Abstract
Chinese Mammalian T-cell responses require synergism between the first signal and co-stimulatory signal. However, whether and how dual signaling regulates the T-cell response in early vertebrates remains unknown. In the present study, we discovered that the Nile tilapia ( Oreochromis niloticus) encodes key components of the LAT signalosome, namely, LAT, ITK, GRB2, VAV1, SLP-76, GADS, and PLC-γ1. These components are evolutionarily conserved, and CD3ε mAb-induced T-cell activation markedly increased their expression. Additionally, at least ITK, GRB2, and VAV1 were found to interact with LAT for signalosome formation. Downstream of the first signal, the NF-κB, MAPK/ERK, and PI3K-AKT pathways were activated upon CD3ε mAb stimulation. Furthermore, treatment of lymphocytes with CD28 mAbs triggered the AKT-mTORC1 pathway downstream of the co-stimulatory signal. Combined CD3ε and CD28 mAb stimulation enhanced ERK1/2 and S6 phosphorylation and elevated NFAT1, c-Fos, IL-2, CD122, and CD44 expression, thereby signifying T-cell activation. Moreover, rather than relying on the first or co-stimulatory signal alone, both signals were required for T-cell proliferation. Full T-cell activation was accompanied by marked apoptosis and cytotoxic responses. These findings suggest that tilapia relies on dual signaling to maintain an optimal T-cell response, providing a novel perspective for understanding the evolution of the adaptive immune system. T细胞是适应性免疫的重要武器，在抵抗细菌、病毒和寄生虫感染以及肿瘤发生中发挥关键作用。哺乳动物中，T细胞免疫需要第一信号和共刺激信号的协同作用。然而，双重信号是否以及如何调控早期脊椎动物的T细胞免疫仍然未知。在该研究中，我们发现硬骨鱼尼罗罗非鱼（ Oreochromis niloticus）编码了LAT信号复合体的关键组分LAT、ITK、GRB2、VAV1、SLP-76、GADS和PLC-γ1。这些组分在进化上是保守的，CD3ε单克隆抗体（mAb）诱导的T细胞活化显著增加了它们的表达。然而，只有ITK、GRB2和VAV1与LAT相互作用，形成信号小体。在第一信号的下游，NF-κB、MAPK/ERK和PI3K-AKT途径在CD3ε mAb的刺激下被激活。此外，用CD28 mAb处理淋巴细胞，触发了共刺激信号下游的AKT-mTORC1途径。使用CD3ε和CD28 mAb的联合刺激，增强了ERK1/2和S6的磷酸化，增加了NFAT1、c-Fos、IL-2、CD122和CD44的表达，表明激活了T细胞。此外， T细胞的增殖需要第一信号、共刺激信号的协同作用，而T细胞的激活伴随着显著的凋亡和细胞毒性作用。因此，我们的研究表明罗非鱼依靠双重信号来维持最佳的T细胞反应，为理解适应性免疫系统的进化提供了新视角。.