慢性HBV感染和慢性乙型肝炎的发病机制、治疗方面的研究进展以及应用前景。     

Effective CD8 T cell priming and tumor protection by enterotoxin B subunit-conjugated peptides targeted to dendritic cells

In our previous studies we have shown that bacterial enterotoxin B subunits are effective vehicles to deliver antigen into the MHC class I processing route. Here we have used the non-toxic Escherichia coli heat labile enterotoxin B subunit (EtxB) conjugated to OVA peptide (EtxB–peptide) to address the impact on induction of specific CD8⁺ T cells in vivo. Although incubation of DCs with these EtxB–peptide conjugates as such did not induce DC maturation in vitro MHC class I antigen presentation was much more efficient as compared to peptide alone. Antigen presentation was further enhanced upon DC maturation with the TLR-4 ligand LPS. Injection of matured DCs incubated with EtxB–peptide conjugates lead to strong induction of OVA-specific CD8⁺ T lymphocytes and fully prevented the outgrowth of lethal B16 melanoma in wild type mice. Our data demonstrate that bacterial non-toxic B subunit–peptide conjugates are potent vaccine vehicles for induction of protective CD8⁺ T cell responses.     

Targeting dendritic cells in humanized mice receiving adoptive T cells via monoclonal antibodies fused to Flu epitopes

The targeting of vaccine antigens to antigen presenting cells (APC), such as dendritic cells (DCs), is a promising strategy for boosting vaccine immunogenicity and, in turn, protective and/or therapeutic efficacy. However, in vivo systems are needed to evaluate the potential of this approach for testing human vaccines. To this end, we examined human CD8⁺ T-cell expansion to novel DC-targeting vaccines in vitro and in vivo in humanized mice. Vaccines incorporating the influenza matrix protein-1 (FluM1) antigen fused to human specific antibodies targeting different DC receptors, including DEC-205, DCIR, Dectin-1, and CD40, elicited human CD8⁺ T-cell responses, as defined by the magnitude of specific CD8⁺ T-cells to the targeted antigen. In vitro we observed differences in response to the different vaccines, particularly between the weakly immunogenic DEC-205-targeted and more strongly immunogenic CD40-targeted vaccines, consistent with previous studies. However, in humanized mice adoptively transferred (AT) with mature human T cells (HM-T), vaccines that performed weakly in vitro (i.e., DEC-205, DCIR, and Dectin-1) gave stronger responses in vivo, some resembling those of the strongly immunogenic CD40-targeted vaccine. These results demonstrate the utility of the humanized mouse model as a platform for studies of human vaccines.     

In vitro stimulation of human influenza-specific CD8 T cells by dendritic cells pulsed with an influenza virus-like particle (VLP) vaccine

The purpose of this in vitro study was to determine if a virus-like particle (VLP) influenza vaccine stimulated human CD8⁺ T cells in a dendritic cell (DC): T cell co-culture system. VLP-pulsed DCs were co-cultured with autologous CD8⁺ T cells from five donors. Functional CD8⁺ T cells were detected via cell surface and intracellular cytokine staining. T cells from four of the five donors showed ≥2-fold increase over background in the % activated CD8⁺ cells. These results indicate that the influenza VLP vaccine can stimulate CD8⁺ T cells via DC antigen presentation, likely through the MHC-I pathway, thus broadening the immunological response induced by this promising influenza vaccine.     

Infection of mouse bone marrow-derived dendritic cells by live attenuated Japanese encephalitis virus induces cells maturation and triggers T cells activation

