CN113005090A - 共表达趋化因子和共刺激分子的dc细胞及其应用 - Google Patents
共表达趋化因子和共刺激分子的dc细胞及其应用 Download PDFInfo
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Abstract
本发明提供共表达趋化因子和共刺激分子的DC细胞及其应用，所述趋化因子优选为CCL5和CXCL9，所述共刺激因子优选为41BBL。本发明的DC细胞能够具有提高的招募T细胞(尤其是记忆性CD8+T细胞)的功能，同时能激活T细胞甚至特异性T细胞的增殖，可用作DC肿瘤疫苗。
Description
技术领域
本发明涉及共表达趋化因子和共刺激分子的DC细胞及其应用。
背景技术
基于DC细胞的癌症免疫治疗的一个中心原则是产生抗原特异性细胞毒性T淋巴细胞(CTL)反应。肿瘤相关抗原(TAA)和DC在这一过程中起着关键作用。DCs是最有效的抗原呈递细胞，具有最强大的抗原呈递能力。不同TAA作用下的DCs在体外和体内都能产生特定的抗肿瘤作用。多种肿瘤抗原已被应用于癌症治疗，包括肿瘤RNA、溶酶体、凋亡体、热休克蛋白、TAA肽和异种肿瘤细胞。
治疗性疫苗的主要目标是诱导细胞免疫反应，其主要是初始T细胞从非激活状态到产生毒性淋巴细胞(CTL，Cytotoxic T lymphocytes)，CTL能够通过抗原特异性的方法识别和消除癌细胞，并产生长期记忆性CD8+T细胞，防止复发。免疫接种中最关键的一步是肿瘤抗原有效的呈递到T细胞上，而DC细胞是最有效的抗原呈递细胞，是改良治疗性疫苗的最有效选择。
由抗原呈递细胞刺激Naive T分化为效应T细胞需要三个信号：信号I：适合的肽-MHC复合物和T细胞表面的TCR受体的结合；信号II：APC细胞表面的共刺激配体(CD80/CD86)和相对应的T细胞受体作用；信号III：DC细胞分泌的细胞因子，特别是IL-12对T细胞的作用。
另外，在信号II的配体-受体中又分为共刺激和共抑制因子，其中41BBL/41BB通路像其它几个共刺激通路(CD70/CD27；OX-40L/OX-40)一样都是TNF受体家族，其依赖于NF-κB的激活。和CD80/86共刺激因子相比，41BBL/41BB通路能够更有效地激活记忆性的CD8+T细胞的活化状态。另外，41BBL/41BB通路显示出刺激Th1细胞因子(IFNγ，IL-12p70)，而抑制Th2细胞因子(IL-4、IL-5、IL-6、IL-9、IL-13和IL-17E)。并且相关的研究表明41BBL/41BB通路的激活显示出较好的抗肿瘤免疫反应和肿瘤组织的减小。41BB同时也是CAR-T细胞的共刺激域分子。相对于CD28共刺激域分子，41BB共刺激域能够较多地刺激中央记忆CD8+T细胞，促进线粒体的呼吸功能，在临床上比CD28共刺激域表现出较强的耐受性。所以在DC疫苗的共刺激策略上，41BBL/41BB通路的激活是较好的选择。
与此同时，DC对T细胞的激活不仅需要三个信号的激活，同时也需要一定的微环境。定位在组织和向淋巴器官的迁徙是DC的免疫功能发挥作用的必需步骤。其中，趋化因子在DC的迁徙中发挥重要的介导作用。比如，受到DC成熟信号的影响，CCR7使DC细胞降低炎症相关的趋化因子，这短暂的适应使DC细胞响应CCR7的激活剂离开组织迁徙到淋巴组织。同时，在肿瘤组织中也有DC细胞产生趋化因子，但其作用有待进一步探索。2019年Dangaj等人在Cancer Cell上发表论文，证明肿瘤细胞表达的CCL5和巨噬细胞、DC细胞表达的CXCL9在T细胞的肿瘤浸润方面起到重要作用，同时证明CD8+浸润T细胞和CCL5和CXCL9的共表达有关。但通常的肿瘤细胞会抑制CCL5的表达，所以也解释了CCL5和CXCL9表达高的肿瘤对免疫检查点抑制剂敏感。另外，Bukczynski等人2004年PNAS上已经证明，转染41BBL受体作为佐剂能够显著增加记忆性CD8+T细胞的激活和多肽特异性T细胞的增殖(如果同时负载多肽)。
发明内容
本发明提供一种DC细胞，其共表达趋化因子和共刺激因子。本发明的DC细胞能够具有提高的招募T细胞(尤其是记忆性CD8+T细胞)的功能，同时能激活T细胞甚至特异性T细胞的增殖。
因此，本发明第一方面提供一种DC细胞，其含有、和/或表达、和/或分泌至少一种共刺激因子和至少两种趋化因子。
在一个或多个实施方案中，所述DC细胞含有所述共刺激因子和趋化因子的编码序列、其表达盒或核酸构建体或表达载体，或含有所述共刺激因子和趋化因子的mRNA序列。
在一个或多个实施方案中，所述至少两种趋化因子中的至少一种选自CC趋化因子亚族的趋化因子，至少另一种选自CXC趋化因子亚族的趋化因子；或所述至少两种趋化因子中的至少一种选自单核/巨噬细胞趋化因子，至少另一种选自T淋巴细胞趋化因子。
在一个或多个实施方案中，所述共刺激因子是DC和T细胞表面共刺激通路中DC表面共刺激配体。
在一个或多个实施方案中，所述趋化因子为CCL5和CXCL9，所述共刺激因子为41BBL。
本发明另一方面提供一种DC肿瘤疫苗或药物组合物，其含有本发明任一实施方案所述的DC细胞。
