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 细胞输出逐渐下降，导致 老年人和小鼠中幼稚 T 细胞的减少。由此导致的新 T 细胞反应下降 随着年龄的增长，病原体、疫苗和癌症会导致免疫力下降和发病率增加。因此，它 对于阐明胸腺功能降低的机制以改善人类健康非常重要 整个生命周期。在过去的项目期间，我们发现早期 T 细胞祖细胞 (ETP) 对于所有下游胸腺细胞亚群来说，数量下降得惊人地早，即在 3 个月 (mo) 年龄时下降。 ETP 在所有年龄段，细胞结构均与下游胸腺细胞亚群成比例，表明 ETP 的早期下降 为胸腺复旧期间胸腺生成的减弱奠定了基础。因为 ETP 是 循环胸腺播种祖细胞（TSP），我们预计功能性 TSP 生态位数量会下降 3个月大时。然而，我们的数据显示，可用的 TSP 利基在至少 12 个月内不会下降。 年龄，提出了早期减少 ETP 的机制是什么的问题。我们的初步数据表明 BM 和胸腺微环境的变化导致 ETP 的早期损失。虽然 潜在机制尚未解决，初步数据表明 BM 中的 NOTCH 信号减弱 淋巴祖细胞和胸腺 ETP 发挥作用。因此，我们将使用遗传小鼠模型和先进的 成像方法来测试Notch配体的表达是否减少，和/或其他信号的变化是否被识别 通过额外的单细胞转录分析（核心 B），导致淋巴早期衰退 BM 中的祖细胞（目标 1）和胸腺中的 ETP（目标 2），以及类似的机制是否会影响 衰老的人类胸腺（核心 C）。我们还将测试恢复 NOTCH 信号是否可以挽救 BM 的细胞结构 衰老小鼠中的淋巴祖细胞、ETP 和胸腺细胞以及下游 T 细胞功能（P3 和 Core D）。 退化过程中胸腺微环境的细胞组成和组织的变化 随着年龄的增长，可能不仅会影响 T 细胞的数量，还会影响其质量。在上一个项目期间 在此期间，我们发现到了中年，胸腺支持中枢耐受的能力受损 通过负选择和调节性 T 细胞生成，特别是响应低亲和力自身 抗原。新发的自身免疫也在人类中年达到顶峰，这表明中枢神经系统受损发挥了作用。 宽容。因此，我们将分析 P2 和 P1 的当前单细胞转录谱数据集（核心 B） 并进行活体 2 光子成像和胸腺切片测定，以识别小鼠和小鼠中与年龄相关的变化 人类（核心 C）胸腺微环境损害中枢耐受性并增加自身免疫易感性 到中年（目标 3）。 P2 与 P1、P3 和内核紧密集成。 P2 成绩对于实现目标至关重要 总体计划目标是阐明 T 细胞产生中与年龄相关的缺陷的机制，以及 选择和设计治疗策略，以随着年龄的增长恢复功能性 T 细胞输出和免疫力。

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.