Visualizing epithelial network connectivity in thymus biology, aging, and regeneration

项目摘要。 T淋巴细胞是免疫系统的细胞，像所有的血细胞一样，在一生中经常丢失， 必须补充。胸腺是T淋巴细胞产生的主要场所。然而，它不包含 淋巴干细胞，而是依赖于在淋巴细胞中循环的干细胞样细胞的持续输入。 血然后，胸腺特有的微环境条件指示这些干细胞样细胞增殖， 分化成T细胞系细胞，识别外来物质，但对自身具有耐受性。持久的胸腺 微环境由其基质细胞建立，主要由上皮细胞（胸腺上皮细胞）组成 细胞，TEC）。TEC形成淋巴细胞发育所依赖的遍布的网状基质（网络）， 并且TEC网络的竞争限制了胸腺中的淋巴细胞构成，从而限制了T细胞输出。为 尽管原因尚不清楚，但胸腺表现出过早和加速的年龄相关性萎缩， 主要是TEC变化的结果。重要的是，胸腺保留了 短暂的再生，这可以通过诸如手术阉割等刺激来诱导。这种机制 这一过程不是众所周知的，但据信需要TEC细胞本身的增殖，或扩增细胞的增殖。 TEC前体人口。我们已经建立了全球基质基因的时空数据库， 在诱导再生过程中转录。多种独立的数据挖掘方法一直以来 表明与细胞过程的投射、延伸和生长相关的基因，例如 特征TEC网络，在再生过程中动态调节。要开始评估 TEC投射在淋巴发育、胸腺老化和胸腺再生中的作用，我们采用了 当代神经科学成像技术（Rosa26[Brainbow 2.1]，a.k.a.五彩纸屑）在厚器官切片 可视化单个TEC和TEC网络。我们发现单个TEC的形态，特别是 皮质中的那些（cTEC），与薄的免疫染色所表明的有很大不同。 截面，整体呈球形，而不是放射状排列的网状形状，每个截面都有精心制作的 包裹大量淋巴细胞的突起。在“老化”胸腺（来自6个月大的Confetti小鼠）中， 这些突起变得既短又薄，从而减少了淋巴接触的表面 这就解释了随着年龄的增长细胞减少的原因。我们预测，手术阉割将不会使TEC增殖， 诱导这些过程的（瞬时）延长和加强，从而增加淋巴细胞 生产测试这个假设是这个项目的一个关键目标，但为了实现这一点，我们首先 有义务为TEC和TEC网络建立健康状态的基线。拟议项目依赖于 在很大程度上建立和新兴的神经科学范式，并与声明的使命的一致， 美国国立卫生研究院“寻求有关生命系统的性质和行为的基本知识，以及 这些知识可以增进健康，减少疾病。”

Project summary. T lymphocytes are cells of the immune system that, like all blood cells, are routinely lost throughout life and must be replenished. The thymus is the primary site for T lymphocyte production. However, it contains no lymphoid stem cells, and instead depends on continuous importation of stem-like cells that circulate in the blood. Microenvironmental conditions unique to the thymus then instruct these stem-like cells to proliferate and differentiate into T lineage cells that recognize foreign substances but are tolerant to self. The durable thymic microenvironment is established by its stromal cells, consisting primarily of epithelial cells (thymic epithelial cells, TEC). TEC form a pervasive reticular matrix (network) upon which lymphocyte development depends, and competition for the TEC network limits lymphoid cellularity in the thymus, and thus T cell output. For reasons that are still not understood, the thymus exhibits premature and accelerated age-related atrophy, a disorder that is primarily a consequence of changes in TEC. Importantly, the thymus retains latent potential for short-lived regeneration, which can be induced by stimuli such as surgical castration. The mechanisms of this process are not well known, but are believed to require proliferation of TEC cells themselves, or expansion of a TEC precursor population. We have generated a large temporo-spatial database of global stromal gene transcription during induced regrowth. Multiple independent data mining approaches have consistently indicated that genes associated with projection, extension, and outgrowth of cellular processes, such as those that characterize the TEC network, are dynamically regulated during regeneration. To begin to evaluate the role of TEC projections in lymphoid development, thymic aging, and thymic regeneration, we have adopted contemporary neuroscience imaging technologies (Rosa26[Brainbow 2.1], a.k.a. Confetti) in thick organ slices to visualize individual TEC and the TEC network. We find that the morphology of individual TEC, particularly those in the cortex (cTEC), is vastly different than what is suggested by conventional immuno-staining of thin sections, with overall globular rather than radially aligned reticular shapes, each possessing elaborate processes that envelop large numbers of lymphocytes. In the `aged' thymus (from 6 month-old Confetti mice), these processes become both shorter and thinner, and thus provide a diminished surface for lymphoid contact that explains reduced cellularity with age. We predict that rather than TEC proliferation, surgical castration will induce the (transient) lengthening and strengthening of these processes, thus increasing lymphocyte production. Testing this hypothesis is a key objective in this project, but in order to accomplish this, we are first obligated to establish a baseline in the healthy state for TEC and the TEC network. The proposed project relies heavily on established and emerging neuroscience paradigms, and is consistent with the stated mission of the NIH to “to seek fundamental knowledge about the nature and behavior of living systems, and the application of that knowledge to enhance health…and reduce illness.”