Age-related changes in the proteome and lipidome of the immunological synapse

描述(由申请人提供)：与年龄相关的T细胞功能下降导致老年人的发病率更高，恢复期更长，以及感染性和肿瘤性疾病的发病率和死亡率增加。T细胞功能受损，以IL-2的产生和细胞增殖来衡量，是免疫衰老的重要原因。与年龄相关的IL-2产生的减少与免疫突触信号分子招募的缺陷有关，免疫突触是T细胞和抗原提呈细胞之间形成的粘连连接。这一点已经通过使用免疫荧光显微镜监测单个蛋白质移位到免疫突触而得到证实。越来越多的证据表明，质膜被横向分割成小的脂质微域，聚集在激活的T细胞受体(TCR)周围，在那里它们促进了产生IL-2所需的信号蛋白的募集。由此产生的结构包括脂筏和蛋白质支架，并以约200 nm的小信号小体存在于免疫突触内。免疫突触的总体成分与年龄相关的差异还没有得到系统的评估。此外，蛋白质向免疫突触募集受损的机制仍不清楚。影响质膜组成的变化，特别是影响鞘脂代谢物神经酰胺的变化，对脂筏结构有重大影响，并可能影响信号小体的组装。质膜鞘脂成分的年龄相关变化还没有被检测到。然而，我们的初步数据表明，老年小鼠的T细胞具有显著更高的特定鞘磷脂水平。这些细胞成分的变化可能导致衰老的T细胞形成有效免疫突触的能力降低。我们假设TCR诱导的信号小体在蛋白质和脂肪募集模式上表现出与年龄相关的差异，并且衰老T细胞鞘磷脂组成的变化影响调节IL-2产生的下游信号级联。我们将使用增强的磁性免疫隔离程序结合最先进的蛋白质组和脂肪组学方法来验证这一假说。我们将通过诱导细胞鞘脂的年龄相关变化以及测量免疫突触的形成和T细胞的激活来进一步检验这一假说。这些研究将首次全面研究在抗原刺激下形成的信号复合体的蛋白质和脂肪含量。从这些免疫突触的蛋白质组学和脂体学分析中获得的信息将形成构建T细胞年龄相关变化的“地图”的基础。这一图谱将增强我们对免疫衰老的分子基础的理解，并将促进逆转与衰老相关的有害变化的策略的发展。T细胞(对抗病原体和肿瘤的重要免疫细胞)功能的年龄相关性下降是老年人健康和生活质量的重要决定因素。这些缺陷的潜在机制尚不完全清楚。这个项目将利用最先进的方法来构建T细胞中与年龄相关的关键脂类和蛋白质变化的“地图”。这一图谱将增强我们对免疫衰老的分子基础的理解，并将促进逆转与衰老相关的有害变化的策略的发展。

DESCRIPTION (provided by applicant): Age-associated declines in T cell function contribute to the higher incidences, longer recovery periods, and elevated morbidity and mortality from infectious and neoplastic diseases in the elderly. Impaired T cell function, as measured by IL-2 production and cell proliferation, contributes significantly to immune senescence. Age-related reductions in IL-2 production are associated with defects in the recruitment of signaling molecules to immune synapses, the adhesive junction that forms between a T cell and an antigen- presenting cell. This has been demonstrated by using immunofluorescence microscopy to monitor the translocation of individual proteins to the immune synapse. Accumulating evidence indicates that the plasma membrane is laterally compartmentalized into small lipid microdomains that coalesce around the activated T cell receptor (TCR), where they facilitate the recruitment of signaling proteins required for IL-2 production. The resulting structures incorporate both lipid rafts and protein scaffolds, and exist as small ~200nm 'signalosomes' within the immune synapse. Age-related differences in overall composition the immune synapse have not been evaluated in a systematic manner. Furthermore, the mechanisms responsible for the impaired recruitment of proteins to the immune synapse remain unknown. Changes affecting the composition of the plasma membrane, particularly changes affecting the sphigolipid metabolite ceramide, have significant impacts on lipid raft structure, and could influence the assembly of signalosomes. Age-related changes in the sphingolipid composition of the plasma membrane have not been examined. However, our preliminary data indicate that T cells from old mice have significantly higher levels of specific sphingolipids. These changes in cellular composition may contribute to the reduced ability of aged T cells to form effective immune synapse. We hypothesize that TCR-induced signalosomes exhibit age-related differences in their patterns of protein and lipid recruitment, and that changes in the sphingolipid composition of aged T cells impact the downstream signaling cascades that regulate IL-2 production. We will test this hypothesis using an enhanced magnetic immunoisolation procedure in conjunction with state-of-the-art proteomic and lipidomic methods. We will further test this hypothesis by inducing age-related changes in cellular sphingolipids and measuring immune synapse formation and T cell activation. These studies will provide the first comprehensive investigation of the protein and lipid content of the signaling complexes formed in response to antigenic stimulation. The information obtained from these proteomic and lipidomic analyses of the immune synapse will form the basis for constructing a 'map' of age-related changes in T cells. This map will enhance our understanding of the molecular bases of immune senescence, and will promote the development of strategies to reverse the detrimental changes associated with aging. The age related decline in T cell (immune cells important for fighting against pathogens and tumors) function is an important determinant of the health and quality of life in elderly. The underlying mechanisms of these defects are not completely known. This project will utilize state-of-the-art methods to construct a 'map' of the key age-related changes in lipids and proteins of T cells. This map will enhance our understanding of the molecular bases of immune senescence, and will promote the development of strategies to reverse the detrimental changes associated with aging.