本发明另一方面提供一种表达盒或由该表达盒转录得到的mRNA序列，其含有依次连接的启动子、共刺激因子的编码序列、IRES或T2A的编码序列、第一趋化因子的编码序列、IRES或T2A的编码序列、第二趋化因子的编码序列、转录后调控序列如WPRE的编码序列；优选地，所述共刺激因子为41BBL，所述第一趋化因子为CXCL9，所述第二趋化因子为CCL5。
本发明另一方面提供一种表达载体，其含有本发明任一实施方案所述的表达盒；优选地，所述表达载体为慢病毒表达载体。
本发明另一方面提供一种细胞培养物，其含有本发明任一实施方案所述的DC细胞和任选的T细胞，以及细胞培养基。
本发明另一方面提供本发明任一实施方案所述的DC细胞在制备治疗癌症用的药物中的应用。
本发明另一方面提供本发明任一实施方案所述的表达盒或由该表达盒转录得到的mRNA序列或表达载体在制备DC肿瘤疫苗中的应用。
附图说明
图1：293细胞转染和41BBL表达示意图。上：293细胞对照组；中：293细胞转染eGFP组；下：293细胞共转染三因子组。
图2：293T细胞转染和T细胞迁徙意图。
图3：DC细胞铺板及DC纯度。
图4：DC细胞转染后EGFP和共刺激因子的表达。上：eGFP组；下：CCL5+CCL9+CD137L组。
图5：HTRF检测DC细胞转染后CCL5的分泌值。
图6：DC细胞病毒质粒转染后细胞形态学的变化。
图7：Transwell示意图和T细胞迁徙数量。
图8：转染后DC细胞和T细胞共孵育3天形态学变化。
图9：表达EGFP的DC-T共孵育组中DC和T细胞的聚集定位。左：转染后4天后有显著的eGFP表达；右：共孵育2天后，T细胞显著的聚集。
图10：DC-T细胞共孵育3天后培养基中细胞因子分泌值。
图11：DC-T细胞共孵育3天后T细胞CD137表达的变化。
图12：DC-T细胞共孵育3天后T细胞自分泌INF-r(ELISOPT)变化。
图13：DC-T细胞共孵育3天后T细胞对PANC-1肿瘤细胞系的毒性作用。
图14：质粒pLV-Y3的结构示意图。
各图中，“activated”指激活，“non-activated”指未激活，“Y”指表达CCL5+CCL9+CD137L的细胞。
具体实施方式
应理解，在本发明范围中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成优选的技术方案。
本发明发现，共表达趋化因子和共刺激因子的DC细胞具有提高的招募T细胞的能力，且能够显著地激活T细胞甚至特异性T细胞的增殖。因此，本发明提供一种DC细胞，其含有、和/或表达、和/或分泌至少一种共刺激因子和至少两种趋化因子。所述“含有”指所述共刺激因子和趋化因子含于间充质干细胞内或表面上；该“表达”指该DC细胞生产所述共刺激因子和趋化因子；该“分泌”指该DC细胞将所表达的共刺激因子和趋化因子分泌出细胞外。一般而言，本发明的DC细胞含有本文所述共刺激因子和趋化因子的编码序列、其表达盒或核酸构建体或表达载体，或含有所述共刺激因子和趋化因子的mRNA序列。在一些实施方案中，所述表达盒整合在所述DC细胞的基因组中。
本文中，DC细胞为树突状细胞，源于造血干细胞，是专职抗原递呈细胞。本领域周知，DC细胞的来源主要有两条途径：(1)髓样干细胞在GM-CSF的刺激下分化为DC细胞，称为髓样DC(MDC)，包括朗格汉斯细胞(LC)、间皮(或真皮)DC以及单核细胞衍生的DC等；(2)来源于淋巴样干细胞，称为淋巴样DC(LDC)或浆细胞样DC。本领域已知的用于制备DC肿瘤疫苗的各种DC细胞均可用于实施本发明。可从PBMC中分离出单核细胞，然后采用本领域常规的方法(例如使用GM-CSF和IL-4)将单核细胞诱导成DC细胞。
本文中，趋化因子具有本领域周知含义和种类。例如，按其N端半胱氨酸的排列方式，趋化因子可分为CC趋化因子亚族和CXC趋化因子亚族，其中CC趋化因子亚族包括CCL1、CCL2、CCL3、CCL4、CCL5、CCL6、CCL7、CCL8、CCL9、CCL10、CCL11、CCL12、CCL13、CCL14、CCL15、CCL16、CCL17、CCL18、CCL19、CCL20、CCL21、CCL22、CCL23、CCL24、CCL25、CCL26、CCL27和CCL28，CXC趋化因子亚族包括CXCL1、CXCL2、CXCL3、CXCL4、CXCL5、CXCL6、CXCL7、CXCL8、CXCL9、CXCL10、CXCL11、CXCL12、CXCL13、CXCL14、CXCL15、CXCL16和CXCL17。按作用细胞分类，包括吸引单核/巨噬细胞到炎症部位的单核/巨噬细胞趋化因子，如CCL2、CCL3、CCL5、CCL7、CCL8、CCL13、CCL17和CCL22，以及T淋巴细胞趋化因子，如CXCL9、CXCL10和CXCL11。
在本发明优选的实施方案中，DC细胞表达至少两种趋化因子；优选地，DC细胞表达一种来自CC趋化因子亚族的趋化因子和一种来自CXC趋化因子亚族的趋化因子，或表达一种单核/巨噬细胞趋化因子和一种T淋巴细胞趋化因子。在更优选的实施方案中，DC细胞表达CCL5和CXCL9。
