CN109439626B - 一种有助于体外获得Th22细胞的组合物及其用途 - Google Patents

一种有助于体外获得Th22细胞的组合物及其用途 Download PDF

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CN109439626B
CN109439626B CN201811330774.5A CN201811330774A CN109439626B CN 109439626 B CN109439626 B CN 109439626B CN 201811330774 A CN201811330774 A CN 201811330774A CN 109439626 B CN109439626 B CN 109439626B
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杨骥
李明
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Zhongshan Hospital Fudan University
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Abstract

本发明提供了一种有助于体外获得Th22细胞的组合物及其用途，所述的有助于体外获得Th22细胞的组合物，其特征在于，包括：幼稚性B细胞或Breg细胞，以及TGF‑β、IL‑6、IL‑1β、抗IL‑4抗体、抗IFN‑γ抗体、抗IL‑2抗体、抗CD3抗体和抗CD28抗体。本发明扩增的Th22细胞可用来治疗一些自身免疫性疾病，如红斑狼疮、皮肌炎、血管炎、干燥综合征、硬皮病、类风湿性关节炎、强直性脊柱炎、多发性硬化、自身免疫性肝炎；慢性炎症性疾病，如糖尿病、冠心病、高脂血症、湿疹、白癜风、异位性皮炎、扁平苔藓等。

Description

一种有助于体外获得Th22细胞的组合物及其用途
技术领域
本发明涉及体外Breg细胞联合细胞因子体外诱导获得Th22细胞的方法，涉 及一种有助于体外获得Th22的组合物、方法及其在红斑狼疮、类风湿性关节炎、 硬皮病等炎症性疾病中的应用。
背景技术
调节性B细胞(Breg)是一群具有免疫调节作用的B细胞，因其膜标记为 CD19+CD5+CD1dhigh并主要分泌抑制性的细胞因子IL-10，故又称为B10细胞。 Breg细胞具有免疫抑制作用，可抑制其他效应性T细胞的增殖和分泌。基于Breg 细胞的负向调控作用，因此有潜力用于治疗自身免疫性疾病如红斑狼疮(SLE)、 胶原诱导关节炎(CIA)、实验性自身免疫性脑脊髓炎(EAE)、慢性结肠炎(CC) 等。但Breg细胞治疗自身免疫性疾病的具体机制并不清楚，进而限制了其临床 的使用。
Th22细胞是一群分泌IL-22的CD4+T细胞，Th17细胞也可以分泌部分的IL-22，TNF-α和IL-6可具有促进Th22分化的作用，其可通过分泌的IL-22起负向的免疫调 节作用，在自身免疫性疾病发病过程可能发挥调控免疫反应的作用。但在自身免 疫性疾病病情活动和发病过程中，Th22细胞减少，分泌IL-22的量减少，进而不 能控制亢进的自身免疫炎症损伤。但Th22细胞的体外诱导分化和扩增较难，目前 并没有找到Th22细胞有效的体外扩增方法。如能找到体外扩增Th22的方法，可 帮助快捷和高效的扩增Th22细胞，扩增后的Th22可用于自身免疫性疾病的细胞 治疗。
发明内容
本发明的目的之一是提供一种有助于体外获得Th22细胞的组合物。
本发明的另一目的是提供上述组合物的用途。
为了达到上述目的，本发明提供了一种有助于体外获得Th22细胞的组合物， 其特征在于，包括：幼稚性B细胞或Breg细胞，以及TGF-β、IL-6、IL-1β、抗 IL-4抗体、抗IFN-γ抗体、抗IL-2抗体、抗CD3抗体和抗CD28抗体。
