CN111593022A - vMIP-Ⅱ诱导CD8+ T细胞去磷酸化为Tcm及其在药物中的应用 - Google Patents
vMIP-Ⅱ诱导CD8+ T细胞去磷酸化为Tcm及其在药物中的应用 Download PDFInfo
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- CN111593022A CN111593022A CN201811616307.9A CN201811616307A CN111593022A CN 111593022 A CN111593022 A CN 111593022A CN 201811616307 A CN201811616307 A CN 201811616307A CN 111593022 A CN111593022 A CN 111593022A
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Abstract
本发明公开了病毒巨噬细胞炎性蛋白vMIP‑Ⅱ诱导CD8+T细胞去磷酸化为Tcm的应用。发明中诱导CD8+T去磷酸化为Tcm的vMIP‑Ⅱ是本实验室研制并通过国家药品和生物制品检定。本发明通过恒河猴SIV感染模型研究CD8+T细胞，发现vMIP‑Ⅱ可使Tcm依赖vMIP‑Ⅱ剂量增殖，且该增殖细胞的差异基因主要富集于趋化因子受体和磷酸化通路，进一步发现该增殖是vMIP‑Ⅱ封闭CD8+T趋化因子受体而低表达G蛋白，降低胞内Ca2+浓度和线粒体膜电位、抑制磷酸化的相关基因，使磷酸化蛋白ERK1/2和Akt低表达，从而使CD8+T磷酸化信号减弱，发生代谢重编程而转化为Tcm。因此，vMIP‑Ⅱ作用机制的这一发现为HIV/SIV感染艾滋病的药物研发提供全新策略，为抗病毒与抗肿瘤的过继性免疫治疗提供新的手段，具有重要的临床应用价值。
Description
技术领域
本发明属病毒病毒巨噬细胞炎性蛋白vMIP-Ⅱ在防治炎症和SIV/HIV感染等方面进行的基础研究领域，更具体地说，本发明涉及病毒巨噬细胞炎性蛋白vMIP诱导CD8+ T细胞去磷酸化为长寿命中枢记忆细胞(CD8+ Tcm)的作用机制。
背景技术
在血液中循环并存在于淋巴器官中的记忆性CD8+ T细胞是长寿命T细胞免疫的重要组成部分。这是由于CD8+ T细胞主要通过其表面的T细胞受体(TCR)识别由MHCⅠ分子递呈的抗原肽而被活化，活化后的CD8+ T细胞会在收缩阶段凋亡90%~95%，仅小部分形成具有抗原特异性的记忆CD8+ T细胞。这些记忆CD8+ T细胞在再次暴露于病原体时仍能迅速发挥效应作用清除抗原，但也有许多与初始细胞相同的特性，包括多能性和迁移到淋巴结和脾脏的能力。因此，记忆细胞体现了初始细胞和效应细胞两者共有的特征，由此而引发了一场围绕记忆T细胞是从效应细胞发展还是直接从初始细胞发展的长期争论。
记忆T细胞通过表达或不表达趋化因子受体CCR7和血管L-选择素 CD62(CD62L)来实现在机体外周血和淋巴组织间的循环，分为中央记忆性T细胞(Central memory Tcells，Tcm)和效应记忆性 T细胞(Effector memory T cells，Tem)。一般情况下Tcm细胞表达CCR7和CD62L，主要分布于外周组织免疫器官和淋巴结，当再次受抗原刺激时可迅速分裂增殖和分化；Tem细胞低水平表达或不表达CCR7和CD62L，主要存在于非淋巴组织和器官，参与周身循环，可迁移至外周炎症组织发生速发性效应功能。组织定居记忆性T细胞(Tissue-resident memory T cells，Trm)则不表达CCR7和CD62L，高表达CD69和/或CD103且不参与体循环。
病毒巨噬细胞炎症蛋白-Ⅱ(vMIP-Ⅱ)是由卡波氏肉瘤疱疹病毒(KSHV) K4基因编码的一种人趋化因子小分子蛋白，与人CC类趋化因子巨噬细胞炎性蛋白Ⅰ在氨基酸序列上有较高的同源性。vMIP-Ⅱ可以利用与其他趋化因子相似的结构骨架与其受体进行相互结合作用，已有研究证明，vMIP-Ⅱ是广谱的趋化因子受体抑制剂，具有结合多种人趋化因子受体亚族的能力，并能竞争性抑制HIV与靶细胞上共受体CCR5、CXCR4、CCR3等的结合，以阻止病毒进入靶细胞，具有抗HIV感染的作用。趋化因子受体其胞内区的羧基末端含丝氨酸/苏氨酸，可发生磷酸化，从而与G蛋白相偶联，参与MAPK、JAK-STAT、NF-κB等信号转导通路的调控。在哺乳动物中，NF-κB与抑制性蛋白IκB紧密结合，以无活性状态存在于细胞质当中。在炎性信号的刺激下(如TNF-α、LPS、IL-1等)，IKK可被激活。接着IKK催化IκB发生磷酸化，进而IκB可被泛素标记，从而被转运到蛋白酶体发生降解，释放出游离的NF-κB。之后，游离的NF-κB便可通过核孔复合物在核转位序列的介导下转位入核，对多种炎症与免疫相关基因(包括IL-1β和TNF-α等)的转录发挥调控作用。
因此，在配体与表达于细胞表面的CCR7结合后，细胞表面整合素大量聚集，激活耦联于细胞浆内的G蛋白，引起Ca2+的快速动员以及进一步磷酸化分裂原活化蛋白激酶(MAPK)、黏着斑激酶(FAK)、蛋白激酶C、鸟苷三磷酸酶等酪氨酸激酶，多种酪氨酸激酶通路介导信号转导，重新组合细胞内的骨架蛋白，产生趋化作用，引起靶细胞运动，参与体内多种生理和病理过程[13]。由于CCR7是中央记忆CD8+ T细胞的表面标记物，而且存在于CD8+ T细胞表面，因此当vMIP-Ⅱ与其结合时，会阻碍CCR7与高亲和力配体CCL21和CCL19结合，可能引发去磷酸化效应。