本发明中，共刺激因子具有本领域周知含义，是指一类参与免疫反应的辅助性分子，存在于T细胞、B细胞、抗原提呈细胞(APC)和靶细胞表面。在细胞对抗原的识别中通过细胞表面共刺激因子的特异结合，可有效增强T细胞与其它细胞的粘附，传导抗原刺激信息，参与细胞的免疫活化过程，在细胞抗原识别及免疫应答过程中起着重要作用。本领域周知的共刺激因子，包括来自41BBL/41BB共刺激通路、CD70/CD27共刺激通路、OX-40L/OX-40共刺激通路中的共刺激因子。在优选的实施方案中，共刺激因子是DC和T细胞表面共刺激通路中DC表面共刺激配体；更优选地，本发明DC细胞表达来自41BBL/41BB共刺激通路的共刺激因子，即41BBL。
在特别优选的实施方案中，本发明的DC细胞表达CCL5、CXCL9和41BBL。应理解，本文所述的各趋化因子和共刺激因子的氨基酸序列均为本领域所周知的序列。
可获得感兴趣趋化因子和共刺激因子的编码序列，将其克隆到合适的载体中，构建得到表达趋化因子和共刺激因子的表达载体，然后将该表达载体转染DC细胞，构建得到表达本文所述趋化因子和共刺激因子的DC细胞。
本文中，“编码序列”为编码多肽产物的氨基酸序列的核酸序列部分。编码序列的边界通常由紧接在编码的mRNA的5'端的开放阅读框上游的核糖体结合位点(对于原核细胞)和编码的mRNA的3'端的开放阅读框下游的转录终止序列决定。编码序列可以包括但不限于DNA、cDNA和重组核酸序列。“表达盒”是指基因表达所需的整个元件，包括启动子、编码序列和转录后调控序列如多聚A尾信号序列或WPRE。在含有两个或多个编码序列的情况下，各编码序列之间可通过常规的连接序列如T2A肽或IRES序列的编码序列进行连接。在本发明的一些实施方案中，表达盒含有依次连接的启动子、共刺激因子的编码序列、IRES或T2A的编码序列、第一趋化因子的编码序列、IRES或T2A的编码序列、第二趋化因子的编码序列、转录后调控序列如WPRE的编码序列。优选地，所述共刺激因子为41BBL，所述第一趋化因子为CXCL9，所述第二趋化因子为CCL5。
本文还包括核酸构建体，其含有本文所述的趋化因子和共刺激因子的编码序列，以及与这些序列操作性连接的一个或多个调控序列。本文中，“操作性连接”指线性DNA序列的某些部分能够调节或控制同一线性DNA序列其它部分的活性。例如，如果启动子控制编码序列的转录，那么它就是可操作地连于编码序列。
调控序列可以是合适的启动子序列。启动子序列通常与待表达蛋白的编码序列操作性连接。启动子可以是在所选择的宿主细胞中显示转录活性的任何核苷酸序列，包括突变的、截短的和杂合启动子，并且可以从编码与该宿主细胞同源或异源的胞外或胞内多肽的基因获得。
调控序列也可以是合适的转录终止子序列，由宿主细胞识别以终止转录的序列。终止子序列与编码该多肽的核苷酸序列的3’末端操作性连接。在选择的宿主细胞中有功能的任何终止子都可用于本文。
在某些实施方案中，所述核酸构建体是载体。具体而言，可将共刺激因子和趋化因子的编码序列克隆入载体，例如载体包括但不限于质粒、噬菌粒、噬菌体衍生物、动物病毒和粘粒。载体可以是表达载体(也称为重组载体)。表达载体可以以病毒载体形式或非病毒形式提供给细胞，优选为病毒载体，更优选为慢病毒载体。通常，慢病毒载体中包括慢病毒基因组及本发明的表达框。
通常，合适的载体包含在至少一种有机体中起作用的复制起点、启动子序列、方便的限制酶位点和一个或多个可选择的标记。
合适的启动子包括但不限于即时早期巨细胞病毒(CMV)启动子序列。该启动子序列是能够驱动可操作地连接至其上的任何多核苷酸序列高水平表达的强组成型启动子序列。合适的启动子的另一个例子为延伸生长因子-1α(EF-1α)。然而，也可使用其他组成型启动子序列，包括但不限于类人猿病毒40(SV40)早期启动子、小鼠乳癌病毒(MMTV)、人免疫缺陷病毒(HIV)长末端重复(LTR)启动子、MoMuLV启动子、鸟类白血病病毒启动子、EB病毒即时早期启动子、鲁斯氏肉瘤病毒启动子、以及人基因启动子，诸如但不限于肌动蛋白启动子、肌球蛋白启动子、血红素启动子和肌酸激酶启动子。进一步地，也可考虑使用诱导型启动子。诱导型启动子的使用提供了分子开关，其能够在期限表达时打开可操作地连接诱导型启动子的多核苷酸序列的表达，而在当表达是不期望的时关闭表达。诱导型启动子的例子包括但不限于金属硫蛋白启动子、糖皮质激素启动子、孕酮启动子和四环素启动子。
可选择的标记包括可选择的标记基因或报道基因中的任一个或两者，以便于从被病毒载体感染的细胞群中鉴定和选择表达细胞。有用的可选择标记基因包括例如抗生素抗性基因，诸如neo等。合适的报道基因可包括编码荧光素酶、β-半乳糖苷酶、氯霉素乙酰转移酶、分泌型碱性磷酸酶或绿色荧光蛋白基因的基因。
本发明的共刺激因子和趋化因子的核苷酸序列全长序列或其片段通常通过PCR扩增法、重组法或者人工合成的方法获得。通常先合成多个小片段，然后将其进行连接可获得序列很长的片段。获得本发明所涉及的DNA序列之后，将该序列引入本领域已知的各种现有的DNA分子中，随后将重组之后的载体转化到适当的宿主细胞中，使其能表达蛋白质。
