Biotechnology Institute Thurgau (BITg), University of Konstanz, Unterseestrasse 47, 8280 Kreuzlingen, Switzerland.
Graduate School for Cellular and Biomedical Sciences, University of Bern, 3012 Bern, Switzerland.
Int J Mol Sci. 2021 Apr 16;22(8):4158. doi: 10.3390/ijms22084158.
Chemokines guide leukocyte migration in different contexts, including homeostasis, immune surveillance and immunity. The chemokines CCL19 and CCL21 control lymphocyte and dendritic cell migration and homing to lymphoid organs. Thereby they orchestrate adaptive immunity in a chemokine receptor CCR7-dependent manner. Likewise, cancer cells that upregulate CCR7 expression are attracted by these chemokines and metastasize to lymphoid organs. In-depth investigation of CCR7 expression and chemokine-mediated signaling is pivotal to understand their role in health and disease. Appropriate fluorescent probes to track these events are increasingly in demand. Here, we present an approach to cost-effectively produce and fluorescently label CCL19 and CCL21 in a semi-automated process. We established a versatile protocol for the production of recombinant chemokines harboring a small C-terminal S6-tag for efficient and site-specific enzymatic labelling with an inorganic fluorescent dye of choice. We demonstrate that the fluorescently labeled chemokines CCL19-S6 and CCL21-S6 retain their full biological function as assessed by their abilities to mobilize intracellular calcium, to recruit β-arrestin to engaged receptors and to attract CCR7-expressing leukocytes. Moreover, we show that CCL19-S6 serves as powerful reagent to monitor CCR7 internalization by time-lapse confocal video microscopy and to stain CCR7-positive primary human and mouse T cell sub-populations.
趋化因子在不同的环境中引导白细胞迁移，包括内稳态、免疫监视和免疫。趋化因子 CCL19 和 CCL21 控制淋巴细胞和树突状细胞的迁移和归巢到淋巴器官。因此，它们以趋化因子受体 CCR7 依赖的方式协调适应性免疫。同样，上调 CCR7 表达的癌细胞被这些趋化因子吸引并转移到淋巴器官。深入研究 CCR7 表达和趋化因子介导的信号转导对于了解其在健康和疾病中的作用至关重要。越来越需要适当的荧光探针来跟踪这些事件。在这里，我们提出了一种在半自动过程中经济有效地生产和荧光标记 CCL19 和 CCL21 的方法。我们建立了一种通用的生产重组趋化因子的方案，该方案带有一个小的 C 末端 S6 标签，用于高效且特异性地用无机荧光染料进行酶标记。我们证明，荧光标记的趋化因子 CCL19-S6 和 CCL21-S6 保留了其全部生物学功能，因为它们能够动员细胞内钙、招募 β-抑制蛋白到已结合的受体，并吸引表达 CCR7 的白细胞。此外，我们表明 CCL19-S6 可作为强大的试剂，通过时程共聚焦视频显微镜监测 CCR7 内化，并染色 CCR7 阳性的原代人和鼠 T 细胞亚群。