本实验室前期研究发现重组vMIP具有明显抑制病毒进入靶细胞和保护靶细胞免受病毒感染的作用，其表明vMIP可能是通过某种机制抑制细胞内SIVmac病毒的产生。因此我们进一步研究了重组vMIP-Ⅱ对食蟹猴免疫系统的影响，结果表明长期大剂量注射重组vMIP-Ⅱ具有刺激食蟹猴免疫系统，并促进CD8+ T细胞增生和增加CD8+ T细胞功能的作用。但重组vMIP-Ⅱ是如何抑制病毒进入靶细胞、如何促进CD8+ T细胞增生的分子机制尚不清楚。
根据vMIP-Ⅱ对CCR5和CXCR4共受体的封闭性，以及CCR7、CXCR5和CX3CR1与CCR5和CXCR4同属于CC类趋化因子受体，所以我们推测vMIP-Ⅱ也能封闭其他趋化因子受体，以提高效应CD8+ T细胞的免疫能力。因此，我们拟以猴艾滋病病毒SIVmac251感染恒河猴为模型实验对象，观察vMIP对效应CD8+ T的亚群分布影响，并通过检测vMIP-Ⅱ诱导后的对靶细胞去磷酸化途径所影响的转录因子，以验证效应CD8+ T细胞是否可以去磷酸化转化为长寿记忆CD8+ T细胞。
发明内容
本发明成功构建恒河猴SIV感染模型分选出能在vMIP-Ⅱ治疗下由去磷酸化增殖为CD8+ Tcm细胞的CD8+ T细胞并研究vMIP-Ⅱ诱导效应CD8+ T细胞去磷酸化为长寿记忆CD8+ Tcm细胞的作用机制，使其在抗HIV/SIV病毒与抗肿瘤的过继性免疫以及验证反应的预防或/和治疗中发挥作用。
本发明构建恒河猴SIV感染模型的方法，是用50 TCID50剂量的SIVmac251静脉注射接种Mamu-A*01阳性恒河猴，并使用超灵敏分支DNA扩增测定法进行病毒载量分析，以确定恒河猴SIV感染模型的构建。
本发明通过恒河猴SIV感染模型进行vMIP-Ⅱ干预治疗，研究vMIP-Ⅱ对淋巴组织、CD8+ T细胞、CD8+ Tcm细胞、CD8+ Tem细胞的影响，对CD8+ T细胞、CD8+ Tcm细胞和CD8+ Tem细胞进行流式细胞仪分选，以确定CD8+ Tcm细胞和CD8+ Tem细胞的比例。
本发明提供的诱导CD8+ T细胞去磷酸化为CD8+ Tcm细胞的vMIP-Ⅱ能促使淋巴组织增生，使淋巴小结生发中心扩大，并使CD8+ Tcm细胞增殖，降低炎症反应的作用，从而对机体免疫产生保护作用。
本发明提供的诱导CD8+ T细胞去磷酸化为CD8+ Tcm细胞的vMIP-Ⅱ可引起CD8+Tcm细胞增殖，基因测序显示该增殖细胞与CD8+ T细胞的差异表达基因主要富集于表面趋化因子受体CCR7、CXCR4、CXCR5和CX3CR1和磷酸化通路相关基因。
本发明提供的诱导CD8+ T细胞去磷酸化为CD8+ Tcm细胞的vMIP-Ⅱ具有如下机制：当vMIP-Ⅱ治疗时，CD8+ T细胞主要通过下调与磷酸化相关的信号通路，包括低表达CD8+ T细胞G蛋白水平、降低细胞Ca2+浓度和线粒体膜电位等，使CD8+ T细胞发生代谢重编程，并通过抑制磷酸化相关基因GNAT1、PI3K、ERK、AKT、BCL-2使CD8+ T细胞磷酸化蛋白ERK1/2和Akt低表达，从而使CD8+ T去磷酸化为CD8+ Tcm细胞，促进CD8+ Tcm细胞增殖。
本发明提供了一种诱导CD8+ T细胞去磷酸化为CD8+ Tcm细胞的vMIP-Ⅱ的作用机制，其可用于制备治疗HIV/SIV感染艾滋病的药物，为抗病毒与抗肿瘤的过继性免疫以及验证反应的预防或/和治疗提供新的手段。
相对于现有技术，本发明经实验发现，vMIP-Ⅱ可以促使淋巴组织增生，淋巴小结中间的生发中心增大。同时vMIP-Ⅱ还能促使CD8+ Tcm细胞增殖，该增殖与CD8+ T细胞表面的趋化因子受体CCR7、CXCR4、CXCR5和CX3CR1的共同作用相关。当vMIP-Ⅱ治疗时，CD8+ T细胞表面的趋化因子受体CCR7、CXCR4、CXCR5和CX3CR1封闭，引起与磷酸化相关的信号通路下调，包括低表达CD8+ T细胞G蛋白水平、降低细胞Ca2+浓度和线粒体膜电位等，使CD8+ T细胞发生代谢重编程，并通过低表达磷酸化基因GNAT1、PI3K、ERK、AKT、BCL-2使CD8+ T细胞去磷酸化为CD8+ Tcm细胞，从而促进CD8+ Tcm细胞增殖。vMIP-Ⅱ的作用机制的这一发现为HIV/SIV感染艾滋病的药物研发提供了全新策略，也为抗病毒与抗肿瘤的过继性免疫治疗提供新的手段。
附图说明
图1为实施例1 vMIP-Ⅱ(800 μg/kg)连续静脉注射13周后恒河猴淋巴组织增生病理组织切片图(注：A为淋巴结滤泡病理切片；B为胸腺滤泡病理切片；C为脾白髓病理切片)。
图2为实施例1不同vMIP-Ⅱ剂量组对血浆病毒载量的影响(注：与阴性对照组相比，*P＜0.05；**P＜0.01；Mean ± SD，n = 4)。
图3为实施例1感染后不同时间点vMIP-Ⅱ诱导CD8+ T细胞增殖的折线图(注：与阴性对照组相比，*P＜0.05；**P＜0.01；Mean ± SD，n = 4)。
图4为实施例1对感染治疗18天不同剂量组CD8+ T细胞的流式细胞仪分析图。
图5为实施例1 PBMC效应CD8+ T细胞的表型(Control为未用SIVmac251感染的对照组；SIVmac251为用SIVmac251感染的试验组)。
图6为实施例1 PBMC效应CD8+ T细胞趋化因子受体表达情况(Control为未用SIVmac251感染的对照组；SIVmac251为用SIVmac251感染的试验组)。
图7为实施例1 PBMC效应CD8+ T细胞归巢受体表达情况。
图8为实施例1差异表达基因MA图。
图9为实施例1 KEGG通路分析(注：bar越长，富集度越高；bar越短，富集度越低)。
图10为实施例1差异表达基因的qRT-PCR验证结果(注：上述数值均为Mean ± SD，n=3；与未用vMIP-Ⅱ治疗组相比，**p＜0.01和***p＜0.001)。
图11为实施例1 Western blot检测vMIP-Ⅱ对胞内G蛋白表达的影响。
图12为实施例1 vMIP-Ⅱ对细胞内钙流的影响。
图13为实施例1流式细胞术分析钙流峰值。
