| Chromium-51 (51Cr) Release Assay to Assess Human T Cells for Functional Avidity and Tumor Cell Recognition
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Author:
Date:
2016-08-20
[Abstract] Cytotoxic CD8+ T cells are able to specifically recognize and kill target cells through specific interaction between their T cell receptors (TCRs) and small immunogenic peptides (antigens) presented by major histocompatibility complex (MHC) molecules. The antigen recognition capacity and in vitro lytic activity of antigen-specific cytotoxic T cells can be assessed functionally in the so-called chromium 51 (51Cr) release assay, which was developed almost 50 years ago in our institution (Brunner et al., 1968). Radioactively-labelled cells deficient for endogenous antigen presentation [e.g., transporter for antigen presentation (TAP)-deficient T2 cells] and stably transfected with the MHC of interest (e.g., HLA-A2+) are ...
[摘要] 细胞毒性CD8 + T细胞能够通过其T细胞受体（TCR）与主要组织相容性复合物（MHC）分子呈递的小免疫原性肽（抗原）之间的特异性相互作用特异性识别和杀死靶细胞。抗原特异性细胞毒性T细胞的抗原识别能力和体外裂解活性可以在所谓的铬51（<51> Cr）释放测定中功能性评估，其是几乎50年前在我们的机构中发展起来的（Brunner等人，1968年）。放射性标记的内源性抗原呈递缺陷的细胞[例如，用于抗原呈递（TAP）缺陷型T2细胞的转运蛋白]，并用感兴趣的MHC稳定转染（例如 ，HLA-A2 sup + +）通常在这个4小时测定期间用作靶标。或者，内源性呈递免疫原性抗原的Cr标记的病毒感染或肿瘤细胞系可以作为靶细胞（例如，用于评估肿瘤识别）。 在肽滴定测定（部分A）中，用抗原性肽的系列稀释物对放射性标记的靶细胞进行脉冲，并在效应物（例如，CD8 + T细胞克隆）与靶细胞（ Cr-T2细胞）比（E:T）为10:1的混合物在96孔V型底板中37℃温育4小时。在肿瘤杀伤试验（B部分）中，将细胞毒性CD8 ...
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| Ex vivo Human Natural Killer (NK) Cell Stimulation and Intracellular IFNγ and CD107a Cytokine Staining
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Author:
Date:
2015-06-20
[Abstract] Natural killer (NK) cells comprise 5–20% of peripheral blood mononuclear cells (PBMC) in humans. In addition to their fundamental roles in the defense against viral infections and tumor surveillance, NK cells help shape adaptive immune responses through their production of cytokines. NK cells are traditionally identified as CD3neg, CD14neg, CD19neg lymphocytes expressing CD56. Using a combination of markers that includes CD56 and CD7 greatly increases the ability to define the phenotype and function of NK cell subsets. Two key markers of NK cell function are the production of IFNγ and the release of cytotoxic granules measured by the expression of CD107a. Here we describe a method to assess IFNγ and CD107a expression in NK cells following stimulation with ...
[摘要] 自然杀伤（NK）细胞在人中包含5-20％的外周血单核细胞（PBMC）。 除了它们在防御病毒感染和肿瘤监测中的基本作用，NK细胞通过其细胞因子的产生帮助形成适应性免疫应答。 NK细胞传统上被鉴定为表达CD56的CD3阴性，CD14阳性，CD19阴性淋巴细胞。 使用包括CD56和CD7的标记物的组合极大地增加了定义NK细胞亚群的表型和功能的能力。 NK细胞功能的两个关键标记是IFNγ的产生和通过CD107a的表达测量的细胞毒性颗粒的释放。 在这里我们描述了一种方法来评估在靶细胞或细胞因子刺激后NK细胞中的IFNγ和CD107a表达。 该方法可用于评估来自广泛的研究参与者的外周血单核细胞中NK细胞的一般功能能力。
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| Ex vivo Natural Killer Cell Cytotoxicity Assay
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Author:
Date:
2013-08-20
[Abstract] Natural Killer (NK) cells are cytotoxic lymphocytes that constitute a major component of the innate immune system. Immunosurveillance of the host by NK cells for malignant and virally-infected cells results in direct cytotoxicity and the production of cytokines to enhance the immune response. This protocol will describe the “gold standard” chromium release assay for measuring the target cell killing capacity of NK cells. Key features of this cytotoxicity assay are that it is performed with sorted NK cells as the effectors and any Major Histocompatibility Class I (MHC-I)-low or deficient tumor cell line can be used as the target cells.
[摘要] 天然杀伤（NK）细胞是细胞毒性淋巴细胞，其构成先天性免疫系统的主要组分。 通过NK细胞对恶性和病毒感染的细胞的宿主的免疫监视导致直接的细胞毒性和细胞因子的产生，以增强免疫应答。 该方案将描述用于测量NK细胞的靶细胞杀伤能力的"金标准"铬释放测定。 该细胞毒性测定的主要特征是其使用分选的NK细胞作为效应物进行，任何主要组织相容性I类（MHC-1） - 低或缺陷的肿瘤细胞系可以用作靶细胞。
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