CD3ζ is part of the T cell receptor (TCR)/CD3 complex that plays a critical role in antigen recognition and subsequent T cell activation. Understanding the mechanisms that regulate CD3ζ can provide new insights into the T cell-mediated immune responses. However, it is challenging to deliver exogenous genes into T cells for functional and mechanistic analyses. To this end, we established a non-T cell transfection system based on HEK293FT cells to screen for candidate regulatory proteins. The transfection was optimized using relatively high confluent cultures and the transfection reagent PolyJet™. Pervanadate (PV) treatment sustained tyrosine phosphorylation of CD3ζ, and facilitated the subsequent activation-dependent ubiquitination by E3 ligase Cbl-b in the HEK293FT system. Lck and Zap70 kinases enhanced the levels of phosphorylated CD3ζ in the presence of PV. We compared the effects of E3 ligases and the corresponding adaptor proteins on activation-dependent ubiquitination of CD3ζ in the PV-stimulated cells, and found that Cbl-b was most effective. Taken together, we have demonstrated that a non-T cell transfection system based on PV-treated HEK293FT cells could effectively mimic CD3ζ phosphorylation and ubiquitination and is a promising model for studying the role of CD3ζ signaling in T cell activation.

中文翻译：

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基于 HEK293FT 细胞的优化非 T 细胞转染系统，用于 CD3ze 磷酸化和泛素化

CD3δ 是 T 细胞受体 (TCR)/CD3 复合物的一部分，在抗原识别和随后的 T 细胞激活中发挥着关键作用。了解调节 CD3ze 的机制可以为 T 细胞介导的免疫反应提供新的见解。然而，将外源基因导入 T 细胞进行功能和机制分析具有挑战性。为此，我们建立了基于HEK293FT细胞的非T细胞转染系统来筛选候选调节蛋白。使用相对较高汇合度的培养物和转染试剂 PolyJet™ 优化转染。过钒酸盐 (PV) 处理可维持 CD3ze 的酪氨酸磷酸化，并促进 HEK293FT 系统中 E3 连接酶 Cbl-b 随后的激活依赖性泛素化。在 PV 存在的情况下，Lck 和 Zap70 激酶增强了磷酸化 CD3z 的水平。我们比较了 E3 连接酶和相应的接头蛋白对 PV 刺激细胞中 CD3ze 激活依赖性泛素化的影响，发现 Cbl-b 最有效。综上所述，我们证明基于 PV 处理的 HEK293FT 细胞的非 T 细胞转染系统可以有效模拟 CD3ze 磷酸化和泛素化，并且是研究 CD3ze 信号传导在 T 细胞激活中的作用的有前途的模型。