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

抗原的获取和呈递是免疫系统的重要组成部分，它使细胞能够区分自我和非自我，并对入侵的病原体产生保护性t细胞反应。细胞使用多种获取抗原的机制，包括细胞内和细胞外物质的摄取。本项目将研究在t细胞选择的背景下，细胞同类相食在抗原获取和呈递中的作用。细胞同类相食是指整个活细胞被其他细胞吞噬，从而形成细胞中细胞结构的一种机制(1)，这是Babraham研究所Florey实验室的核心兴趣所在（http://www.babraham.ac.uk/our-research/signalling/oliver-florey）。细胞中细胞结构的例子可以在多种疾病中发现（2,3），但重要的是，在胸腺T细胞发育过程中胸腺细胞和上皮护理细胞之间的生理环境中也会发生(4)，甚至在着床过程中胚胎和子宫之间(5)。在这些例子中，内化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，这涉及非典型自噬途径的激活，该途径促进蛋白LC3脂化到容纳内化细胞的液泡（6-8）。该项目将测试细胞同类相食可以作为抗原呈递途径的假设，并可以通过非典型自噬途径进行调节。这可能对通过控制T细胞选择来形成免疫库具有潜在的重大意义，因此参与产生能够在维持免疫耐受的同时对感染作出反应的免疫系统。主要目的将包括利用基于显微镜技术的细胞同类相食模型系统中细胞-细胞相互作用的发展和表征。这将利用现有的培养人上皮细胞自相残杀模型和建立小鼠原代胸腺细胞与胸腺护理细胞系相互作用的新模型。这些系统随后将被操纵，由内化细胞装载卵清蛋白抗原，随后由宿主细胞呈递抗原，使用卵清蛋白特异性T细胞进行监测。细胞内细胞形成和命运的不同方面将被操纵，以测试对抗原呈递的影响。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基因编辑试剂进行测试，该试剂专门抑制非典型自噬。该项目将结合免疫学和细胞生物学专业知识，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。预期的结果将对我们理解T细胞是如何被选择的以及这一重要稳态事件背后的分子途径产生影响。

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.