Role of the microenvironment in regulating early stages of thymic involution and central tolerance

摘要-项目2 在与年龄相关的胸腺退化过程中，T细胞输出进行性下降，导致 老年人和小鼠中幼稚T细胞的减少。由此导致的新的T细胞反应的下降， 病原体、疫苗和癌症，导致免疫力受损，发病率随年龄增长而增加。因此 对于阐明胸腺功能降低的潜在机制以改善人类健康是重要的 在整个生命周期中。在过去的项目期间，我们发现，早期T细胞祖细胞（ETP），它引起 对于所有下游胸腺细胞亚群，在3个月龄时数量惊人地早下降。ETP 在所有年龄，细胞构成与下游胸腺细胞亚群成比例，表明ETP的早期下降 在胸腺退化期间为减少胸腺生成奠定了基础。因为ETP是 循环胸腺播种祖细胞（TSP），我们预计功能TSP小生境的数量下降 3个月的年龄。然而，我们的数据表明，可用的TSP生态位不会通过至少12个月的下降， 年龄，提出了什么机制的基础上早期减少ETP的问题。我们的初步数据显示 BM和胸腺微环境的变化有助于ETP的早期丢失。虽然 潜在机制尚未解决，初步数据表明BM中NOTCH信号传导减少 淋巴祖细胞和胸腺ETP起作用。因此，我们将使用遗传小鼠模型和先进的 检测Notch配体表达的降低和/或其他信号的变化的成像方法 通过额外的单细胞转录谱（核心B），负责淋巴细胞的早期下降， BM中的祖细胞（目标1）和胸腺中的ETP（目标2），以及是否存在类似的机制影响 衰老的人类胸腺（核心C）。我们还将测试恢复NOTCH信号传导是否能挽救BM的细胞结构， 淋巴祖细胞、ETP和胸腺细胞以及衰老小鼠中的下游T细胞功能（P3和核心D）。 退化过程中胸腺微环境细胞组成和组织的变化 随着年龄的增长，不仅会影响T细胞的数量，还会影响T细胞的质量。在上一个项目中 我们发现，到中年时，胸腺支持中枢耐受的能力受损 通过负选择和调节性T细胞的产生，特别是在应答低亲合力自身免疫时， 抗原新发自身免疫也在人类中年时达到峰值，这表明受损的中枢神经系统在其中的作用。 宽容因此，我们将分析来自P2和P1（核心B）的当前单细胞转录谱数据集。 并进行活体双光子成像和胸腺切片测定以鉴定小鼠中与年龄相关的变化， 损害中枢耐受性和增加自身免疫易感性的人类（核心C）胸腺微环境 中年（目标3）P2与P1、P3和核心紧密集成。P2结果将是实现 阐明与年龄相关的T细胞产生缺陷的机制的总体计划目标， 选择和设计治疗策略，以随着年龄的增长恢复功能性T细胞输出和免疫。

ABSTRACT-Project 2 During the process of age-associated thymus involution, T cell output progressively declines, contributing to a reduction in naive T cells in older humans and mice. The resultant decline in new T-cell responses to pathogens, vaccines, and cancers, leads to compromised immunity and increased morbidity with age. Thus, it is important to elucidate mechanisms underlying reduced thymic function in order to improve human health throughout the lifespan. In the past Project period, we found that early T-cell progenitors (ETPs), which give rise to all downstream thymocyte subsets, decline in numbers surprisingly early, by 3 months (mo) of age. ETP cellularity remains proportional to downstream thymocyte subsets at all ages, indicating the early decline in ETPs sets the stage for diminished thymopoiesis during thymic involution. Because ETPs are the direct progeny of circulating thymic seeding progenitors (TSPs), we anticipated a decline in the number of functional TSP niches by 3mo of age. However, our data reveal that the available TSP niches do not decline through at least 12mo of age, raising the question of what mechanisms underlie the early reduction in ETPs. Our preliminary data suggest that changes in the BM and thymus microenvironments contribute to the early loss in ETPs. Although the underlying mechanisms are not yet resolved, preliminary data suggest diminished NOTCH signaling in BM lymphoid progenitors and thymic ETPs contribute. Thus, we will use genetic mouse models and advanced imaging approaches to test if reduced expression of Notch ligands, and/or changes in other signals identified through additional single-cell transcriptional profiling (Core B), are responsible for the early decline in lymphoid progenitors in the BM (Aim 1) and ETPs in the thymus (Aim 2), and whether comparable mechanisms impact the aging human thymus (Core C). We will also test if restoring NOTCH signaling rescues cellularity of BM lymphoid progenitors, ETPs and thymocytes, and downstream T cell function in aging mice (P3 and Core D). Changes in the cellular composition and organization of the thymic microenvironment during involution may impact not only the quantity, but also the quality of developing T cells with age. During the previous Project period, we found that by middle age, the thymus becomes impaired in its ability to support central tolerance through both negative selection and regulatory T cell generation, particularly in response to low avidity self- antigens. New onset autoimmunity also peaks at middle age in humans, suggesting a role for impaired central tolerance. Thus, we will analyze current single-cell transcriptional profiling datasets from P2 and P1 (Core B) and perform live 2-photon imaging and thymic slice assays to identify age-associated changes in the mouse and human (Core C) thymic microenvironments that impair central tolerance and increase autoimmune susceptibility by middle-age (Aim 3). P2 is tightly integrated with P1, P3, and the Cores. P2 results will be integral to achieving the overall Program goals of elucidating mechanisms underlying age-associated defects in T-cell production and selection and devising therapeutic strategies to restore functional T cell output and immunity with age.