Involvement of Nonclassical MHC in Early T Cell Ontogeny in Xenopus

描述(由申请人提供)：本申请的总体目标是通过比较非洲爪哇的方法，探索非经典MHC Ib类(Ib类)分子在胸腺细胞早期发育过程中的参与。越来越多的证据表明，在新生儿中缺乏或不理想地表达经典的MHC Ia类(Ia类)时，Ib类分子可以产生分化胸腺细胞的生存信号。关于这一替代胸腺选择过程的生理相关性，人们知之甚少。假设在发育早期缺乏Ia类基因的表达(或次优水平)，胸腺分化和T细胞教育严重依赖于Ib类基因。在颌骨脊椎动物中，T细胞的胸腺依赖性和在调节T细胞发育中发挥关键作用的因子都被广泛保守。因此，研究人员建议使用非洲爪哇及其免疫活性但自然缺乏Ia类的幼虫阶段作为模型，因为它提供了早期发育的幼虫胸腺的外部可见、容易接近和易于实验操作的优势。重要的是，涉及Ib类的假设可以在自然缺乏Ia类的情况下得到检验，T细胞的发育可以在生物学背景下进行研究。最后，研究人员最近鉴定了一个新的非经典MHC类Ib(XNC)基因亚家族XNC10，它的表达自个体发育早期起就与胸腺细胞分化过程中的CD8 T细胞谱系密切相关。因此，他们提出了一项假设，即Ib类基因在非洲爪哇个体发育过程中对胸腺分化和T细胞教育起关键作用。这将通过以下两个目标来实现：1.确定XNC10和一组选定的免疫基因在个体发育过程中的表达模式：这将涉及原位杂交以及免疫组织学和流式细胞术分析，它们将产生针对XNC10和其他胸腺细胞标记物(如CD4、CD8？、TCR？)的新抗体；以及2.评估XNC10在T细胞分化和胸腺教育中的功能，这将涉及通过RNA干扰利用转基因在体内敲除XNC10，以揭示XNC10在CD8 T细胞发育中的功能。 这一应用将提供基本的且不受系统发育限制的洞察，了解Ia类与Ib类介导的胸腺细胞教育途径在个体发育过程中的相对贡献。开发的技术和获得的试剂将为科学界带来极大的好处，并将极大地增加莱氏X.laevis作为非哺乳动物替代模型的潜力。 公共卫生相关性：该项目的目标是更好地了解某些MHC非经典I类分子在生命早期帮助完全成熟的T细胞发育并最终帮助它们对容易逃脱宿主免疫防御的病原体或肿瘤做出反应的可能作用。为此，研究人员将使用一种进化的方法，利用青蛙X.laevis，这是一种独特的非哺乳动物比较模型，允许在发育早期轻松访问胸腺。

DESCRIPTION (Provided by Applicant): The overall goal of this application is to explore the involvement of non-classical MHC class Ib (class Ib) molecules in thymocyte differentiation during early development by a comparative approach in the Xenopus. Increasing evidence suggests that in the absence or suboptimal expression of 'classical' MHC class Ia (class Ia) as it occurs in neonates, class Ib molecules can generate survival signals to differentiating thymocytes. Not much is known about the physiological relevance of this alternative thymic selection process. It is hypothesized that in the absence (or suboptimal level) of class Ia expression early in development, thymic differentiation and T cell education critically depends on class Ib. Both the thymic dependence of T cells and the factors that play key roles in the regulation of T cell development are broadly conserved among jawed vertebrates. Therefore, the investigators propose to use the Xenopus and its immunocompetent but naturally class Ia-deficient larval stage as a model because it provides the advantage of having the early developing thymus of larvae externally visible, easily accessible, and amenable to experimental manipulation. Importantly, the hypothesis involving class Ib can be tested in the natural absence of class Ia, and the development of T cells can be studied in a biological context. Finally, the investigators have recently characterized a novel Xenopus nonclassical MHC class Ib (XNC) gene subfamily, XNC10, whose expression is tightly associated with the CD8 T-cell lineage during thymocyte differentiation since early in ontogeny. Therefore, they propose to test the hypothesis that class Ib genes are critically involved in thymic differentiation and T cell education during ontogeny of Xenopus. This will be achieved by the two following aims: 1. To determine the expression pattern of XNC10 and a selected set of immune genes during ontogeny: This will involve in situ hybridization as well as immunohistology and flow cytometry analysis for which they will generate new antibodies against XNC10 and other thymocytes markers (e.g., CD4, CD8¿, TCR¿, a); and 2. To assess the function of XNC10 in T cell differentiation and thymic education, which will involve in vivo knockdown by RNA interference using transgenesis to reveal the function of XNC10 in CD8 T cell development. This application will provide fundamental, and not phylogenetically restricted, insight on the relative contributions of class Ia versus class Ib-mediated thymocyte education pathways during ontogeny. Technologies developed and reagents obtained will be of high benefit for the scientific community and will considerably increase the potential of X. laevis as a non-mammalian alternative model. PUBLIC HEALTH RELEVANCE: The goal of this project is to better understand the possible role of certain MHC nonclassical class I molecules in assisting the development of fully mature T cells during early stage of life and ultimately helping their response against pathogens or tumors prone to escape the host's immune defenses. For this, the investigators will use an evolutionary approach that takes advantage of the frog X. laevis, which is a unique non-mammalian comparative model that permits easy access of the thymus early during development.