CN110423723A - 一种外周血b细胞系的构建方法及其应用 - Google Patents
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Abstract
本发明公开了一种外周血B细胞系的构建方法及其应用，本发明中构建的BCL具有永生化生长的特点，BCL中CD19+细胞占比升高，且CD19+BCL中CD138、CD38、CD27表达水平显著高于EBV转化前的CD19+B细胞，且保留了B细胞分泌抗体、提呈抗原的能力。本发明中的BCL可用于筛选优势T细胞免疫反应抗原表位，或制备高效价anti‑HBsAg单克隆抗体，及诱导HBV特异性T细胞系，为“清除乙肝”提供新方向。
Description
技术领域
本发明涉及一种外周血B细胞系的构建方法及其应用，具体涉及一种可用于提呈HBV抗原肽的外周血B细胞系的构建方法及其应用。
背景技术
2013年全球疾病负担研究提示，肝炎病毒感染是全球第七大死亡原因，其中乙型肝炎病毒(Hepatitis B virus，HBV)和丙型肝炎病毒感染所致死亡占96％[1]。在HBV感染中，机体针对HBV的免疫可分为两部分，一为固有免疫反应，是免疫系统非特异性的免疫应答；二为适应性免疫反应，为T细胞和B细胞针对HBV抗原的特异性免疫应答。在慢性HBV感染中，HBV特异性T细胞的功能状态与病毒清除与否关系密切。其中，CD8+T细胞可通过细胞毒性作用清除病毒，以及通过产生IFN-γ、TNF-α等抗病毒细胞因子非细胞毒性杀伤感染的肝细胞[2,3]。此外，CD4+T细胞的功能也与患者疾病转归相关[4]。然而，慢性HBV感染者中T细胞呈现耗竭状态，表现为细胞毒性减弱[5]，分泌IL-2、IFN-γ、TNF-α的能力受损，抑制性共刺激信号分子表达水平升高[6]。因此，恢复T细胞功能可有助于慢性HBV感染者获得更好的临床结局。
既往研究HBV感染状态下T细胞功能通常将整个T细胞亚群作为整体来分析，精确靶向HBV特异性T细胞的研究较少。这是因为，本身特异性T细胞占外周血的频数低[7]，使研究常受限于标本量。其次，T细胞的活化需要抗原提呈细胞。我们的研究经验表示，冻融后复苏的标本，抗原提呈细胞活力差。而且慢性HBV感染时，T细胞处于功能耗竭状态，频数进一步减低，且在体外实验中对额外给予的刺激反应差，难以测出。因此，亟需发展高效的检验特异性T细胞功能的方法。
EB病毒(Epstein-Barr virus，EBV)转化外周血B细胞系(B cell lines，BCL)可作为B细胞的替代细胞用于提呈抗原。既往文献报道利用BCL提呈破伤风毒素(Tetanus，TT)给TT特异性T细胞克隆，发现BCL提呈作用具有MHC限制性，且相比于非特异性BCL，特异性BCL可更高效率提呈低浓度抗原[8,9]。另外，利用转基因技术构建表达巨细胞病毒(Cytomegalovirus，CMV)抗原的BCL，可以诱导同时针对CMV和EBV抗原的特异性T细胞免疫应答[10]。然而，既往对BCL的研究通常聚集于其提呈肿瘤细胞抗原或TT的能力，缺乏在炎症性疾病研究中的应用。
[1]Stanaway J D,Flaxman A D,Naghavi M,et al:The global burden ofviral hepatitis from 1990to 2013:findings from the Global Burden of DiseaseStudy 2013,Lancet,2016,388(10049):1081-1088.
[2]Thimme R,Wieland S,Steiger C,et al:CD8(+)T cells mediate viralclearance and disease pathogenesis during acute hepatitis B virus infection,JVirol,2003,77(1):68-76.
[3]Phillips S,Chokshi S,Riva A,et al:CD8(+)T cell control ofhepatitis B virus replication:direct comparison between cytolytic andnoncytolytic functions,J Immunol,2010,184(1):287-295.
[4]Ferrari C,Penna A,Bertoletti A,et al:Cellular immune response tohepatitis B virus-encoded antigens in acute and chronic hepatitis B virusinfection,J Immunol,1990,145(10):3442-3449.
[5]Wherry E J,Ha S J,Kaech S M,et al:Molecular signature of CD8+Tcell exhaustion during chronic viral infection,Immunity,2007,27(4):670-684.
[6]Boni C,Fisicaro P,Valdatta C,et al:Characterization of hepatitis Bvirus(HBV)-specific T-cell dysfunction in chronic HBV infection,J Virol,2007,81(8):4215-4225.