图14为实施例1 vMIP-Ⅱ导致效应CD8+ T细胞线粒体膜电位下降。
图15为实施例1效应CD8+ T细胞磷酸化总体水平。
图16为实施例1 vMIP-Ⅱ对MAPK/ERK、Akt磷酸化通路的影响。
具体实施方式
以下结合实施例，对本发明进行进一步详细说明。
实施例1重组病毒巨噬细胞炎性蛋白诱导效应CD8+ T细胞去磷酸化为长寿记忆细胞
1材料与方法
1.1 实验材料
实验动物：SPF级Mamu-A*01健康成年中国恒河猴20只，5~9 kg，雌雄各半，购自北京中国医学科学院医学生物科学研究所。血清测试呈SIV、SRV、STLV-1和HBV反应阴性。采用如前所述的基于序列特异性引物聚合酶链反应(PCR)的MHC分型方法，以确定恒河猴中存在MAMU-A*01主要组织相容性复合体(MHC)Ⅰ类等位基因。本动物实验参照“实验动物护理和使用指南”(美国国家科学院出版社，华盛顿特区出版)中规定的标准进行。
病毒和细胞：SIVmac251在人外周血单个核细胞(PBMC)中增殖。病毒滴度滴定显示接种的SIV在人PBMC中大约包含50 TCID50/ml。
实验试剂：vMIP-Ⅱ注射用原液和冻干粉针剂，vMIP-Ⅱ抗原标准品(理化对照品)由暨南大学基因组药物研究所研制并通过国家药品和生物制品检定。vMIP-Ⅱ单克隆抗体、猴CD3、CD4、CD8单克隆抗体购自美国R&D公司。抗CD8抗体-APC(allophycocyanin，别藻蓝蛋白)、抗CD3抗体-FITC (fluorescein isothiocyanate，异硫氰酸荧光素)、抗CD4抗体-ECD、抗CCR7抗体-PE(phycoerythrin，藻红蛋白)等，均购自Biolegend公司；Real-time PCRMaster Mix试剂盒(日本TOYOBO公司)；BD FACS流式细胞仪、荧光定量PCR系统(美国Bio-Rad公司)；人CC趋化因子受体7 (CCR7) ELISA试剂盒；1640完全培养基、二硫苏糖醇(DTT)；1 mmol/L乙二胺四乙酸(EDTA)等。
感染和动物分组给药方法
用50 TCID50剂量的SIVmac251静脉注射接种恒河猴。在感染前，将Mamu-A*01阳性恒河猴20只随机分为5组(每组4只)。第Ⅰ组为阴性未治疗对照组，即动物静脉注射9 g/L的NaCl；第Ⅱ组为50 μg/kg剂量组；第Ⅲ组为200 μg/kg剂量组；第Ⅳ组为800 μg/kg剂量组；第Ⅴ组为阳性治疗对照组，静脉注射(AZT+3TC) (Glaxo，AZT 100 mg/kg，3TC 50 mg/kg)。其中Ⅱ、Ⅲ、Ⅳ组分别静脉注射相应剂量的vMIP-Ⅱ(由本实验室制备，在大肠杆菌中表达并纯化。SIVmac251感染后第3~4周，实验动物按照分组分别给药，每天一次，连续两周。然后停药一周，第6~7周重复用药两周。
猴PBMC、血浆的制备
猴外周血样品经EDTA处理后，如前所述的用梯度离心法分离PBMCs，于-80℃储存；血浆分离按常规方法自收集后3 h内的外周血样品中制备，冻存于-80℃。
淋巴组织病理学检查
全面细致观察并记录淋巴组织的肉眼变化并作常规病理切片检查。淋巴组织包括淋巴结、脾脏、胸腺和小肠黏膜。
病毒载量分析
将冻存的血浆在Ficoll密度梯度离心后从上层收集血浆样品。使用超灵敏分支DNA扩增测定法(Bayer Diagnostics，Berkeley，CA)测量病毒RNA水平。检测下限为200拷贝每毫升。
四聚体染色和效应CD8+ T淋巴细胞亚群的分选
特异性肽p11C (CTPYDINQM)由New England Peptide LLC合成，并且如所述那样制备Mamu-A*01/p11C四聚体复合物。将PE偶联的Mamu-A*01/p11C复合物与抗CD8-FITC(BectonDickinson，San Jose，CA)、抗CD4-ECD(Beckman Coulter，Miami，FL)和抗恒河猴CD3-APC(Dako，Glostrup，Denmark)一起使用，对所有动物外周血单个核细胞(PBMCs)进行染色。在位于专用BSL-3区域的Coulter EPICS Elite ESP (Beckman Coulter)上进行分选，以分别获得各个动物的CD8+ T淋巴细胞亚群。该分选器通过电子方式设置，以达到>98％的所选细胞亚群的富集。在每次流式细胞术分析中均包括由SIV感染的Mamu-A*01阴性或未感染的Mamu-A*01阳性动物组成的阴性对照。
+细胞分选
对从各组动物PBMC中分选获得的效应CD8+ T细胞进行抗体标记(抗CD8抗体-APC、抗CD45RA抗体-FITC和抗CCR7抗体-PE)，实验中添加gp120抗原肽，然后立即用流式细胞术检测CD8+ Tcm细胞和CD8+ Tem细胞所占的比例。根据实验结果确定vMIP-Ⅱ治疗下效应CD8+ T细胞与CD8+ Tcm比例最高的剂量组，然后确定将该组动物用于后续验证实验。
效应CD8+ T细胞培养
将分选出来的各组效应CD8+ T细胞中纯度较高的效应CD8+ T细胞悬浮于含10%胎牛血清的RPMI-1640培养基中，同时加入gp120抗原肽，然后均匀铺于96孔板中(每孔细胞数为1×105)；将细胞培养板置于5% CO2、饱和湿度和37 ℃的细胞培养箱中培养。
转录组测序由上海康成生物科技有限公司完成。测序平台为Illumina Hiseq2500 V4，测序模式为125PE，样品为800 μg/kg vMIP-Ⅱ治疗组的效应CD8+ T & Tcm细胞亚群的RNA-seq文库。RNA-seq文库的构建：总RNA经过利用Oligo (dT)富集RNA，将RNA随机打断成200 nt，随机引物六聚体反转录成cDNA，进行末端修复，加A，加接头后PCR扩增实现文库的构建。
