【作者】 赵娜；

【导师】 陈政良； 左大明；

【作者基本信息】 南方医科大学 ， 免疫学， 2017， 博士

【摘要】 MBL属于C型凝集素超家族中胶凝素(collectins)家族成员,是肝细胞分泌的血浆蛋白,对于抗御病原体感染和清除凋亡细胞及免疫复合物具有重要意义。我们近期研究发现MBL可调节人单核细胞(Mo)、树突状细胞(DC)等抗原提呈细胞的分化发育,然而,MBL对T淋巴细胞是否具有直接的调节作用,目前尚无相关文献报道。为进一步研究MBL对获得性免疫的调节作用,本课题以T细胞为切入点,初步探索MBL对T细胞活化、增殖、分化的影响及其分子机制。第一章MBL对T淋巴细胞活化增殖的调节作用及其机制随着天然免疫研究的深入,发现胶凝素家族的一些成员和获得性免疫之间的联系越来越紧密。研究表明表面活性物质-A(SP-A),表面活性物质-D(SP-D)可直接抑制T细胞增殖活化。MBL与SP-A、SP-D的同源性,尤其是其与SP-A在结构上的高度相似,我们推测MBL很有可能参与获得性免疫应答。我们使用MACS负选法分离人纯化T细胞,在CD3/CD28共刺激T细胞活化体系中加入血浆纯化MBL蛋白可减弱T细胞的活化与增殖,外源性的IL-2可以逆转MBL的作用;MBL抑制CD3/CD28共刺激T细胞活化过程中ZAP-70蛋白和活化T细胞接头蛋白及NF-kappa B及MAPK信号通路的活化。MBL还可使活化T细胞处于无应答状态。MBL可以结合活化T细胞而非初始T细胞;MBL通过其CLR结构域与活化T细胞表面的CRT蛋白直接结合,使用抗CRT抗体封闭T细胞表面的CRT蛋白,可以逆转MBL对T细胞的抑制作用。我们还发现与MBL结构高度同源的C1q及凝集素家族新成员CL-11具有与MBL同样的作用方式,即通过与活化T细胞表面CRT作用来抑制T细胞的增殖。总而言之,本部分研究确定了 MBL可通过其CLR结构域与活化T细胞表面CRT蛋白作用,这可能是MBL抑制T细胞活化与增殖的机制。第二章MBL对T淋巴细胞功能的调节作用及其在矽肺中的意义负向免疫调控机制对于维持体内免疫系统自稳和预防免疫系统过度活化具有重要作用。我们首次报道了矽肺患者血清中MBL浓度升高,且血清MBL浓度与CD45RO+T细胞比率呈负相关,与肺功能呈正相关;MBL亦能抑制矽肺病人T细胞活化与增殖;早期矽肺患者中MBL浓度与Th17细胞呈负相关,与Treg细胞呈正相关。我们亦发现MBL-L-小鼠对SiO2诱导的肺损伤较为敏感,表现为严重肺泡炎和肺水肿;与WT小鼠相比,MBL-/-小鼠受SiO2攻击后体内Th17细胞比率较高,Treg细胞比率较低,这可能与MBL-L-小鼠受致病因素攻击后,体内STAT3磷酸化水平较高有关。在体外Th17分化培养系统中,MBL可抑制人naive CD4+ T细胞向Th17分化,促进其向Treg分化,这可能与MBL抑制IL-6诱导的STAT3磷酸化有关。综上,本课题研究发现MBL可抑制T细胞的增殖、活化及相关细胞因子的分泌,并提出MBL可能通过调节T细胞的功能在矽肺中发挥免疫调节作用。本研究将为天然免疫及其指导获得性免疫应答的基础理论提供实验依据,并为MBL和T细胞功能紊乱有关疾病的诊治研究提供新的研究基础。更多还原

