【作者】 刘蓓；

【导师】 金伯泉； 张赟； 庄然；

【作者基本信息】 第四军医大学 ， 免疫学， 2013， 博士

【摘要】 汉滩病毒（hantaan virus, HTNV）属于布尼亚病毒科（family Bunyaviridae），汉坦病毒属（genus Hantavirus），可引起人类严重致死性的肾综合征出血热（hemorrhagicfever with renal syndrome，HFRS）全球每年约有10万病例，其中90%以上发生在我国，死亡率达2%-10%。HFRS患者主要临床表现为发热、出血、血小板数量下降和急性肾功能损害，病情不一，可从亚症状一直到危及生命。典型HFRS分为发热期、低血压休克期、少尿期、多尿期和恢复期五个病期。尽管已知HTNV感染后免疫应答涉及免疫复合物的产生，补体活化，B细胞应答，T细胞应答以及HTNV诱导的细胞因子分泌，但无论是该病病理损伤机制还是杀伤性细胞（cytoxic T lymphocyte，CTL）介导的免疫保护作用均未完全搞清。CD100/Sema4D为Ⅳ型信号素家族成员，分子量150kD，是发现的首个免疫信号素，参与体液免疫应答和细胞免疫应答。CD100存在膜型（mCD100）和可溶型（sCD100）两种形式。mCD100在T细胞表面高表达，B细胞和APC上表达水平较低。细胞活化时可引起mCD100的脱落，产生sCD100。 sCD100具有生物学活性，可协同CD40诱导B细胞的增殖和Ig产生，调节APC细胞产生促炎因子。CD100的主要受体：高亲和力受体Plexin-B1主要表达于非淋巴样组织；低亲和力受体CD72主要表达于免疫系统。大量研究表明，CD100在生理性和病理性免疫应答中发挥重要作用。CD100-/-小鼠能存活，但T细胞致敏和B细胞应答存在缺陷；而CD100转基因小鼠适应性免疫应答水平明显升高。重要的是，CD100与某些临床疾病的发生发展有关。人热带痉挛性截瘫/HTLV-1相关脊髓病患者的脑脊液中可以检测到sCD100，提示CD100可能参与了中枢神经系统疾病的病理损伤。在系统性硬化症患者血清中能检测到sCD100，并且患者淋巴细胞mCD100表达异常，表明CD100在系统性硬化症的发生和/或维持中发挥作用。新近，有报道在HIV感染患者中，存在CD8~+CD100-亚群，该亚群功能降低，提示CD100的丢失可能在HIV感染后免疫功能异常中起重要作用。但是，在其它疾病中是否也存在CD100参与疾病病理损伤，或者淋巴细胞mCD100表达的异常还不清楚，有关CD100与急性传染病的关系也鲜有报道。鉴于CD100在免疫应答中的重要作用，本研究集中在以下两个方面展开研究：一是HTNV感染后是否存在着sCD100的释放，血浆sCD100水平和HFRS疾病病情有何关系；二是CTLmCD100的表达变化是否与CTL的亚群及其免疫功能有关。首先，我们收集了HFRS住院患者和正常人的PBMC和血浆。采用本文建立的sCD100酶联免疫吸附试验（ELISA）定量检测了不同病程和病情患者血浆sCD100水平，对血浆sCD100水平的动态变化以及与指示病情的实验室参数的相关性进行了分析。然后，我们检测了HFRS患者PBMC中CD4+T细胞、CD8~+T细胞和其它亚群mCD100表达水平的变化。首次发现并研究了一个新的CD8lowCD100-亚群的表型和功能，以及在病程中该亚群所占PBMC的比例与HTNV病毒载量及病情的相关性。根据病历资料和HFRS诊断标准，将99名HFRS患者划分为轻型（17例），中型（25例），重型（29例），危重型（28例）四组。HFRS患者发热/低血压休克期、少尿期、多尿期和恢复期血浆sCD100的中位数（四分位间距）分别为42.8(26.5-55)ng/ml,34.8(21.3-52.6) ng/ml、12.1(8.5-18.2) ng/ml和15.9(8.6-21.4) ng/ml。与正常人相比，患者急性期（包括发热期、低血压/休克期及少尿期）血浆sCD100水平明显升高（P<0.0001），恢复期（多尿期和恢复期）迅速下降，但仍然高于正常对照组。不同病情组患者血浆sCD100水平呈现出急性期高、恢复期低的相同趋势，但以重型/危重型变化更为明显。38例轻/中型患者急性期血浆sCD100水平>50ng/ml的有7例（18.4%），而61例重型/危重型患者sCD100水平值>50ng/ml的有26例（42.6%），是轻型/中型组的2.32倍。此外，我们用Western Blot方法检测了HFRS患者血浆中sCD100水平，发现无论是患者还是正常人，在还原条件下，血浆中sCD100的分子量为120kD。我们对HFRS早期（发热期或低血压期）血浆sCD100水平与指示HFRS严重程度的实验室参数相关性进行了分析，发现升高的血浆sCD100与血小板计数呈显著负相关（r=-0.50, P=0.0001），与白细胞计数（r=0.54, P <0.0001），血清肌酐（r=0.49, P=0.0001），尿素氮（r=0.56, P=0.0002）呈显著正相关。HFRS患者急性期PBMC中CD4+T细胞、CD8~+T细胞、B细胞、NK细胞和单核细胞膜型CD100表达水平有不同程度的下降。我们在HFRS患者急性期PBMC中发现了一个新的CD8lowCD100-T细胞亚群，平均占PBMC10.0%，至恢复期下降至2.6%，正常人中不存在这个亚群。该亚群高表达CD38、HLA-DR、Ki67、颗粒酶B及穿孔素，表面标志为CD45RA-CCR7+/-CD127int/highCD27intCD62L-，符合效应T细胞的特征。急性期HFRS患者PBMC中CD8lowCD100-亚群比例越高，血浆中病毒载量越低，呈显著负相关（p<0.0001），轻型组CD8lowCD100-亚群所占PBMC百分率高于中型、重型、危重型组（P<0.05）。在HTNV-NP特异性9肽混合肽的刺激下，CD8lowCD100-亚群产生大量TNF-及IFN-γ，而其他两群细胞（CD8highCD100+, CD8lowCD100+）仅产生少量TNF-及IFN-γ。提示CD8lowCD100-亚群主要可能是与清除病毒及免疫保护有关的效应T细胞亚群。随着HTNV的清除以及CD8~+T细胞数量降低到正常水平，CD8lowCD100-亚群细胞数量明显下降，至痊愈出院后消失。该群细胞在病程后期陡然下降可能是通过PD-1途径克隆收缩而引起，此动态变化可能反映了HFRS病毒（抗原）清除后免疫系统自身稳定的一个途径。总之，HFRS患者急性期血浆中升高的sCD100可能与病情相关，外周血中CD8lowCD100-亚群比例与血浆病毒载量和病情呈负相关。这些结果有助于我们加深认识病毒感染后病理损伤的机制、CD8~+T细胞介导的免疫保护作用，以及病毒清除后免疫自身稳定的途径。更多还原