按照Illumina标准进行样本文库混合，制备Cluster：复制链其一端固定在芯片上，另一端随机与附近的另一个引物互补被固定，形成“桥”。形成的桥单链以周边的引物为扩增引物，在芯片的表面扩增而变成双链，然后经过变性而形成单链，于是又会再次形成桥，即可进行下一轮的扩增反应，扩增若干次后，每个单分子就都能得到大量扩增，形成Cluster。
将得到的数据结果按照质量控制标准去掉包含接头的短序列，去掉N的比例> 10%的短序列，同时也去掉低质量的短序列，最终得到的数据(Q30 > 85%)，用于后续分析。
基因差异表达分析
800 μg/kg vMIP-Ⅱ治疗组和未用vMIP-Ⅱ治疗组的效应CD8+ T & Tcm细胞亚群样品的基因差异表达分析采用DESeq软件进行。结果的P值均使用Benjamini和Hochberg的方法控制错误发现率并进行调整。调整后的P值< 0.01且差异表达倍数＞2 (|log2|＞1)即被DESeq筛选出并标记为差异表达基因。
差异表达基因Gene Ontology (http://www.geneontology.org/)功能富集分析采用GOseq R软件包进行。KEGG是了解和利用生物系统如细胞、生物体和生态系统的高级功能的数据库资源，从分子水平信息，特别是由基因组测序和其他高通量实验技术产生的大规模分子数据集(http://www.genome.jp/kegg/)中挖掘信息。KEGG通路中差异表达基因的统计富集分析采用KOBAS软件进行。分析差异表达基因在某一通路上是否过出现(over-presentation)即为差异表达基因的Pathway富集分析，并利用富集因子(EnrichmentFactor)分析Pathway的富集程度。
荧光定量PCR
为了验证RNA-Seq数据的准确性，我们挑选部分差异表达基因，对其表达量进行了相对荧光定量PCR分析。将分选出的效应CD8+ T细胞和CD8+ Tcm细胞置于5%的CO2细胞培养箱中，37 ℃下培养24 h。根据细胞总RNA提取试剂盒操作说明进行效应CD8+ T细胞和Tcm细胞总RNA提取，然后使用SuperScriptTM Preamplification System for First Strand cDNASynthesis试剂盒进行cDNA合成。荧光定量PCR反应的所有引物序列列于表1中，反应按照操作手册说明的方法在荧光定量PCR仪：MiniOpticonTM (BIO-RAD, Laboratories，Inc. USA)系统中进行。反应总体积25.0 μL，包括总RNA 2.0 μL，RNase-free H2O 8.5 μL，正向和反向引物(10 mM/L)各0.5 μL，2×One-step SYBR RT-PCR Buffer Ⅲ 12.5 μL，TaKaRa ExTaq HS(5 U/μL) 0.5μL，以及PrimeScript RT Enzyme Mix Ⅱ 0.5μL。PCR反应条件为95℃ 5 min，95 ℃ 30 s，60 ℃ 45 s，72 ℃ 45 s，共33个循环。所有实验均进行3次独立的生物学重复。标准的相对转录水平用2-ΔΔCt法估算。
表1 荧光定量PCR反应的引物序列
1.12 PBMC效应CD8+ T细胞表型测定
分别采集上述确定的剂量组和对照组恒河猴PBMC 2 ml，EDTA抗凝后加入淋巴细胞分离液，密度梯度离心后收集中间悬浮的细胞，用免疫荧光抗体分别标记CD3、CD4、CD8、CD44、CD45、CD69，及趋化因子受体CCR3、CCR4、CCR5、CCR7、CXCR2、CXCR3、CXCR4、CXCR5、XCR1、CX3CR1，归巢受体α4β7、CD62L。然后分别孵育免疫荧光抗体20 min，加入破膜剂后继续孵育5 min，最后在流式细胞仪上测定。
检测G蛋白α的表达
将800 μg/kg剂量组动物PBMC分选所得的效应CD8+ T细胞经超声破碎后，离心10 min，收集上清液，弃沉淀。将上清液进行SDS-PAGE电泳，获得电泳条带后，剪取Tcm、CCR7对应的条带转膜，将膜用1×丽春红染液染5 min，水洗后将膜晾干备用。将膜用TBS从下向上浸湿后，移至含有封闭液的平皿中，室温下脱色摇床上摇动封闭1 h。将G蛋白α一抗用TBST稀释至适当浓度并加至膜上；室温下孵育1~2 h后，用TBST在室温下脱色摇床上洗两次，每次10min；再用TBS洗一次，10 min。接着准备二抗稀释液并与膜接触，室温下孵育1~2 h后，用TBST在室温下脱色摇床上洗两次，每次10 min；再用TBS洗一次，10 min，进行化学发光反应。将胶片进行扫描或拍照，用凝胶图象处理系统分析目标带的分子量和净光密度值。
钙流实验
将5 mg/L聚羟基脂肪酸酯(PHA)加入从800 μg/kg剂量组动物PBMC分选所得的效应CD8+ T细胞悬液，于37℃、50 ml/L CO2中培养24 h，按实验分组分别加入Fractalkine或vMIP-Ⅱ，4℃处理30 min，加入Fluo-3 (6×10-3 mol/ml)探针，室温避光孵育30 min后用PBS洗涤，过滤上样流式细胞仪，激发波长488 nm，检测波长530 nm，每管样品检测1×104个细胞，获取数据用CELLQuest分析结果。
流式细胞术检测细胞线粒体膜电位水平
取1×106个细胞，重置于0.5 ml细胞培养液中，加入0.5 ml JC-1染色工作液，颠倒数次混匀。细胞培养箱中37 ℃孵育20 min。在孵育期间，按照每1 ml JC-1染色缓冲液(5×)加入4 ml蒸馏水的比例，配置适量的JC-1染色缓冲液(1×)，并放置于冰浴。孵育结束后，600 g 4 ℃离心3~4 min，沉淀细胞，弃上清。再加入1 ml JC-1染色缓冲液(1×)重悬细胞，600 g 4 ℃离心3~4 min，沉淀细胞，弃上清。再加入适量JC-1染色缓冲液(1×)重悬后，立刻使用流式细胞仪检测分析。
