MOLECULAR MECHANISMS OF T-LYMPHOCYTE ACTIVATION

本研究的目的是探讨这一耦合机制。 淋巴细胞的T细胞受体(TCR)/CD3复合体与 磷脂酶C(PLC)--肌醇磷脂的关键酶 (InsPL)水解途径。这条途径产生了第二信使 这可能是诱导T细胞活化的关键。一种对 淋巴细胞活化的机制可能为设计提供依据 针对信号关键成分的免疫调节策略 转导途径。一种细胞通透性的策略是 这是我们实验室开发的，它允许访问小分子(例如， 核苷酸、多肽等)的细胞内环境 淋巴细胞，同时保持TCR/CD3复合体与PLC的偶联。 通过使用这一策略，我们获得了有关监管的信息 InsPL水解度和src解离的化学计量 激酶，Fyn，与CD3复合体的多肽。通过使用这一点 战略，我们将寻求关于PLC控制机制的信息 活动。抗PLC单抗与src家族产物的共沉淀 (Fyn、lck、ves等)将会被使用。短暂或“弱”的相互作用 将通过使用同源双官能化学交联剂在 将细胞通透化，然后进行免疫沉淀。这一策略是 成功获取CD3/FYN的化学计量学数据 互动。通过使用蛋白酪氨酸磷酸的抑制剂 磷酸酶(包括可能具有潜在功能的多肽 竞争性抑制物，如磷酸化FYN或LCK多肽)在 细胞的完好和通透性，会注意其作用 这些酶，包括CD45磷酸酶，在调节InsPL中 水解液。由于先前的证据表明信号的异质性 不同T细胞亚群之间的转导，将受到关注 潜在的差异及其功能影响。首字母A 将尝试构建PLC中有缺陷的T细胞克隆(PLC- Y1)通过基因“敲除”这种酶。

The objective of this study is to investigate the coupling mechansism between the T cell receptor (TCR)/CD3 complex of lymphocytes and phospholipase C (PLC), the key enzyme in the inositol phospholipid (InsPL) hydrolysis pathway. This pathway generates second messengers that may be critical in inducing T cell activation. An understanding of the mechanism of lymphocyte activation may offer a basis for designing immunomodulatory strategies targeted to critical elements of the signal transducion pathway. A strategy of cell permeabilization has been developed in our laboratory which allows access to micromolecules (e.g., nucleotide, peptides, etc.) to the intracellular enviromnment of lymphocytes, while maintaining coupling of the TCR/CD3 complex with PLC. By using this strategy, we have obtained information on the regulation of InsPL hydrolysis and on the stoichiometry of the assoication of a src kinase, fyn, with polypetides of the CD3 complex. By the use of this strategy, we will seek information on the mechanism of control of PLC activity. Coprecipitation using anti-PLC Ab and src-family products (fyn, lck.ves, etc.) will be used. Transient or "weak" interactions will be "stabilized" by using homobifunctional chemical cross-linker in permeabilized cells followed by immunoprecipitation. This strategy was previously used successfully to obtain stoichionetry data of CD3/fyn interation. By using inhibitors of protein tyrosine phosphate phosphatase (including peptides which may function as potential competitive inhibitors, such as phosphorylated fyn or lck peptides) in intact and permeabilized cells, attention will be paid to the role of these enzymes, including the CD45 phosphatase, in modulating insPL hydrolysis. Since previous evidence suggests heterogeneity in signal transduction among different T cell subsets, attention will be paid to potential differences and their functional implications. AN initial attempt will be made to construct T cell clones defective in PLC (PLC- y1) by genetically "knocking out" the enzyme.