【Abstract】 Hantaan virus (HTNV), which belongs to the genus Hantavirus of the familyBunyaviridae, could cause a severe lethal hemorrhagic fever with renal syndrome (HFRS)in human. More than100,000cases of HFRS, over90%of which were documented inmainland of China, occurred annually worldwide with a mortality rate of2-10%. Peoplewith HFRS are clinically characterized by sudden fever, hemorrhage, thrombocytopenia,and acute renal failure, leading from an asymptomatic to a severe, life-threatening illness.Typically, the course of HFRS undergoes five sequential stages: febrile, hypotensive,oliguric, diuretic, and convalescent. Although the importance of immune responses afterHTNV infection has been widely recognized including immune complexes, complementactivation, B cell response, T cell response, and HTNV-induced cytokine production, notonly the pathogenesis of HFRS but also the protective immunity dediated by cytotoxic Tlymphocyte (CTL) during the cause of the diease are considerably far from beingcompletely understood. The150kDa transmembrane protein CD100/Sema4D belongs to group IV of thesemaphorin family, which is the first known semaphorin identified in the immune system,and is involved in several aspects of both humoral and cellular immunity. It exists in bothmembrane-bound and soluble forms. The membrane CD100is preferentially expressed onT cells and weakly on B cells and APC. Cellular activation can cause the release ofsCD100. sCD100is demonstrated to retain biological activities such as acting as acostimulator for CD40-induced B-cell proliferation and Ig production, affectingpro-inflammatory cytokines production by APCs. There are two types of receptors thatCD100used to bind: Plexin-B1mainly expressed in nonlymphoid tissues, and CD72inthe immune system. Accumulating evidence indicates that CD100plays a relevant role inphysiological and pathological immune responses. CD100-/-mice are viable, but showdefective T cell priming and B cell responses, whereas adaptive immune responses aresignificantly enhanced in CD100transgenic mice. Furthermore, CD100is believed to beinvolved in some clinical diseases. In the spinal cords of patients with human T-celllymphotropic virus type1-associated myelopathy, the presence of sCD100in the spinalcord suggested the potential pathological effect of sCD100in the central nervous system.In the systemic sclerosis patients, the frequently detectable levels of sCD100in sera andthe dysregulations of CD100expression on lymphocytes were observed, suggesting therole of sCD100in the systemic sclerosis development and/or maintenance. Recently,Eriksson et al investigated the consequence of HIV-1infection on CD100expression by Tcells, and observed a subset of CD8~+T cell lacking of membrane CD100with decreasedfunctional capacity, which suggested that loss of CD100expression would probably playan important role in dysfunctional immunity in HIV-1infection. However, there is stilllimited information on the functional role of CD100in infectious disease. Whether thispathogenetic role of CD100or involvement of CD100expression in CTL subset andfunction could extend to other acute infectious diseases mediated by immune responses isalso unclear.Since the important