【作者】 王明永；

【导师】 陈政良；

【作者基本信息】 南方医科大学 ， 免疫学， 2008， 博士

【摘要】 甘露聚糖结合凝集素（mannan-binding lectin,MBL）是由肝细胞产生的血浆蛋白,为C型凝集素超家族中胶凝素（collectins）家族成员,具有补体C1q的“郁金香”样结构。成熟MBL肽链自N端至C端依次有4个结构域:富含Cys的N端区、胶原样区（collagen-like region,CLR）、颈区和C端糖识别域（carbohydrate-recognition domain,CRD）。人的完整MBL分子是同质三肽链结构单位的寡聚体,多至六聚体。只有高寡聚体MBL分子才具有生物学活性。MBL可选择性识别多种病原体的糖结构,通过激活补体凝集素途径而发挥溶破和间接调理功能,并能与吞噬细胞胶凝素受体（即C1qR）结合而启动调理吞噬,还可介导MBL依赖的细胞介导的细胞毒作用;而且,MBL也是一个重要的粘膜表面防御分子。多种著名杂志如Science、Jounal of Immunology等刊文称MBL为“天然免疫系统中的关键分子”,凝集素途径是“天然免疫的关键成份”。十余年来,随着对天然免疫研究的深入,特别是初步研究提示天然免疫对获得性免疫应答还有一定的影响,人们对天然免疫系统的传统认识发生了根本性的转变。现代免疫学认为,天然免疫系统不仅仅能够通过诱导必要的急性反应来清除病原微生物,而且还能够通过配体识别和分泌细胞因子等方式触发适当的获得性免疫应答,并认为天然免疫系统对外来入侵病原体的初次应答状态,决定了后续获得性免疫应答的性质。天然免疫模式识别受体（patten recognitionreceptor,PRR）通过分析外来入侵病原体的危险程度,启动对宿主的保护程序。单核细胞（monocyte,Mo）、巨噬细胞（macrophage,Mφ）及树突状细胞（dendriticcell,DC）作为某些应答的直接介导者,能够感知外来危险信号并在其刺激下分泌一些炎性细胞因子,细胞因子环境进一步影响后续的获得性免疫应答。正常情况下,机体天然免疫分子能够清除自身凋亡的细胞或坏死的组织进行组织修复,维持机体代谢平衡,因此在没有危险信号情况下,天然免疫分子能够通过鉴别自身凋亡细胞,避免诱导一些能够引起自身免疫病的获得性免疫应答,扮演获得性免疫应答调节者的角色。现已发现,胶凝素家族的其他成员如肺表面活性物质蛋白A和D（surfactantProtein A、surfactant protein D,SP-A、SP-D）及补体C1q具有多种免疫调节功能,如SP-A能够抑制DC的成熟和分化,通过与CD14分子相互作用触发细胞应答,并能够抑制脂多糖（lipopolysaccharide,LPS）诱导的单核巨噬细胞分泌TNF-α等细胞因子,还可以直接与Toll样受体-4（Toll-like receptor 4,TLR4）、MD-2分子相互作用来调节炎性细胞反应;SP-D能够加强抗原提呈;C1q则可以直接调节DC的成熟与分化并能够抑制LPS诱导的细胞因子产生。MBL作为天然免疫的关键分子,已被报道能够调节多种细胞因子的表达,但是到目前为止,对MBL在获得性免疫应答中的作用几无所知。根据MBL与SP-A、SP-D的同源性、都能与胶凝素受体结合及已发现Mφ表达特异性胶凝素受体并介导多种免疫调节功能的情况,推测MBL必定参与获得性免疫应答。针对此设想,本课题组陈月博士已经作了大量相关研究,并得出结论:MBL对DC的分化成熟有一定的影响。联想到DC作为机体内最重要的抗原提呈细胞（antigen presentingcell,APC）在启动获得性免疫应答起着关键作用,提示MBL极有可能参与调节获得性免疫应答。因此,在现有研究基础之上,本课题继续探讨MBL在调节获得性免疫应答中所扮演的角色。我们首先联合应用配体和抗体亲和层析柱子,从人新鲜冰冻血浆中提取高纯度的具有生物学活性的天然MBL,并原核表达了可溶性TLR4胞外段蛋白（sTLR4）,然后对MBL负向调控LPS诱导的DC功能效应做了初步的机理分析;又选择人B细胞系Raji细胞作为研究对象,分析MBL与其相互作用的特性及其对细胞功能的影响;又原核表达了模式抗原破伤风毒素C蛋白（TTC）,探讨了MBL对PHA、PMA或PMA/ionomycin活化的人T细胞系Jurkat细胞、外周血单个核细胞（peripheral blood monoeuclear cell,PBMC）、外周血新鲜分离的CD3⁺T淋巴细胞的增殖及分泌细胞因子的影响,并分析了MBL对DC提呈TTC抗原能力的影响。本研究分4部分,兹分述如下。一、联合应用配体和单克隆抗体亲和层析纯化人血浆天然MBL蛋白MBL是天然免疫系统的一个重要分子,许多有关凝集素途径及其相关疾病的理论研究、临床疾病病人血清中MBL水平测定和抗MBL单克隆抗体（monoclonal antibody,mAb）制备等实际工作中,都需要高度纯化的MBL制剂。然而,血浆中MBL浓度很低,正常人在40～3500μg/L之间,平均约900μg/L,血浆成份又非常复杂,获得高纯度MBL比较困难。为获取高纯度天然MBL,本研究优化了传统提纯MBL的方法。制备了抗人MBL-CRD mAb-Sepharose 4B亲和层析柱,联合采用配体甘露聚糖-Sepharose4B亲和层析柱,将预处理人血浆3次上柱亲和层析分离,分别用EDTA、D-甘露糖、Gly-HCl（pH 2.4）溶液洗脱结合蛋白。以SDS-PAGE、Western blot和ELISA等技术鉴定纯化产物。试验结果显示,所获纯化MBL为28 KD和32 KD肽链构成的功能性寡聚体,其纯度较高,可与配体甘露聚糖结合并有效凝集酵母菌,还能与U937细胞胶凝素受体结合。本文报道的血浆预处理技术然后采用甘露聚糖-Sepharose 4B亲和层析柱和抗人MBL-CRD mAb-Sepharose 4B亲和层析柱联合层析技术,可以获得高纯度、高活性的MBL蛋白,为后续研究奠定了坚实的物质基础。二、MBL调节DC功能的机制初步研究TLR是天然免疫系统中重要的模式识别受体,不仅能够介导对病原微生物及其产物的识别,而且能够参与获得性免疫应答,被视为联系天然免疫和获得性免疫的桥梁。在所有的TLR家族中,TLR4在未成熟DC（immature dendriticcell,imDC）、Mo及Mφ广泛表达,而且在识别LPS及其所介导的信号途径中起着关键作用。本课题组陈月博士研究发现,MBL对LPS诱导的imDC的成熟、细胞因子产生具有负向调控作用,但其作用机理并不明了。本研究继续陈月博士的工作,以TLR4为切入点,探讨MBL对LPS/TLR4介导的信号途径的影响,进一步分析MBL对DC功能调节的机理。实验中首先原核表达了可溶性TLR4胞外段蛋白（sTLR4）,以蛋白质印迹试验（Western blot）鉴定其特异性;接着用Western blot和ELISA两种方法分析MBL是否与sTLR4相互作用,并用流式细胞术（FCM）分析MBL与imDC的结合情况,竞争性抑制试验分析MBL是否结合imDC表面的TLR4受体及影响后续LPS/TLR4介导的信号途径。