CELL BIOLOGY OF IMMUNE INTERACTIONS

免疫相互作用的细胞生物学

• 批准号: 7216404
• 负责人: Abraham Kupfer
• 金额: $ 44.28万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-09-01 至 2010-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7216404
• 关键词: 1-Phosphatidylinositol 3-Kinase; 3-Dimensional; Adhesions; Antigens; Area; Biochemical; CD28 gene; Cells; Cellular biology; Contracts; Cytoplasmic Tail; DNA Sequence Rearrangement; Etiology; Event; Image; Immune; Immune response; Immunofluorescence Immunologic; Immunologic Surveillance; Immunology; Knowledge; Length; Ligation; Molecular; Outcome; PTPRC gene; Physiological; Proteins; Receptor Signaling; Research Proposals; Role; SHPS-1 protein; Signal Transduction; Signaling Molecule; Structure; System; T-Cell Receptor; T-Lymphocyte; Talin; ZAP-70 Gene; base; cell mediated immune response; design; improved; insight; molecular rearrangement; mutant; novel; novel vaccines; ras GTPase-Activating Proteins; receptor; research study; response

The long term objective of this research proposal is to gain new insights into the molecular and cellular events that are caused by encountering foreign antigens and generate the appropriate immune responses. The ligation of receptors on T cells with their counter receptors on APCs initiates cell-mediated immune responses. The molecular mechanisms by which multiple extracellularly engaged receptors regulated intracellular biochemical response is of prime importance in modern immunology. We have recently developed a highly sensitive and quantitative 3-dimensional (3- D) immunofluorescence workstation to study at the single cell level physiological immune interactions. Our previous studies at the single cell level showed that engaged receptors and their associated proteins cluster at the T-APC contact area. Early 3-D studies indicate that multiple adhesion and signaling molecules cluster ind distinct domains at the cell-cell contacts. In the present proposal we will use the new imaging system to study at the single cell level the detailed spatial and temporal molecular rearrangements that occur at the T-APC contacts during physiological interactions. We will then attempt to determine the mechanisms that are responsible for these regulated rearrangements and their functional significance for the relevant immune responses. The specific aims are: Aim 1. To study the temporal and 3-dimensional spatial redistribution of T cell receptors (TCR, CD4, FLA-1, CD28, CD45) and their colocalization with intracellular signaling and regulatory proteins that are implicated in regulating T cell responses (talin, PKCtheta, P-Tyr, grb2, lck, fyn, zap-70, PI-3K, PLCgamma1, and ras-GAP) in Ag-induced T-APC conjugates in order to determine their functional involvement in physiological immune interactions. Aim 2. To determine the role of CD28 and to identify its associated proteins during physiological Th-APC by repeating the experiments in Aim 1 with a cloned Th cell that does not express CD28 but retained expression of TCR, CD4, LFA-1 and CD45. To confirm causality by re- expressing either full length CD28 or a truncated CD28 that lacks its entire cytoplasmic domain. Aim 3. To determine by structure-function analysis the structural basis for the selective clustering of PKCtheta, but not any of the other expressed PKCs, at the cell contract along with CD28. To identify signals that cause this unique PKC translocation with the aid of well-defined PDGF-receptor signaling mutants. It is expected that these novel studies at the single cell level will significantly increase our understanding of the molecular events that occur early in the immune response and determine its outcome. This knowledge may be useful in the long term in enhancing immune surveillance and in improving the design of new vaccines.

该研究计划的长期目标是获得新的见解 进入因遭遇而引起的分子和细胞事件 外来抗原并产生适当的免疫反应。这 T 细胞上的受体与其 APC 上的反受体连接 启动细胞介导的免疫反应。其分子机制为 多个细胞外参与受体调节细胞内 生化反应在现代免疫学中至关重要。我们有 最近开发了一种高灵敏度和定量的 3 维（3- D) 免疫荧光工作站在单细胞水平上进行研究 生理免疫相互作用。我们之前的单项研究 细胞水平表明参与受体及其相关蛋白 聚集在 T-APC 接触区域。早期的 3D 研究表明 多个粘附和信号分子聚集在不同的域中 在细胞与细胞的接触处。在本提案中，我们将使用新的 成像系统在单细胞水平上研究详细的空间和 在 T-APC 接触过程中发生的时间分子重排 生理相互作用。然后我们将尝试确定 负责这些受监管的重排的机制和 它们对相关免疫反应的功能意义。这 具体目标是： 目标 1. 研究时间和 3 维空间重新分布 T 细胞受体（TCR、CD4、FLA-1、CD28、CD45）及其共定位 与相关的细胞内信号传导和调节蛋白 调节 T 细胞反应（talin、PKCtheta、P-Tyr、grb2、lck、fyn、 Ag 诱导的 T-APC 缀合物中的 zap-70、PI-3K、PLCgamma1 和 ras-GAP） 为了确定它们在生理免疫中的功能参与 互动。 目标 2. 确定 CD28 的作用并确定其相关的 通过重复 Aim 中的实验来检测生理 Th-APC 期间的蛋白质 1 具有不表达 CD28 但保留的克隆 Th 细胞 TCR、CD4、LFA-1 和 CD45 的表达。通过重新确认因果关系 表达全长 CD28 或缺乏其长度的截短 CD28 整个细胞质结构域。 目标 3. 通过结构功能分析确定结构基础 用于 PKCtheta 的选择性聚类，但不适用于其他任何聚类 在细胞收缩时与 CD28 一起表达 PKC。识别信号 在明确定义的帮助下导致这种独特的 PKC 易位 PDGF 受体信号传导突变体。 预计这些单细胞水平的新颖研究将 显着增加我们对分子事件的理解 发生在免疫反应的早期并决定其结果。这 从长远来看，这些知识可能有助于增强免疫监视 以及改进新疫苗的设计。