Molecular Interactions in the Aging Immunological Synapse

描述(由申请人提供)：在衰老过程中，淋巴细胞的发育和功能受到损害，导致对感染性物质的易感性随年龄增加。T细胞功能的下降是与衰老有关的免疫系统缺陷中最明显和最引人注目的，导致免疫反应降低和缓慢。本研究将使用活细胞成像和Forster(或荧光)共振能量转移(FRET)显微镜来研究衰老过程中免疫突触的形成和突触内分子相互作用的变化。TCR和辅助受体(CD4或CD8)之间的相互作用的诱导将在衰老过程中进行研究。免疫突触的形式以及突触细胞内TCR和辅助受体之间的相互作用将在老年或年轻TCR转基因动物的T细胞中进行研究。这些细胞将被导入表达荧光嵌合蛋白的基因，并将使用活细胞成像和FRET显微镜来分析TCR-辅助受体相互作用的诱导和动力学。可逆蛋白高亮，结合可光激活的荧光蛋白，将被用来测量TCR或辅助受体在细胞表面或突触中的运动动力学。T细胞需要识别自体多肽-MHC(PMHC)复合体，以便通过限制抗原性pMHC的数量来帮助激活。这一要求在T细胞群体老化过程中的变化将被分析。将测量T细胞对有限数量的抗原的敏感性的变化。一种用于脂筏的荧光标记蛋白将被用来比较年轻和老年T细胞的脂筏动力学。将同时分析TCR和/或共受体蛋白的运动，以便RAFT动力学可以与突触的形成相关。可逆蛋白质突出显示将用于可视化TCR或辅助受体蛋白质进出木筏的运动。T细胞膜成分从抗原提呈细胞转移到T细胞的可能与年龄相关的变化(“巨噬细胞增多症”)将被分析为T细胞膜特性变化的一种衡量标准。免疫反应，特别是T细胞的免疫反应，随着年龄的增长而下降，使个人更容易受到感染。该项目旨在确定T细胞识别结构在衰老过程中如何发生变化，这对于了解如何预防与年龄相关的缺陷非常重要。

DESCRIPTION (provided by applicant): During aging, lymphocyte development and functions become compromised, resulting in age- dependent increases in susceptibility to infectious agents. The decline in T cell function is the most clear and striking of the immune system defects related to aging, resulting in lower and slower immune responses This study will use live cell imaging and Forster (or fluorescence) Resonance Energy Transfer (FRET) microscopy to investigate changes in formation of the immunological synapse and molecular interactions within the synapse during aging. The induction of the interaction between the TCR and co-receptor, CD4 or CD8, will be investigated during aging. The form of the immunological synapse and interactions between TCR and coreceptor within the synapse cells will be investigated in T cells from aged or young TCR transgenic animals. The cells will be transfected with genes to express fluorescent chimeric proteins and live cell imaging and FRET microscopy will be used to analyze the induction and kinetics of the TCR-coreceptor interaction. Reversible protein highlighting, with a photo-reactivatable fluorescent protein will be used to measure the movement kinetics of TCR or coreceptor on the cell surface or in the synapse. T cells require recognition of self-peptide-MHC (pMHC) complexes in order to aid in activation by limiting quantities of antigenic pMHC. Changes in this requirement during aging of the T cell population will be assayed. Changes in the sensitivity of the T cells to limited quantities of antigen will be measured. A fluorescent marker protein for lipid rafts will be used to compare raft dynamics in young or aged T cells. Movement of TCR and or co-receptor proteins will be analyzed at the same time, so that raft dynamics can be related to formation of the synapse. Reversible protein highlighting will be used to visualize movement of TCR or coreceptor proteins into and out of rafts. Possible age-related changes in the transfer of membrane components from the antigen presenting cell to the T cell ("trogocytosis") will be analyzed as a measure of changes in T cell membrane properties. The immune response particularly that of T cells, declines with age, leaving the individual more susceptible to infection. This project aims to determine how changes in the T cell's recognition structures occur during aging, which is important in understanding how age-related defects could be prevented.