悬液芯片系统检测效应CD8+ T细胞的磷酸化总体水平
根据基因芯片结果显示，效应CD8+ T &Tcm细胞两细胞亚群存在明显的去磷酸化，因此，我们取效应CD8+ T细胞和CD8+ Tcm细胞混合亚群进行Bio-Plex悬液芯片系统检测，以验证其磷酸化总体水平变化情况。
实验流程按照Bio-Plex pro assay说明书进行。(1) 打开Bio-Plex System系统并校正。真空吸板机的真空度调节为1~2 Hg压力，用96孔板盖住。(2) 将标准品稀释后加入96孔板中，然后加入微珠。轻轻盖上封板膜和铝簿，使每个小孔完全遮住。室温下以1100rpm/s下振动30 s，再以600 rpm/s下振动30 min。(3) 振动结束后，以100 μL Bio-PlexAssay Buffer润湿滤板，轻轻打开封板膜和铝簿，用100 μL Bio-Plex wash buffer洗2次，吸去buffer，使之完全吸净以防止交叉污染。(4) 每孔加入50 μL标准品，轻轻盖上封板膜和铝薄，使每个小孔完全遮住。室温下以1100 rpm/s下振动30 s，再以600 rpm/s下振动30min。然后将封板膜和铝薄打开，过程中要避免溅出，真空抽去buffer，再100μL Bio-Plexwash buffer洗2次，吸去buffer，使之完全吸净以防止交叉污染。(5) 漩涡振动检测抗体数秒，每孔加入detection antibodies 25 μL，轻轻盖上封板膜和铝簿，使每个小孔完全遮住。室温下以1100 rpm/s下振动30 s，再以600 rpm/s下振动30 min。然后将封板膜和铝薄打开，过程中要避免溅出，真空抽去buffer，再100 μL Bio-Plex wash buffer洗2次，吸去buffer，使之完全吸净以防止交叉污染。(6) 漩涡混合Streptavidin-PE荧光色素，每孔加入50μL，轻轻盖上封板膜和铝簿，使每个小孔完全遮住。室温下以1100 rpm/s下振动30 s，再以600 rpm/s下振动30 min。然后将封板膜和铝薄打开，过程中要避免溅出，真空抽去buffer，再100μL Bio-Plex wash buffer洗2次，吸去buffer，使之完全吸净以防止交叉污染。(7) 每孔加入125μL Bio-Plex assay buffer微珠重悬，轻轻用密封袋盖住，1100 rpm/s室温摇床30秒后立即放入仪器中进行读盘检测。
检测磷酸化蛋白表达
将800 μg/kg剂量组动物PBMC分选所得的效应CD8+ T细胞在0.1%牛血清RPMI 1640培养液饥饿1 h，加入或不加CCL21 (200 ng/ml)进行刺激，收集细胞后用冷PBS洗3次，细胞裂解后抽提总蛋白，Bradford法定量，加热变性，等量蛋白上样行SDS-PAGE。电转至PVDF膜，用TBS配制的5%脱脂奶粉封闭过夜。加入兔抗磷酸化Akt (1:1000)、Akt (1:1000)、ERK1/2(1:1000)一抗室温孵育2 h，TTBS(TBS加入1‰ Tween-20)洗膜，羊抗兔IgG二抗(1:2000)室温孵育1 h，洗膜后经ECL放射自显影，曝光于Kodak胶片。以Akt和ERK2为参照，用图像分析软件GELpro3.0对条带吸光度值进行分析。
统计分析
使用Prism软件在三个或更多个生物学重复样品上测定除WGBS分析之外的所有体内和体外研究的统计学显著性。使用双尾t检验确定P值。*P<0.05；**P<0.01。
结果与分析
2.1 vMIP-Ⅱ长期静脉注射对恒河猴血液指标的影响
在观察期间，各组动物的一般状况、体温、心电图、尿常规和血液生化指标均无明显异常。外周血成分检查显示：低剂量组动物各项指标均在正常范围内；与阴性对照组相比，中、高剂量组动物外周血淋巴细胞总数和分类在给药6、13周时显著性增高(P<0.05)，恢复期2周中剂量组恢复至正常范围，如表1所示。
表1 vMIP-Ⅱ连续给药13周期间恒河猴外周血淋巴细胞总数的改变(Mean ± SD,n = 4,109 L-1）
注：*表示与阴性对照组相比，P＜0.05；**表示与阴性对照组相比，P＜0.01。
Ⅱ长期静脉注射引起的恒河猴淋巴器官组织学改变
vMIP-Ⅱ连续静脉注射13周后，我们发现淋巴器官出现明显的增生，且呈现剂量依赖效应，即：50 μg/kg剂量组增生较少；200 μg/kg剂量组淋巴结淋巴滤泡增生；800 μg/kg剂量组动物淋巴结淋巴滤泡、脾白髓、胸腺、小肠黏膜固有层淋巴滤泡均出现增生，后者并伴有较明显的中心扩大，见图1。恢复期第2周与第13周相比，增生程度显著性减轻。
Ⅱ对血浆病毒载量的影响
在整个研究期间或直至各动物死亡时监测血浆病毒载量。如图2所示，虽然所有20只恒河猴均显示出可变的血浆病毒载量，但急性血浆病毒血症的峰值相当，而且50 μg/kg、200μg/kg和800 μg/kg剂量组动物的血浆病毒载量测定值均普遍低于阴性对照组动物血浆中的载量，并且50 μg/kg、200 μg/kg和800 μg/kg剂量组动物的血浆病毒载量与vMIP-Ⅱ呈依赖关系，但略高于AZT+3TC阳性对照组。血浆病毒载量结果显示趋化因子受体抑制剂，尤其是vMIP-Ⅱ的施用有助于控制体内SIV复制。
Ⅱ对四聚体阳性CD8+ T淋巴细胞亚群扩增的影响
使用MHCⅠ类四聚体和荧光激活细胞分选染色检测四聚体阳性CD8+ T细胞。正如所料，感染前Mamu-A*01阳性动物的Mamu-A*01/p11C四聚体结合细胞检测不到(<0.1％)(图3)。但感染后，来自50 μg/kg、200 μg/kg和800 μg/kg剂量组恒河猴的PBMC显示Mamu-A*01/p11C四聚体结合CD8+ T细胞水平显着增加，且CD8+ T细胞的增殖与vMIP-Ⅱ治疗剂量存在正比关系，与流式细胞仪图谱分析所得结果一致(图4)。相反，在阴性对照组和AZT+3TC组动物中，四聚体结合CD8+ T细胞的水平较低，CD8+ T细胞仅为2％左右，且AZT+3TC组动物CD8+ T细胞数量略低于阴性对照组。表明AZT+3TC引起病毒载量降低并不是通过增殖CD8+ T细胞来实现的。这些数据表明趋化因子受体抑制剂vMIP-Ⅱ可通过保持SIV表位特异性CD8+ T细胞的扩增来控制SIV复制。
