Determining optimal cytotoxic activity of human Her2neu specific CD8 T cells by comparing the Cr51 release assay to the xCELLigence system.
Determining optimal cytotoxic activity of human Her2neu specific CD8 T cells by comparing the Cr51 release assay to the xCELLigence system.
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DOI：
10.3791/3683
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发表时间：
2012-08-08
期刊：
影响因子：
--
通讯作者：
Knutson KL
中科院分区：
文献类型：
--
作者：
Erskine CL;Henle AM;Knutson KL
细胞毒性CD8 T细胞是T细胞的一个亚群，能够诱导感染或恶性宿主细胞死亡。这些细胞表达一种特殊的受体，称为T细胞受体（TCR），它可以识别与HLA I类分子结合的特定抗原肽。感染细胞或肿瘤细胞通过它们的HLA I类分子接触，导致颗粒酶和穿孔蛋白等溶解分子的产生，从而导致靶细胞死亡。虽然使用ELIspot或流式细胞术测定抗原特异性CD8 T细胞的频率是有用的，但使用细胞毒性测定也有助于确定CD8 T细胞反应的强度。用于评估细胞毒性功能的最知名的分析是铬释放分析（CRA），这被认为是一个标准的分析。CRA有几个局限性，包括细胞暴露于伽马辐射，缺乏可重复性，以及需要大量细胞。在过去的十年中，人们对采用新的战略来克服这些限制产生了兴趣。较新的方法包括测量caspase释放，BLT酯酶活性和CD107表面表达。使用罗氏xCelligence系统的基于阻抗的分析，在本文中研究了其作为CRA替代方案的潜力。当附着的肿瘤细胞与电极板结合时，就会产生电流的阻抗或对立。肿瘤细胞在杀死后分离，电阻抗降低，可通过xCelligence系统测量。适应基于阻抗的方法来评估细胞介导的杀伤的能力取决于观察到T细胞不紧密粘附在大多数表面上，并且似乎对阻抗没有太大影响，因此减少了T细胞与测量直接干扰的任何担忧。结果表明，基于阻抗的检测可以检测抗原特异性细胞毒性CD8 T细胞水平的变化，相对于标准CRA，灵敏度更高。基于这些结果，基于阻抗的方法可能是cra或其他旨在测量细胞毒性CD8 T细胞功能的方法的良好替代方法。
Cytotoxic CD8 T cells constitute a subgroup of T cells that are capable of inducing the death of infected or malignant host cells. These cells express a specialized receptor, called the T cell receptor (TCR), which can recognize a specific antigenic peptide bound to HLA class I molecules. Engagement of infected cells or tumor cells through their HLA class I molecule results in production of lytic molecules such as granzymes and perforin resulting in target cell death. While it is useful to determine frequencies of antigen-specific CD8 T cells using assays such as the ELIspot or flow cytometry, it is also helpful to ascertain the strength of CD8 T cell responses using cytotoxicity assays. The most recognizable assay for assessing cytotoxic function is the Chromium Release Assay (CRA), which is considered a standard assay. The CRA has several limitations, including exposure of cells to gamma radiation, lack of reproducibility, and a requirement for large numbers of cells. Over the past decade, there has been interest in adopting new strategies to overcome these limitations. Newer approaches include those that measure caspase release, BLT esterase activity and surface expression of CD107. The impedance-based assay, using the Roche xCelligence system, was examined in the present paper for its potential as an alternative to the CRA. Impedance or opposition to an electric current occurs when adherent tumor cells bind to electrode plates. Tumor cells detach following killing and electrical impedance is reduced which can be measured by the xCelligence system. The ability to adapt the impedance-based approach to assess cell-mediated killing rests on the observation that T cells do not adhere tightly to most surfaces and do not appear to have much impact on impedance thus diminishing any concern of direct interference of the T cells with the measurement. Results show that the impedance-based assay can detect changes in the levels of antigen-specific cytotoxic CD8 T cells with increased sensitivity relative to the standard CRA. Based on these results, impedance-based approaches may be good alternatives to CRAs or other approaches that aim to measure cytotoxic CD8 T cell functionality.
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影响因子：
15.9
作者：
Knutson, KL;Schiffman, K;Disis, ML
通讯作者：
Disis, ML
影响因子：
2.2
作者：
STRIJBOSCH, LWG;BUURMAN, WA;GROENEWEGEN, G
通讯作者：
GROENEWEGEN, G
DOI：
10.1084/jem.187.9.1383
发表时间：
1998-05-04
期刊：
The Journal of experimental medicine
影响因子：
--
作者：
Gallimore A;Glithero A;Godkin A;Tissot AC;Plückthun A;Elliott T;Hengartner H;Zinkernagel R
通讯作者：
Zinkernagel R
影响因子：
32.4
作者：
Zhang N;Bevan MJ
通讯作者：
Bevan MJ
影响因子：
4.3
作者：
Aktas, Esin;Kucuksezer, Umut Can;Deniz, Gunnur
通讯作者：
Deniz, Gunnur