实验结果显示:（1） MBL可以直接以浓度依赖方式与可溶性TLR4蛋白胞外段结合;（2） MBL能够以Ca²⁺依赖方式结合imDC,这种结合能被sTLR4蛋白及抗TLR4 mAb所抑制;（3） MBL能够竞争性抑制LPS结合imDC;（4） MBL能够抑制LPS诱导的核转录因子-κB（nuclear factor-κB,NF-κB）活性。资料表明,MBL可通过结合imDC表面TLR4受体来调节LPS/TLR4介导的信号通路,提示MBL和TLR4之间存在联系,在免疫调节中扮演重要的角色。三、MBL对Raji细胞作用特性的研究作为关键的天然免疫分子之一,MBL还能以钙离子依赖、糖特异的方式结合自身的Mo、DC,影响细胞因子的表达,提示MBL在免疫调节中发挥重要作用,并极有可能参与获得性免疫应答。前文报道了MBL对DC的调节作用,而T、B淋巴细胞是获得性免疫应答的介导者,但迄今未见MBL直接与淋巴细胞相互作用而参与调节获得性免疫应答的报道。因此,研究MBL与淋巴细胞之间的相互作用具有重要意义。本实验中,我们选择人B淋巴系Raji细胞作为对象,研究MBL是否与其结合及其结合特性,并对这种相互作用的后果进行了简单分析,为探索MBL直接参与B细胞介导的获得性免疫应答提供初步的依据。实验中首先采用细胞ELISA方法分析MBL能否与Raji细胞结合,并采用FCM着重研究其与Raji细胞结合特性。结果显示,MBL以浓度依赖方式结合Raji细胞;MBL与Raji细胞的结合是Ca²⁺依赖的,且能被甘露糖、葡萄糖、N-乙酰葡糖胺所抑制;C1q或抗C1qR mAb能部分抑制MBL与Raji细胞结合;重组人MBL-CRD蛋白或MBL-CLR蛋白均能抑制MBL与Raji细胞结合,两者联合应用则可完全阻断这种结合。资料表明,B淋巴细胞系Raji细胞表达Ca²⁺依赖性、糖敏感的MBL受体（或结合蛋白）,包括对CLR特异和CRD特异的两种受体,前者为MBL和C1q的共同受体。进一步的功能研究显示,高浓度MBL（10～50 mg/L）对Raji细胞的生长具有明显抑制作用,且呈剂量依赖关系。提示MBL作为一种重要的模式识别分子,不仅发挥天然免疫功能,而且可能在调节B细胞介导的获得性免疫应答中起一定作用。四、MBL调节T淋巴细胞功能的初步研究近年发现,与MBL同为C型凝集素超家族中胶凝素家族成员的SP-A和SP-D可抑制丝裂原激活的T细胞增殖,并下调其分泌IL-2;SP-A对T细胞增殖的抑制作用发生在丝裂原处理T细胞24 h之后;SP-A和SP-D也能调节DC的功能,如SP-A抑制DC的分化成熟,SP-D增强DC的提呈抗原能力,促进抗感染获得性免疫应答。然而,有关MBL免疫调节作用的报道极少,尤其未见其对T淋巴细胞直接调节作用的报道。根据胶凝素家族各成员之间的结构和功能同源性,我们推测MBL很可能对T细胞的增殖及功能也有影响。因此,本实验探讨了在体外MBL对T细胞的调节的可能性。破伤风毒素（tetanus toxoid,TT）能激活CD4⁺T淋巴细胞,因而被作为一种模式抗原,在CD4⁺ T细胞介导免疫应答的研究中广为应用。然而,传统的破伤风类毒素（tetanus toxoid,TToid）是经甲醛脱毒而成,而破伤风杆菌可形成芽孢且毒性高,生产过程中有一定危险;甲醛处理TT易造成污染,TToid还可能发生毒性逆转。因此,TToid的研究应用受到一定限制。而TT的C片段（TTC）在动物实验中能有效诱导产生中和抗体并可耐受外毒素攻击,也可激活CD4⁺T细胞,是理想的破伤风亚单位候选疫苗,且具较好的安全性。因此,本研究选择TTC作为模式抗原,研究MBL调节T细胞介导的获得性免疫应答。本实验中,首先原核表达了模式抗原TTC蛋白,并对其抗原性、免疫原性进行分析,得到高度纯化、免疫原性良好的TTC蛋白,为后续研究提供了物质基础。然后,采用密度梯度离心法自成年健康自愿者外周血分离单个核细胞（peripheral blood mononuclear cells,PBMC）,并采用尼龙毛柱分离CD3⁺T淋巴细胞。用非特异性刺激物PHA、PMA或PMA/ionomycin等分别活化人T淋巴细胞株Jurkat细胞、PBMC及CD3⁺T淋巴细胞,FCM测MBL对PHA、PMA活化的Jurkat细胞及CD3⁺T淋巴细胞结合能力,WST-1和ELISA检测MBL对PHA、PMA或PMA/ionomycin活化的Jurkat细胞、CD3⁺T淋巴细胞及PBMC的增殖及分泌细胞因子IL-2或IFN-γ的影响;然后体外诱导DC和TTC特异性CD4⁺T淋巴细胞,WST-1法分析MBL对APC刺激同种异体T淋巴细胞及提呈模式抗原TTC能力的影响。结果表明,MBL能够结合PHA、PMA活化Jurkat细胞及CD3⁺T淋巴细胞,MBL能够以浓度依赖的方式抑制PHA、PMA或PMA/ionomycin活化的上述各组细胞增殖及其分泌细胞因子IL-2或IFN-γ,同时MBL下调APC刺激同种异体T淋巴细胞增殖并减弱DC对TTC抗原的提呈能力,提示MBL能够负向调节T细胞介导的获得性免疫应答。上述研究结果显示,MBL可直接与DC表面的TLR4相互作用,调节后续的NF-κB信号通路,提示MBL可能通过与TLR4相互作用来调节免疫应答。MBL还能直接调控T淋巴细胞、B淋巴细胞的功能,并负向调节控APC的抗原提呈能力,提示MBL可能通过T淋巴细胞、B淋巴细胞和抗原提呈细胞三个环节来调控获得性免疫应答。目前,国内外尚未见与之类似的报道,尽管其详细的作用机制有待于进一步的实验研究加以阐明。这些发现为进一步探索MBL参与获得性免疫应答的作用及其机制提供了新的切入点,后续MBL免疫调节作用的特性分析、分子机制及信号传导途径的阐明,将进一步揭示MBL这种重要天然免疫分子的生物学意义。更多还原

【Abstract】 Mannan-binding lectin（MBL）,a member of the collectin family in the C-type lectin superfamily,is a multimeric protein containing collagen-like sequences.It is synthesized and excreted into blood by hepatocytes.It has the overall ’bundle-of-tulips’ structure first described for C1q.The polypeptide of MBL is composed of four domains,a cysteine-rich N-terminal domain,a collagen-like region （CLR） that contains Gly-X-Y repeats（where X is any amino acid,and Y is often hydroxyproline or hydroxylysine）,a neck region and a C-terminal carbohydrate-recognition domain（CRD）.The MBL polymer is composed of homogenous subunits,which can be as many as six,but only the high polymeric forms of MBL have the