优选地，所述的有助于体外获得Th22细胞的组合物还包括抗IgM抗体和 抗CD40L抗体。
优选地，所述的有助于体外获得Th22细胞的组合物包括：
幼稚性B细胞，以及抗CD3抗体1.8-2.2μg/ml、抗CD28抗体1.8-2.2μg/ml、 TGF-β0.5-5ng/ml、IL-6 20-100ng/ml、IL-1β 10-20ng/ml、抗IL-4抗体8-12 μg/ml、抗IFN-γ抗体8-12μg/ml、抗IL-2抗体8-12μg/ml，抗IgM抗体1.8-2.2 μg/ml、抗CD40L抗体1.8-2.2μg/ml；
或包括：Breg细胞，以及抗CD3抗体1.8-2.2μg/ml、抗CD28抗体1.8-2.2 μg/ml、TGF-β 0.5-5ng/ml、IL-6 20-100ng/ml、IL-1β 10-20ng/ml、抗IL-4 抗体8-12μg/ml、抗IFN-γ抗体8-12μg/ml、抗IL-2抗体8-12μg/ml。
更优选地，所述的有助于体外获得Th22细胞的组合物包括：
幼稚性B细胞，以及抗CD3抗体2μg/ml、抗CD28抗体2μg/ml、TGF-β0.5-5 ng/ml、IL-6 20-100ng/ml、IL-1β 10-20ng/ml、抗IL-4抗体10μg/ml、抗IFN-γ 抗体10μg/ml、抗IL-2抗体10μg/ml、抗IgM抗体2μg/ml和抗CD40L抗体2μg/ml；
或包括：Breg细胞，以及抗CD3抗体2μg/ml、抗CD28抗体2μg/ml、TGF-β 0.5-5ng/ml、IL-6 20-100ng/ml、IL-1β 10-20ng/ml、抗IL-4抗体10μg/ml、抗 IFN-γ抗体10μg/ml、抗IL-2抗体10μg/ml。
更优选地，所述的幼稚性B细胞或Breg细胞的个数为1×106-5×106个/ml。
本发明还提供了上述的有助于体外获得Th22细胞的组合物在体外获得 Th22细胞中的用途。
本发明还提供了一种体外获得Th22细胞的方法，其特征在于，应用上述的 有助于体外获得Th22细胞的组合物，具体步骤包括：向幼稚性T细胞的培养液 中加入上述任一组合物进行诱导分化。
本发明还提供了一种体外获得Th22细胞的方法，其特征在于，应用上述的 有助于体外获得Th22细胞的组合物，具体步骤包括：将幼稚性B细胞在含有抗 IgM抗体1-3μg/ml和抗CD40L抗体1-3μg/ml的培养基中预先培养1-3天，诱导 向Breg细胞分化，然后与T细胞共培养，加入抗CD3抗体1.8-2.2μg/ml、抗CD28 抗体1.8-2.2μg/ml、TGF-β0.5-5ng/ml、IL-620-100ng/ml、IL-1β 10-20ng/ml、 抗IL-4抗体8-12μg/ml、抗IFN-γ抗体8-12μg/ml、抗IL-2抗体8-12μg/ml，继续 培养。
本发明还提供了一种体外获得Th22细胞的方法，其特征在于，应用上述的 有助于体外获得Th22细胞的组合物，具体步骤包括：将幼稚性B细胞在含有抗 IgM抗体2μg/ml和抗CD40L抗体2μg/ml的培养基中预先培养2天，诱导向Breg 细胞分化，然后与T细胞共培养，加入抗CD3抗体2μg/ml、抗CD28抗体2μg/ml、 TGF-β0.5ng/ml、IL-6 50ng/ml、IL-1β10ng/ml、抗IL-4抗体10μg/ml、抗IFN-γ 抗体10μg/ml、抗IL-2抗体10μg/ml，继续培养。
本发明还提供了上述的有助于体外获得Th22细胞的组合物在制备治疗自 身免疫性疾病或免疫炎症性疾病药物中的应用。
优选地，所述的自身免疫性疾病或免疫炎症性疾病为红斑狼疮、皮肌炎、 血管炎、干燥综合征、硬皮病、类风湿性关节炎、强直性脊柱炎、多发性硬化和 自身免疫性肝炎；慢性炎症性疾病，如糖尿病、冠心病、高脂血症、湿疹、白癜 风或异位性皮炎。