可采用常规的方法将本文的载体导入DC细胞中，这些方法包括显微注射法、基因枪法、电穿孔法、病毒介导的转化法、电子轰击法、磷酸钙沉淀法等。在某些实施方案中，本文采用电穿孔法将本文的核酸构建体导入间充质干细胞中。电穿孔法常用的设备和试剂为本领域所周知。例如，可参照LONZA的2b-Nucleofector细胞核电转仪操作方法实施电穿孔法。在一些实施方案中，本发明病毒介导的方法将重组载体转到DC细胞中。化学转染法的试剂为本领域所周知，包括但不限于脂质体转染试剂(Takara的X-fect转染试剂)或者聚阳离子转染试剂等。
在一些实施方案中，本发明直接将编码本发明所述共刺激因子和趋化因子的mRNA序列转入DC细胞中，使其在DC细胞中表达所述共刺激因子和趋化因子。该mRNA序列可以是共刺激因子和趋化因子各自独立的mRNA序列，也可以是以任意排列组合连接在一起的mRNA序列。可采用本领域周知的mRNA转染方法将感兴趣的核酸序列转入DC细胞中。
获得转染后的DC细胞后，可采用常规的方法培养该DC细胞。例如，在转染后可加入TNF-α、IL-1β、IL-6、PGE2进行成熟诱导，从而可获得成熟DC细胞。然后在常规的条件下培养该细胞。
本发明的DC细胞具有提高的招募T细胞(尤其是记忆性CD8+T细胞)的功能，同时能激活T细胞甚至特异性T细胞的增殖。
本发明还提供一种药物组合物，其含有本发明所述的DC细胞。药物组合物中还可含有适于DC细胞递送的药学上可接受的载体。
药物组合物中通常含有治疗有效量的DC细胞。“治疗有效量”指可以在对象中实现治疗、改善或减轻疾病或病症的剂量。本文中，“对象”可以指接受本发明的药物组合物以治疗、改善和/或减轻本发明的疾病或病症的患者或其他动物，特别是哺乳动物，例如人、狗、猴、牛、马等。可根据不同的对象、疾病种类等因素由本领域专业技术人员确定治疗有效量。
本发明的药物组合物可以以药物制剂的形式提供。合适的药物制剂可以是具有治疗或预防作用的医药学上可以接受的任何药物剂型，包括但不限于液体剂型、固体剂型、半固体剂型等。该药物制剂还可辅以其他活性成分，如玻璃酸钠等。
还提供的是一种DC肿瘤疫苗，其含有本发明所述的DC细胞。疫苗中还可含有本领域周知的细胞疫苗中所用的佐剂。
本发明还提供一种试剂盒，所述试剂盒含有本文任一实施方案所述的DC细胞。该试剂盒中的DC细胞可以药物制剂的形式提供。
还提供本文任一实施方案所述的本发明也包括本发明所述的DC细胞在制备治疗癌症的药物中的应用。本发明对癌症的种类并无特殊限制，只要适合DC细胞进行肿瘤免疫治疗的癌症都可用本发明的DC细胞、其药物组合物或疫苗进行治疗。本文中，“肿瘤免疫治疗”具有本领域周知的含义，即激活对象(哺乳动物，尤其指人)的免疫系统，以识别并清除肿瘤微环境中的肿瘤细胞，从而治疗目的。示例性的可采用肿瘤免疫治疗的癌症包括但不限于黑色素瘤、非小细胞肺癌、肾癌、霍奇金淋巴瘤、头颈鳞癌、前列腺癌、膀胱癌、胰腺癌、非霍奇金淋巴瘤、乳腺癌、直肠癌、淋巴癌、急性淋巴细胞白血病等。在某些实施方案中，所述癌症是胰腺癌。
治疗癌症、尤其是适合进行肿瘤免疫治疗的癌症的方法也包括在本文的范围之内，所述方法包括将本文所述的间充质干细胞或其药物组合物给予有需要的个体的步骤。给予的方法可以是细胞治疗中常用的方法。给予的剂量可根据病患性别、年龄、所患疾病、身体状况等因素加以考虑。
还提供一种体外刺激T细胞的方法，所述方法包括使本发明的DC细胞与T细胞接触的步骤。T细胞可以是本领域已知的各种来源的T细胞，包括分离自人体的天然T细胞、肿瘤浸润T细胞(Tumor-infiltrating Lymphocyte,TIL)、也包括已经经过基因工程改造的T细胞，如CAR-T细胞。优选的T细胞是用于治疗目的、尤其是用于肿瘤治疗的T细胞，如本领域周知的各类用于肿瘤治疗的CAR-T细胞。所述接触包括使本发明的DC细胞与感兴趣的T细胞共孵育的步骤。例如，可在含有IL-2但不含FBS的AIM-V培养基中共孵育。DC细胞与感兴趣T细胞的比例可以在1：1到1：20的范围内
在一些实施方案中，本发明还提供一种细胞培养物，其含有本发明的DC细胞以及相应的培养基。在其他一些实施方案中，所述细胞培养物中还含有T细胞。
下面将结合实施案例对本发明所涉及的实施方案进行详细描述。本领域技术人员将会理解，下面的实施案例仅用于说明本发明，而不应视为限定本发明的范围。实施案例中未注明具体技术或条件者，按照本领域内的文献所描述的技术或条件(例如参考J.萨姆布鲁克等著，黄培堂等译的《分子克隆实验指南》，第三版，科学出版社)或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
一.试验/检测方法
1.1 共表达趋化因子和共刺激因子质粒构建及病毒包装流程
(1)质粒构建