biologic activity and can fix complements.MBL can selectively recognize sugars presented on pathogens through the pattern-recognition function.On binding to pathogens,MBL may activate the complement cascade via lectin pathway,leading to the killing of microbes by exerting lysis effect and indirect opsonizing function.At the same time,MBL has a direct opsonization when binding to lectin receptors on the phagocytic cells,and it can also induce the MBL-dependent cell-mediated cytotoxic reaction.MBL also plays an important role in surfactant defenses of mucous membrane.Furtherore,MBL and MBL-mediated lectin pathway have beeen considered as the key molecule and key element in innate immunity, respectively,by such important magazines as Science,Jounnal of Immunology.The basic understanding of the immune system has undergone a substantial paradigm shift in the past decade,as an awareness of the power and influence of the innate immune system has emerged.It is now being recognized that the innate immune system is not only responsible for the induction of acute responses necessary for elimination of pathogens but is also able to activate appropriate adaptive immune responses via ligand interaction and secretion of cytokines and chemokines,and that the nature of the first response to invasion（i.e.,the innate immune response） has significant influence in determining the nature of the subsequent adaptive immune response.Both soluble and membrane bound pattern recognition molecules of the innate immune system assess the level of danger of a particular intrusion and initiate a protection program for the host.When activated by pathogens,monocytes, macrophages and dendritic cells often initiate the synthesis of proinflammatory cytokines by mediating some of these changes.The cytokine environment then influences the subsequent specific immune responses.Conversely,soluble and cellular components of the innate system remove dead or degenerated cells or tissue debris and participate in systems of tissue repair.Thus,in the absence of perceived danger,the innate system seems to be able to remove dying cells and avoid the induction of an adaptive immune response,which would lead to autoimmunity or further tissue damage.Thus,in the absence of perceived danger,the innate system appears to be able to discriminate removal of dying cells and avoid the induction of an adaptive immune response,which would result in autoimmunity and/or further tissue damage,and play roles in regulation of adaptive immune responses.It has been reported that other members of the soluble defensive collagen family, such as surfactant protein A（SP-A）,surfactant protein D（SP-D）,complement C1q, have some roles in immune regulation by interaction with various cells.For instance, SP-A inhibits the differentiation and maturation of DCs and modulates cellular responses induced by LPS through interacting with CD14;SP-A suppresses TNF-αsecretion from alveolarΜΦstimulated with LPS,and SP-A-deficient mice produce significantly more TNF-αthan wild-type mice after intratracheal LPS-administration. SP-A