Nonclassical MHC-dependent Innate T Cell Ontogeny and Function in the Amphibian Xenopus

众所周知，T细胞在产生适应性免疫系统识别和应答多种非自身抗原的能力中起作用。T细胞的选择性来自于它在其表面显示的T细胞受体变体，个体？s T细胞群体含有大量不同的T细胞受体变体。然而，T细胞亚群显示有限或不变的T细胞受体集。这些所谓的先天性T细胞（iT细胞）被认为是对感染的快速反应者，通过其T细胞受体识别病原体中的保守分子决定簇，并在适应性免疫应答之前（或不存在）提供防御。然而，人们对iT细胞如何分化或它们在免疫反应中的作用知之甚少。初步数据表明，iT细胞在蛙爪蟾蝌蚪中有很好的代表性，该项目解决了一般假设，即蝌蚪的外部发育需要有能力对病毒和细菌进行快速免疫反应，这些反应由一小群免疫活性细胞介导，这些要求由iT细胞满足。这项研究将扩展我们对iT细胞在宿主/病原体相互作用中的进化、生物学和重要性的理解。它也有可能提出免疫学上知情的战略，以控制病原体及其在两栖动物下降的作用。本科生，研究生和研究生将广泛参与研究的各个方面，其中包括学习基础免疫学，发育，微生物学和进化的机会，以及学习一系列现代分子，细胞和反向遗传技术。PI将在学年期间招募一到两名本科生，并与NSF BIO REU计划和夏季本科生研究计划合作，每年夏天至少增加一名少数民族本科生。主要研究者还指导非洲爪蟾免疫生物学研究资源，该资源为科学家和公众提供试剂、动物、信息、援助、培训和外展。两个具体的假设指导了这个项目：（i）具有快速外部发育的变温脊椎动物严重依赖iT细胞作为先天的类T细胞库;和（ii）Ib类限制性iT细胞在协调针对病毒和细菌病原体的免疫应答中是关键的。这一假设将在X. laevis蝌蚪和成年人的创新反向遗传功能丧失方法，该方法将联合收割机转基因与RNAi或CRISPr/Cas基因组编辑技术相结合，以确定Ib类分子在X中iT细胞的发育和功能中的作用。光滑。该研究还将使用并进一步开发Ib类四聚体和抗体，以识别不同的iT细胞亚群。具体目标是：（1）使用qPCR、免疫组织学和原位杂交以及Ib类缺陷转基因系来鉴定和表征调节不同iT细胞谱系的发育和功能的Ib类基因;（2）使用Ib类四聚体和单克隆抗体的组合以及流式细胞术、细胞分选和荧光显微镜来表征蝌蚪和成体中不同的Ib类限制性iT细胞谱系;（3）阐明蝌蚪和成体不同的Ib类限制性iT细胞谱系在对生态相关病原体（包括蛙病毒3型、海洋分枝杆菌和嗜水气单胞菌）的免疫应答中的作用。工具（Ib类四聚体和抗体）的成功应用和转基因技术的创新功能丧失将进一步增强非洲爪蟾的研究模式。

T cells are well known for their role in generating the capacity of the adaptive immune system to identify and respond to diverse non-self antigens. The selectivity of a T cell arises from the T cell receptor variant it displays at its surface, with an individual?s T cell population containing a massive repertoire of distinct T cell receptor variants. However, a subgroup of T cells displays a limited or invariant set of T cell receptors. These so called innate T cells (iT cells) are thought to be rapid responders to infections, recognizing via their T cell receptors conserved molecular determinants in pathogens and providing defense in advance (or absence) of an adaptive immune response. However little is known about how iT cells differentiate or the role they play in immune responses. With preliminary data demonstrating that iT cells are well represented in tadpoles of the frog Xenopus, this project addresses the general hypothesis that the external development of tadpoles requires a capacity to mount rapid immune responses to viruses and bacteria, that the responses be mediated by a small population of immune competent cells, and that these requirements are met by iT cells. This research will extend our understanding of the evolution, biology and importance of iT cells in host/pathogen interactions. It also has the potential to suggest immunologically informed strategies for controlling pathogens and their role in amphibian decline. Undergraduate, graduate and post-graduate students will be extensively involved in all aspects of the research, which includes opportunities to study fundamental immunology, development, microbiology and evolution, as well as to learn a range of modern molecular, cellular and reverse genetic techniques. The PI will recruit one or two undergraduate students during the academic year, and in collaboration with the NSF BIO REU program and the Summer Undergraduate Research Program, at least one additional minority undergraduate each summer. The PI also directs the Xenopus laevis Research Resource for Immunobiology, which provides reagents, animals, information, assistance, training and outreach to scientists and the public.Two specific hypotheses guide this project: (i) ectothermic vertebrates with rapid external development critically rely on iT cells for an innate-like T cell repertoire; and (ii) that class Ib restricted iT cells are pivotal in orchestrating immune responses against viral and bacterial pathogens. This hypothesis will be addressed in vivo in X. laevis tadpoles and adults with innovative reverse genetic loss-of-function approaches that combine transgenesis with RNAi or CRISPr/Cas genome editing technologies to determine the roles of class Ib molecules in the development and function of iT cells in X. laevis. The research will also use and further develop class Ib tetramers and antibodies to identify distinct iT cell subsets. The specific aims are: (1) To identify and characterize class Ib genes regulating the development and function of distinct iT cell lineages using qPCR, immunohistology and in situ hybridization as well as class Ib-deficient transgenic lines; (2) To characterize distinct class Ib-restricted iT cell lineages in tadpoles and adults using a combination of class Ib tetramers and monoclonal antibodies with flow cytometry, cell sorting and fluorescence microscopy; (3) To elucidate the roles of distinct class Ib-restricted iT cell lineages of tadpoles and adults in vivo during immune response against ecologically relevant pathogens including the ranavirus Frog virus 3, Mycobacterium marinum, and Aeromonas hydrophila. The successful application of tools (class Ib tetramers and antibodies) and innovative loss-of-function by transgenesis technologies will further empower the Xenopus research model.