| Efficient Production of Functional Human NKT Cells from Induced Pluripotent Stem Cells − Reprogramming of Human Vα24+iNKT Cells
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Author:
Date:
2017-05-20
[Abstract] Antigen-specific T cell-derived induced pluripotent stem cells (iPSCs) have been shown to re-differentiate into functional T cells and thus provide a potential source of T cells that could be useful for cancer immunotherapy. Human Vα24+ invariant natural killer T (Vα24+iNKT) cells are subset of T cells that are characterized by the expression of an invariant Vα24-Jα18 paired with Vβ11, that recognize glycolipids, such as α-galactosylceramide (α-GalCer), presented by the MHC class I-like molecule CD1d. Vα24+iNKT cells capable of producing IFN-γ are reported to augment anti-tumor responses, which affects both NK cells and CD8+ cytotoxic T lymphocytes to eliminate MHC- and MHC+ tumor cells, respectively. Here we describe a ...
[摘要] 抗原特异性T细胞来源的诱导多能干细胞（iPSCs）已显示重新分化为功能性T细胞，从而提供可用于癌症免疫治疗的T细胞的潜在来源。不变性自然杀伤T（Vα24 + iNKT）细胞的人Vα24 + 细胞是T细胞的子集，其特征在于与Vβ11配对的不变Vα24-Jα18的表达，其识别糖脂，如α-半乳糖神经酰胺（α-GalCer），由MHC I类分子CD1d呈递。据报道能够产生IFN-γ的Vα24 + i / KT细胞增加抗肿瘤反应，其影响NK细胞和CD8 +细胞毒性T淋巴细胞以消除MHC - 和MHC + 肿瘤细胞。在这里，我们描述了将人Vα24 + iNKT细胞重编程到iPSC中的鲁棒方案，然后将其重新分化为Vα24 + iNKT细胞（iPS-Vα24功能的iNKT）。我们进一步提供了测定iPS-Vα24 + iNKT细胞活性的方案。背景 以前有报道说，针对晚期非小细胞肺癌（NSCLC）和头颈部癌症的Vα24 + iNKT细胞癌免疫治疗的临床试验显示疗效，耐受性良好（Motohashi et al。等人，2009; Yamasaki等人，2011）。然而，已知来自外周血单核细胞（PBMC）的Vα24 ...
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| Quantitative Measurements of HIV-1 and Dextran Capture by Human Monocyte-derived Dendritic Cells (MDDCs)
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Author:
Date:
2016-11-20
[Abstract] The aim of this protocol is to describe how to measure and quantify the amount of HIV-1 particles and dextran molecules internalized in human monocyte derived dendritic cells (MDDCs), using three different techniques: flow cytometry, quantitative PCR and confocal microscopy.
[摘要] 本协议的目的是描述如何使用三种不同的技术：流式细胞术，定量PCR和共聚焦显微镜来测量和量化人类单核细胞衍生树突细胞（MDDCs）中内在的HIV-1颗粒和葡聚糖分子的量。
[背景] 开发此协议是为了评估在人单核细胞衍生的树突细胞中肌动蛋白成核破坏时HIV-1内化的变化。在shRNA筛选后，识别对于HIV-1从树突状细胞转移到T细胞重要的基因，我们观察到肌动蛋白成核的中断导致从富含肌动蛋白的树突到水泡的转变，由于过量的肌动球蛋白收缩。结果，观察到HIV-1转移的减少和由于水泡收缩驱动的巨噬细胞增多引起的HIV-1内化的增加。我们的结论是，肌动蛋白成核和稳定的效应器是维持艾滋病毒1对肌动蛋白丰富的树突和限制其内吞作用，有效转移到T淋巴细胞的关键（Menager和Littman，2016）。
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| Analysis of Phagosomal Antigen Degradation by Flow Organellocytometry
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Author:
Date:
2016-11-20
[Abstract] Professional phagocytes internalize self and non-self particles by phagocytosis to initiate innate immune responses. After internalization, the formed phagosome matures through fusion and fission events with endosomes and lysosomes to obtain a more acidic, oxidative and hydrolytic environment for the degradation of its cargo. Interestingly, phagosome maturation kinetics differ between cell types and cell activation states. This protocol allows to quantify phagosome maturation kinetics on a single organelle level in different types of phagocytes using flow cytometry. Here, ovalbumin (OVA)-coupled particles are used as phagocytosis model system in dendritic cells (DC), which are internalized by phagocytosis. After different time points, phagosome maturation parameters, such as phagosomal ...
[摘要] 专业吞噬细胞通过吞噬作用内化自身和非自身颗粒以启动先天免疫应答。内化后，形成的吞噬体通过与内体和溶酶体的融合和裂变事件成熟，以获得更酸性，氧化和水解的环境用于其货物的降解。有趣的是，吞噬体成熟动力学在细胞类型和细胞活化状态之间不同。该协议允许使用流式细胞术定量不同类型的吞噬细胞中单个细胞器水平上的吞噬体成熟动力学。在这里，卵白蛋白（OVA）耦合的颗粒用作吞噬作用模型系统在树突状细胞（DC），其通过吞噬内化。在不同的时间点之后，吞噬体成熟参数，例如OVA的吞噬体降解和溶酶体蛋白（例如LAMP-1）的获得，可以通过流式细胞器细胞计数以高度定量的方式同时测量。这些读出可以与其他吞噬体功能相关，例如抗原降解，在DC中的加工和负载。
[背景] ...
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