directly interacts with TLR4 and MD-2 and regulates inflammatory cellular response.SP-D enhances bacterial antigen presentation by bone marrow-derived dendritic cells.Complement C1q regulates LPS-induced cytokine production in bone marrow-derived dendritic cells,and can induce maturation of human dendritic cells. As a key soluble pattern recognition molecule in the innate immune system,MBL has been reported to influence the cytokines network after stimulation by various microorganisms.However,so far,little is known concerning the role of MBL in the regulation of adaptive response.The hypothesis that MBL must participate in acquired immune response is based on the following three facts:the structure homology among MBL,SP-A and SP-D,their binding to collectin-receptor,and the findings thatΜΦexpress specific collectin-receptor and mediate lots of immune regulatory processes.A conclusion that MBL may play certain roles in regulating dendritic cell differentiation and maturation has been drawn from Dr.Chen’s research work,which suggests that MBL most likely be involved in the regulation of immune responses,considering the key roles of antigen-presenting cells in initiating of adaptive immune response.In the present research,we continued our previous studies to explore the role of MBL in regulating adaptive immune responses.Firstly,we established a convenient procedure for the purification of MBL through the ligand-and monoclonal antibodyaffinity chromatography,and obtained natural MBL with high purity and bioactivity from freshly frozen human plasma.Then we obtained soluble extracellular-TLR4 protein（sTLR4） by prokaryotic expression system,and investigated possible mechanisms underlying MBL’s effects on DC maturation and cytokine expression. Thirdly,we investigated the interaction of MBL with human B cell line Raji cells. Fourthly,we analyzed the role of MBL in the proliferation and cytokine secretion in PHA-,PMA- or PMA/ionomycin-activated human T-cell line Jurkat cells,peripheral blood mononuclear cells（PBMCs）,and the freshly isolated peripheral blood CD3⁺ T lymphocytes.Finally,we investigated role of MBL in APC stimulators of allogeneic T cells in mixed lymphocyte reaction（MLR） and in its presentation of tetanus toxin protein C（TTC） antigen to specific T cell lines.ⅠPurification of MBL from Human Plasma through the Ligand- and Monoclonal Antibody-Affinity ChromatographyMBL is a central component of the innate immune system.MBL preparation with high purity is required in many practical works such as theory researches concerned with the lectin pathway and its related diseases,measurement of MBL level in patients’ serum and preparation of anti-MBL monoclonal antibody.However, the concentration of plasma MBL is quite low（mean 900μg/L,40～3 500μg/L in normal subjects）,and the components of the plasma is very complex,therefore,it is quite difficult to obtain MBL with high purity.In order to optimize the method for purifying mannan-binding lectin（MBL） from human plasma,we firstly prepared anti-human MBL-CRD monoclonal antibody-Sepharose 