CELL BIOLOGY OF IMMUNE INTERACTIONS

这项研究计划的长期目标是获得新的见解。 分子和细胞事件是由相遇引起的 并产生适当的免疫反应。这个 T细胞表面受体与其在APC上的对位受体的连接 启动细胞介导的免疫反应。分子机制是通过 哪个多个细胞外参与的受体调节细胞内 生化反应在现代免疫学中占有非常重要的地位。我们有 最近开发了一种高度灵敏和定量的三维(3- D)在单细胞一级进行研究的免疫荧光工作站 生理免疫相互作用。我们之前在Single的研究 细胞水平显示参与的受体及其相关蛋白 聚集在T-APC联系区域。早期的3D研究表明 多个黏附和信号分子聚集在不同的结构域 在细胞-细胞接触处。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接触处发生的暂时性分子重排 生理上的相互作用。然后我们将尝试确定 负责这些受监管的重新安排和 它们对于相关免疫反应的功能意义。这个 具体目标是： 目的1.研究大鼠的时间和空间再分布。 T细胞受体(TCR、CD4、FLA-1、CD28、CD45)及其共定位 与细胞内信号和调节蛋白有关 在调节T细胞反应中(talin，PKCtheta，P-Tyr，Grb2，Lck，Fyn， ZAP-70、PI-3K、PLC-Gamma1和ras-GAP)在Ag诱导的T-APC结合物中的表达 以确定它们在生理免疫中的功能参与 互动。 目的2.确定CD28的作用并确定与其相关的 AIM重复实验研究生理性Th-APC过程中的蛋白质 1克隆的Th细胞不表达CD28，但仍保留 TCR、CD4、LFA-1和CD45的表达。通过重新-确认因果关系 表达全长CD28或缺少其基因的截短CD28 整个细胞质区域。 目的3.通过结构-功能分析确定结构基础 用于PKCtheta的选择性聚类，而不是其他任何 在细胞内表达PKCs，与CD28一起收缩。识别信号 导致了这种独特的PKC易位 PDGF受体信号突变体。 预计这些在单细胞水平上的新颖研究将 显著增加了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，知识可能有助于加强免疫监测 以及改进新疫苗的设计。

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.