[7]Maini M K,Boni C,Ogg G S,et al:Direct ex vivo analysis ofhepatitis B virus-specific CD8(+)T cells associated with the control ofinfection,Gastroenterology,1999,117(6):1386-1396.
[8]Lanzavecchia A:Antigen-specific interaction between T and B cells,Nature,1985,314(6011):537-539.
[9]Lanzavecchia A,Bove S:Specific B lymphocytes efficiently pick up,process and present antigen to T cells,Behring Inst Mitt,1985,(77):82-87.
[10]Sun Q:Simultaneous Ex Vivo Expansion of Cytomegalovirus andEpstein-Barr Virus–Specific Cytotoxic T Lymphocytes Using B-LymphoblastoidCell Lines Expressing Cytomegalovirus pp65,transplantation,1999.
发明内容
本发明的目的在于提供一种可用于提呈HBV抗原肽的外周血B细胞系的构建方法及其应用。
本发明所采取的技术方案是：
一种外周血B细胞系的构建方法，其步骤包括：收集HBV感染者外周血，分离得到外周血单个核细胞；培养基重悬外周血单个核细胞，离心后弃上清；EBV上清重悬外周血单个核细胞后进行孵育，孵育3-4h后，加入含环孢霉素的培养基继续培养。
进一步地，EBV上清重悬外周血单个核细胞至0.5*10^6/ml～1*10^6/ml。
进一步地，培养基中环孢霉素的工作浓度为1μg/ml。
进一步地，培养基中环孢霉素的工作时间为2周。
上述的构建方法制备得到的外周血B细胞系。
上述的外周血B细胞系在筛选优势T细胞免疫反应抗原表位中的应用。
上述的外周血B细胞系在制备anti-HBsAg单克隆抗体中的应用。
一种anti-HBsAg单克隆抗体，由上述的外周血B细胞系制备得到。
上述的外周血B细胞系在诱导HBV特异性T细胞系中的应用。
一种单克隆T细胞系，由上述的外周血B细胞系诱导得到。
本发明的有益效果是：
本发明提供一种可用于提呈HBV抗原肽的外周血B细胞系，该BCL具有永生化生长的特点，BCL中CD19+细胞占比升高，且CD19+BCL中CD138、CD38、CD27表达水平显著高于EBV转化前的CD19+B细胞，且保留了B细胞分泌抗体、提呈抗原的能力。
本发明中的BCL可用于筛选优势T细胞免疫反应抗原表位，或制备高效价anti-HBsAg单克隆抗体，及诱导HBV特异性T细胞系，为“清除乙肝”提供新方向。
附图说明
图1 FACS检测BCL构建过程中在各个不同时间点的CD19+水平；
图2 FACS检测基线PBMC及BCL的CD138、CD38、CD27的平均荧光强度(MFI)(A)和CD138、CD38、CD27阳性细胞分别占CD19+阳性细胞的比例(B)；
图3 ELISA检测PBMC及BCL培养上清人总IgG；
图4 ICS检测PBMC及BCL分泌IL-6、IL-10、IFN-γ的能力；
图5 ICS检测HBV特异性T细胞IFN-γ分泌。
具体实施方式
下面进一步列举实施例以详细说明本发明。同样应理解，以下实施例只用于对本发明进行进一步说明，不能理解为对本发明保护范围的限制，本领域技术人员根据本发明阐述的原理做出的一些非本质的改进和调整均属于本发明的保护范围。下述示例具体的工艺参数等也仅是合适范围中的一个示例，即本领域技术人员可以通过本文的说明做合适范围内的选择，而并非要限定于下文示例的具体数据。
实施例1
一种外周血B细胞系的构建方法
收集200例HBV感染者肝素钠抗凝血，10mL外周血加入10mL PBS(磷酸盐缓冲液)稀释，加入到装有5mL淋巴细胞分离液的15mL离心管中，800g，25min，加减速为0，常温，密度梯度离心。吸白膜到50mL离心管中，补加PBS至50mL，1800rpm，10min，离心；打散细胞，加5mL培养基重悬，计数；根据计数结果取2million细胞，用含15％FBS的RPMI 1640培养基(R15)将PBMC(外周血单个核细胞)重悬至0.5*10^6～1*10^6/mL，500g 5min离心后弃干净上清，100μL EBV上清/0.5*10^6～1*10^6细胞重悬细胞，将悬液转移至96孔U底板中，孵箱中(37℃，5％二氧化碳)孵育3～4h后，加入100μl含2μg/mL CsA(环孢霉素，CsA终浓度为1μg/mL)的R15，培养两周，必要时换液或传代。两周后可使用不含CsA的R15进行培养。
