MODULATION OF IMMUNE SYNAPSE BY ENGINEERED AAPC

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。所列机构为中心，不一定是研究者所在机构。我们先前已经描述了（船体GW，Mccurdy MA，Nasu Y，Bangma CH，Yang G，Shimura S，Lee HM，Wang J，Albani J，Ebara S，Sato T，Timme TL，Thompson TC.（1999）前列腺癌基因治疗：腺病毒介导的白细胞介素12表达与白细胞介素12加B7-1原位基因治疗和基因修饰的细胞疫苗的比较。EUR.免疫学杂志29（12）：3826-36）鉴定II类限制性低亲和力抗原特异性T细胞的方法，所述T细胞结合带有选择的MHC/肽组合的标记的人工抗原呈递细胞（aAPC）。该方法基于对含有微膜结构域的aAPC进行工程改造，所述微膜结构域表达鉴定和调节抗原特异性T细胞所必需的分子（即MHC/肽、共刺激分子）。aAPC和T细胞之间的相互作用允许跨膜蛋白向初始相互作用位点迁移的生理现象，从而允许免疫突触的形成（参见Nature Medicine，2000 Dec; 6（12），1406-1410）。 这项工作旨在评估aAPC在三个不同水平上对T细胞的调节机制：a）通过抗原特异性T细胞的FACS分析测量的免疫T细胞应答、IL-2产生和活化标志物CD 69的表达。这部分研究已经完成。B）通过TPEM对T细胞/aAPC相互作用位点处免疫突触的形成进行真实的时间分析。这种分析将探索在aAPC和T细胞之间相互作用的亲和力变化的条件下免疫突触的时间和平均表面。研究的这一部分需要国家CMIR的协助。c）点a）和B）中获得的结果与WASP表达的相关性，WASP是一种控制细胞骨架运动的看门蛋白。这部分研究已经完成。总而言之，该项目通过在一个全新的系统中关联免疫学，形态学和功能数据来探索免疫突触的形成和调节机制。该知识对于开发旨在成功地离体调节抗原特异性T细胞的方法至关重要。这个项目已经完成，现在正在写报告。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. We have previously described (Hull GW, Mccurdy MA, Nasu Y, Bangma CH, Yang G, Shimura S, Lee HM, Wang J, Albani J, Ebara S, Sato T, Timme TL, Thompson TC. (1999) Prostate cancer gene therapy: comparison of adenovirus-mediated expression of interleukin 12 with interleukin 12 plus B7-1 for in situ gene therapy and gene-modified, cell-based vaccines. Eur. J. Immunol Dec;29(12):3826-36) a method to identify class II restricted, low affinity antigen specific T-cells, which bind tagged artificial antigen presenting cells (aAPC) bearing an MHC/peptide combination of choice. This method is based on the engineering of aAPC containing micromembrane domains expressing the molecules (i.e. MHC/peptide, costimulatory molecules) necessary for identification and modulation of antigen specific T-cells. Interaction among aAPC and T-cells allows the physiologic phenomenon of migration of transmembrane proteins toward the initial interaction site, thus allowing the formation of the immune synapse (see Nature Medicine, 2000 Dec; 6 (12), 1406-1410). This work is aimed at evaluating modulation mechanisms of T-cells by aAPC at three different levels: a) Immunological T-cell responses, measured by FACS analysis of antigen specific T-cells, IL-2 production and expression of activation marker CD69. This part of the study has been completed. b) Real time analysis by TPEM of formation of the immune synapse at the T-cell/aAPC interaction site. Such analysis will explore timing and average surface of the immune synapse under conditions in which the affinity of interaction between aAPC and T-cells is varied. This part of the study required NCMIR assistance. c) Correlation of results obtained in points a) and b) with expression of WASP, a gatekeeper protein, which controls cytoskeletal movements. This part of the study is complete. Altogether, this project explores the mechanisms of formation and modulation of the immune synapse by correlating immunological, morphological, and functional data in an entirely novel system. This knowledge is crucial for developing methods aimed at successfully modulating antigen specific T-cells ex vivo. The project is concluded and is now being written up.