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

两栖类非洲爪蟾非经典 MHC 依赖性先天 T 细胞个体发育和功能

• 批准号: 1456213
• 负责人: Jacques Robert
• 金额: $ 66.5万
• 依托单位: University of Rochester
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-05-01 至 2019-04-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1456213&HistoricalAwards=false
• 关键词: Nonclassical; MHC; dependent; Innate; Cell

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.

T细胞以其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的作用而闻名。 T细胞的选择性来自其表面显示的T细胞受体变体，其中一个个体的T细胞种群包含大量的T细胞受体变体的大量曲目。但是，T细胞的一个亚组显示了一组有限或不变的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者，通过其T细胞受体保守的病原体中保守的分子决定因素，并提前（或不存在）适应性免疫反应。然而，关于IT细胞如何区分或它们在免疫反应中起作用的作用知之甚少。通过初步数据，表明IT细胞在青蛙爪蟾的t骨中得到很好的表示，该项目解决了以下一般假设：t的外部发育需要能够实现对病毒和细菌的快速免疫反应的能力，而反应是由少量的免疫胜任细胞介导的，并且这些需求符合这些需求，并且这些需求是由IT细胞满足的。这项研究将扩展我们对宿主/病原体相互作用中IT细胞的进化，生物学和重要性的理解。它还有可能提出免疫学知情的策略来控制病原体及其在两栖动物衰落中的作用。本科，研究生和研究生将广泛参与研究的各个方面，其中包括研究基本免疫学，发展，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将在学年期间招募一两个本科生，并与NSF Bio REU计划和夏季本科研究计划合作，每年夏天至少有一个少数少数族裔本科生。 PI还指导Xenopus laevis免疫生物学研究资源，该研究资源为科学家和公众提供试剂，动物，信息，辅助，培训和外展。两个特定的假设指导该项目：（i）敏捷的外部发育迅速依赖于IT的快速外部发育，以使IT细胞依赖于先天性的T Cell Ectertoire； （ii）IB类限制IT细胞在策划针对病毒和细菌病原体的免疫反应方面至关重要。该假设将在X. laevis t和成年人中的体内解决，具有创新的反向遗传功能丧失方法，这些方法将转世与RNAi或Crispr/Caspr/Caspr/Caspr/Caspr/Caspr/Caspr/Caspr/Caspr/Caspr/Caspr/cas genome编辑技术确定，以确定IB类分子在X. laevis中IT细胞开发和功能中的作用。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细胞子集。具体目的是：（1）识别和表征IB类基因，使用QPCR，免疫组织学和原位杂交以及IB类缺乏的转基因线来调节不同IT细胞谱系的发育和功能； （2）使用IB类四聚体和单克隆抗体与流式细胞仪，细胞分配和荧光显微镜的组合来表征t和成年人中不同类IB限制的IT细胞谱系； （3）在免疫反应期间，促t和成年人在体内对生态相关病原体（包括ranavirus frog病毒3，虫害菌群和空气果中的生态相关病原体）的不同生态相关病原体的体内t和成年人在体内的角色的作用。转基因技术的工具（IB类四聚体和抗体）的成功应用以及创新的功能丧失将进一步增强Xenopus研究模型。