外送公司进行序列的合成，并克隆至慢病毒表达载体，得到质粒pLV-Y3，进行酶切鉴定及全序列测序鉴定，中抽纯化。质粒pLV-Y3的结构如图14所示，其中5’LTR(truncated)指来自HIV-1的截短的5’长末端重复序列(LTR)；HIV-1ψ为来自HIV-1的包装信号；RRE为HIV-1的Rev应答元件，用于实现使mRNA Rev依赖性地从核中转运到胞浆；cPPT/CTS指HIV-1的中心多嘌呤区和中心终止序列；IRES脑心肌炎病毒的内核糖体进入点；WPRE为土拨鼠肝炎病毒转录后调控元件；T2A为来自马齿苋病毒衣壳蛋白的2A肽；3’LTR(△U3)指HIV-1的自激活3’长末端重复序列。其中，4-1BBL(本文也称为41BBL或CD137L)的编码序列如SEQ IDNO:1所示，氨基酸序列如SEQ ID NO:2所示，CXCL9的编码序列如SEQ ID NO:3所示，氨基酸序列如SEQ ID NO:4所示，CCL5的编码序列如SEQ ID NO:5所示，氨基酸序列如SEQ ID NO:6所示，T2A的编码序列如SEQ ID NO:7所示，氨基酸序列如SEQ ID NO:8所示，IRES的编码序列如SEQ ID NO:9所示。
(2)慢病毒包装
慢病毒悬液的生产需要以下三个质粒共转染293T细胞：
1)同时编码慢病毒基因组和携带目标基因的慢病毒载体(质粒pLV-Y3)；
2)CMV△R8.91(表达HIV-1所需的三种蛋白)；
3)MD.G(表达VSV-G包膜蛋白)。
293T细胞生长到密度大约为70-80％时，在5ml或15ml管内进行转染操作。首先在管内加入水、CaCl2和DNA。加入HBSS后，会产生气泡，用移液枪吹打10-20次，直到溶液微微出现雾状。将混合液室温静置10-20min。将混合液加入上述的细胞培养基内，漩涡平行晃动培养基。孵育6h后，将培养基更换成含胰岛素包的装培养基。第2-4天，收集病毒悬液。2500g离心10min除去细胞和碎片，储存备用。
此转染病毒未检测病毒滴度，在功能验证的过程中同时转染eGFP为对照确定转染效率。
1.2 DC细胞培养及诱导
(1)PBMC的分离
1)取50ml离心管，加17.5ml生理盐水，再加17.5ml血。
2)取另一支50ml离心管(内有15ml Ficoll淋巴细胞分离液)缓慢沿管壁加入步骤1的血液和生理盐水混合液，然后800g、20min、升降速为1的离心。
3)吸白色细胞层至另一支50ml离心管中，并加生理盐水，离心清洗1500r/min，10min，升降速为9。
4)倒掉废液，不倒尽，加生理盐水，再次清洗，离心(同步骤3)。
5)倒尽废液，加培液悬浮细胞，并加进培养瓶中。
6)贴壁2小时或过夜，用移液管吹一下，收细胞，计数，离心。
冻存细胞，1支1×107个细胞。由此得到的贴壁细胞基本为单核细胞，加入50ng/mLGM-CSF和1000U/mL IL-4诱导成DC细胞，悬浮的基本为淋巴细胞。
(2)DC细胞培养及诱导
Day0：PBMC加入不含血清的AIM-V培养基，贴壁2h，将悬浮细胞与贴壁细胞分离；悬浮细胞吸取1X106到1.5mL EP管中，寄送检测HLA分型。剩余细胞离心并冻存备用。贴壁细胞继续使用不含血清的AIM-V培养基培养，以一定比例添加入培养液；使培养液中的GM-CSF和IL-4因子的终浓度分别为50ng/mL和1000U/mL。
Day3：贴壁细胞半量换液，补加GM-CSF和IL-4因子。
Day6：部分DC细胞收集后检测纯度，同时更换培养基中加入包装质粒的慢病毒进行转染。12孔板1ml的培养基中加入100ul，滴度未检测。如果同时负载多肽，多肽终浓度为40μg/mL。加入培养基4小时后，清洗去除未负载的多肽，然后加入慢病毒转染24小时，洗去。
Day7：贴壁细胞加入TNF-α、IL-1β、IL-6、PGE2因子(工作浓度为：20ng/mL TNF-α、10ng/mL IL-1β、1000U/mL IL-6、1μg/mL PGE2)。
Day8：收获成熟DC，与相应供体T细胞以1：5的比例共孵育，采用不含FBS的AIM-V培养基，加入100U/mL的IL-2，培养3天。
Day11：测试DC-T共孵育后T细胞的功能，包括：
1)DC细胞对T细胞的招募和T细胞的迁徙；
2)T细胞激活及细胞因子分泌；
3)细胞毒性/杀伤。
1.2 HLA分型检测
HLA分型检测为江苏伟禾生物科技有限公司代为检测，使用TBG MorganTM HLA SSPABCDRDQ Typing Kit进行检测。基本方法流程：PCR序列特异性引物(sequence specificprimer，SSP)分析法是使用能够特异识别特定等位基因的引物通过PCR扩增检测序列多态性的方法，也称作等位基因特异性引物PCR法。只有引物完全和DNA序列匹配才能在PCR条件下扩增，扩增的DNA片段能够指示特异性的基因型。扩增后的DNA片段通过琼脂糖电泳区别大小。首先进行DNA的提取，本实验从培养的细胞(T细胞)中获取DNA，经含有混合引物的PCR管进行扩增后加入琼脂糖确定是否扩增，不同的孔扩增标识不同的TCR基因型(由于特异性的引物)。所以，最后判读出不同的HLA分型。
1.3 流式细胞检测
流式检测基本步骤如下：
(1)每管中加入1×106个细胞。
(2)加入1ml PBS磷酸盐缓冲液进行洗涤2次，400g离心5min，弃去上清；加入100μLPBS磷酸盐缓冲液进行重悬；
