人正常记忆 b 细胞与长寿浆细胞体外分化模型
Summary
利用多步培养系统, 我们报告了体外 b 细胞对血浆细胞分化模型。
Abstract
血浆细胞 (pc) 分泌大量抗体, 并从被激活的 b 细胞中产生。pc 是位于骨髓或粘膜中的罕见细胞, 可确保体液免疫。由于其频率和位置较低, 对个人电脑的研究在人类中很困难。我们报告了 b 到 pc 体外分化模型使用选定的细胞因子和活化分子的组合, 允许重现发生在体内的细胞分化。在这个体外模型中, 记忆 b 细胞 (mbc) 将分化为前血浆 ab面 (前 pbs)、血浆 abc (pbs)、早期 pc, 最后, 分化为长寿 pc, 表型接近健康个体中的对应基因。我们还构建了一个开放访问生物信息学工具, 以分析与 pc 分化有关的 gep 数据中最突出的信息。这些资源可用于研究人 b 对 pc 分化, 在目前的研究中, 我们研究了人 b 到 pc 分化过程中表观遗传因子的基因表达调控。
Introduction
b 细胞与血浆细胞 (pc) 的分化对体液免疫和保护宿主免受感染至关重要1。b 至 pc 分化与转录能力和代谢的主要变化有关, 以适应抗体分泌。对控制 b 到 pc 分化的转录因子进行了广泛的研究, 揭示了包括 b-和 pc 特异性转录因子 (tf)2在内的专属网络。在 b 细胞中, pox5、bcl6 和 bach2 tf 是 b 细胞身份2、3 的监护人。irf4、 prdm1编码 blimp1 和xbp1 pc tf 的诱导将消灭 b 细胞基因, 并诱导一个协调的抗体分泌细胞转录程序 3,4,5。这些协调的转录变化与 ig 基因转录激活以及从膜结合形式到免疫球蛋白重链 2,3的分泌形式的转换有关,4. b 至 pc 分化与内质网和 golgi 仪器功能相关的基因诱导, 同时进行展开的蛋白质反应 (upr) 激活, 已知通过适应合成分泌免疫球蛋白6,7。tf xbp1 在这种细胞适应 8,9,10中起着重要作用。
b 细胞和 pc 是体液免疫的关键参与者。了解控制正常血浆细胞产生和存活的生物过程对于需要确保有效免疫反应和防止自身免疫或免疫缺陷的治疗干预至关重要。pc 是罕见的细胞, 早期分化阶段发生在解剖位置, 妨碍充分的生物表征, 特别是在人类。利用多步培养系统, 我们报告了一个体外 b 到 pc 分化模型。该模型再现了体内不同器官中发生的顺序细胞分化和成熟11、 12、13。第一步, 记忆 b 细胞首先被 cd40 配体、寡核苷酸和细胞因子结合激活四天, 并分化为前质体 (prepbs)。第二步, 通过去除 cd40l 和寡核苷酸刺激并改变细胞因子组合, 诱导前质体分化为质粒 (pbs)。第三步, 通过改变细胞因子组合11,12,诱导血浆 ab面体分化为早期 pc。第四步是用骨髓基质细胞条件培养基或选定的生长因子13培养这些早期的 pc, 从而获得完全成熟的 pc。这些成熟的 pc 可以在体外存活数月, 并分泌大量免疫球蛋白 (图 1)。有趣的是, 我们的体外模型重述了可在体内检测到的不同 b 到 pc 阶段的协同转录变化和表型11、12、13、14 ,15。pc 是稀有细胞, 我们的体外分化模型可以研究人的 b 到 pc 分化。
Protocol
该议定书遵循《赫尔辛基宣言》和蒙彼利埃大学生物资源医院中心的协议。 1. 体外正常血浆细胞分化模型 请注意:pc 是通过 11、12、13的四步文化生成的。 b 细胞扩增和分化 使用健康志愿者的外周血细胞进行记忆 b 细胞纯化。 用 rpmi 1640…
Representative Results
体外正常 pc 分化的总体过程如图 1所示。利用这里提出的协议, 我们可以产生足够数量的细胞, 而这些细胞无法从体内人体样本中获得。尽管研究了复杂的转录因子网络在 pc 分化中的作用, 但调节关键 pc 分化转录网络的机制仍然鲜为人知。细胞分化主要是由表观遗传和转录的变化驱动的。利用我们的体外模型和 b 到 pc gep 图集, 研究了表观遗传因子在?…
Discussion
在人类中, pc 是罕见的细胞, 分化阶段发生在解剖的地方, 妨碍充分的生物表征。我们已经开发了一个体外 b 到 pc 分化模型使用多步骤培养系统, 其中激活分子和细胞因子的各种组合随后应用, 以再现发生在体内不同的器官组织 11,12,13。
其他有效的体外 b 对 pc 分化模型报告为18,<sup …
Divulgations
The authors have nothing to disclose.
Acknowledgements
这项工作得到了法国国家癌症研究所 (PLBIO15-256)、anr (tie-leot 跳过) 和 itmo 癌症 (mm & tt) 的资助。
Materials
| anti-CD2 magnetic beads | Invitrogen | 11159D | |
| Anti-CD138-APC | Beckman-Coulter | B49219 | |
| Anti-CD19-APC | BD | 555415 | |
| Anti-CD20-PB | Beckman-Coulter | B49208 | |
| Anti-CD27-PE | BD | 555441 | |
| Anti-CD38-PE | Beckman-Coulter | A07779 | |
| Anti-histidine | R&D Systems | MAB050 | |
| CpG ODN(PT) | Sigma | T*C*G*T*C*G*T*T*T*T*G*T*C* G*T*T*T*T*G*T*C*G*T*T |
|
| human Transferin | Sigma-Aldrich | T3309 | |
| IFN-α | Merck | Intron A | |
| IMDM | Gibco | 31980-022 | |
| Recombinan Human CD40L-hi | R&D Systems | 2706-CL | |
| Recombinant Human APRIL | R&D Systems | 5860-AP-010 | |
| Recombinant Human IL-10 | R&D Systems | 217-IL- | |
| Recombinant Human IL-15 | Peprotech | 200-15-10ug | |
| Recombinant Human IL-2 Protein | R&D Systems | 202-IL- | |
| Recombinant Human IL-6 | Peprotech | 200-06 |
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Citer Cet Article
Jourdan, M., de Boussac, H., Viziteu, E., Kassambara, A., Moreaux, J. In Vitro Differentiation Model of Human Normal Memory B Cells to Long-lived Plasma Cells. J. Vis. Exp. (143), e58929, doi:10.3791/58929 (2019).