4B affinity chromatography column,then three steps of affinity chromatography on mannan-Sepharose 4B and anti-human MBL-CRD monoclonal antibody-Sepharose 4B were in turn employed and eluateed with EDTA,D-mannose and Gly-HCl（pH 2.4） solution independently to gather the target protein,which was identified by SDS-PAGE,Western blot and ELISA.Our dates showed that the high-purified MBL was found as a functional multimer composed of 28KD and 32KD peptide chains in SDS-PAGE and Western blot,and it was not only able to bind to mannan and to agglutinate baker yeast but also capable of binding to the collectins receptor on U937 cells.Natural MBL protein with high purity and activity was gained from human plasma by combined appfication of ligand affinity chromatography and monoclonal antibody affinity chromatography,laying a solid foundation for the following study.ⅡPreliminary Study on the Role of MBL in Regulating the Differentiation and Function of Dendritic CellsToll-like receptors（TLRs）,one of important pattern-recognition receptors （PAMP） of the innate immune system,can distinguish infectious nonself from noninfectious self and control the initiation of adaptive responses,which have been considered as a linkage of innate and adaptive immunity.Among the TLR family, TLR4 is expressed very extensively（e.g.,Monocytes,Macrophages,Immature DCs, T cells and B cells）,and plays a critical role in recognition and signaling of bacterial lipopolysaccharid.Dr.Chen of our team found that MBL could regulate dendritic cell maturation and cytokine production induced by lipopolysaccharid.However the regulatory mechanism remains unclear.In this study,we continued our previous studies and evaluated the impact of MBL on LPS/TLR4-mediated signaling pathway.We further investigated the possible mechanistic insight into the inhibitory effect of MBL on DC.We initiallly obtained prokaryotically expressed soluble extracellular-TLR4 protein（sTLR4） identified by SDS-PAGE and Western blot.Then,Western blot and ELISA were used to evaluate whether MBL interact with sTLR4,flow cytometry （FCM） to analyze the binding of MBL to immature dendritic cells（imDCs）,and competitive inhibition test to the binding of MBL to TLR4 receptor on the surface of imDCs and its role in the consequent LPS-induced nuclear factor-κB（NF-κB） signaling pathway.Our date showed that MBL could bind to monocyte-derived immature DCs（imMDCs） at physiological calcium concentrations but optimal at superphysiological calcium concentrations.Furthermore,MBL could directly bind to extracellular domain of TLR4 in a dose-dependent manner and attenuate the binding of LPS to cell surface,resulting in decreased LPS-induced nuclear factor-κB（NF-κB） activity in imDCs.All these suggest that MBL could affect the functions of DCs by modifying LPS/TLR-signaling pathways.This study supports an important role of MBL in the regulation of adaptive immune responses.