Continuous Culture of Mouse Primary B Lymphocytes by Forced Expression of Bach2.
Continuous Culture of Mouse Primary B Lymphocytes by Forced Expression of Bach2.
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强制表达Bach2对小鼠原代B淋巴细胞的连续培养
DOI：
10.4049/jimmunol.2100172
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发表时间：
2021-09-01
期刊：
影响因子：
--
通讯作者：
Kelsoe G
中科院分区：
文献类型：
--
作者：
Finney J;Kelsoe G
原代 B 淋巴细胞的稳定、长期培养具有许多潜在的科学和医学应用，但仍然是一项难以捉摸的壮举。长期培养的一个主要障碍是体外有丝分裂原快速驱动 B 细胞分化为短命的浆细胞 (PC)。 PC 分化由相反的转录因子组控制：Pax5、Bach2 和 Bcl6 抑制 PC 定向，而 IRF4 和 Blimp1 则促进 PC 定向。为了确定转录编程是否可以通过阻断 PC 定型来延长 B 细胞培养，我们生成了关键转录因子功能获得或丧失的小鼠原代 B 细胞，用 CD154 和 IL-21 持续刺激这些细胞，并在体外测定生长潜力和表型。我们发现 Bach2 的转基因表达可抑制 PC 定型，并赋予 B 细胞响应外部增殖和生存信号的非凡生长潜力。长期 Bach2 转基因 B 细胞系具有遗传稳定的 BCR（即不会获得 V(D)J 突变），表达高水平的 MHCII 和用于共刺激 T 细胞的分子，并在与 BCR 配体孵育时转导细胞内信号。在已建立的转基因细胞系中沉默 Bach2 转基因会导致细胞分泌大量 Ig。该系统在 mAb 生产、BCR 信号传导研究、Ag 呈递给 T 细胞以及用于过继细胞转移的离体克隆扩增方面具有潜在应用。此外，我们的结果提供了对活化 B 细胞命运的分子控制的见解，并表明体内强制 Bach2 表达可能会增强生发中心 B 细胞或记忆 B 细胞的分化，但会损害 PC 的承诺。
Stable, long-term culture of primary B lymphocytes has many potential scientific and medical applications, but remains an elusive feat. A major obstacle to long-term culture is that in vitro mitogens quickly drive B cells to differentiate into short-lived plasmacytes (PCs). PC differentiation is governed by opposing teams of transcription factors: Pax5, Bach2, and Bcl6 suppress PC commitment, while IRF4 and Blimp1 promote it. To determine whether transcriptional programming could prolong B-cell culture by blocking PC commitment, we generated mouse primary B cells harboring gain- or loss-of-function in the key transcription factors, continuously stimulated these cells with CD154 and IL-21, and determined growth potential and phenotypes in vitro. We found that transgenic expression of Bach2 prohibits PC commitment and endows B cells with extraordinary growth potential in response to external proliferation and survival cues. Long-term Bach2-transgenic B cell lines have genetically stable BCRs (i.e., do not acquire V(D)J mutations), express high levels of MHCII and molecules for co-stimulation of T cells, and transduce intracellular signals when incubated with BCR ligands. Silencing the Bach2 transgene in an established transgenic cell line causes the cells to secrete large quantities of Ig. This system has potential applications in mAb production, BCR signaling studies, Ag presentation to T cells, and ex vivo clonal expansion for adoptive cell transfer. Additionally, our results provide insight into molecular control over activated B-cell fate, and suggest that forced Bach2 expression in vivo may augment germinal center B cell or memory B cell differentiation at the expense of PC commitment.
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影响因子：
3.7
作者：
Verkoczy L;Moody MA;Holl TM;Bouton-Verville H;Scearce RM;Hutchinson J;Alam SM;Kelsoe G;Haynes BF
通讯作者：
Haynes BF
影响因子：
32.4
作者：
Kallies, Axel;Hasbold, Jhagvaral;Nutt, Stephen L.
通讯作者：
Nutt, Stephen L.
影响因子：
5.3
作者：
Li, Feng;Vijayasankaran, Natarajan;Amanullah, Ashraf
通讯作者：
Amanullah, Ashraf
影响因子：
56.9
作者：
Lin, Y;Wong, KK;Calame, K
通讯作者：
Calame, K
DOI：
10.1073/pnas.1102571108
发表时间：
2011-07-12
影响因子：
11.1
作者：
Kuraoka, Masayuki;Holl, T. Matt;Kelsoe, Garnett
通讯作者：
Kelsoe, Garnett