Development of an Inducible Conditional Gene Deletion Mouse Model to Study Plasma Cell Development and Longevity

项目概要/摘要 衰老与适应性免疫反应的整体下降有关，大部分研究都集中在 关于幼稚B和T淋巴细胞的功能和产生的缺陷。在 相比之下，人们对衰老如何改变浆细胞的功能知之甚少。浆细胞代表 成熟B细胞的终末分化步骤，由短寿命的增殖性浆母细胞（PB） 和更成熟的非增殖性浆细胞（PC）。这些细胞是体液免疫的关键促进者 通过其强大的抗体（Ab）生产以及其长寿的潜力。在人类中，这 寿命可长达数十年。从历史上看，这些细胞被认为只不过是抗体工厂; 然而，最近的工作已经证明了浆细胞调节多种生理功能的能力。 流程.在衰老的背景下，PC已被证明进化为促炎表型，并且是关键因素。 通常在老年人中观察到的骨髓（BM）骨髓生成增加的驱动因素。这部分是通过 造血干细胞和髓系祖细胞数量的PC依赖性扩增， 与老年人群中髓性白血病发病率升高有关。最近，我们有 确定了一种存在于幼胸腺（THY）内PB/PC群体的性别差异，其中3 月龄的雌性小鼠与雄性小鼠相比，这些细胞的数量明显更高。有趣的 这些THY PB/PC表达THY T细胞选择所需的关键蛋白质及其数量 与总体THY细胞构成显著相关。与BM相似，我们观察到THY PB/PC的变化 人口老龄化的背景下。特别是，雄性小鼠随着年龄的增长而积累THY PB/PC， 在女性中保持不变。这与BM和脾脏（SPL）形成对比，其中PB/PC在两者中均随年龄增加。 性别因此，了解PB/PC如何老化将使我们能够更好地评估它们在适应性免疫中的作用。 在老年人的反应，以及他们不断发展的剧目的抗体独立的功能。最终，这可能 导致了改变PB/PC行为和促进老年人健康寿命增加的疗法的发展。 个体目前很难识别真正的长寿或老化的浆细胞。虽然各种细胞 表面标志物已被证明可以识别浆细胞，但这些决定因素不一定与 长寿，而不是成熟。或者，细胞标记技术依赖于上游B细胞的有效标记 以及在随后产生的PB/PC中的标记保留。在某些情况下，检测这些标记需要 细胞透化，从而消除了进一步研究活的、老化的PB/PC的能力。目标1侧重于 开发诱导系统，使用荧光报告物对浆细胞进行不可消除的标记或时间戳标记 从而促进评估群体动态和潜在地分离不同年龄的细胞的能力。目标2将 证明该报告系统特异性确定差异群体周转的可行性，和/或 维持女性与男性THY浆细胞。

Project Summary/Abstract Aging is associated with an overall decline in the adaptive immune response with a bulk of the research focusing on the deficiencies surrounding not only the function, but also the generation, of naive B and T lymphocytes. In contrast, not much is known about how aging alters the functionality of plasmacytes. Plasmacytes represent the terminal differentiation step of mature B cells and consist of both short-lived, proliferative plasmablasts (PBs) and more mature, non-proliferative plasma cells (PCs). These cells are key facilitators of humoral immunity through their robust production of antibodies (Abs) as well as their potential to be long-lived. In humans, this longevity can span decades. Historically, these cells have been considered nothing more than Ab factories; however, recent work has demonstrated the ability of plasmacytes to regulate a variety of physiological processes. In the context of aging, PCs have been shown to evolve a pro-inflammatory phenotype and be key drivers of the increased bone marrow (BM) myelopoiesis normally observed in the elderly. This is in part through the PC-dependent expansion in the numbers of hematopoietic stem cells and myeloid progenitors which has been linked to the elevated incidence of myeloid leukemias in the aged population. More recently, we have identified a sex disparity that exists in regard to PB/PC populations within the young thymus (THY) in which 3 months old female mice have significantly higher numbers of these cells compared to males. Interestingly enough, these THY PBs/PCs express key proteins required for THY T cell selection and their numbers significantly correlate with overall THY cellularity. Similar to BM, we have observed changes in THY PB/PC populations in the context of aging. In particular, male mice accumulate THY PBs/PCs with age while numbers remain constant in females. This is in contrast to BM and spleen (SPL) where PBs/PCs increase with age in both sexes. Thus, understanding how PBs/PCs age will allow us to better evaluate their role(s) in adaptive immune responses in the elderly as well as their evolving repertoire of Ab-independent functions. Ultimately, this may lead to the development of therapies that alter PB/PC behavior and promote increased healthspan in aged individuals. It is currently difficult to identify bona fide long-lived, or aged, plasmacytes. While a variety of cell surface markers have been shown to identify plasmacytes, these determinants do not necessarily correlate with longevity but rather maturity. Alternatively, cell labeling techniques rely on efficient labeling of upstream B cells and label retention in the subsequently generated PBs/PCs. In some instances, detection of these labels requires cell permeabilization thus eliminating the ability to further study living, aged PBs/PCs. Aim 1 focuses on the development of an inducible system to indelibly mark, or timestamp, plasmacytes using a fluorescence reporter thus facilitating the ability to assess population dynamics and potentially isolate cells of various ages. Aim 2 will demonstrate the feasibility of this reporter system to specifically determine differential population turnover and/or maintenance in female versus male THY plasmacytes.