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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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