效应CD8+ T细胞的表型特征
取恒河猴PBMC制备单细胞悬液，染色后通过流式细胞仪检测效应CD8+ T细胞表面分子CD44、CD69、CD103、CD62L和CCR7的表达水平。结果显示，SIVmac251感染后，恒河猴PBMC效应CD8+ T细胞高表达CD62L(94.6%)、CD45RO(90.2%)、CCR7(79.8%)，低表达CD44(7.9%)、CD69(3.3%) (图5)。该结果说明PBMC效应CD8+ T细胞主要为中枢记忆型T细胞，即Tcm细胞。
效应CD8+ T 细胞趋化因子受体和归巢受体的表达情况
基于趋化因子受体和归巢受体的表达与细胞募集密切相关，本实验采用流式细胞术对SIVmac251感染恒河猴PBMC效应CD8+ T细胞的趋化因子受体和归巢受体进行检测。结果显示与对照组相比，感染组恒河猴PBMC效应CD8+ T细胞高表达趋化因子受体CXCR5、CXCR4、CX3CR1和CCR7，具有统计学差异(图6)。归巢受体α4β7和L-selectin在PBMC效应CD8+ T细胞的表达相对较低，并且与对照组相比没有显著的统计学差异(图7)。这说明PBMC效应CD8+ T细胞主要是通过趋化因子受体募集到PBMC的而不是通过归巢受体。
+和CD8+ Tem细胞纯度和所占比例分析
在感染治疗18天时，对上述使用MHCⅠ类四聚体和荧光激活细胞染色分选所得的各组效应CD8+ T细胞分别进行抗体标记，分别检测其中CD8+ Tcm细胞和CD8+ Tem细胞所占的比例。结果表明(表2)，在未用vMIP-Ⅱ治疗时，CD8+ Tcm细胞含量较少，仅占2.33%；但在vMIP-Ⅱ治疗的Ⅱ、Ⅲ和Ⅳ组中，CD8+ Tcm细胞发生明显的增殖，并存在剂量依赖性。而且，我们发现CD8+ Tcm细胞增殖的同时，CD8+ T细胞的纯度在增加，而CD8+ Tem细胞的含量呈现出减少的趋势。这些数据显示出CD8+ Tcm细胞的增殖与效应CD8+ T细胞存在某种必然的联系，但还需作进一步的研究。因此，我们认为vMIP-Ⅱ可以诱导效应CD8+ T细胞向CD8+ Tcm细胞分化，以增强细胞和机体的免疫。
表2 感染治疗18 d后CD8+ T细胞的纯度以及CD8+ Tcm和CD8+ Tem所占的比例(%.Mean ± SD. n = 4)
| Treatment | Purity of effector CD8<sup>+</sup> T cells | Percentage of CD8<sup>+</sup> Tcm | Percentage of CD8<sup>+</sup> Tem |
| Negative control | 98.36 ± 0.42 | 2.33 ± 0.54 | 46.29 ± 1.18 |
| 50 μg/kg | 98.72 ±0.33 | 3.02 ± 0.75 | 59.73 ± 2.17 |
| 200 μg/kg | 99.01 ± 0.61 | 3.95 ± 0.12* | 68.17 ± 1.44 |
| 800 μg/kg | 99.38 ± 0.43 | 5.69 ± 0.44* | 50.19 ± 2.55 |
| AZT+3TC | 97.99 ± 0.56 | 2.19 ± 0.48 | 44.33 ± 0.62 |
注：与阴性对照组相比，*P＜0.05
2.8 RNA-seq测序检测结果
2.8.1 组间差异表达基因
我们应用RNA-seq测序检测800 μg/kg vMIP-Ⅱ治疗组和未用vMIP-Ⅱ治疗组的效应CD8+ T & Tcm细胞亚群基因的差异表达，通过对比，根据筛选标准共筛选出79个显著差异基因(差异倍数≥2，p＜0.01)，其中包括48个基因显著上调，31个基因显著下调(图8)。
差异表达基因GO本体分析
我们对效应CD8+ T & Tcm细胞亚群进行分析，在800 μg/kg vMIP-Ⅱ治疗组和未用vMIP-Ⅱ治疗组之间确定了97个显著差异基因，对这些基因进行GO本体分析，结果显示(表3、4、5)：这些基因的生物过程(Biological process)主要集中在细胞分化(Celldifferentiation)、细胞凋亡(Apoptosis)、细胞定位和代谢(Cell localization andmetabolism)以及磷酸化过程(Phosphorylation process)等；细胞组分(Cellularcomponent)主要集中在线粒体(Mitochondrion)、细胞质(Cytoplasm)、内质网(Endoplasmic reticulum)、膜组分(Membrane component)和细胞器(Organelle)等；分子功能(Molecular function)主要包括信号通路分子(Signaling pathway molecule)、核酸结合(Nucleic acid binding)、跨膜受体调节蛋白(Transmembrane receptor regulatoryprotein)、序列特异性DNA绑定(Sequence-specific DNA binding)和转录因子活性(Transcription factor activity)等。由此可知vMIP-Ⅱ在感染SIVmac251病毒的机体免疫反应中主要参与调控效应CD8+ T细胞凋亡和磷酸化途径分化为CD8+ Tcm细胞的相关过程，尤其是对磷酸化信号通路的影响最为显著，同时差异表达基因在信号通路分子和细胞质的大量富集，也从另一方面说明了上述观点。
