Soluble T Cell Receptor Studies on MHC Restriction

MHC 限制的可溶性 T 细胞受体研究

• 批准号: 8075341
• 负责人: NICHOLAS R GASCOIGNE
• 金额: $ 1.1万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-06-01 至 2010-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8075341
• 关键词: Affinity; Antigens; Autoimmunity; Avidity; Binding; Biochemical; C-Peptide; Cell surface; Cells; Chimera organism; Collaborations; Color; Communicable Diseases; Confocal Microscopy; DNA Sequence Rearrangement; Data; Defect; Fluorescence; Fluorescence Resonance Energy Transfer; Grant; Image; Immune; Investigation; Kinetics; Ligands; Lipid Bilayers; MHC Interaction; Malignant Neoplasms; Measures; Membrane; Methods; Microscopy; Molecular Conformation; Peptides; Phase; Preparation; Property; Proteins; Receptor-CD3 Complex, Antigen, T-Cell; Reporter; Rest; Series; Signal Transduction; Signaling Molecule; Staining method; Stains; Synapses; T-Cell Receptor; T-Lymphocyte; TCR Activation; Techniques; Testing; Thymocyte Development; Thymocyte Selection; Thymus Gland; Time; Universities; Virus; Western Blotting; antigen binding; cancer cell; defined contribution; intermolecular interaction; mutant; response; thymocyte

DESCRIPTION (provided by applicant): A series of peptides that have similar abilities to activate thymocytes, but very different abilities to positively and negatively select thymocytes will be tested for their ability to induce TCR-CD8 interaction by FRET microscopy. Positive selecting ligands may have a slower rate of induction of this interaction than negative selecting ligands, but not necessarily a weaker response. Signaling will be compared between these ligands using a FRET Erk reporter construct, classical western blot techniques, and intracellular staining for phospho-proteins. Binding kinetics for MHCp to T cell derived membrane preparations will be measured to define the contribution of TCR-MHCp and CD8-MHC interactions in the different binding phases. A membrane-proximal region of the TCR ?-chain (?-CPM) is required for positive but not negative selection. The limits of positive and negative selection for thymocytes bearing ?-CPM mutant TCRs will be tested to see if they recognize negative selecting ligands of the same potency as wild type TCR, and if they can positively select with higher affinity ligands. The ability of ?-CPM TCRs to bind MHCp of varying avidities will be compared, and FRET microscopy will be used to quantify the likely defect in CD8-TCR interaction. Recruitment of signaling molecules to the immune synapse of ?-CPM mutant T cells will be imaged. T cells expressing different components of the TCR-CD3 complex as fluorescent chimeras will be made to investigate inter-subunit distances in the TCR, and how or if these are altered during antigen recognition. This strategy will allow investigation of potential clustering of TCRs on the cell surface in the resting state and during MHCp recognition. These strategies will allow investigation of potential conformation changes either within or between TCRs during antigen recognition. The TCR is crucial for recognizing virus-infected cells and cancer cells. Understanding its function, and how T cells develop in the thymus is of primary importance to controlling infectious disease, cancer and autoimmunity.

描述（由申请人提供）：一系列具有类似能力激活胸腺细胞的肽，但将测试其积极和负选择胸腺细胞的能力，以测试其通过FRET显微镜诱导TCR-CD8相互作用的能力。阳性选择配体的诱导速率可能比选择配体的负相互作用较慢，但不一定是较弱的响应。使用FRET ERK报告构建体，经典的蛋白质印迹技术和磷酸蛋白质的细胞内染色，将比较这些配体之间的信号传导。将测量MHCP与T细胞衍生的膜制剂的结合动力学，以定义不同结合阶段中TCR-MHCP和CD8-MHC相互作用的贡献。阳性选择但不是负选择的TCR？ - 链（？-CPM）的膜斑区域。胸腺细胞阳性和阴性选择的限制将测试 - cpm突变体TCR，以查看它们是否识别与野生型TCR具有相同效力的阴性配体，并且是否可以使用较高的亲和力配体进行积极选择。将比较？-CPM TCR结合不同狂热的MHCP的能力，将使用FRET显微镜来量化CD8-TCR相互作用中的可能缺陷。将募集信号分子对？-CPM突变T细胞的免疫突触。将制作表达TCR-CD3复合物不同成分的T细胞作为荧光嵌合体，以研究TCR中的亚基间距离，以及在抗原识别过程中如何或是否改变这些距离。该策略将允许研究静止状态和MHCP识别期间TCR的潜在聚类。这些策略将允许调查抗原识别过程中TCR内或之间的潜在构象变化。 TCR对于识别感染病毒的细胞和癌细胞至关重要。了解其功能以及胸腺中T细胞的发展方式对于控制传染病，癌症和自身免疫性至关重要。