Imaging-based methods for NK cell studies at the single-cell level

The immune system provides defense against infectious agents such as viruses, bacteria and parasites. Besides eliminating extracellular agents, the immune system also constantly monitors our own cells for signs of transformation, including tumor development and virus infection. This process, called immune surveillance, is mediated in part by natural killer (NK) cells. NK cells sense transformation through the interaction of surface receptors with proteins on the surface of the diseased cell. The efficient binding of these receptors results in the formation of a tight contact between the two cells, called an immune synapse. If danger signals dominate in the synapse, the NK cell has the potential to deliver toxic compounds and to bind to specific death receptors at the target cell surface, resulting in the induction of target cell death. Apart from the ability to eliminate transformed cells, NK cells also have an immuno-regulatory function by directly killing other immune cells and by secreting pro- and anti-inflammatory cytokines. Because of these roles, NK cells are of special interest in the growing field of cancer immunotherapy, where the function of immune cells is enhanced to defeat tumor cells. Clinical trials using NK cell-centered therapy have shown promising results against blood-borne cancer, yet progress has been limited against solid tumors. One possible explanation is related to the locally immuno-suppressive environment created by the solid tumor, for which improved research models are necessary. Besides, there is growing evidence of pronounced heterogeneity in the function of individual cells amidst the NK cell pool. Improving our understanding of NK cell biology thus requires advances in dedicated single-cell assays. For this purpose, our research group has previously developed miniaturized multi-well chips where individual cells can be confined and followed by microscopy over periods of several days. Using these microchips, a peculiar group of highly potent NK cells has been identified, which are able to kill several target cells in a row and contribute disproportionately to the overall cytotoxicity, and are therefore referred to as serial-killing NK cells. The work presented in this thesis is focused on developing and applying microscopy-based single-cell assays to the study of NK cell functional heterogeneity, with a particular focus on the mechanistic aspects of cytotoxicity. In Paper I, we investigated the formation and outcome of immune synapses in single cells, using micro-patterning to create distinct spatial distributions of ligands. We observed that synapse formation was guided by the overall shape of the ligands while local signaling regulated the final steps of exocytosis. Paper II is dedicated to the study of the cytotoxic mechanisms used by individual NK cells and their regulation, in particular comparing serial-killing NK cells and moderate killers. Using dedicated fluorescent reporters, we identified a switch between two commonly used killing pathways, degranulation and death ligand engagement, and proposed a model for the underlying process. This topic was further detailed in Paper III, where the contribution of these cytotoxic mechanisms under additional antibody stimulation was studied. The investigation was conducted in a newly developed single-use plastic microchip, designed to enable the generation of multiple simultaneous two- and three-dimensional cell cultures while retaining high imaging performance. In Paper IV, we implemented single-cell retrieval from the silicon-glass microwells. We characterized the performance of our setup and demonstrated its potential at identifying and retrieving rare populations defined by functional readouts.  Together, these studies further demonstrate the importance of single-cell analysis in the field of immunology. Besides advancing our understanding of NK cell biology, these developments may prove valuable in developing improved immunotherapies

Mesure de vitesses de fissuration en mode III dans les élastomères

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Taxonomic revision of Mantispidae (Neuroptera) from Taiwan with special reference to their biology