本发明采用了以下两种技术方案：
第一种方案为：一种体外获得Th22细胞的方法，所述的方法包括以下步骤： 向幼稚性T细胞的培养液中加入如上任一所述的组合物进行诱导分化。
另一种方案为：B细胞在含有抗IgM抗体和抗CD40L抗体的培养基中预先 培养，诱导向Breg细胞分化，然后与幼稚性T细胞共培养，在培养液加入抗CD3 抗体、抗CD28抗体、TGF-β、IL-6、IL-1β、抗IL-4抗体、抗IFN-γ抗体和抗IL-2 抗体。
本发明中首次创新性发现Breg细胞具有促进Th22细胞分化和IL-22分泌的作 用，Breg细胞通过促进Th22细胞分化发挥对狼疮的治疗作用。
本发明以Th22细胞为靶点，发现一种体外促进幼稚性T细胞向Th22细胞 分化的新方法，即Breg细胞联合细胞因子刺激可有效促进Th22细胞的分化和具 有免疫抑制作用的细胞因子的分泌。而Breg细胞体外扩增后回输的也是通过促 进Th22细胞的方式发挥对自身免疫性疾病的治疗作用。
与现有技术相比，本发明的有益效果是：
1)新颖：本发明中使用Breg细胞，以及TGF-β+IL-6+IL-1β可有效促进 CD4+IL-22+Th22细胞的分化和扩增。Breg细胞联合TGF-β+IL-6+IL-1β扩大了Th22细胞的分化以及IL-22的分泌。该方法可开发用于体外Th22细胞的扩增。 即便根据现有的技术很难想到将Breg细胞，抗CD3抗体、抗CD28抗体、抗IL-4 抗体、抗IFN-γ抗体、抗IL-2抗体、TGF-β、IL-6和IL-1β组化起来诱导Th22 细胞的分化。此外，本发明创新性发现合适的抗体、细胞因子和细胞组合，恰当 的配伍比例可有效的诱导出大量的Th22细胞。
2)可靠：本发明中Th22细胞主要由幼稚性T细胞诱导分化而来，而幼稚 性T细胞可由脾脏和外周血通过免疫磁珠分选获得，分选方式方便快捷，分选可 获得大量幼稚性T细胞，为后续Th22诱导分化提供保障。且诱导Th22细胞扩 增的Breg细胞也可体外由幼稚性B细胞诱导分化而来，诱导方法成熟、诱导效 率可靠。因此可为体外大量的扩增Th22细胞提供保障。
3)方便快捷：临床应用过程中只需要抽取患者外周血，免疫磁珠分选幼稚 性T和B细胞，体外诱导向Th22细胞分化，然后将扩增的Th22细胞回输到患 者体内即可。
4)应用范围广：Th22细胞具有免疫抑制作用，可B细胞介导的体液免疫和 T细胞介导的细胞免疫发挥免疫抑制作用。本发明证实了Breg细胞联合TGF-β、 IL-6、IL-1β、抗IL-4抗体、抗IFN-γ抗体、抗IL-2抗体、抗CD3抗体和抗CD28 抗体可有效诱导体外Th22细胞的分化和IL-22的分泌。通过该方法体外扩增的 Th22细胞可广泛应用于治疗自身免疫性疾病或免疫炎症性疾病，如红斑狼疮、 皮肌炎、血管炎、干燥综合征、硬皮病、类风湿性关节炎、强直性脊柱炎、多发 性硬化和自身免疫性肝炎；慢性炎症性疾病，如糖尿病、冠心病、高脂血症、湿 疹、白癜风、异位性皮炎等。
附图说明
图1：Breg细胞联合细胞因子促进Th22细胞分化；
图2：Breg细胞联合细胞因子诱导的Th22细胞体外抑制Th1细胞的作用；
图3：Breg细胞联合细胞因子诱导的Th22细胞体外抑制Th17细胞的作用；
图4：Breg细胞联合细胞因子诱导的Th22细胞体外抑制B细胞的作用。
具体实施方式
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说 明本发明而不用于限制本发明的范围。此外应理解，在阅读了本发明讲授的内容 之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于 本申请所附权利要求书所限定的范围。
实施例
一、材料
C57BL/6(B6)鼠：购自上海斯莱克实验动物有限责任公司。
