Soluble T Cell Receptor Studies on MHC Restriction

描述（由申请人提供）：一系列肽具有相似的激活胸腺细胞的能力，但对胸腺细胞的阳性和阴性选择能力非常不同，将通过FRET显微镜测试它们诱导TCR-CD8相互作用的能力。与负选择配体相比，正选择配体诱导这种相互作用的速率可能较慢，但不一定反应较弱。将使用FRET Erk报告结构、经典western blot技术和磷酸化蛋白的细胞内染色来比较这些配体之间的信号。将测量MHCp与T细胞衍生膜制剂的结合动力学，以确定TCR-MHCp和CD8-MHC在不同结合阶段的相互作用的贡献。TCR的近端膜区？链(?-CPM)是积极选择而非消极选择所必需的。胸腺细胞阳性和阴性选择的界限？将测试-CPM突变TCR，看它们是否能识别与野生型TCR相同效力的负选择配体，以及它们是否能积极选择具有更高亲和力的配体。的能力？将比较-CPM tcr结合不同活性的MHCp，并使用FRET显微镜来量化CD8-TCR相互作用中可能存在的缺陷。向免疫突触募集信号分子？-CPM突变T细胞成像。T细胞表达TCR- cd3复合物的不同组分作为荧光嵌合体，将研究TCR的亚基间距离，以及这些距离在抗原识别过程中如何或是否被改变。该策略将允许在静息状态和MHCp识别期间研究细胞表面潜在的tcr聚集。这些策略将允许在抗原识别期间调查tcr内部或之间的潜在构象变化。TCR对于识别病毒感染细胞和癌细胞至关重要。了解它的功能，以及T细胞如何在胸腺中发育，对控制传染病、癌症和自身免疫至关重要。

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.