Department of Microbiology and Immunology, University of Melbourne, Parkville, VIC 3010, Australia.
J Immunol Methods. 2012 Aug 31;382(1-2):150-9. doi: 10.1016/j.jim.2012.05.019. Epub 2012 Jun 7.
NKT cells are key mediators of antiviral and anticancer immunity. Experiments in mice have demonstrated that activation of NKT cells in vivo induces the expression of multiple effector molecules critical to successful immunity. Human clinical trials have shown similar responses, although in vivo activation of NKT cells in humans or primate models are far more limited in number and scope. Measuring ex vivo activation of NKT cells by the CD1d-restricted glycolipid ligand α-Galactosylceramide (α-GalCer) through cytokine expression profiles is a useful marker of NKT cell function, but for reasons that are unclear, this approach does not appear to work as well in humans and non-human primate macaque models in comparison to mice. We performed a series of experiments on human and macaque (Macaca nemestrina) fresh whole blood samples to define optimal conditions to detect NKT cell cytokine (TNF, IFNγ, IL-2) and degranulation marker (CD107a) expression by flow cytometry. We found that conditions previously described for mouse splenocyte NKT cell activation were suboptimal on human or macaque blood NKT cells. In contrast, a 6h incubation with brefeldin A added for the last 4h, in a 96-well plate based assay, and using an α-GalCer concentration of 1 μg/ml were optimal methods to stimulate NKT cells in fresh blood from both humans and macaques. Unexpectedly, we noted that blood NKT cells from macaques infected with SIV were more readily activated by α-GalCer than NKT cells from uninfected macaques, suggesting that SIV infection may have primed the NKT cells. In conclusion, we describe optimized methods for the ex vivo antigen-specific activation of human and macaque blood NKT cells. These assays should be useful in monitoring NKT cells in disease and in immunotherapy studies.
自然杀伤 T（NKT）细胞是抗病毒和抗癌免疫的关键介质。在小鼠中的实验表明，体内激活 NKT 细胞可诱导多种对成功免疫至关重要的效应分子的表达。人体临床试验也显示出类似的反应，尽管在人类或灵长类动物模型中，体内激活 NKT 细胞的数量和范围要有限得多。通过细胞因子表达谱测量 CD1d 限制性糖脂配体 α-半乳糖基神经酰胺（α-GalCer）对 NKT 细胞的体外激活是评估 NKT 细胞功能的有用标志物，但由于原因尚不清楚，与小鼠相比，这种方法在人类和非人类灵长类猕猴模型中似乎效果不佳。我们在人类和猕猴（Macaca nemestrina）新鲜全血样本中进行了一系列实验，以确定通过流式细胞术检测 NKT 细胞细胞因子（TNF、IFNγ、IL-2）和脱颗粒标志物（CD107a）表达的最佳条件。我们发现，以前描述的用于激活小鼠脾 NKT 细胞的条件在人类或猕猴血液 NKT 细胞上并不理想。相比之下，在基于 96 孔板的测定中，在添加了布雷菲德菌素 A 的情况下孵育 6 小时，最后 4 小时添加，使用 1μg/ml 的 α-GalCer 浓度是刺激人类和猕猴新鲜血液中 NKT 细胞的最佳方法。出乎意料的是，我们注意到感染 SIV 的猕猴血液 NKT 细胞比未感染的猕猴血液 NKT 细胞更容易被 α-GalCer 激活，这表明 SIV 感染可能使 NKT 细胞被预先激活。总之，我们描述了优化的方法，用于体外刺激人类和猕猴血液 NKT 细胞的抗原特异性激活。这些检测方法应该有助于监测疾病中的 NKT 细胞，并用于免疫治疗研究。
