A Versatile Toolkit for Semi-Automated Production of Fluorescent Chemokines to Study CCR7 Expression and Functions

Abstract

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^Dy649P1 and CCL21-S6^Dy649P1 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^Dy649P1 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.

Keywords: CCL19; CCL21; CCR7; cell migration; chemokine production; flow cytometry; fluorescent chemokine; leukocytes.

Figure 1

Design and production of the recombinant chemokines CCL19 and CCL21. (a) Schematic representation of the size matched expression constructs and the corresponding final fluorescently labeled chemokine proteins. A cleavable His₆-SUMO-tag is fused to the N-terminus of mature human CCL19 and CCL21 followed by a short, flexible linker and the S6-tag for enzymatic labelling with the dye Dy^649P1. Human CCL19-mRFP and CCL21-mRFP are included for size comparison. (b) Workflow overview of the chemokine production process. IMAC purification, chemokine refolding, and CIEX purification can be performed in an automated fashion on a chromatography system.

Figure 2

Purification and fluorescent labelling of human CCL19 and CCL21. (a,b) Representative Coomassie-stained SDS-PAGE gels with samples derived from the bacterial cell lysate before loading on the IMAC column, the IMAC flow through (FT), the IMAC eluate, the CIEX-C load after cleaving the His₆-SUMO-tag, the CIEX-C flow through (FT) and CIEX-C eluate for the CCL19-S6 (a) and CCL21-S6 (b) purification steps illustrated in Figure 1b. (c,d) Representative HPLC chromatograms displaying the volume of gradient elution of CCL19-S6 (c) or CCL21-S6 (d) from a C-18 column used to separate correctly folded from misfolded chemokines (left panels) and to separate the fluorescently labeled chemokine from unlabeled chemokine, free substrate and the Sfp-CoA-Dy^649P1 (marked with *). Protein concentration was determined by measuring its absorbance at 280 nm (OD280); fluorescence was measured at 652 nm (OD652) and illustrated in red. (e,f). Representative Coomassie-stained SDS-PAGE gels (upper panels) and corresponding Western blots (WB, lower panels) of the same samples of native, S6-tagged and S6-tagged, as well as fluorescently labeled CCL19 (e) and CCL21 (f).

Figure 3

Fluorescent CCL19-S6^649P1 and CCL21-S6^649P1 efficiently elicit CCR7-mediated signaling and migration. (a,b) Pre-B 300-19 cells stably expressing CCR7 were stimulated with 50 nM of the indicated CCL19 (a) and CCL21 (b) chemokine variants and changes in intracellular calcium levels were recorded by flow cytometry over time. The time point of stimulation (Stim.) is indicated. Stimulation with PBS, the chemokine solvent, served as negative control. Mean values ± SD of three independent experiments are shown. (c,d) HeLa cells transiently transfected with CCR7-EGFP and β-arrestin2-Nluc were stimulated with 500 nM of CCL19, CCL19-S6, or CCL19-S6^649P1 (c), and 1.5 µM CCL21, CCL21-S6, or CCL21-S6^649P1 (d), respectively, and chemokine-mediated β-arrestin2 recruitment to CCR7 was determined by BRET. Mean values ± SD of three independent experiments are depicted. (e,f) Pre-B 300-19 cells stably expressing CCR7 were allowed to migrate towards graded concentrations of CCL19, CCL19-S6^649P1 (e), CCL21, or CCL21-S6^649P1 (f) for 3h in a Transwell migration assay. Migrated cells were counted by flow cytometry; random migration in the absence of chemokine was subtracted. Mean values ± SD of three independent experiments are shown.

Figure 4

CCL19-S6^649P1 readily and specifically binds to CCR7-expressing cells. (a,b) HeLa cells transiently transfected with CCR7-EGFP or its vector control (pcDNA3) were incubated at indicated temperature and time periods with 25 nM of CCL19-S6^649P1 (a), or CCL21-S6^649P1 (b). Cell associated, chemokine-derived mean fluorescence intensities (MFI) were recorded by flow cytometry. Mean values ± SD of three independent experiments are shown. (c) HeLa cells transiently transfected with CCR7-EGFP or its vector control (pcDNA3) were incubated at 22 °C with 25 nM of CCL19-S6^649P1 for indicated time periods. Representative dot plots of one out of three experiments depicting CCL19-S6^649P1 binding to CCR7-EGFP expressing and pcDNA3 vector control transfected cells.

Figure 5

CCL19-S6^649P1 binds to and is internalized specifically by CCR7-expressing cells. Time-lapse confocal video microscopy of HeLa cells transiently expressing CCR7-YPet stimulated at time point 10 min with 10 nM of CCL19-S6^649P1 at 37 °C. Inlets (white rectangles) in merge images illustrate magnification, BF shows corresponding bright field images. Scale bar: 20 µm. Note that the neighboring cell lacking CCR7-YPet expression does not interact with CCL19-S6^649P1. Images are representative for two independent experiments.

Figure 6

CCL19-S6^649P1 is a versatile tool to stain CCR7-expressing primary human and mouse T cell sub-populations. Primary human peripheral blood CD3-sorted T cells (a,b) and mouse splenic T cells (c) were stained with 50 nM CCL19-S6^649P1 for 20 min at 22 °C together with either anti-huCD4-FITC, anti-huCD45RA-FITC, anti-huCD45RO-FITC, anti-huCCR7-APC, anti-huCCR7-PacificBlue, anti-muCD3-PE, or anti-muCCR7-APC conjugated antibodies, respectively, and analyzed by flow cytometry. One (a) out of the two (b) human donors, or three (c) independent experiments with comparable results are shown.