(3)加入需检测的流式抗体，混匀，2-8℃冰箱放置，避光孵育30min；设置一组空白对照，不加试剂或者加入对应的同种型；
(4)加1ml PBS磷酸盐缓冲液，400g离心5min，清洗2次，弃去上清，吸取400μl PBS磷酸盐缓冲液重悬细胞，用流式细胞仪进行检测，设置所有细胞收集1×104个。使用KaluzaAnalysis软件分析数据。
检测如上述所示步骤，加入的不同抗体如下表所示：
| 检测项目抗体 | 发光信号 | 流式通道 |
| 抗人CD3(Biolegend) | APC | FL6 |
| 抗人CD137L(4-1BB Ligand)(Biolegend) | APC | FL6 |
| 抗人CD14(Biolegend) | FITC | FL1 |
| 抗人CD11b(Biolegend) | APC | FL6 |
| 抗人CD209(DC-SIGN)(Biolegend) | PE | FL2 |
| 抗人CD137(4-1BB)(Biolegend) | PE/Cy5 | FL4 |
1.4 IFN-γELISPOT
1)调整细胞密度2.5×106/ml在培养基中，在每孔BD ELISPOT平板中加入200ul，这样每孔细胞总数维持在50万。
2)在37℃5％CO2培养箱中孵育过夜(16小时)，此步骤后可以在非无菌条件操作。
3)用配置好的洗涤缓冲液清洗3次，加入检测抗体，100ul每孔，在室温中孵育2小时。
4)弃检测抗体溶液，用洗涤缓冲液清洗3次，每孔可以震荡1-2min或吸管吸取1-2次。
5)加入链霉亲和素-HRP溶液，100ul/孔，在室温孵育1小时。
6)弃链霉亲和素-HRP溶液，用洗涤缓冲液洗4次。
7)用PBS清洗2次。
8)加入100ul AEC底物溶液，观察斑点形成过程，一般5-10min即可稳定，不需要让颜色过度而导致较高的背景值。
9)用去离子水清洗各孔，停止底物反应。
10)在ELISPOT Plate reader上读数分析。
1.5 RTCA细胞毒性实验
1)先取16孔的E-plate加入50ul靶细胞培养基，打开RTCA软件进行保存路径及校准。
2)针对不同的靶细胞，需要对靶细胞进行铺板，在Trypsin消化后调节靶细胞的密度在2×105在杀伤板中每孔加入50ul，使每孔细胞保持在10000个，过夜或20小时左右后，根据电阻的细胞指数(Cell index)信号确定是否加入效应细胞。一般细胞指数超过1.0数值即可加入效应细胞。
3)根据不同的E:T确定需要加入的效应细胞的量，本次试验中调节细胞密度为2×106加入50ul到E-Plate板中(E:T为10:1)。一般的杀伤时间为10-20小时，杀伤结束时，停止软件并在RTCA软件中进一步的分析。
1.6 多重细胞因子CBA方法检测
按说明书的操作步骤进行，首先梯度稀释标准品，2mL重悬标准品后，取出9根流式上样管，分别标记梯度稀释的倍数1:2、1:4、1:8、1:16、1:32、1:64、1:128、1:256。为保证每个实验管都含有6种微球，每种捕获微球需各取10μl，混合微球体积为60μl，具体实验过程中每管检测样本或标准品加入微球的量为50，按此比例配置微球。如，待测样本为8个，9个标准品、1个阴性对照，共18管。所以，需要每种微球的量是180μl，6×180μl混合后，加入稀释后的样本/标准品(50μl)每管中50μl，再加入50ul PE检测试剂室温避光孵育3小时。在上机之前需要结合磁珠对仪器进行校准和电压调节，用对照磁珠进行补偿的调节，孵育后上机获取后用CBA分析软件FCAP结合标准曲线分析计算检测样本的浓度。
1.7 HTRF方法检测培养基中CCL-5
HTRF技术是CISBIO公司注册的产品商标，其基本原理：当标记的抗体与相同的抗原结合时，用光源激发供体向受体发出荧光共振能量转移(FRET)，从而使受体发出特定波长(665nm)的荧光。这两种抗体与样品中的CCL-5结合，从而产生FRET。信号强度与抗原-抗体复合物的数量成正比，因此与CCL-5浓度成正比。其基本操作步骤包括将供体和受体的标记的抗体4μl加入到16μl待测样本或者标准品中，孵育2小时后读取HTRF的值，根据标准曲线拟合计算待测样本中的CCL-5的浓度。
二.结果
2.1 质粒/病毒的功能
首先在293细胞系上证明质粒/慢病毒的蛋白表达水平。由于293T细胞用脂质体转染试剂即可转入，所以用lip3000转染试剂共表达质粒。如图1所示，12孔板中293细胞用病毒转染后，实验组41BBL的表达显著增加，阳性率达到了75％。
其次在293T细胞上验证是否具有T细胞迁徙特性。具体而言，通过T细胞在Transwell培养体系中24小时后，收集板孔中的所有细胞计数，经流式检测计算CD3+T细胞的比例，乘以细胞的总数即可得到板孔中CD3+T细胞的绝对数量。其中均为通过半透膜迁徙的T细胞。结果如图2所示。图2中，A1为293T细胞对照；B1和B2为相同载体的eGFP对照组，B1为低剂量组，B2为高剂量组，由图中可显著见eGFP表达的剂量关系；A2、C1、C3为表达共刺激因子的实验组。有图2的数据可见，转染趋化因子和共刺激因子的293T细胞对CD3+(非匹配)T细胞具有显著的招募作用，能够促使T细胞向高浓度的方向迁徙。