ⅢCharacterization of the Action of MBL onto Raji CellsAs a key soluble pattern recognition molecule in the innate immune,MBL can not only recognize pathogens or autologous apoptotic cells,but also bind to healthy autologous cells in a specific and sugar-sensitive manner,and it has been shown that cytokine expression can be altered by interaction of the cells with MBL,suggesting that MBL may play important roles in immune regulation and do some roles in adaptive immune responses.Studies of our previous section showed that MBL be involved in the regulation of DC;however T and B lymphocytes are the mediator of acquired immune responses,thus,it is very informative to investigate whether the soluble defense collagen family could regulate lymphocytes.In this study,we investigated the interaction of MBL with Raji cells by ELISA, and the characteristics of MBL binding to Raji cells by FACS.Our results showed that MBL could bind to Raji cells in a dose-dependent manner.Furthermore,the binding of MBL to Raji cells was evident in a Ca²⁺-dependent manner,which was partially inhibited by some saccharides（mannose,glucose,or N-acetylglucosamine）, C1q and anti-C1qR mAb.Similarly,it was also incompletely inhibited by recombinant human MBL carbohydrate recognition domains（rhMBL-CRD） protein and recombinant human MBL collagen-like region（rhMBL-CLR） protein, respectively,but completely inhibited by both.These data indicated that Raji cells express Ca²⁺-dependent and sugar-sensitive CLR-specific and CRD-specific MBL receptors,of which,the former was also shared with C1q.Further investigation showed that MBL could significantly inhibit Raji cell proliferation directly at higher concentrations（10～50 mg/L） in a dose-dependent manner,suggesting that MBL might also play certain roles in adaptive immune responses.ⅣPreliminary Study on the Role of MBL in Regulation of T lymphocytesIt is reported that both SP-A and SP-D can not only inhibit mitogen-induced T cell proliferation,but also attenuate interleukin-2（IL-2） secretion from these cells in a dose-dependent manner.The inhibitory effects of SP-A on T cell proliferation occurs at 24 hours after the treatment of lymphocytes with mitogen.The functions of DC can also be regulated by SP-A through its inhibitory effect on DC differentiation and maturation,and by SP-D through enhancing DC’s antigen-presenting capacity,which promotes its acquired immune response to infection.However,so far,little is known about the role of MBL in the regulation of lymphocytes,especially in its direct action on T cells.We speculate that MBL might have regulatory roles in T lymphocytes based on the structural homology among MBL,SP-A and SP-D.In the present study, we explored the feasibility of the in vitro regulation of MBL in T lymphocytes.Tetanus toxin（TT） has been widely used as a model antigen in the study of CD4+ T cell-mediated immune responses due to its activation of CD4+ T lymphocytes.However,the traditional tetanus toxoid（TToid） is detoxified tetanus bacilli by formaldehyde.Since tetanus bacilli can form spores with high toxicity,the preparation of TToid appears to be dangerous to certain extent.Furthermore,it is easy to cause pollution during processing TT by formaldehyde,and