Arimilli S, Nag B
Anergen Inc., Redwood City, CA 94063, USA.
Recept Signal Transduct. 1997;7(3):151-63.
Recently it has been shown that purified complexes of major histocompatibility (MHC) class II and antigenic peptide can recognize T-cell receptors (TCRs) on virally transformed CD4+ T-cells in vitro. It is not clearly understood whether peptide bound to purified MHC II molecules (MHC-P), or to MHC II molecules on the surface of antigen-presenting cells (APC-peptide), initiate similar or different signals in transformed human T-cells. To address this question, the expression of protein tyrosine kinases (PTKs), their phosphorylation, and the effect of various kinase inhibitors were investigated using transformed T- and B-cells. HLA-DR2- and MBP(84-102)-restricted cloned T-cells (SS8T) immortalized with herpes saimiri virus (HSV) and DR2-expressing lymphoblastoid B cells transformed with Epstein-Barr virus (EBV) were utilized in this study. The expression and phosphorylation of three major PTKs (1ck-56, fyn-59, zap-70) involved in signaling through the TCR were analyzed by enhanced chemiluminescence blots. T-cells exposed to soluble MHC-P complex did not show altered expression of 1ck-56 protein. In contrast, a decrease in 1ck expression was observed in SS8T cells when TCRs were engaged with APC-peptide. Upon interaction with the TCR, both MHC-P complex and APC-peptide showed increased fyn-59 protein expression and phosphorylation. In our experiments using immortalized T- and B-cells, the expression of zap-70 protein remained unchanged. When T-cells were exposed to herbimycin and H-7, inhibitors of PTKs and protein kinase C (PKC) pathways, respectively, a dose-dependent decrease in gamma-IFN levels was observed with both systems. However, in the presence of genestein, another PTK inhibitor, such decrease in gamma-IFN was observed only in the case in which T-cells were exposed to MHC-P complexes. These results together suggest that the occupancy of TCRs in transformed T-cells by soluble MHC-P complex and APC-peptide differs with respect to the 1ck expression, although both can induce signals that lead to increased fyn activity and its phosphorylation. In addition, genestein showed differential inhibitory effect on gamma-IFN production by T-cells exposed to APC-peptide and MHC-P complexes, suggesting that the TCR occupancy by MHC-P complex and APC-peptide have subtle differences in PTK pathways.
最近研究表明，纯化的主要组织相容性复合体（MHC）II类分子与抗原肽复合物在体外能够识别病毒转化的CD4+ T细胞上的T细胞受体（TCR）。目前尚不清楚与纯化的MHC II类分子结合的肽（MHC-P），或与抗原呈递细胞表面的MHC II类分子结合的肽（APC-肽），在转化的人T细胞中引发的信号是相似还是不同。为了解决这个问题，我们使用转化的T细胞和B细胞研究了蛋白酪氨酸激酶（PTK）的表达、它们的磷酸化以及各种激酶抑制剂的作用。本研究利用感染疱疹病毒（HSV）永生化的HLA-DR2和髓鞘碱性蛋白（84-102）限制性克隆T细胞（SS8T），以及感染EB病毒（EBV）转化的表达DR2的淋巴母细胞样B细胞。通过增强化学发光印迹分析参与TCR信号传导的三种主要PTK（1ck-56、fyn-59、zap-70）的表达和磷酸化。暴露于可溶性MHC-P复合物的T细胞未显示1ck-56蛋白表达改变。相反，当TCR与APC-肽结合时，SS8T细胞中观察到1ck表达下降。与TCR相互作用后，MHC-P复合物和APC-肽均显示fyn-59蛋白表达和磷酸化增加。在我们使用永生化T细胞和B细胞的实验中，zap-70蛋白的表达保持不变。当T细胞分别暴露于PTK抑制剂赫曲霉素和蛋白激酶C（PKC）途径抑制剂H-7时，两个系统中γ-干扰素水平均出现剂量依赖性下降。然而，在存在另一种PTK抑制剂染料木黄酮的情况下，仅在T细胞暴露于MHC-P复合物时观察到γ-干扰素水平的下降。这些结果共同表明，可溶性MHC-P复合物和APC-肽在转化T细胞中占据TCR的情况在1ck表达方面有所不同，尽管两者都能诱导导致fyn活性及其磷酸化增加的信号。此外，染料木黄酮对暴露于APC-肽和MHC-P复合物的T细胞产生γ-干扰素具有不同的抑制作用，这表明MHC-P复合物和APC-肽占据TCR在PTK途径中存在细微差异。
