Molecular Immunology of MHC-Restricted T Cell Responses

MHC 限制性 T 细胞反应的分子免疫学

• 批准号: 6869531
• 负责人: Luc Teyton
• 金额: $ 77.66万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-12-01 至 2008-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6869531
• 关键词: CD3 molecule; CD8 molecule; MHC class I antigen; SDS polyacrylamide gel electrophoresis; T cell receptor; biophysics; chemical structure; crystallization; cytotoxic T lymphocyte; dimer; fluorescence resonance energy transfer; intermolecular interaction; laboratory mouse; lipid bilayer membrane; major histocompatibility complex; mass spectrometry; membrane reconstitution /synthesis; molecular cloning; protein engineering; protein structure function; proteomics; receptor binding; receptor expression; recombinant proteins; surface plasmon resonance; transfection /expression vector

DESCRIPTION (provided by applicant): Our project entitled "Molecular Immunology of MHC-Restricted T Cell Responses" aims at understanding the initial events of T cell activation. In order to do so, we will attempt the structure determination of a number of molecules retained in the TCR complex such as CD8ab and CD3 ge and de. Protein engineering will be used to produce single chain CD8-MHC molecules to facilitate crystallization of the CD8-MHC and CD8ab/TCRp-MHC complexes. In addition new crystallization techniques will be attempted for CD8 and CD3 molecules with the usage of viral display. The platform of the N-w virus and CPMV virus will be used for these studies. Also, a large number of Fab-CD3, and Fab- CD8ab complexes will be produced and used for crystallization. The second aim is to characterize the lateral interactions of CD3 dimers with T cell receptors by surface plasmon resonance and FRET using monomers and tetramers of the CD3 dimers and purified TCR molecules. Similar studies will be carried out with the pre-TCR and gd TCR. The first two aims will converge and help us into our attempt at reconstituting artificial TCRC in membranes. Observation of these structures will be done by single molecule microscopy in supported bilayers. The association of the different subunits of the TCRC will be studied in supported membranes by total internal reflection microscopy. Recombinant molecules will be labeled in vitro with fluorophores and reattached to membranes through chelating lipids. Interactions between individual components will be measured and compared to affinity numbers in solution. Behavior of reconstituted TCRC will be observed upon ligation with appropriate p-MHC complexes. These dynamic studies will be complemented by attempts at 2-dimensional crystallization of the TCRC. Finally, studies of the pre-TCR and the characterization of its ligand will aim at determining its structure and the structure of its ligand. Recombinant pre-TCR monomers and tetramers will be used to isolate a ligand using advanced proteomics approaches. Expression cloning will be attempted if biochemical techniques fail. Confirmation of ligand identity and binding properties will be studied by using transfection, expression of soluble recombinant forms of the molecule and surface plasmon resonance.

描述（由申请人提供）：我们的项目标题为“ MHC限制的T细胞反应的分子免疫学”旨在了解T细胞激活的初始事件。为此，我们将尝试测定保留在TCR复合物中的许多分子，例如CD8AB和CD3 GE和DE。蛋白质工程将用于生产单链CD8-MHC分子，以促进CD8-MHC和CD8AB/TCRP-MHC复合物的结晶。另外，将尝试使用使用病毒显示的CD8和CD3分子进行新的结晶技术。 N-W病毒和CPMV病毒的平台将用于这些研究。此外，将生产大量的FAB-CD3和Fab-CD8AB复合物，用于结晶。第二个目的是使用CD3二聚体的单体和四聚体和纯化的TCR分子来表征CD3二聚体与T细胞受体与T细胞受体的横向相互作用。类似的研究将使用前TCR和GD TCR进行。前两个目标将融合并帮助我们尝试在膜中重新建立人造TCRC。这些结构的观察将通过受支持的双层中的单分子显微镜完成。 TCRC不同亚基的关联将通过总内反射显微镜在支持的膜上进行研究。重组分子将在体外用荧光团标记，并通过螯合脂质重新安装到膜上。将测量各个组件之间的相互作用，并将其与溶液中的亲和力数进行比较。与适当的P-MHC复合物结扎后，将观察到重构TCRC的行为。这些动态研究将通过TCRC的二维结晶的尝试来补充。最后，对TCR前的研究及其配体的表征将旨在确定其结构和配体的结构。重组前TCR单体和四聚体将使用先进的蛋白质组学方法分离配体。如果生化技术失败，将尝试进行表达克隆。通过转染，分子的可溶性重组形式的表达和表面等离子体共振，将研究配体同一性和结合特性的确认。