Dept. of Biochemistry & Cell Biology, Faculty of Veterinary Medicine, Yalelaan 2, Utrecht, The Netherlands.
J Leukoc Biol. 2013 Mar;93(3):395-402. doi: 10.1189/jlb.0911480. Epub 2012 Dec 17.
Nano-sized membrane vesicles are secreted by many cell types. These vesicles can serve as carriers of cellular information. DC-derived vesicles can be targeted to other immune cells and modify their function. Accurate analysis of quantitative and qualitative changes in EV production by DC upon different activation stimuli is needed to further reveal the immune regulatory properties of DC-derived EVs. However, methods for reliable quantification of individual EVs and for analysis of the heterogeneity of EV populations are limited. With our recently developed high-resolution flow cytometry-based method, we can perform a high-throughput, multiparameter, and quantitative analysis of individual EVs. With the use of this novel technique, we show that despite previous assumptions, stimulation with bacterial LPS increases EV release by DC. Furthermore, we demonstrate heterogeneity in DC-derived EVs regarding their buoyant density and MHC class II content. Finally, we show that cognate interaction between LPS-stimulated DC and CD4(+) T cells affects both the quantity and quality of LPS DC-derived EVs present in the culture supernatant. These data indicate that flow cytometry-based analysis of individual EVs is a valuable, novel tool to study the dynamics of EV secretion and composition, offering great opportunities to unveil the function of immune cell-derived EVs.
纳米大小的膜囊泡由许多细胞类型分泌。这些囊泡可以作为细胞信息的载体。树突状细胞衍生的囊泡可以被靶向到其他免疫细胞，并改变它们的功能。需要对不同激活刺激下 DC 产生的 EV 的数量和质量变化进行准确分析，以进一步揭示 DC 衍生 EV 的免疫调节特性。然而，用于可靠定量单个 EV 并分析 EV 群体异质性的方法有限。使用我们最近开发的基于高分辨率流式细胞术的方法，我们可以对单个 EV 进行高通量、多参数和定量分析。使用这项新技术，我们表明，尽管存在先前的假设，但细菌 LPS 的刺激会增加 DC 释放 EV。此外，我们证明了 DC 衍生 EV 在其浮密度和 MHC Ⅱ类含量方面存在异质性。最后，我们表明，LPS 刺激的 DC 与 CD4(+)T 细胞之间的同源相互作用会影响培养上清液中存在的 LPS DC 衍生 EV 的数量和质量。这些数据表明，基于流式细胞术的单个 EV 分析是研究 EV 分泌和组成动力学的一种有价值的新工具，为揭示免疫细胞衍生 EV 的功能提供了巨大的机会。
