Molecular Interactions in the Aging Immunological Synapse

描述（由申请方提供）：在衰老过程中，淋巴细胞发育和功能受损，导致对感染因子的敏感性随年龄增加。T细胞功能的下降是与衰老相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。这项研究将使用活细胞成像和福斯特（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形成和突触内分子相互作用的变化。将在老化过程中研究TCR与辅助受体CD 4或CD 8之间相互作用的诱导。免疫突触的形式和突触细胞内TCR与辅助受体之间的相互作用将在来自老年或年轻TCR转基因动物的T细胞中进行研究。将用表达荧光嵌合蛋白的基因转染细胞，并将使用活细胞成像和FRET显微镜分析TCR-辅助受体相互作用的诱导和动力学。可逆的蛋白质突出显示，与光可再活化的荧光蛋白将用于测量细胞表面或突触中的TCR或辅助受体的运动动力学。T细胞需要识别自身肽-MHC（pMHC）复合物，以便通过限制抗原性pMHC的量来帮助激活。将测定T细胞群老化期间该要求的变化。将测量T细胞对有限量抗原的敏感性的变化。脂筏的荧光标记蛋白将用于比较年轻或老年T细胞中的筏动力学。同时分析TCR和/或辅助受体蛋白的运动，使得筏动力学可以与突触的形成相关。可逆的蛋白质突出显示将用于可视化TCR或辅助受体蛋白进出筏的运动。将分析膜组分从抗原呈递细胞转移到T细胞（“胞刺”）中可能的年龄相关变化，作为T细胞膜特性变化的指标。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，使个体更容易受到感染。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.