一种有助于体外获得Th22细胞的组合物，包括：幼稚性B细胞或Breg细 胞，以及TGF-β、IL-6、IL-1β、抗IL-4抗体、抗IFN-γ抗体、抗IL-2抗体、抗 CD3抗体和抗CD28抗体；其中，抗-CD3/CD28抗体购自Life Dynabeads公司； TGF-β、IL-6、IL-1β、抗IL-4抗体、抗IFN-γ抗体、抗IL-2抗体、抗IgM抗体 和抗CD40L抗体购自Peprotech公司。
幼稚性T细胞和幼稚性B细胞分选磁珠购自Miltenyi Biotec公司。
二、方法和结果
1、Breg细胞联合细胞因子促进Th22细胞分化：
从B6鼠脾脏中按照常规方法分选单个核细胞，通过免疫磁珠分选幼稚性T 细胞和幼稚性B细胞进行体外培养。第0天将分选到的1×106幼稚性B细胞重 悬于24孔板中，培养液为含10％胎牛血清的RPMI1640培养基，加入1μg/ml 抗IgM抗体和1μg/ml抗CD40L抗体，培养2天向Breg细胞诱导分化。将分选 到的1×106幼稚性T细胞重悬于24孔板中，培养液为含10％胎牛血清的 RPMI1640培养基，将上一步体外诱导分化得到的Breg细胞与T细胞共培养， Breg细胞和T细胞个数均为1×106个/ml，加入抗CD3抗体2μg/ml、抗CD28 抗体2μg/ml、TGF-β0.5ng/ml、IL-6 50ng/ml、IL-1β10ng/ml、抗IL-4抗体10μg/ml、 抗IFN-γ抗体10μg/ml、抗IL-2抗体10μg/ml，继续培养3天。
流式检测CD4+IL-22+Th22细胞的比例，酶联免疫吸附实验检测上清中IL-22 的分泌。结果如图1所示。该图说明了Breg细胞可以诱导Th22细胞的扩增和 IL-22的分泌。
2、Breg细胞联合细胞因子诱导的Th22细胞体外抑制Th1细胞的作用：
第0天，通过流式细胞仪分选第一步中体外诱导分化的约1×106/ml Th22细 胞，然后与免疫磁珠分选的1×106/ml CD45.2+鼠来源的幼稚性CD4+T细胞共培 养在RPMI1640培养基中，培养液中加入2μg/ml anti-CD3,2μg/ml anti-CD28诱 导向Th1细胞分化3天。酶联免疫吸附实验检测Th1细胞上清中相关的IFN-γ 和TNF-α细胞因子的含量。结果如图2所示，该图说明了Breg细胞联合细胞因 子诱导的Th22细胞体外抑制Th1细胞的作用。
3、Breg细胞联合细胞因子诱导的Th22细胞体外抑制Th17细胞的作用：
第0天，通过流式细胞仪分选第一步中体外诱导分化的约1×106/mlTh22细 胞，然后与免疫磁珠分选的1×106/ml CD45.2+鼠来源的幼稚性CD4+T细胞共培 养在RPMI1640培养基中(加入1ng/ml TGF-β和50ng/ml IL-6，诱导向Th17细 胞分化)3天。通过流式检测Th17细胞相关的CD4+IL-17+细胞的比例,酶联免 疫吸附实验检测上清中IL-17的分泌。结果如图3所示，该图说明了Breg细胞 联合细胞因子诱导的Th22细胞体外抑制Th17细胞的作用。
4、Breg细胞联合细胞因子诱导的Th22细胞体外抑制B细胞的作用：
第0天，通过流式细胞仪分选第一步中体外诱导分化的约1×106/mlTh22细 胞，然后与约1×106/ml幼稚性B细胞共培养(在培养液中加入抗2μg/ml IgM抗 体和2μg/ml抗CD40L抗体，诱导向效应性B细胞分化)3天。通过酶联免疫 吸附试验检测上清中IgM和IgG的分泌。结果如图3所示。该图说明了Breg细 胞联合细胞因子诱导的Th22细胞体外抑制B细胞的作用。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技 术人员，在不脱离本发明方法的前提下，还可以做出若干改进和补充，这些改进 和补充也应视为本发明的保护范围。

