Department of Immunology, SA Pathology at Women's and Children's Hospital, North Adelaide, Australia.
The Robinson Research Institute and School of Medicine, University of Adelaide, Adelaide, Australia.
Immunology. 2023 Nov;170(3):359-373. doi: 10.1111/imm.13674. Epub 2023 Jun 20.
A significant number of babies present transiently with low protein kinase C zeta (PKCζ) levels in cord blood T cells (CBTC), associated with reduced ability to transition from a neonatal Th2 to a mature Th1 cytokine bias, leading to a higher risk of developing allergic sensitisation, compared to neonates whose T cells have 'normal' PKCζ levels. However, the importance of PKCζ signalling in regulating their differentiation from a Th2 to a Th1 cytokine phenotype propensity remains undefined. To define the role of PKCζ signalling in the regulation of CBTC differentiation from a Th2 to a Th1cytokine phenotype we have developed a neonatal T cell maturation model which enables the cells to develop to CD45RA /CD45RO T cells while maintaining the Th2 immature cytokine bias, despite having normal levels of PKCζ. The immature cells were treated with phytohaemagglutinin, but in addition with phorbol 12-myristate 13-acetate (PMA), an agonist which does not activate PKCζ. This was compared to development in CBTC in which the cells were transfected to express constitutively active PKCζ. The lack of PKCζ activation by PMA was monitored by western blot for phospho-PKCζ and translocation from cell cytosol to the membrane by confocal microscopy. The findings demonstrate that PMA fails to activate PKCζ in CBTC. The data show that CBTC matured under the influence of the PKC stimulator, PMA, maintain a Th2 cytokine bias, characterised by robust IL-4 and minimal interferon gamma production (IFN-γ), and lack of expression of transcriptional factor, T-bet. This was also reflected in the production of a range of other Th2/Th1 cytokines. Interestingly, introduction of a constitutively active PKCζ mutant into CBTC promoted development towards a Th1 profile with high IFN-γ production. The findings demonstrate that PKCζ signalling is essential for the immature neonatal T cells to transition from a Th2 to a Th1 cytokine production bias.
相当数量的婴儿在脐带血 T 细胞（CBTC）中短暂表现出低蛋白激酶 C ζ（PKCζ）水平，这与从新生儿 Th2 向成熟 Th1 细胞因子偏向的过渡能力降低有关，导致过敏致敏的风险增加，与 T 细胞具有“正常”PKCζ 水平的新生儿相比。然而，PKCζ 信号在调节其从 Th2 向 Th1 细胞因子表型偏向的分化中的重要性仍未确定。为了确定 PKCζ 信号在调节 CBTC 从 Th2 向 Th1 细胞因子表型分化中的作用，我们开发了一种新生儿 T 细胞成熟模型，该模型允许细胞发育为 CD45RA/CD45RO T 细胞，同时保持 Th2 不成熟细胞因子偏向，尽管 PKCζ 水平正常。幼稚细胞用植物血球凝集素处理，但另外用佛波醇 12-肉豆蔻酸 13-乙酸酯（PMA）处理，PMA 是一种不激活 PKCζ 的激动剂。这与 CBTC 中的发育进行了比较，其中细胞被转染以表达组成型激活的 PKCζ。通过 Western blot 监测 PMA 对磷酸化 PKCζ 的缺乏激活和通过共聚焦显微镜从细胞质向膜的易位。结果表明，PMA 未能在 CBTC 中激活 PKCζ。数据表明，在 PKC 刺激物 PMA 的影响下成熟的 CBTC 保持 Th2 细胞因子偏向，其特征是强烈的 IL-4 和最小的干扰素γ产生（IFN-γ），并且缺乏转录因子 T-bet 的表达。这也反映在一系列其他 Th2/Th1 细胞因子的产生中。有趣的是，将组成型激活的 PKCζ 突变体引入 CBTC 促进了向高 IFN-γ产生的 Th1 表型的发育。研究结果表明，PKCζ 信号对于不成熟的新生儿 T 细胞从 Th2 向 Th1 细胞因子产生偏向的过渡是必不可少的。
