| Multiplex T-cell Stimulation Assay Utilizing a T-cell Activation Reporter-based Detection System
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Author:
Date:
2021-01-20
[Abstract] Immune tolerance and response are both largely driven by the interactions between the major histocompatibility complex (MHC) expressed by antigen presenting cells (APCs), T-cell receptors (TCRs) on T-cells, and their cognate antigens. Disordered interactions cause the pathogenesis of autoimmune diseases such as type 1 diabetes. Therefore, the identification of antigenic epitopes of autoreactive T-cells leads to important advances in therapeutics and biomarkers. Next-generation sequencing methods allow for the rapid identification of thousands of TCR clonotypes from single T-cells, and thus there is a need to determine cognate antigens for identified TCRs. This protocol describes a reporter system of T-cell activation where the fluorescent reporter protein ZsGreen-1 is driven by nuclear ...
[摘要] [摘要] 免疫耐受和应答都很大程度上由抗原呈递细胞（APC）表达的主要组织相容性复合物（MHC），T细胞上的T细胞受体（TCR）及其同源抗原之间的相互作用驱动。相互作用障碍导致自身免疫性疾病（例如1型糖尿病）的发病机理。因此，鉴定自身反应性T细胞的抗原表位导致治疗和生物标志物的重要进展。下一代测序方法可从单个T细胞快速鉴定数千种TCR克隆型，因此需要确定已鉴定TCR的同源抗原。该协议描述了T细胞活化的报告系统，其中荧光报告蛋白ZsGreen-1由活化T细胞的核因子（NFAT）信号驱动并通过流式细胞仪读取。记者T细胞也组成性表达额外的一对荧光素tein作为识别物，允许同时多路复用多达8种不同的报告T细胞系，每种表达不同的目标TCR，可通过流式细胞仪区分。一旦制成TCR表达细胞系，仅需一个转导步骤即可将其无限期用于制备新的T细胞系。这种多路复用系统允许筛选TCR-抗原相互作用的数量，否则这些相互作用将是不切实际的，可在多种情况下使用（即，筛选单个抗原或抗原库），并可用于研究任何T细胞-MHC-抗原三分子相互作用。
[背景] T细胞，抗原呈递细胞（APC）及其同源抗原之间的相互作用是自身免疫性疾病（例如1型糖尿病）的主要事件（Michels等，2017； ...
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| In vitro Regulatory T cells Differentiation From Naïve T Cells
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Author:
Date:
2014-03-20
[Abstract] In the past years, a subset of regulatory T cells (Tregs) expressing CD4, CD25 and the transcription factor FoxP3 has gained considerable attention as key regulators of T-cell tolerance and homeostasis (Sakaguchi, 2004). This population of T cells is specifically engaged in the maintenance of immune self-tolerance and the control of aberrant immune responses to foreign antigens. Remarkably, regulatory T cells have been implicated in tumor cell evasion of immune responses (Curiel et al., 2004; Zou, 2006) by suppressing T cell mediated antitumor immunity. The study of the signals that promote the differentiation of this suppressive population in the tumor microenvironment has become a central issue. Here we described a detailed method to in vitro differentiate Tregs using ...
[摘要] 在过去的几年中，表达CD4，CD25和转录因子FoxP3的调节性T细胞（Treg）的一个子集已经作为T细胞耐受性和体内平衡的关键调节剂获得了相当大的关注（Sakaguchi，2004）。 这种T细胞群特别参与维持免疫自身耐受性和控制对外来抗原的异常免疫应答。 值得注意的是，调节性T细胞通过抑制T细胞介导的抗肿瘤免疫参与了肿瘤细胞逃避免疫应答（Curiel等人，2004; Zou，2006）。 促进这种抑制群体在肿瘤微环境中的分化的信号的研究已经成为一个中心问题。 在这里，我们描述了使用来自小鼠天然T细胞的肿瘤细胞条件培养基来体外鉴别Treg的详细方法，并且基于它们的特异性标记物来鉴定它们（Dalotto-Moreno等人， >，2013）。
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| T Cell Calcium Mobilization Study (Flow Cytometry)
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Author:
Date:
2012-05-05
[Abstract] Antigen recognition and activation of T cell receptor (TCR) triggers transient calcium release from intracellular compartments and subsequent sustained calcium influx through cell surface Icrac channels. Sustained elevation of the cytoplasmic calcium level activates many calcium-dependent enzymes and transcription factors, which are essential for T cell activation and function. This protocol uses non-ratiometric dyes, in combination with flow cytometry, to monitor TCR-triggered calcium changes over time, and is a simple assay to examine the existence of T cell calcium mobilization defects in transgenic mice.
[摘要] 抗原识别和T细胞受体（TCR）的激活触发从细胞内区室瞬时钙释放和随后通过细胞表面Icrac通道的持续钙流入。 细胞质钙水平的持续升高激活许多钙依赖性酶和转录因子，其对于T细胞活化和功能是必需的。 该协议使用非比率染料，结合流式细胞术，监测TCR触发的钙随时间的变化，是一个简单的测定，检查转基因小鼠的T细胞钙动员缺陷的存在。
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