Claims (8)

1.一种有助于体外获得Th22细胞的组合物，其特征在于，所述的有助于体外获得Th22细胞的组合物包括：
幼稚性B细胞，以及抗CD3抗体1.8-2.2μg/ml、抗CD28抗体1.8-2.2μg/ml、TGF-β0.5-5ng/ml、IL-6 20-100ng/ml、IL-1β10-20ng/ml、抗IL-4抗体8-12μg/ml、抗IFN-γ抗体8-12μg/ml、抗IL-2抗体8-12μg/ml，抗IgM抗体1.8-2.2μg/ml和抗CD40L抗体1.8-2.2μg/ml；
或包括：Breg细胞，以及抗CD3抗体1.8-2.2μg/ml、抗CD28抗体1.8-2.2μg/ml、TGF-β0.5-5ng/ml、IL-6 20-100ng/ml、IL-1β10-20ng/ml、抗IL-4抗体8-12μg/ml、抗IFN-γ抗体8-12μg/ml和抗IL-2抗体8-12μg/ml。
2.如权利要求1所述的有助于体外获得Th22细胞的组合物，其特征在于，所述的有助于体外获得Th22细胞的组合物包括：
幼稚性B细胞，以及抗CD3抗体2μg/ml、抗CD28抗体2μg/ml、TGF-β0.5-5ng/ml、IL-6 20-100ng/ml、IL-1β10-20ng/ml、抗IL-4抗体10μg/ml、抗IFN-γ抗体10μg/ml、抗IL-2抗体10μg/ml、抗IgM抗体2μg/ml和抗CD40L抗体2μg/ml；
或包括：Breg细胞，以及抗CD3抗体2μg/ml、抗CD28抗体2μg/ml、TGF-β0.5-5ng/ml、IL-620-100ng/ml、IL-1β10-20ng/ml、抗IL-4抗体10μg/ml、抗IFN-γ抗体10μg/ml和抗IL-2抗体10μg/ml。
3.如权利要求1所述的有助于体外获得Th22细胞的组合物，其特征在于，所述的幼稚性B细胞或Breg细胞的个数为1×106-5×106个/ml。
4.权利要求1-3中任一项所述的有助于体外获得Th22细胞的组合物在体外获得Th22细胞中的用途。
5.一种体外获得Th22细胞的方法，其特征在于，应用权利要求1-3中任一项所述的有助于体外获得Th22细胞的组合物，具体步骤包括：向幼稚性T细胞的培养液中加入上述任一组合物进行诱导分化。
6.一种体外获得Th22细胞的方法，其特征在于，应用权利要求1或3中任一项所述的有助于体外获得Th22细胞的组合物，具体步骤包括：将幼稚性B细胞在含有抗IgM抗体1.8-2.2μg/ml和抗CD40L抗体1.8-2.2μg/ml的培养基中预先培养1-3天，诱导向Breg细胞分化，然后与T细胞共培养，加入抗CD3抗体1.8-2.2μg/ml、抗CD28抗体1.8-2.2μg/ml、TGF-β0.5-5ng/ml、IL-620-100ng/ml、IL-1β10-20ng/ml、抗IL-4抗体8-12μg/ml、抗IFN-γ抗体8-12μg/ml、抗IL-2抗体8-12μg/ml，继续培养。
7.一种体外获得Th22细胞的方法，其特征在于，应用权利要求1-3中任一项所述的有助于体外获得Th22细胞的组合物，具体步骤包括：将幼稚性B细胞在含有抗IgM抗体2μg/ml和抗CD40L抗体2μg/ml的培养基中预先培养2天，诱导向Breg细胞分化，然后与T细胞共培养，加入抗CD3抗体2μg/ml、抗CD28抗体2μg/ml、TGF-β0.5ng/ml、IL-6 50ng/ml、IL-1β10ng/ml、抗IL-4抗体10μg/ml、抗IFN-γ抗体10μg/ml、抗IL-2抗体10μg/ml，继续培养。
8.权利要求1-3中任一项所述的有助于体外获得Th22细胞的组合物在制备治疗自身免疫性疾病或免疫炎症性疾病药物中的应用。
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