Lu Y, Rodriguez R, Bjorndahl J, Phillips C A, Trevillyan J M
Department of Veterans Affairs Medical Center, Amarillo, USA.
Eur J Immunol. 1996 Jun;26(6):1278-84. doi: 10.1002/eji.1830260615.
CD28/B7 interactions have been demonstrated to provide a co-stimulatory signal for the generation of CD8+ cytotoxic T lymphocytes in the absence of CD4+ T helper cells. The CD28 signals required for induction of cytotoxicity have yet to be described. To investigate further the biochemical signaling pathways associated with CD28-dependent cytotoxicity, we have studied the human thymic leukemia cell line, YT. YT cells kill B7+ targets in a non-major histocompatibility complex (MHC)-restricted, CD28-dependent manner. CD28 ligation on the surface of YT cells caused a rapid increase in the tyrosine phosphorylation of four major cellular substrates with masses estimated to be 110, 95, 85, and 44 kDa. The 110 and 85 kDa substrates were identified as the catalytic and regulatory subunits, respectively, of phosphatidylinositol 3-kinase (PI3-K). Engagement of CD28 caused the rapid receptor association and activation of PI3-K but did not activate phospholipase C gamma. CD28-induced tyrosine phosphorylation and PI3-K activation was independent of p56lck protein tyrosine kinase (PTK) activity (previously reported to be associated with CD28) and was insensitive to inhibition by the PTK inhibitor herbimycin A. Two structurally and mechanistically dissimilar inhibitors of PI3-K, wortmannin and 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002) also failed to block CD28-dependent tyrosine phosphorylation events or the association of PI3-K with the CD28 receptor. However, both drugs inhibited CD28-dependent cytotoxicity and CD28 receptor associated PI3-K activity with IC50 values similar to the reported IC50 values for PI3-K inhibition. Although herbimycin A did not significantly block the observed CD28-dependent tyrosine phosphorylation or PI3-K activation, herbimycin did block CD28-dependent cytotoxicity in a dose-dependent manner. These data support a role for PI3-K activation in the CD28-dependent initiation of cytotoxic effector function and suggest that a herbimycin sensitive step(s) is either CD28-independent, resides within a PI3-K-independent CD28 signaling pathway, or is downstream of CD28-dependent PI3-K activation.
CD28/B7相互作用已被证明在缺乏CD4+辅助性T细胞的情况下，为CD8+细胞毒性T淋巴细胞的产生提供共刺激信号。诱导细胞毒性所需的CD28信号尚未得到描述。为了进一步研究与CD28依赖性细胞毒性相关的生化信号通路，我们研究了人胸腺白血病细胞系YT。YT细胞以非主要组织相容性复合体（MHC）限制、CD28依赖性的方式杀伤B7+靶细胞。YT细胞表面的CD28连接导致四种主要细胞底物的酪氨酸磷酸化迅速增加，其质量估计分别为110、95、85和44 kDa。110 kDa和85 kDa的底物分别被鉴定为磷脂酰肌醇3激酶（PI3-K）的催化亚基和调节亚基。CD28的结合导致PI3-K的快速受体缔合和激活，但未激活磷脂酶Cγ。CD28诱导的酪氨酸磷酸化和PI3-K激活独立于p56lck蛋白酪氨酸激酶（PTK）活性（先前报道与CD28相关），并且对PTK抑制剂赫曲霉素A的抑制不敏感。两种结构和机制不同的PI3-K抑制剂渥曼青霉素和2-（4-吗啉基）-8-苯基-4H-1-苯并吡喃-4-酮（LY294002）也未能阻断CD28依赖性酪氨酸磷酸化事件或PI3-K与CD28受体的缔合。然而，这两种药物均抑制CD28依赖性细胞毒性和与CD28受体相关的PI3-K活性，其IC50值与报道的PI3-K抑制IC50值相似。尽管赫曲霉素A没有显著阻断观察到的CD28依赖性酪氨酸磷酸化或PI3-K激活，但赫曲霉素确实以剂量依赖性方式阻断了CD28依赖性细胞毒性。这些数据支持PI3-K激活在CD28依赖性细胞毒性效应功能启动中的作用，并表明一个对赫曲霉素敏感的步骤要么独立于CD28，存在于一个独立于PI3-K的CD28信号通路中，要么在CD28依赖性PI3-K激活的下游。
