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: 6562166
• 负责人: Luc Teyton
• 金额: $ 66.72万
• 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/6562166
• 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）：我们的项目名为“MHC 限制性 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，我们将尝试确定 TCR 复合物中保留的许多分子的结构，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。此外，还将尝试利用病毒展示技术对 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，将产生大量Fab-CD3和Fab-CD8ab复合物并用于结晶。第二个目标是使用 CD3 二聚体的单体和四聚体以及纯化的 TCR 分子，通过表面等离子共振和 FRET 来表征 CD3 二聚体与 T 细胞受体的横向相互作用。类似的研究还将针对 pre-TCR 和 gd TCR 进行。前两个目标将融合在一起，帮助我们尝试在膜中重建人工 TCRC。这些结构的观察将通过单分子显微镜在支持的双层中完成。将通过全内反射显微镜在支撑膜中研究 TCRC 不同亚基的关联。重组分子将在体外用荧光团标记，并通过螯合脂质重新附着到膜上。将测量各个组分之间的相互作用并与溶液中的亲和力数进行比较。在与适当的 p-MHC 复合物连接后，将观察重建的 TCRC 的行为。这些动态研究将通过 TCRC 二维结晶的尝试得到补充。最后，对前TCR及其配体表征的研究将旨在确定其结构及其配体的结构。重组前 TCR 单体和四聚体将用于通过先进的蛋白质组学方法分离配体。如果生化技术失败，将尝试表达克隆。将通过使用转染、分子可溶性重组形式的表达和表面等离振子共振来研究配体身份和结合特性的确认。