the toxicity of TToid can be recovered sometimes.Therefore,the application of TToid in researches subjects to certain restrictions.TTC can not only stimulate the body to produce sufficient neutralizing antibodies against toxin-mediated attacks,but also activate CD4⁺ T cells,which has been considered as a safe and optimal tetanus subunit vaccine candidate.In the present study,we used TTC as a model antigen to investigate the role of MBL in regulation of T cell-mediated acquired immune response.We investigated the possibility of MBL in regulating T lymphocytes in vitro. Firstly,we cloned,expressed and analysed the immunogenicity of TTC and obtained TTC with high purity and immunogenicity,which provides an ideal model antigen for studying antigen presentation and immune responses.Then,PBMCs were isolated by sequential centrifugation on Ficoll-Hypaque and Percoll gradients from blood drawn from normal adult donors in our laboratory and CD3⁺ T lymphocytes were obtained from PBMCs by passing a nylon wool column.Jurkat cells,PBMCs and CD3⁺ T lymphocytes were activated by non-specific stimulation of phytohemagglutinin （PHA）,PMA or PMA/ionomycin.The binding of MBL to PHA- or PMA- activated Jurkat cells and CD3⁺ T lymphocytes were measured by FCM.WST-1 and ELISA were usued to analyse the impact of MBL on the proliferation and IL-2 or IFN-γsecretion in PHA-,PMA- or PMA/ionomycin- activated Jurkat cells,CD3⁺ T lymphocytes and PBMCs.And finally,we investigated the role of MBL in APC stimulators of allogeneic T cells in mixed lymphocyte reaction（MLR） and presentation of TTC antigen to specific T cell lines.Our date showed that MBL could bind to PHA- or PMA- activated Jurkat cells and CD3⁺ T lymphocytes;that MBL could inhibit the proliferation and IL-2 or IFN-γsecretion in a concentration-dependent manner in PHA-,PMA- or PMA/ionomycinactivated cell;and that MBL could reduce APC’s ability to stimulate allogeneic T lymphocyte proliferation and DC’s ability to present TTC to specific T cell lines.All these date suggest that MBL could down-regulate T cell-mediated acquired immune responses.The findings mentioned above indicate that MBL could directly interact with TLR4 expressed on DCs,and that MBL could alter the downstream of NF-κB signaling pathway,suggesting that MBL and TLR might establish a large network for host defense and regulate the subsequent immune responses.In addition,MBL can directly regulate the functions of T and B lymphocytes,and down-regulate the antigen-presenting ability of APC,suggesting that MBL may regulate acquired immune responses through T cells,B cells and APCs.So far,no similar researches have been reported,although the detailed mechanism remains to be elucidated.What we found in this study proposed a novel idea for MBL’s role in acquired immune responses and its mechanism.The interpretation of the future analyses of the properties of MBL regulation,elucidations of the molecular mechanism and the signal pathway in which MBL is involved will further reveal the biological significance of this key innate molecule,MBL.更多还原