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响应各种非自身抗原的能力方面起着重要作用。T细胞的选择性来自其表面显示的T细胞受体变异体，单个S T细胞群体包含大量不同的T细胞受体变异体。然而，T细胞亚群显示有限或不变的一组T细胞受体。这些所谓的先天T细胞(it细胞)被认为是对感染的快速反应，通过它们的T细胞受体识别病原体中保守的分子决定因素，并提前(或不)提供适应性免疫反应的防御。然而，人们对it细胞如何分化或它们在免疫反应中扮演的角色知之甚少。通过初步数据显示，在非洲爪蛙的蝌蚪中，it细胞得到了很好的表现，这个项目解决了普遍的假设，即蝌蚪的外部发育需要对病毒和细菌产生快速免疫反应的能力，这些反应是由一小群免疫活性细胞介导的，这些要求是由it细胞满足的。这项研究将扩大我们对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。它还有可能为控制病原体及其在两栖动物衰落中的作用提出免疫知情的策略。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、微生物学和进化论的机会，以及学习一系列现代分子、细胞和反向遗传技术。PI将在本学年招收一到两名本科生，并与NSF Bio REU计划和暑期本科生研究计划合作，每年夏天至少增加一名少数族裔本科生。PI还指导非洲爪哇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、培训和推广。两个特定的假说指导着这个项目：(I)外部快速发育的温带脊椎动物严重依赖it细胞来获得先天的T细胞库；(Ii)Ib类限制的it细胞在协调针对病毒和细菌病原体的免疫反应中起关键作用。这一假说将通过创新的反向遗传功能丧失方法在莱氏X.laevis蝌蚪和成虫体内得到解决，这种方法将转基因与RNAi或CRISPR/Cas基因组编辑技术相结合，以确定Ib类分子在X.laevis it细胞的发育和功能中的作用。这项研究还将使用并进一步开发Ib类四聚体和抗体来识别不同的it细胞亚群。其具体目标是：(1)利用qPCR、免疫组织学和原位杂交以及缺乏Ib类基因的转基因株鉴定和鉴定调控不同it细胞系发育和功能的Ib类基因；(2)使用Ib类四聚体和单抗与流式细胞术、细胞分选和荧光显微镜相结合的方法，鉴定蝌蚪和成虫中Ib类限制性it细胞系；(3)阐明蝌蚪和成虫Ib类限制性it细胞系在体内对包括蛙类病毒3、海洋分枝杆菌和嗜水气单胞菌在内的生态相关病原体的免疫反应中所起的作用。工具(Ib类四聚体和抗体)的成功应用和转基因技术创新的功能丧失将进一步增强非洲爪哇研究模型的能力。

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.