台灣的螳蛉科 (Mantispidae) 紀錄始於二十世紀初期，至今國內未有針對此類群的全面性分類學報告。本研究回顧台灣螳蛉科分類學研究的歷史與現況，並修訂台灣螳蛉科物種，重新描述外部形態與雄蟲外生殖器。本研究共記錄台灣的螳蛉科5屬14種，包含2個新種 (Mantispa flavipennis sp. n. 與Orientispa bisnota sp. n.)、1個新紀錄種 (Mantispa japonica McLachlan, 1875)、2個新組合 (Orientispa formosana (Okamoto, 1910) 與 Orientispa orientalis (Esben-Petersen, 1913)) 以及1個物種恢復(Mantispa nigra Stitz, 1913)，並提出7個同物異名，即台灣的Austroclimaciella quadrituberculata (Westwood, 1852) 為A. habutsuella (Okamoto, 1910) 之同物異名， Euclimacia badia Okamoto, 1910以及E. fusca Stitz, 1913為E. vespiformis Okamoto, 1910之同物異名，Ceromantispa formosana (Okamoto, 1910)、Necyla formosana minor (Stitz, 1913)以及Necyla formosana chiaiensis Ohl, 2004 為O. formosana (Okamoto, 1910) 之同物異名，Ceromantispa orientalis (Esben-Petersen, 1913) 為O. orientalis (Esben-Petersen, 1913) 之同物異名。由於梯螳蛉屬 (Euclimacia) 的成員除了體色以外，無其他可供區別的外部形態特徵，本研究以粒線體COI序列探討螳蛉科種級的分子特徵變異情形，建立螳蛉科種間遺傳距離界限為5%，確認台灣不同表型的梯螳蛉屬成員為相同物種。此外，本研究透過室內飼養，記錄台灣東螳蛉與擬蜂梯螳蛉之生活史、幼蟲取食策略以及成蟲求偶行為，建立基礎生物學資訊。Although the record of Mantispidae in Taiwan dated back to the early 20th centrury, up to date, there is no thorough taxonomic report. In this study, I reviewed the taxonomic history and revised the species of Mantispidae from Taiwan. Adult morphology and male genitalia are redescribed. Fourtheen species of 5 genera from Taiwan are recoreded, inculding 2 new species, 3 new combinations, 1 revived species, 7 new synonyms and 1 species first record from Taiwan. The new species are Mantispa falvipennis sp. n., Orientispa bisnota sp. n.; the new combination are Mantispa nigra (Stitz, 1913), Orientispa formosana (Okamoto, 1910), Orientispa orientalis (Esben-Petersen, 1913); the revived species is M. nigra (Stitz, 1913); the new record species is Mantispa japonica MacLachlan, 1875; Austroclimaciella quadrituberculata (Westwood, 1852) from Taiwan is revised as a synonym of A. habutsuella (Okamoto, 1910); Euclimacia badia Okamoto, 1910 and E. fusca Stitz, 1913 are revised as a synonym of E. vespiformis Okamoto, 1910; Ceromantispa formosana (Okamoto, 1910), Necyla formosana minor (Stitz, 1913) and Necyla formosana chiaiensis Ohl, 2004 are revised as a synonym of O. formosana (Okamoto, 1910); Ceromantispa orientalis (Esben-Petersen, 1913) is revised as a synonym of O. orientalis (Esben-Petersen, 1913). Body color are usually the only characters used for species discrimination in Euclimacia. To solve this problem, mitochondrial COI gene is used to investigate the molecular characteristics of species-level variation in Mantispidae. The result of molecular analysis shows interspecific divergence is higher than 5% and Euclimacia species from Taiwan with different color types are the same species. In addition, I conducted labotorial experiments and established the biological information of O. formosana and E. vespiformis with recording the life cycle, larval feeding strategies and adult courtship behavior.摘要 i Abstract ii 目次 iv 表次 vi 圖次 vii 前言 1 往昔研究 3 一、螳蛉科的分類歷史 3 二、台灣的螳蛉科分類研究 4 三、螳蛉科成蟲形態特徵 6 四、螳蛉科的生物學 11 材料與方法 13 一、標本來源 13 二、標本檢查 13 三、形態術語 14 四、分子特徵 15 五、室內飼養與觀察 17 結果 20 一、分類學 20 澳蜂螳蛉屬Austroclimaciella 20 梯螳蛉屬Euclimacia 23 優螳蛉屬Eumantispa 32 螳蛉屬Mantispa 37 東螳蛉屬Orientispa 49 二、分子分析 65 三、擬蜂梯螳蛉生物學 66 討論 70 一、分類修訂 70 二、分子分析 73 三、生物學 74 參考文獻 79 表 85 圖 86 附錄 11

Modulation of lytic molecules restrain serial killing in γδ T lymphocytes

Abstract γδ T cells play a pivotal role in protection against various types of infections and tumours, from early childhood on and throughout life. They consist of several subsets characterised by adaptive and innate-like functions, with Vγ9Vδ2 being the largest subset in human peripheral blood. Although these cells show signs of cytotoxicity, their modus operandi remains poorly understood. Here we explore, using live single-cell imaging, the cytotoxic functions of γδ T cells upon interactions with tumour target cells with high temporal and spatial resolution. While γδ T cell killing is dominated by degranulation, the availability of lytic molecules appears tightly regulated in time and space. In particular, the limited co-occurrence of granzyme B and perforin restrains serial killing of tumour cells by γδ T cells. Thus, our data provide new insights into the cytotoxic arsenal and functions of γδ T cells, which may guide the development of more efficient γδ T cell based adoptive immunotherapies