2.2 质粒/病毒的DC转染功能
由于DC细胞的转染效率不高，所以首选慢病毒载体。12孔板中加入含1×106DC细胞的1mL培养基，进行图3所示的分组。在分组前同时取部分DC细胞测定DC细胞的纯度，由CD14、CD11B和CD209抗体染色，并确定DC细胞的纯度在50％以上。在加入病毒转染前，可以用PBS洗去部分T细胞以提高DC细胞的纯度。
病毒转染48小时后，取出部分细胞，检测这些细胞中eGFP和共刺激因子CD137L的表达。结果如图4所示，EGFP的表达效率在65％左右，而DC细胞表面表达CD137L的阳性率在80％左右，所以可以进行下一步的实验。
同时，DC分泌到培养基中的CCL5和CXCL9也通过HTRF的检测方法检测。具体而言，DC细胞转染后24、48小时收集培养基，用HTRF的方法检测CLL5的分泌量。结果如图5所示，转染CCL5/CXCL9/CD137L的DC细胞在24、48小时后CCL5显著增加。结合图4中CD137L和EGFP的表达可以证明：病毒质粒是正确的并在DC细胞内表达稳定。
此外，在DC转染48小时后观察细胞形态学的区别。结果如图6所示，在表达CCL5/CXCL9和CD137L的DC细胞中显示出更好的细胞聚集效应。可能为残留的T细胞的迁徙导致。
综上所述：病毒质粒能够很好地转染DC细胞，并且CCL5/CD137L表达稳定，实验组和对照组对比具有一定的差异。
2.3 DC转染后对T细胞的功能影响
DC细胞在转染48小时后在Transwell板孔中加入预先用CD3+CD28抗体激活的T细胞，过夜后去除吊篮里的T细胞，收集下层的所有细胞计数并流式检测T细胞的比例，最后得出下层孔板中的T细胞绝对数量。
结果如图7所示，DC细胞表达了CCL5和CXCL9后显著地对T细胞具有招募作用。
在病毒质粒转染DC细胞48小时后，将另一部分激活和未激活的T细胞(相匹配)与DC细胞按DC:T＝1:5比例共孵育3天后测试T细胞的功能。结果如图8所示。观察到DC-T共孵育3天后细胞培养形态方面的变化。DC和未刺激和刺激后的T细胞共孵育后，实验组(表达CCL5、CXCL9和CD137L)显著地招募T细胞的聚集，同时，T细胞激活后细胞的增殖较未激活组比较显著的增加。同时也可以观察到对照组DC细胞的间隙更加明显。
同时，在DC和T细胞共孵育2天时，观察T细胞和DC细胞的共定位情况。如图9所示，在表达EGFP的同时可以观察到更多T细胞的聚集(黑色箭头所示)。
另外，DC-T细胞共培养3天后，收集培养基检测细胞因子分泌：IL-2、IL-4、IL-6、IL-10、TNF-a、INF-r。结果如图10所示。可见IL-2和INF-r具有显著的增加，未激活的T细胞和DC细胞共孵育检测IL-2的分泌是免疫原性的标准检测方法，主要是CD4+细胞的激活，并且相对于对照组，转染eGFP和实验组(表达CCL5，CXCL9，CD137L)培养基中IL-2分泌具有显著的增加，并且提示eGFP的免疫原性反应可能高于实验组。此结果与相关免疫原性报道一致。同时一般情况下，在T细胞经过CD3/28抗体刺激后，T细胞被激活后INF-r分泌均有增加，但eGFP组却显著下降，可能的机制是eGFP的对细胞的毒性作用。
T细胞在共孵育3天后，需检测T细胞的功能变化，1)T细胞CD147表达的变化；2)自然情况下T细胞分泌INF-r的变化；3)T细胞杀伤肿瘤细胞系的作用。结果如图11所示，未激活T细胞组在共培养3天后均无显著区别，而激活后的T细胞在实验组(表达CCL5、CXCL9和CD137L)的MFI(平均荧光强度)显著右移，提示可能实验组的T细胞CD137表达增强。
DC-T细胞共孵育3天后，清洗T细胞，然后加入ELISPOT板，过夜检测在自然情况下T细胞分泌INF-r的变化。结果如图12所示，eGFP组和实验组INF-r分泌的量均显著增加，其中实验组平均值略高于eGFP组，可见DC细胞对T细胞的刺激是显著的。
细胞毒性采用RTCA的实时监测贴壁细胞电阻的方法进行检测。杀伤的靶细胞均为PANC-1细胞系，E:T比为10:1。结果如图13所示，非激活T细胞组eGFP对DC细胞的毒性作用可以显著显现，eGFP导致的DC细胞死亡在杀伤板中没有使信号具有初始的增加；最后，在未激活组和激活组表达CCL5、CXCL9和CD137L的DC-T较其他组均有一定的强细胞毒性作用。因此，此种DC疫苗具有较强的肿瘤细胞杀伤功能。
序列表
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Leu Cys Ala Pro Ala Ser Ala Ser Pro Tyr Ser Ser Asp Thr Thr Pro
20 25 30
Cys Cys Phe Ala Tyr Ile Ala Arg Pro Leu Pro Arg Ala His Ile Lys
35 40 45
