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

描述（由申请人提供）：年龄相关的T细胞功能下降导致老年人感染性和肿瘤性疾病的发病率更高，恢复期更长，发病率和死亡率更高。通过IL-2的产生和细胞增殖来测量，T细胞功能受损对免疫衰老有重要作用。与年龄相关的IL-2生成减少与信号分子向免疫突触募集的缺陷有关，免疫突触是T细胞和抗原呈递细胞之间形成的粘附连接。这已经通过使用免疫荧光显微镜监测单个蛋白质到免疫突触的易位得到证实。越来越多的证据表明，质膜被横向划分成小的脂质微域，这些微域在活化的T细胞受体（TCR）周围结合，在那里它们促进了IL-2生产所需的信号蛋白的募集。所得结构包括脂筏和蛋白质支架，并以小的~200nm的“信号体”存在于免疫突触内。免疫突触总体组成的年龄相关差异尚未得到系统的评估。此外，导致免疫突触蛋白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产物神经酰胺的变化，对脂筏结构有显著影响，并可能影响信号素体的组装。细胞膜鞘脂组成的年龄相关变化尚未被研究。然而，我们的初步数据表明，来自老年小鼠的T细胞具有明显更高水平的特异性鞘脂。细胞组成的这些变化可能导致老年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质募集模式上表现出与年龄相关的差异，并且衰老T细胞鞘脂成分的变化影响了调节IL-2产生的下游信号级联反应。我们将使用增强磁免疫分离程序结合最先进的蛋白质组学和脂质组学方法来验证这一假设。我们将通过诱导细胞鞘脂的年龄相关变化和测量免疫突触形成和T细胞活化来进一步验证这一假设。这些研究将首次全面研究抗原刺激下形成的信号复合物的蛋白质和脂质含量。从免疫突触的蛋白质组学和脂质组学分析中获得的信息将构成构建T细胞年龄相关变化“图谱”的基础。这张图谱将增强我们对免疫衰老分子基础的理解，并将促进逆转与衰老相关的有害变化的策略的发展。与年龄相关的T细胞（对抗病原体和肿瘤的重要免疫细胞）功能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。该项目将利用最先进的方法构建T细胞脂质和蛋白质中与年龄相关的关键变化的“地图”。这张图谱将增强我们对免疫衰老分子基础的理解，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.