制备得到的BCL的生物学特性
BCL中CD19+细胞的比例
分别在加入EBV孵育的基线(Day0)、首次传代(Day41)、第二次传代(Day60)、第三次传代(Day72)以及稳定传代后(Day129)FACS检测细胞中CD19+细胞的比例(图1)。发现从第二次传代(Day60)始，CD19+细胞比例显著高于基线PBMC中CD19+细胞比例(P＜0.05)，且在稳定传代后CD19+细胞稳定占细胞系98％以上(图1)，证明转化成功的细胞确实为B细胞。
CD138、CD38、CD27表达水平
FACS检测BCL种CD9+细胞的CD138、CD38、CD27的表达水平，发现相比于EBV孵育前的外周血CD19+B细胞，稳定传代后的BCL中CD9+细胞的CD138、CD38、CD27的平均荧光强度(MFI)均显著升高(图2A，P＜0.05)。进一步分析CD138、CD38、CD27阳性细胞分别占CD19+细胞的比例，发现BCL中CD19+细胞中，CD27+细胞比例明显升高(P＜0.05)，而CD138+、CD38+细胞的比例均有升高的趋势(图2B)，提示BCL中记忆性B细胞以及浆细胞的比例升高。
分泌抗体能力
PWM联合SAC刺激BCL以及对应来源的PBMC，培养12天后，用人总IgG ELISA试剂盒检测细胞培养上清中人总IgG水平，实验发现BCL仍具有分泌抗体的能力(图3)。利用乙肝核心抗体ELISA试剂盒进一步上清中的HBV core抗原特异性抗体(anti-HBcAg)，未能检测到anti-HBcAg，提示由总PBMC转化的BCL分泌特异性抗体的能力低。
分泌细胞因子能力
检测方法
将BCL以及对应来源的PBMC以0.3*10^6/200μL R10接种于96孔U底板，加入PMA(50ng/mL)、离子霉素(0.75μg/mL)、CpG(10μg/mL)、CD40L(1μg/mL)和BFA，刺激6h，所得细胞用于胞内因子染色(Intracellular staining，ICS)检测胞内IFN-γ、IL-10和IL-6水平。
检测结果
通过比较CD19+BCL及其对应患者来源的未经EBV孵育的外周血CD19+B细胞分泌IFN-γ、IL-6、IL-10的能力，发现CD19+BCL分泌IFN-γ、IL-10的水平与未孵育前外周血CD19+B细胞相当，但其而分泌IL-6水平则显著下降(图4，P＜0.01)。
BCL抗原提呈能力的检测
检测方法
复苏PBMC后体外诱导HBV特异性T细胞扩增，即将PBMC以0.3*10^6/200μL接种于96孔U底板，同时加入HBV抗原肽(2μg/mL)以及IL-7(25ng/mL)、CD28L(1μg/mL)、CD49L(1μg/mL)，在第3天和第7天视细胞增殖集落形成情况补加IL-2(10ng/mL)，或予以换液、传代，第10天将细胞洗涤3次后以R10重悬至1*10^6/mL，置于6孔板中静息3天备用。将BCL重悬至1*10^6/mL，加入MMC(10μg/mL)孵箱中孵育30min，洗涤三次备用。实验组BCL加载抗原肽1h后与对应PBMC混匀，加入BFA后孵育5h；对照组PBMC加载抗原肽，同时加入BFA，孵育5h。本实验中使用HIV肽作为阴性对照。
检测结果
将PBMC复苏后加入HBV肽及IL-2、IL-7，刺激诱导HBV肽特异性T细胞扩增，以提高PBMC中特异性T细胞比例。BCL为同PBMC经EBV转化来源。直接使用HBV抗原肽刺激扩增后的PBMC，可检测到低水平的分泌IFN-γ的T细胞(图5)，提示复苏的PBMC仍残有少量抗原提呈细胞，但是其提呈效率较低。加入加载了HBV抗原肽的BCL后，具有分泌IFN-γ能力的T细胞频数显著升高(图5，P＜0.005)，提示本体系中BCL具有提呈HBV抗原肽的能力，可增强HBV特异性T细胞反应。
实施例2 BCL用于筛选优势T细胞免疫反应抗原表位
