CELL BIOLOGY OF IMMUNE INTERACTIONS

这项研究计划的长期目标是获得新的见解 进入分子和细胞的事件， 并产生适当的免疫反应。的 T细胞上的受体与其APC上的反受体的连接 启动细胞介导的免疫反应。的分子机制 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是最重要的。我们有 最近开发了一种高度灵敏和定量的三维（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。我们以前在单身时的研究 细胞水平显示，参与受体及其相关蛋白 在T-APC接触区聚集。早期的3D研究表明， 多种粘附和信号分子聚集在不同的结构域中 细胞间的接触在本提案中，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 发生在T-APC接触的时间分子重排， 生理互动然后我们将尝试确定 负责这些受管制的重新安排的机制， 它们对相关免疫反应的功能意义。的 具体目标是： 目标1。为了研究在时间和三维空间上的再分布， T细胞受体（TCR、CD 4、弗拉-1、CD 28、CD 45）及其共定位 细胞内信号和调节蛋白 在调节T细胞应答中（talin，PKC θ，P-Tyr，grb 2，lck，fyn， zap-70、PI-3 K、PLC γ 1和ras-GAP）在 为了确定它们在生理免疫中的功能参与， 交互. 目标二。为了确定CD 28的作用，并确定其相关的 通过重复Aim中的实验， 1与克隆的Th细胞，不表达CD 28，但保留 TCR、CD 4、LFA-1和CD 45的表达。通过重新检查确认因果关系- 表达全长CD 28或缺乏其表达的截短的CD 28， 整个胞质结构域。 目标3：通过结构-功能分析确定结构基础 对于PKC θ的选择性聚集，而不是任何其他的 表达的PKC，在细胞中与CD 28沿着收缩。来识别信号 导致这种独特的PKC易位， PDGF受体信号突变体。 预计这些单细胞水平的新研究将 大大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，这些知识可能有助于加强免疫监视， 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.