表3 差异表达基因的GO分析-生物过程富集分析
Table 3 Gene ontology analysis for significantly altered genes-biologicalprocess
| Biological Progress | Count | P-value |
| Phosphorylation process | 20 | 3.3E-06 |
| Cell apoptosis | 15 | 4.8E-04 |
| Cell differentiation | 11 | 6.1E-04 |
| Glucose metabolic process | 9 | 1.2E-03 |
| Cell localization and metabolism | 9 | 3.4E-03 |
| Regulation of transcription, DNA-templated | 9 | 5.3E-03 |
| Regulation of mitophagy | 7 | 4.1E-03 |
| Cellular response to mechanical stimulus | 6 | 2.8E-02 |
| Systerm process | 6 | 3.2E-02 |
| Transcription from RNA polymerase II promoter | 5 | 3.8E-02 |
表4 差异表达基因的GO分析-细胞组分富集分析
Table 4 Gene ontology analysis for significantly altered genes-cellularcomponent
| Cellular Component | Count | P-value |
| Cytoplasm | 26 | 5.3E-03 |
| Intracellular membrane-bounded organelle | 16 | 2.8E-04 |
| Endoplasmic reticulum | 12 | 4.5E-04 |
| Mitochondrion | 12 | 6.2E-04 |
| Extracellular exosome | 11 | 3.1E-03 |
| Nucleoplasm | 10 | 3.3E-03 |
| ER to Golgi transport vesicle membrane | 6 | 1.2E-02 |
| RNA polymerase II transcription factor complex | 6 | 3.1E-02 |
| Receptor complex | 5 | 2.8E-02 |
| Peroxisome | 4 | 6.1E-02 |
表 5 差异表达基因的GO分析-分子功能富集分析
Table 5 Gene ontology analysis for significantly altered genes-molecularfunction
| Molecular function | Count | P-value |
| Protein binding | 28 | 8.5E-06 |
| Sequence-specific DNA binding | 11 | 2.0E-05 |
| Transcription factor activity | 10 | 1.3E-05 |
| DNA binding | 10 | 3.1E-04 |
| Identical protein binding | 8 | 2.8E-04 |
| Transcription regulatory region DNA binding | 7 | 4.6E-04 |
| Cytokine activity | 6 | 4.9E-03 |
| Receptor binding | 6 | 1.5E-02 |
| Oxidoreductase activity | 6 | 3.6E-02 |
| Steroid hormone receptor activity | 5 | 1.7E-02 |
| RNA polymerase II transcription factor activity | 5 | 7.9E-02 |
2.8.3 差异表达基因KEGG通路分析结果
我们对差异基因进行了在线KEGG生物通路注释，发现它们主要富集与细胞凋亡通路、能力代谢(TCA循环)通路、磷酸化通路以及一些细胞信号通路等(图9)。通过综合分析效应CD8+ T细胞和CD8+ Tcm细胞在机体中的成分和比例以及基因芯片通路分析，我们得出差异表达基因最富集的通路是磷酸化信号通路。
确定候选差异表达基因
根据GO本体分析的结果，磷酸化途径、TCA循环和调控细胞凋亡是差异表达基因最显著富集的三个GO-term，我们将这3组生物过程中涉及基因的差异表达倍数按照从大到小进行降序排列，依次筛选差异倍数＞3(即P-value＜0.001；log2Ration＞1.5)的差异基因，最后确定了GNAT1、PI3K、ERK、AKT、NF-κB、BCL-2、FAS、PUMA、BAX、p53AIP1这10个基因作为我们后续研究的重点目标基因。
反向验证
我们对这10个基因用qRT-PCR进行反向验证，结果如图10所示，相比未用vMIP-Ⅱ治疗的效应CD8+ T & Tcm细胞组，用800 μg/kg vMIP-Ⅱ治疗组中GNAT1、PI3K、ERK、AKT、BCL-2的表达量均明显减少，而NF-κB、BAX、FAS、PUMA、p53AIP1的表达明显增加，这与RNA-seq的测序结果一致。
Ⅱ对效应CD8+ T细胞内G蛋白受体α的影响
