Multiplexed detection and isolation of viable low-frequency cytokine-secreting human B cells using cytokine secretion assay and flow cytometry (CSA-Flow).
Multiplexed detection and isolation of viable low-frequency cytokine-secreting human B cells using cytokine secretion assay and flow cytometry (CSA-Flow).
复制标题
使用细胞因子分泌测定和流式细胞术 (CSA-Flow) 多重检测和分离活的低频细胞因子分泌人 B 细胞。
DOI：
10.1038/s41598-020-71750-z
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发表时间：
2020
影响因子：
4.6
通讯作者：
Bar-Or,Amit
中科院分区：
文献类型：
--
作者：
Rezk,Ayman;Li,Rui;Bar-Or,Amit
对分泌细胞因子的免疫细胞进行功能表征的能力对健康以及一系列免疫介导和自身免疫疾病具有广泛的影响。低频细胞因子定义的免疫细胞亚群可以发挥关键的免疫调节作用，但其详细研究往往受到有限的临床样本可用性的阻碍。常用的技术（包括细胞内细胞因子染色）需要细胞固定，从而排除了后续的功能询问。细胞因子分泌测定（CSA）可以克服这一限制，但主要用于检测频率相对较高的单细胞因子分泌细胞。我们研究了 CSA 与多参数流式细胞术 (CSA-Flow) 相结合如何能够同时分离多个低频细胞因子分泌细胞。我们重点关注人类 B 细胞（传统上认为比 T 细胞更难检测），我们发现单次捕获 CSA-Flow 可以分离高度纯化的低频（IL-10+；GM-CSF+）和高频（TNF+）细胞因子定义的 B 细胞群。同时检测和分离多达三种活的、高度纯化的细胞因子分泌 B 细胞亚群是可行的，尽管存在一些信号损失，并且随后的组分可用于基因表达分析和体外细胞培养。这种多重 CSA-Flow 方法将在许多人类细胞免疫学环境中引起人们的兴趣，旨在对细胞因子分泌免疫细胞进行功能表征，特别是当样本量和细胞数量有限时。
The ability to functionally characterize cytokine-secreting immune cells has broad implications in both health and a range of immune-mediated and auto-immune diseases. Low-frequency cytokine-defined immune-cell subsets can play key immune-regulatory roles, yet their detailed study is often hampered by limited clinical sample availability. Commonly used techniques including intracellular cytokine staining require cell fixation, precluding subsequent functional interrogation. The cytokine-secretion assay (CSA) can overcome this limitation, though has mostly been used for detection of relatively high-frequency, single-cytokine secreting cells. We examined how adaptation of the CSA in combination with multiparametric flow-cytometry (CSA-Flow) may enable simultaneous isolation of multiple, low-frequency, cytokine-secreting cells. Focusing on human B cells (traditionally recognized as harder to assay than T cells), we show that single-capture CSA-Flow allows for isolation of highly-purified populations of both low-frequency (IL-10+; GM-CSF+) and high-frequency (TNF+) cytokine-defined B cells. Simultaneous detection and isolation of up to three viable and highly-purified cytokine-secreting B-cell subpopulations is feasible, albeit with some signal loss, with fractions subsequently amenable to gene expression analysis and in vitro cell culture. This multiplexing CSA-Flow approach will be of interest in many human cellular immunology contexts aiming to functionally characterize cytokine-secreting immune cells, especially when sample volumes and cell numbers are limited.