Glu Tyr Phe Tyr Thr Ser Gly Lys Cys Ser Asn Pro Ala Val Val Phe
50 55 60
Val Thr Arg Lys Asn Arg Gln Val Cys Ala Asn Pro Glu Lys Lys Trp
65 70 75 80
Val Arg Glu Tyr Ile Asn Ser Leu Glu Met Ser
85 90
<210> 7
<211> 54
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
gagggcaggg gaagtcttct aacatgcggg gacgtggagg aaaatcccgg ccca 54
<210> 8
<211> 18
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 8
Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val Glu Glu Asn Pro
1 5 10 15
Gly Pro
<210> 9
<211> 574
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
cccctctccc tccccccccc ctaacgttac tggccgaagc cgcttggaat aaggccggtg 60
tgcgtttgtc tatatgttat tttccaccat attgccgtct tttggcaatg tgagggcccg 120
gaaacctggc cctgtcttct tgacgagcat tcctaggggt ctttcccctc tcgccaaagg 180
aatgcaaggt ctgttgaatg tcgtgaagga agcagttcct ctggaagctt cttgaagaca 240
aacaacgtct gtagcgaccc tttgcaggca gcggaacccc ccacctggcg acaggtgcct 300
ctgcggccaa aagccacgtg tataagatac acctgcaaag gcggcacaac cccagtgcca 360
cgttgtgagt tggatagttg tggaaagagt caaatggctc tcctcaagcg tattcaacaa 420
ggggctgaag gatgcccaga aggtacccca ttgtatggga tctgatctgg ggcctcggtg 480
cacatgcttt acatgtgttt agtcgaggtt aaaaaaacgt ctaggccccc cgaaccacgg 540
ggacgtggtt ttcctttgaa aaacacgatg ataa 574
Claims (10)
1.一种DC细胞，其含有、和/或表达、和/或分泌至少一种共刺激因子和至少两种趋化因子。
2.如权利要求1所述的DC细胞，其特征在于，所述DC细胞含有所述共刺激因子和趋化因子的编码序列、其表达盒或核酸构建体或表达载体，或含有所述共刺激因子和趋化因子的mRNA序列。
3.如权利要求1或2所述的DC细胞，其特征在于，
所述至少两种趋化因子中的至少一种选自CC趋化因子亚族的趋化因子，至少另一种选自CXC趋化因子亚族的趋化因子；或所述至少两种趋化因子中的至少一种选自单核/巨噬细胞趋化因子，至少另一种选自T淋巴细胞趋化因子；
所述共刺激因子是DC和T细胞表面共刺激通路中DC表面共刺激配体。
4.如权利要求1或2所述的DC细胞，其特征在于，所述趋化因子为CCL5和CXCL9，所述共刺激因子为41BBL。
5.一种DC肿瘤疫苗或药物组合物，其含有权利要求1-4中任一项所述的DC细胞。
6.一种表达盒或由该表达盒转录得到的mRNA序列，其含有依次连接的启动子、共刺激因子的编码序列、IRES或T2A的编码序列、第一趋化因子的编码序列、IRES或T2A的编码序列、第二趋化因子的编码序列、转录后调控序列如WPRE的编码序列；优选地，所述共刺激因子为41BBL，所述第一趋化因子为CXCL9，所述第二趋化因子为CCL5。
7.一种表达载体，其含有权利要求6所述的表达盒；优选地，所述表达载体为慢病毒表达载体。
8.一种细胞培养物，其含有权利要求1-4中任一项所述的DC细胞和任选的T细胞，以及细胞培养基。
9.权利要求1-4中任一项所述的DC细胞在制备治疗癌症用的药物中的应用。
10.权利要求6所述的表达盒或由该表达盒转录得到的mRNA序列或权利要求7所述的表达载体在制备DC肿瘤疫苗中的应用。
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| PCT/CN2020/137627 WO2021121392A1 (zh) | 2019-12-20 | 2020-12-18 | 共表达趋化因子和共刺激分子的dc细胞及其应用 |
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