Role of cell cannibalism and non-canonical autophagy in antigen presentation: implications in T-cell selection and immune homeostasis

主题：生物科学促进健康抗原的获取和呈现是免疫系统的重要组成部分，它允许细胞区分自我和非自我，并针对入侵的病原体建立保护性T细胞反应。细胞使用多种获得抗原的机制，包括摄取细胞内和细胞外的物质。该项目将在T细胞选择的背景下研究细胞自相残杀在抗原的获取和呈递中的作用。细胞自相残杀是一种整个活细胞被其他细胞吞噬的机制，导致细胞内细胞结构的形成(1)，这是巴普拉姆研究所(http://www.babraham.ac.uk/our-research/signalling/oliver-florey)的弗洛里实验室的核心兴趣。细胞内细胞结构的例子可以在各种疾病中发现(2，3)，但重要的是，在胸腺T细胞发育期间胸腺细胞和上皮性哺育细胞之间的生理环境中也会发生(4)，甚至在植入期间胚胎和子宫之间也会发生(5)。在这些例子中，内化的细胞可以逃脱它们的吞噬，但更常见的是它们的命运是死亡，这涉及到一个非规范的自噬途径的激活，该途径促进了蛋白质Lc3的脂化到容纳内化的细胞的空泡(6-8)。这个项目将检验这样的假设，即细胞自相残杀可以作为一种抗原呈递途径，并且可以由非典型的自噬途径调节。这可能会对通过控制T细胞选择来形成免疫系统具有重要意义，从而参与生成能够在保持免疫耐受性的同时对感染做出反应的免疫系统。主要目标将包括利用基于显微镜的技术在细胞相食的模型系统中发展和表征细胞与细胞的相互作用。这将利用现有的培养的人类上皮细胞的自相残杀模型，以及建立原代小鼠胸腺细胞与胸腺哺育细胞系相互作用的新模型。然后，这些系统将被操纵，通过内化的细胞装载卵白蛋白抗原，并随后由宿主细胞利用卵白蛋白特异性T细胞监测抗原的呈递。细胞内细胞形成和命运的不同方面将被操纵，以测试对抗原提呈的影响。自噬在细胞内介导的抗原呈递中的作用将使用实验室开发的专门抑制非典型自噬的基因编辑试剂进行测试。这个项目将结合免疫学和细胞生物学的专业知识，并提供细胞生物学技术方面的强大培训，重点是显微镜和分子生物学，包括CRISPR/Cas9基因编辑。预期的结果将影响我们对T细胞是如何选择的，以及这一重要的体内平衡事件背后的分子途径的理解。

Theme: Bioscience for HealthAntigen acquisition and presentation is a vital part of the immune system which allows cells to distinguish self from non-self and mount a protective T-cell response against invading pathogens. Cells use a variety of mechanisms of acquiring antigens, involving the uptake of intracellular and extracellular material. This project will study the role of cell cannibalism in the acquisition and presentation of antigen in the context of T-cell selection. Cell cannibalism is a mechanism in which entire live cells are engulfed by other cells, resulting in the formation of cell-in-cell structures (1), and is a core interest of the Florey lab at the Babraham Institute (http://www.babraham.ac.uk/our-research/signalling/oliver-florey) . Examples of cell-in-cell structures are found in a variety of diseases (2, 3), but importantly also occur in physiological settings between thymocytes and epithelial nurse cells during T cell development in the thymus (4) and even between the embryo and uterus during implantation (5). In these examples, internalized cells can escape their engulfment, but more commonly their fate is death which involves the activation of a non-canonical autophagy pathway that promotes the lipidation of the protein LC3 to the vacuole housing the internalized cell (6-8).This project will test the hypothesis that cell cannibalism can act as a route of antigen presentation and can be regulated by a non-canonical autophagy pathway. This would potentially have major implications in shaping the immune repertoire by controlling T cell selection and therefore participate in generating an immune system capable of responding to infection while maintaining immune tolerance.The main aims will include the development and characterization of cell-cell interactions in model systems of cell cannibalism using microscopy based techniques. This will utilize existing cell cannibalism models of cultured human epithelial cells and the establishment of new models of primary mouse thymocytes interacting with a thymic nurse cell line. These systems will then be manipulated where by internalized cells are loaded with ovalbumin antigen, and subsequent presentation of antigen by hosts cells monitored using ovalbumin-specific T cells. Different aspects of cell-in-cell formation and fate will be manipulated to test for an effect on antigen presentation. The role of autophagy in cell-in-cell mediated antigen presentation will be tested using gene editing reagents developed by the lab that specifically inhibit non-canonical autophagy.This project will combine immunology and cell biology expertise and provide a strong training in cell biology techniques, with an emphasis on microscopy, and molecular biology, including CRISPR/Cas9 gene editing. Expected results will have impacts on our understanding of how T cells are selected and the molecular pathways underlying this important homeostatic event.