通过基因芯片和qRT-PCR，我们的结果显示vMIP-Ⅱ治疗组可影响效应CD8+ T细胞/CD8+ Tcm细胞的G蛋白受体α。因此，我们进行Western blot检测效应CD8+ T细胞内G蛋白受体α的表达情况，以确定vMIP-Ⅱ对效应CD8+ T细胞内G蛋白受体α是否有影响。根据Westernblot结果(图11)，与未用vMIP-Ⅱ治疗组相比，vMIP-Ⅱ治疗后的效应CD8+ T细胞内G蛋白受体α的表达明显抑制，表明vMIP-Ⅱ存在时，能抑制效应CD8+ T细胞内的G蛋白受体α的表达。
Ⅱ对钙离子信号通路的影响
相对于正常对照组，vMIP-Ⅱ不仅不能明显诱导效应CD8+ T细胞内钙离子浓度的升高，反而明显诱导效应CD8+ T细胞内钙离子浓度降低(P＜0.05)。同时，相对于阳性对照组，vMIP-Ⅱ预处理细胞可使Fractalkine诱导的钙离子浓度明显下降(P<0.01，图12、13)。
Ⅱ对效应CD8+ T细胞线粒体膜电位的影响
vMIP-Ⅱ对效应CD8+ T细胞线粒体膜电位的影响见图14。JC-1荧光染料聚集在线粒体的基质中，在线粒体膜电位较高时，产生红色荧光；当细胞线立体膜电位下降时，JC-1开始变为单体，并产生绿色荧光。对从800 μg/kg剂量组恒河猴分选出的效应CD8+ T细胞进行线粒体膜电位分析，与对照组相比，vMIP-Ⅱ治疗后的效应CD8+ T细胞线粒体膜电位下降极显著(P＜0.01)。
悬液芯片系统检测磷酸化总体水平
如图15所示，在未用vMIP-Ⅱ治疗组和用800 μg/kg vMIP-Ⅱ治疗组的效应CD8+ T细胞磷酸化总体水平中，两组之间的差异有统计学意义(P＜0.01)。因此，vMIP-Ⅱ可引起效应CD8+ T细胞磷酸化总体水平下降，即发生去磷酸化，与RNA-seq所得结果一致。
磷酸化通路分析
自800 μg/kg剂量组恒河猴效应CD8+ T细胞在0.1%牛血清培养液饥饿1 h，加入或不加CCL21 (200 ng/ml)刺激，以未用vMIP-Ⅱ治疗的阴性对照组作对照，分别于刺激60 min检测磷酸化ERK1/2、Akt 蛋白表达。图16结果显示，只在CCL21作用下，CD8+ Tcm细胞可检测到高水平磷酸化ERK1/2和Akt。而当在vMIP-Ⅱ存在时，磷酸化ERK1/2和Akt表达水平显著降低。这表明vMIP会抑制磷酸化ERK1/2和Akt蛋白的表达，而且vMIP-Ⅱ能够拮抗CCL21对ERK1/2和Akt 的活化。
Claims (9)
1.一种诱导CD8+ T细胞去磷酸化为CD8+ Tcm细胞的病毒巨噬细胞炎性蛋白vMIP-Ⅱ，其特征在于，所述的病毒巨噬细胞炎性蛋白vMIP-Ⅱ包括可诱导CD8+ T细胞去磷酸化为CD8+Tcm细胞的vMIP活性成分，还包括其可药用的盐和酯、选择性取代的类似物或者包含vMIP-Ⅱ的一种或多种化合物的组合，还包括vMIP-Ⅱ的衍生物，或者其衍生物在药学上可接受的介质或载体。
2.权利要求1所述能诱导CD8+ T细胞去磷酸化为CD8+ Tcm细胞的病毒巨噬细胞炎性蛋白vMIP-Ⅱ的应用，其特征在于，所述的vMIP-Ⅱ作用机制包括vMIP-Ⅱ通过封闭CD8+ T细胞表面趋化因子受体CCR7、CXCR4、CXCR5和CX3CR1，以降低其下游信号通路的靶标分子。
3.权利要求1所述vMIP-Ⅱ诱导CD8+ T细胞去磷酸化为CD8+ Tcm细胞的作用机制，其特征在于，vMIP-Ⅱ在恒河猴SIV感染模型中能促使淋巴组织增生，淋巴小结生发中心扩大，病毒载量降低，CD8+ Tcm细胞增殖。
4.权利要求3所述vMIP-Ⅱ诱导CD8+ T细胞去磷酸化为CD8+ Tcm细胞而引起CD8+ Tcm细胞增殖，其特征在于，vMIP-Ⅱ诱导增殖的CD8+ Tcm细胞在基因测序中与CD8+ T细胞的差异表达基因主要富集于表面趋化因子受体CCR7、CXCR4、CXCR5和CX3CR1和磷酸化通路相关基因。
5.权利要求1所述vMIP-Ⅱ诱导CD8+ T细胞去磷酸化为CD8+ Tcm细胞的作用机制，其特征在于，vMIP-Ⅱ封闭CD8+ T细胞的CCR7、CXCR4、CXCR5和CX3CR1通过下调其与磷酸化相关的信号通路，包括低表达CD8+ T细胞G蛋白水平、降低细胞Ca2+浓度和线粒体膜电位等，使CD8+ T细胞发生代谢重编程，并通过抑制磷酸化相关基因GNAT1、PI3K、ERK、AKT、BCL-2使CD8+ T细胞磷酸化蛋白ERK1/2和Akt低表达，从而使CD8+ T发生重编程而去磷酸化为CD8+Tcm细胞，促进CD8+ Tcm细胞增殖。
6.权利要求1所述的vMIP-Ⅱ诱导CD8+ T细胞去磷酸化为CD8+ Tcm细胞，其特征在于，vMIP-Ⅱ在HIV/SIV感染艾滋病的药物研发或在抗HIV/SIV等病毒与抗肿瘤的过继性免疫预防或/和治疗药物中的应用。
7.一种诱导CD8+ T细胞去磷酸化为CD8+ Tcm细胞的病毒巨噬细胞炎性蛋白vMIP-Ⅱ，其特征在于，包括有效剂量的作为活性成分的权利要求5所述具有诱导CD8+ T细胞去磷酸化为CD8+ Tcm细胞作用机制的抗HIV/SIV感染艾滋病的药物成分的vMIP-Ⅱ。
8.一种诱导CD8+ T细胞去磷酸化为CD8+ Tcm细胞的病毒巨噬细胞炎性蛋白vMIP-Ⅱ，其特征在于，包括有效剂量的作为活性成分的权利要求5所述具有诱导CD8+ T细胞去磷酸化为CD8+ Tcm细胞作用机制的抗炎症反应的vMIP-Ⅱ药物。
9.一种诱导CD8+ T细胞转化为CD8+ Tcm细胞的病毒巨噬细胞炎性蛋白vMIP-Ⅱ，其特征在于，包括有效剂量的作为活性成分的权利要求5所述具有诱导CD8+ T细胞去磷酸化为CD8+Tcm细胞作用机制的抗肿瘤过继性免疫治疗的vMIP-Ⅱ药物。
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