A rapid method for assessment of natural killer cell function after multiple receptor crosslinking
A rapid method for assessment of natural killer cell function after multiple receptor crosslinking
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DOI：
10.1016/j.jim.2011.01.007
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发表时间：
2011-03-07
影响因子：
2.2
通讯作者：
Goodier, M. R.
中科院分区：
文献类型：
--
作者：
Al-Hubeshy, Z. B.;Coleman, A.;Goodier, M. R.
NK细胞的功能是由活化和抑制受体的信号整合调节的。我们开发了一种测定来研究成对组合中共交联NK细胞受体的作用，而不需要纯化NK细胞。将识别抑制性和活化性受体的单克隆抗体包被到平底组织培养板上，并通过流式细胞术分析CD 107 a表达，在未分级分离的新鲜分离的静息或细胞因子活化的外周血单核细胞内测量脱粒。测量的脱粒应答是NK细胞特异性的，因为在门控T细胞中没有诱导CD 107 a的表达。我们检测到，与与同种型匹配的对照抗体组合时的每种抗体相比，响应于针对活化NK细胞受体（包括CD 16、NKG 2D、NKp 30和NKp 46）的抗体组合的脱粒增强。在静息或细胞因子预活化NK细胞中，NKG 2A的共交联导致对抗NKp 30或抗NKp 46单药的脱粒抑制，但对CD 16或NKG 2D介导的应答无影响。通过细胞内细胞因子染色和受体交联后的细胞培养上清液测定干扰素γ的产生。在受体交联后，从静息NK细胞中未检测到IFN-γ，而在细胞因子预活化的NK细胞中IFN-γ产生的模式反映了所观察到的脱粒。我们的结论是，该试验是适合的NK细胞受体共交联对多种NK细胞功能的影响的分析，并有可能应用于病理条件下，有限数量的细胞可用于研究。出版社：Elsevier BM
NK cell function is regulated by the integration of signals from activating and inhibitory receptors. We developed an assay to study the effect of co-crosslinking NK cell receptors in pair-wise combinations without the need to purify NK cells. Monoclonal antibodies recognising inhibitory and activating receptors were coated to flat bottomed tissue culture plates and degranulation was measured within unfractionated, freshly isolated resting or cytokine activated peripheral blood mononuclear cells by flow cytometric analysis of CD107a expression. Measured degranulation responses were NK cell specific, since no expression of CD107a was induced in gated T cells. We detected enhancement of degranulation in response to combinations of antibodies against activating NK cell receptors, including CD16, NKG2D, NKp30 and NKp46 compared to each antibody when combined with an isotype matched control antibody. Co-crosslinking of NKG2A resulted in the inhibition of degranulation measured in response to anti-NKp30 or anti-NKp46 alone in both resting or cytokine preactivated NK cells, but had no effect on CD16 or NKG2D mediated responses. Interferon gamma production was assayed by intracellular cytokine staining and in cell culture supernatants after receptor crosslinking. No IFN-gamma could be detected from resting NK cells after receptor crosslinking whereas the pattern of IFN-gamma production in cytokine pre-activated NK cells reflected that observed for degranulation. We conclude that this assay is suitable for the analysis of the impact of NK cell receptor co-crosslinking on multiple NK cell functions and has the potential for application to pathologic conditions where limited numbers of cells are available for study. Published by Elsevier BM.