【Abstract】 MBL,an evolutionary conserved circulating host defense protein produced mainly by liver,is a member of the family of collectins(C-type lectins with a collagen-like domain),which is important for resisting pathogen infections and removing of apoptosis cells and immune complex.Previous studies by our lab showed that MBL can upregulate the differentiation and development of antigen presenting cell,as monocyte and dendritic cells.However,whether MBL wound directly modulate the T cell,there is no literature report.The present study aimed to determine the regulatory roles of MBL on adaptive immunity,especially its regulatory roles on T cell.We investigated the effects of MBL in regulating T cell activation,proliferation and diffrerentiation and its mechanisms.Part Ⅰ Effect of MBL on Activation and Proliferation of T cells and its mechanismsIt is clear that the proteins of collectin family such as surfactant-associated proteins SP-A and SP-D can initiate and shape adaptive immunity.The structural homology of the collectins led us to hypothysize that MBL may have effect on T cells activation that is similar to SP-A and SP-D.In this study,we sought to determine if MBL have functional similarity with SP-A and SP-D.PBMCs were isolated from peripheral blood of healthy adult volunteers by Ficoll-Hypaque density gradient centrifugation and further purified into T cells with MACS,then cultured with anti-CD3/28 antibodies with MBL.MBL could attenuate anti-CD3/28-induced T cell proliferation and activation and exogenous IL-2 could restore the MBL-mediated inhibiton of T cell proliferation.Western blot analysis showed that the presence of MBL during initiation of TCR signal transduction was associated with reduced phosphorylation of ZAP-70,NF-κB and MAPK pathway.MBL also induced T cell unresponsiveness.The result showed that FITC-MBL protein was slightly bound to naive T cells.Surprisingly,anti-CD3/28-activated T cells were much firmly bound to FITC-MBL protein compared with naive T cells.MBL interacted with CRT on T cell surface via its CLR domain.FACS study showed that the anti-CRT neutralizing antibody completely blocked the association of FITC-labeled MBL with activated T cells.Moreover,the proliferation of T cells was comparable when T cells co-cultured with the anti-CRT antibody during TCR activation either with or without MBL treatment.The Clq and CL-11 also significantly inhibited anti-CD3/28-stimulated T cell proliferation and the effect can be abolished by treatment with the anti-CRT antibody.These data suggested that it might be a common feature that the interaction of serum collectin with CRT on T cells triggered the suppressive effect on T cell activation.Totally,This section studies confirmed that MBL interacted with CRT on T cell surface via its CLR domain,which might be the mechanism of inhibitory roles of MBL.Part Ⅱ Mechanisms of regulatory roles of MBL on T cell and its significant in silicosisNegative feedback immune mechanisms are essential for maintenance of immune system homestasis and prevention of over-response.ELISA analysis of the sera from silicosis patients showed the MBL serum levels in these patients were significantly higher than those in HCs.The serum level of MBL was negative correlated with CD45RO+ T cells and positively correlated with the pulmonary function in patients with silicosis at the early stage.MBL has inhibitory effects on the activation of T cells from silicosis patients.The serum level of MBL was positively correlated with Th17 and positively correlated with Treg in patients with silicosis at the early stage.Strikingly,ablation of MBL strongly sensitizes mice to SiO2-induced lung injury,showing sever pulmonary alveolitis and edema,which depends on excessive production of Th17 and lower production of Treg.In Th17 polarization condition,MBL inhibited the differentiation of Th17 from naive CD4+ and promoted differentiation of Treg,which may relate to the inhibitory effect of MBL on IL-6-induced phosphorylation of STAT3.In summary,results above have showed that MBL suppressed proliferation,activation and proliferation of T cells.Inhibition was associated with a decreased production of IL-2.Our results uncover an essential mechanism of MBL-mediated protection of lung damage,which provides insight into lung pathophysiology and offers new opportunities to develop future therapeutic strategies.Our findings support the notion that MBL on APCs as well as MBL function as an immunosuppressive molecule capable of restricting T cell activation and may serve as a basis for the rational design of novel strategies targeting MBL to achieve improved immunotherapeutic efficacy against cancer and other diseases.更多还原