CD8-LYT-2 RECOGNITION OF THE CLASS I ALPHA 3 DOMAIN

CD8-LYT-2 对 I 类 ALPHA 3 结构域的识别

• 批准号: 3141824
• 负责人: JANET M CONNOLLY
• 金额: $ 11.52万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-09-30 至 1992-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3141824
• 关键词: CD8 molecule; T cell receptor; antigen presenting cell; cell cell interaction; clone cells; cytotoxic T lymphocyte; flow cytometry; histocompatibility antigens; laboratory mouse; protein sequence; receptor binding; site directed mutagenesis; tissue /cell culture

The CD8 molecule on the surface of class I reactive T cells was initially believed to function as an accessory molecule, increasing the affinity of the TCR for class I MHC. Recent evidence suggests that CD8 molecules may associate with the TCR for antigen and thereby function in signal transduction and activation. It appears now that the CD8 receptor is also involved in both positive and negative selection of class I reactive T cells during thymic maturation. The recent observation by the principal investigator the CD8 binds to residues in the class I alpha3 domain was the first identification of the monomorphic determinant recognized by CD8. Of paramount importance in these studies was the use of the Dd alpha 3 domain mutant, Ddlys, that had lost the ability to be recognized by CD8. The primary objectives of this proposal are to probe questions concerning the role of CD8 in T cell recognition and to extend the observation that the CD8 molecule o class I-specific CTL: recognized residues in the class I alpha3 domain. Whereas the previous studies involved heterogeneous populations of CTL the new investigations will employ clonal populations of CD8-dependent and CD8-independent CTL. These clones will be analyzed for their ability to lyse cells expressing Ddlys and other alpha3 domain mutants and for the ability of antibodies to the alpha3 domain and CD8 to inhibit their function. They will also be used in cell:cell binding assays to determine their dependency on CD8 for cell binding and the effect of alpha3 domain mutants of cell binding. Additional alpha3 domain mutants obtained by site directed mutagenesis are being provided by Dr. T. Potter. These new mutants will be compared in a variety of assays and will define the extent of the class I ligand recognized by CD8. In addition, the CTL response against the class I alpha3 domain mutants will be analyzed to determine if additional residues are involved in recognition by CD8. Finally, the involvement of CD8 in the generation and effector function of MHC-restricted CTL responses will be analyzed. Thus several salient properties of CD8 will be investigated using a variety of immunologic assays and a unique collection of site-specific class I mutants.

I类反应性T细胞表面上的CD8分子最初是 被认为是作为辅助分子，增加了 I类MHC的TCR 最近的证据表明，CD8分子可能 与抗原的TCR结合，从而在信号传导中起作用。 转导和激活。 现在看来，CD8受体也是 参与I类反应性T细胞的阳性和阴性选择 胸腺成熟过程中的细胞。 校长最近的观察 研究人员发现，CD8与I类α 3结构域中的残基结合是 首次鉴定出CD8识别的单态决定簇。 的 在这些研究中最重要的是使用Dd α 3结构域 突变体，Ddlys，已经失去了被CD8识别的能力。 的 该提案的主要目标是探讨有关 CD8在T细胞识别中的作用，并扩大观察， I类特异性CTL的CD8分子：I类中识别的残基 α 3结构域。 而以前的研究涉及异质性 新的研究将采用克隆群体的CTL， CD8依赖性和CD8非依赖性CTL。 这些克隆将被分析， 它们裂解表达Ddlys和其它α 3结构域的细胞的能力 突变体的能力，以及针对α 3结构域和CD8的抗体 抑制其功能。它们也将用于细胞：细胞结合试验 以确定它们对CD8细胞结合的依赖性以及 细胞结合的α 3结构域突变体。 另外的α 3结构域突变体 通过定点诱变获得的由T.波特 这些新的突变体将在各种测定中进行比较，并将确定 I类配体被CD8识别的程度。 此外，CTL 将分析针对I类α 3结构域突变体的应答， 确定是否有其他残基参与CD8的识别。 最后，CD8参与的产生和效应功能， 将分析MHC限制性CTL应答。 因此，几个突出的 CD8的特性将使用各种免疫学方法进行研究。 分析和位点特异性I类突变体的独特集合。