Soluble T Cell Receptor Studies on MHC Restriction

描述（由申请方提供）：将通过FRET显微镜检查检测一系列具有相似的活化胸腺细胞能力但具有非常不同的阳性和阴性选择胸腺细胞能力的肽诱导TCR-CD 8相互作用的能力。阳性选择配体可以具有比阴性选择配体更慢的这种相互作用的诱导速率，但不一定是更弱的应答。将使用FRET Erk报告构建体、经典蛋白质印迹技术和磷酸蛋白的细胞内染色来比较这些配体之间的信号传导。将测量MHC β与T细胞衍生的膜制备物的结合动力学，以确定TCR-MHC β和CD 8-MHC相互作用在不同结合相中的贡献。TCR的近膜区？链（？- CPM）是阳性选择而非阴性选择所必需的。胸腺细胞的正负选择界限将测试CPM突变TCR以观察它们是否识别与野生型TCR具有相同效力的阴性选择配体，以及它们是否可以用更高亲和力的配体进行阳性选择。能力？-将比较CPM TCR结合不同亲合力的MHCp，并且将使用FRET显微术来量化CD 8-TCR相互作用中的可能缺陷。募集信号分子到免疫突触？-将对CPM突变T细胞进行成像。将产生表达TCR-CD 3复合物的不同组分作为荧光嵌合体的T细胞，以研究TCR中的亚基间距离，以及这些在抗原识别期间如何或是否改变。该策略将允许研究在静息状态和MHCp识别期间TCR在细胞表面上的潜在聚集。这些策略将允许研究抗原识别期间TCR内或TCR之间的潜在构象变化。TCR对于识别病毒感染的细胞和癌细胞至关重要。了解其功能以及T细胞如何在胸腺中发育对于控制传染病，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.