MOLECULAR CHARACTERIZATION OF CD4-CLASS II INTERACTION

CD4-II 类相互作用的分子表征

• 批准号: 3468525
• 负责人: CAROLYN F DOYLE
• 金额: $ 11.89万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-09-30 至 1996-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3468525
• 关键词: B lymphocyte; CD4 molecule; CHO cells; MHC class II antigen; T cell receptor; T lymphocyte; biological signal transduction; cell adhesion; cell cell interaction; gene expression; genetic polymorphism; histocompatibility gene; human genetic material tag; leukocyte activation /transformation; mutant; site directed mutagenesis; tissue /cell culture; transfection; B lymphocyte; CD4 molecule; CHO cells; MHC class II antigen; T cell receptor; T lymphocyte; biological signal transduction; cell adhesion; cell cell interaction; gene expression; genetic polymorphism; histocompatibility gene; human genetic material tag; leukocyte activation /transformation; mutant; site directed mutagenesis; tissue /cell culture; transfection

The major goal of this proposal is to understand the molecular mechanism by which CD4+ T lymphocytes are restricted to antigen presenting cells bearing class II major histocompatibility complex (MHC) antigens. It is evident that both CD4 and CD8 are involved in the selection of T cells in the thymus. Further, these accessory molecules can act to enhance the avidity of low affinity T cell receptor responses and may also be involved in signalling. Using a cell-binding assay, we have shown that B lymphocytes which express MHC class II antigens bind to monolayers of adherent cells which express high levels of CD4. However, the molecular mechanism by which the class II molecule interacts with CD4 on the T cell has not been defined. Our approach to further characterization of the CD4-class II interaction will be as follows: (1) Identification of residues in the human DRbeta 1 molecule important for attachment to CD4. Several experimental approaches will be used. Initially, we will test a panel of EBV-transformed B cells homozygous for the HLA=DR1 allele in the cell binding assay to look for natural polymorphisms which might affect binding to CD4. This will be followed by site-directed mutagenesis of the DR 1beta polypeptide. Mutant molecules will be re- expressed in class II negative murine B cells for analysis of CD4 binding. In addition, recognition of mutant class II molecules will be analyzed using class II DR1 restricted influenza-specific helper cell clones. (2) Identification of cellular signals mediating adhesion and de- adhesion. The kinetics of adhesion and de-adhesion in the cell binding assay will be studies. The role of p56 lck in class II antigen binding to CD4 will also be analyzed by transfection and co-expression of normal and mutant lck genes in a CD4 transfectant in Chinese hamster ovary (CHO) cells which binds class II-bearing lymphocytes.

该提议的主要目标是了解分子机制 CD4+ T淋巴细胞仅限于抗原呈现细胞 轴承II类主要组织相容性复合物（MHC）抗原。 这是 显然，CD4和CD8都参与T细胞的选择 在胸腺中。 此外，这些附件分子可以起作用以增强 低亲和力T细胞受体反应的亲和力，也可能是 参与信号。 使用细胞结合测定法，我们已经表明 表达MHC II类抗原的B淋巴细胞与单层结合 表达高水平CD4的粘附细胞。 但是，分子 II类分子在T上与CD4相互作用的机制 细胞尚未定义。 我们进一步表征的方法 CD4级II相互作用将如下： （1）鉴定人Drbeta 1分子中的残基很重要 用于附着CD4。 将使用几种实验方法。 最初，我们将测试 HLA = Dr1等位基因的EBV转换的B细胞面板 细胞结合分析以寻找可能的自然多态性 影响与CD4的结合。 接下来是站点定向的 Dr 1beta多肽的诱变。 突变分子将被重新 在II类负鼠B细胞中表达，用于分析CD4 结合。 此外，识别突变II类分子将是 使用II类DR1限制了流感特定的辅助辅助细胞进行分析 克隆。 （2）鉴定细胞信号介导粘附和de- 粘附。 细胞结合测定中的粘附和去粘附的动力学将 研究。 p56 LCK在II类抗原与CD4结合的作用将 也可以通过正常和突变体的转染和共表达来分析 中国仓鼠卵巢（CHO）细胞中CD4转染剂中的LCK基因 结合II类戴淋巴细胞。