An attenuated Japanese encephalitis virus (JEV) strain SA14-14-2, generated from the wild strain SA14, is an effective live vaccine against JEV infection. It has led to a significant decrease in JEV infection around the world. Although it is highly effective, the mechanism for its robust immunity was not well investigated. In this study, the interaction of SA14-14-2 with bone marrow-derived dendritic cells (bmDCs) was investigated. Our results showed that the infection of bmDCs with SA14-14-2 resulted in viral replication and upregulation of bmDC maturation marker molecules (CD40, CD80, CD83 and MHC I). SA14-14-2 infection also stimulated the production of interferon-α (IFN-α), monocyte chemoattractant protein-1 (MCP-1/CCL2), tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) of bmDC. Both MLR and ELISPOT assay showed an enhanced allostimulatory capacity of SA14-14-2-infected bmDCs. Furthermore, the SA14-14-2-infected bmDCs impaired the expansion of Foxp3+ regulatory T (Treg) cells with immunosuppressive potential, suggesting that SA14-14-2 infection induced antiviral immunity rather than immunosuppression. Taken together, our results indicated that SA14-14-2 infection caused bmDC maturation, changed the expression profiles of several cytokines, and triggered T cell activation. This offered an insight in the immunologic mechanisms associated with the high efficiency of the SA14-14-2 vaccine.     

乙型肝炎病毒感染者体内树突细胞及树突细胞疫苗研究现状

树突细胞(DC)是目前已知功能最强的专职抗原提呈细胞(APC),为当前抗肿瘤、抗病毒等疾病免疫治疗研究的热点.近年来,对DC疫苗的探索和研究为多种疾病的免疫治疗提供了新的治疗前景.此文就DC在慢性乙型肝炎中的研究现状进行综述.     

DNA疫苗抗原提呈机制研究进展

DNA疫苗是基因治疗研究中衍生并发展起来的一个新的研究领域。虽然该疫苗的免疫效应在动物实验及部分临床工作中得到验证,但其免疫效果仍不够理想,免疫效价低于现有疫苗。深入研究DNA疫苗免疫后抗原提呈机制成为近年研究热点内容,此文就近年来这方面的研究进展综述如下。     

Immunization with adenovirus LIGHT-engineered dendritic cells induces potent T cell responses and therapeutic immunity in HBV transgenic mice

LIGHT, a TNF superfamily member (TNFSF14), is a type II transmembrane protein expressed on activated T cells and immature dendritic cells (DCs). However, the expression of LIGHT on mature DCs is down-regulated. Recent studies demonstrated that LIGHT provides potent costimulatory activity for T cells, enhancing proliferation and the production of Th1 cytokines independently of the B7-CD28 pathway. Here, we evaluated the effectiveness of peptide-pulsed DC-mediated antiviral immunity in HBV transgenic mice and the immunoadjuvant effect of LIGHT. The bone marrow-derived DCs were modified in vitro with an adenovirus (Ad) vector expressing mouse LIGHT (Ad-LIGHT), the expression of costimulatory molecules was up-regulated and the secretion of cytokines IL-12 and IFN-γ increased. LIGHT-modified DCs enhanced allostimulation for T cells in mixed lymphocyte reaction (MLR). HBV peptide-pulsed DCs elicited HBV specific CD8+ T cell response and reduced the level of HBsAg and HBV DNA in sera of HBV transgenic mice. Importantly, LIGHT-modified DCs could induce stronger antiviral immunity. These results support the concept that genetic modification of DCs with a recombinant LIGHT adenovirus vector may be a useful strategy for antiviral immunotherapy.     

Considerations in the design of vaccines that induce CD8 T cell mediated immunity

The protective capacity of many currently used vaccines is based on induction of neutralizing antibodies. Many pathogens, however, have adapted themselves in different ways to escape antibody-based immune protection. In particular, for those infections against which conventional neutralizing antibody-based vaccinations appear challenging, CD8 T-cells are considered to be promising candidates for vaccine targeting. The design of vaccines that induce robust and long-lasting protective CD8 T-cell responses however imposes new challenges, as many factors such as kinetics and efficiency of antigen-processing and presentation by antigen presenting cells, T-cell repertoire and cytokine environment during T cell priming contribute to the specificity and functionality of CD8 T-cell responses. In the following, we review the most prominent aspects that underlie CD8 T-cell induction and discuss how this knowledge may help to improve the design of efficient CD8 T-cell inducing vaccines.