role of CD100in immune response, we focused on two aspects:whether sCD100release after HTNV infection exist and changed level of plasma sCD100 correlate with the outcome of HFRS, and whether the changes of mCD100expression onCTL are related to CTL subsets and function.Plasma and peripheral blood mononuclear cell (PBMC) samples from a large cohortof HFRS patients and health controls were collected. First, the plasma sCD100levels indifferent disease stages or severities of HFRS patients were quantified by enzyme-linkedimmunosorbent assay (ELISA) which has been established successfully in this study. Thecorrelations between sCD100and disease course, disease severity-indicating parameterswere also analyzed. Second, the changes of mCD100on CD4+T cells, CD8~+T cells andother population in PBMC were investigated by multi-color staining and flow cytometry(FCM) analysis. Particularly, we payed more attention on a novel CD8lowCD100-subsetand detected its phenotype, function as well as the relationship between the distribution ofCD8lowCD100-subset in PBMC and plasma HTNV RNA load or disease severity duringthe course of HFRS.According to the clinical records and diagnostic criteria,17,25,29, and28patientswere diagnosed as mild, moderate, severe, and critical HFRS, respectively. The median(IQR) of sCD100for febrile/hypotensive, oliguric, diuretic and convalescent stage was42.8(26.5-55),34.8(21.3-52.6),12.1(8.5-18.2), and15.9(8.6-21.4) ng/ml, respectively.The elevated plasma sCD100level of HFRS patients was observed in acute phase(including febrile, hypotensive, or oliguric stage) when compared with healthy controlsand decreased, but still higher than that of healthy controls in convalescent phase(including diuretic or convalescent stage). Patients with different disease severity showedthe same tendency of the plasma sCD100change, but more dramatic decline in patients ofsevere/critical group. When plasma sCD100concentrations in mild/moderate group werecompared with those in severe/critical group, only7cases whose plasma sCD100levelswere over50ng/ml measured among38cases of mild/moderate group (18.4%), while26cases whose plasma sCD100levels were over50ng/ml in61cases of severe/critical groupcould be detected (42.6%,2.32fold high vs. mild/moderate group). In addition, wedetected the plasma sCD100by Western Blot, which showed that the sCD100level washigher in acute phase than in convalescent phase apparently. The molecular weight of the sCD100in plasma from both patients and normal controls is120kDa in the reducedcondition which is consistent with the size of extracellular region of CD100. Furthermore,the relationships between plasma sCD100levels at febrile or hypotensive stages (the timeof admission generally about3-7days after the onset of disease) and four clinicalparameters that could represent the severity of the disease were analyzed. The resultrevealed a significant negative correlation between plasma sCD100levels and the plateletcount (r=-0.50, P=0.0001) and significant positive correlations between plasma sCD100levels