上述实施例中已经证明，加入加载了HBV抗原肽的BCL后，具有分泌IFN-γ能力的T细胞频数显著升高，说明BCL仍保留有B细胞的抗原提呈能力。但是不同患者来源的T细胞对同一HBV抗原肽的反应性存在差异(表现为具有分泌IFN-γ能力的T细胞频数的不同)，同时，同一患者来源的T细胞对不同HBV抗原肽的反应性也存在差异，说明对不同患者来说，能诱导最强T细胞免疫应答的HBV抗原肽是不一样的。因此，可利用BCL提呈抗原肽给T细胞，通过检测T细胞中具有分泌IFN-γ能力的T细胞频数，判断该患者的优势T细胞免疫反应抗原表位。
具体方法如下：
复苏PBMC后R10重悬至1*10^6/mL备用；将BCL用R10重悬至1*10^6/mL，加入MMC(10μg/mL)孵箱中孵育30min，洗涤三次备用。实验组BCL加载不同HBV抗原肽(2μg/ml)1h后与对应PBMC混匀，加入BFA后孵育5h；5h后收集细胞，胞内因子染色检测T细胞中具有分泌IFN-γ能力的T细胞频数，其中分泌IFN-γ能力的T细胞频数高者对应的HBV抗原肽为该患者的优势肽。
实施例3BCL用于制备高效价anti-HBsAg单克隆抗体
B细胞经过V(D)J基因重排、抗体类别转换、体细胞高频突变，分化成为能分泌高亲和力抗体的浆细胞，由于等位排斥和同种型排斥，每个浆细胞只能分泌一种抗体。本发明中发现BCL仍具有分泌抗体的能力，因此可推定BCL中含有“浆细胞”，且这种浆细胞具有永生化特点。通过单细胞分选，或有限稀释法，使一个或几个浆细胞克隆增生形成单克隆细胞系，分泌单克隆抗体。收集单克隆细胞系培养上清，检测上清中抗体，可筛选得产生高效价anti-HBsAg单克隆抗体的BCL细胞系。
具体方法如下：
将BCL用R10重悬至250个/mL，200μl/孔接种到96孔U底板，培养2周，ELISA检测上清中anti-HBsAg，选择anti-HBsAg含量高的细胞孔继续培养，每3d传代一次(1：2传代)，每次传代培养3d后收集上清，ELISA检测anti-HBsAg含量，当上清中anti-HBsAg含量不再升高时得到的BCL细胞系为产生anti-HBsAg单克隆抗体的细胞系。
实施例4BCL用于诱导HBV特异性T细胞系
T细胞离体后，由于缺乏持续活化信号，将走向凋亡。T细胞活化信号包括特异性抗原刺激产生的第一信号以及共刺激分子刺激产生的第二信号。由于T细胞无法直接识别抗原表位，第一信号需要抗原提呈细胞以MHC-肽复合物的形式提呈抗原表位刺激产生。而BCL可给活化的T细胞提供第一信号。通过与加载了HBV特异性抗原肽的BCL共培养，HBV特异性T细胞可持续活化，而其他T细胞则在培养过程中逐渐凋亡，最终纯化得到HBV特异性T细胞系。因此，BCL可用于诱导HBV特异性T细胞系。
具体方法如下：
复苏PBMC后R10重悬至1*10^6/mL备用；将BCL用R10重悬至1*10^6/mL，加入MMC(10μg/mL)孵箱中孵育30min，洗涤三次备用。BCL加载HBV抗原肽(2μg/ml)1h后与对应PBMC混匀，同时加入IL-7(25ng/mL)、CD28L(1μg/mL)、CD49L(1μg/mL)，在第3天和第7天视细胞增殖集落形成情况补加IL-2(10ng/mL)，或予以换液、传代。第10天再次加入MMC处理过、加载了HBV抗原肽的BCL，同时加入IL-7(25ng/mL)、CD28L(1μg/mL)、CD49L(1μg/mL)。第14天收集细胞计数，磁珠正选CD19+细胞(操作按试剂盒说明书)，剩余的细胞为HBV特异性T细胞系。
Claims (10)
1.一种外周血B细胞系的构建方法，其步骤包括：收集HBV感染者外周血，分离得到外周血单个核细胞；培养基重悬外周血单个核细胞，离心后弃上清；EBV上清重悬外周血单个核细胞后进行孵育，孵育3-4h后，加入含环孢霉素的培养基继续培养。
2.根据权利要求1所述的构建方法，其特征在于：EBV上清重悬外周血单个核细胞至0.5*10^6/ml～1*10^6/ml。
3.根据权利要求1所述的构建方法，其特征在于：培养基中环孢霉素的工作浓度为1μg/ml。
4.根据权利要求3所述的构建方法，其特征在于：培养基中环孢霉素的工作时间为2周。
5.权利要求1～4任一项所述的构建方法制备得到的外周血B细胞系。
6.权利要求5所述的外周血B细胞系在筛选优势T细胞免疫反应抗原表位中的应用。
7.权利要求5所述的外周血B细胞系在制备anti-HBsAg单克隆抗体中的应用。
8.一种anti-HBsAg单克隆抗体，其特征在于：由权利要求5所述的外周血B细胞系制备得到。
9.权利要求5所述的外周血B细胞系在诱导HBV特异性T细胞系中的应用。
10.一种单克隆T细胞系，其特征在于：由权利要求5所述的外周血B细胞系诱导得到。
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