and the white blood cell count (r=0.54, P <0.0001), or the level of serumcreatinine (r=0.49, P=0.0001), or the level of blood urea nitrogen (r=0.56, P=0.0002).The membrane CD100on PBMCs was also investigated. We found the expressionof membrane CD100on PBMCs including CD4+T cells, CD8~+T cells, B cells, naturalkiller cells and monocytes all decreased to different extents in the acute phase of HFRScompared with that of the normal controls and recovered in the convalescent phase.For the first time, we revealed a novel functional subpopulation in CD8~+T cells inHFRS patients characterized by the phenotype of CD8lowCD100-. The average distributionof CD8lowCD100-subpopulation in PBMC was10%in acute phase and2.6%inconvalescent phase, whereas this subset did not exist in health controls. Furthermulti-color staining and flow cytomety analysis demonstrated that CD8lowCD100-subsetexpressed higher level of CD38+HLA-DR+Ki-67+than the other two subsets did, highlyexpressed cytolytic effector molecules including perforin and granzyme B, displayed thephenotype CD45RA-CD127int/highCD27lowCD62L-, which were consistent with the majorfeature of effector CD8~+T cells. This observation was supported by the investigations ofthe relationship between the distribution of CD8lowCD100-subset in PBMC and plasmahantaan virus load of HFRS patients, and by cytokine production of different CD8~+T cellsubsets when stimulated by specific HTNV-NP derived9mer peptide pool. Thepercentage of CD8lowCD100-subset in PBMC was negatively correlated with plasmaHTNV virus load (P<0.0001) in acute phase of HFRS. In addition, the percentage ofCD8lowCD100-subset in PBMC of mild group was higher than that of moderate, severe orcritical gruops (P<0.05). TNF-and IFN-γ are key effector cytokines for effector CD8~+ cells. In vitro CD8lowCD100-subset produce little TNF-and IFN-γ without thestimulation. However, when stimulated by the HTNV-NP9mer peptide pool, theCD8lowCD100-cells produced a large amount of TNF-and IFN-γ identified byintracellular cytokine staining and FCM, whereas other two subsets of CD8~+T cells(CD8highCD100+and CD8lowCD100+) produced small amount of TNF-and IFN-γ. Withthe clearance of HTNV virus and the number of CTL returned to normal level, and thenumber of CD8lowCD100-T cells decreased dramatically. This kinetic change ofCD8lowCD100-T cells may be due to clonal contraction of effecter T cells via PD-1pathway and reflects the homeostasis pathway of immune system after virus infection.In summary, the elevated sCD100in plasma seemed to be a characteristic in HFRSpatients especially in the acute phase of the disease indicating a possible associationbetween increased release of sCD100and different disease severity in HFRS. Forthermore,a novel functional CD8lowCD100-subset in PBMC from HFRS patients was negativelycorrelated with plasma HTNV virus load and severity of the disease severity. These resultsare useful for understanding the pathogenesis and CD8~+T cell mediated immunity as wellas immune homeostasis after HTNV infetion in human.更多还原