Selective Boosting of CCR7-Acting Chemokines; Short Peptides Boost Chemokines with Short Basic Tails, Longer Peptides Boost Chemokines with Long Basic Tails

Abstract

The chemokine receptor CCR7 and its ligands CCL19 and CCL21 regulate the lymph node homing of dendritic cells and naïve T-cells and the following induction of a motile DC-T cell priming state. Although CCL19 and CCL21 bind CCR7 with similar affinities, CCL21 is a weak agonist compared to CCL19. Using a chimeric chemokine, CCL19^{CCL21N-term|C-term}, harboring the N-terminus and the C-terminus of CCL21 attached to the core domain of CCL19, we show that these parts of CCL21 act in a synergistic manner to lower ligand potency and determine the way CCL21 engages with CCR7. We have published that a naturally occurring basic C-terminal fragment of CCL21 (C21TP) boosts the signaling of both CCL19 and CCL21. Boosting occurs as a direct consequence of C21TP binding to the CCR7 N-terminus, which seems to free chemokines with basic C-termini from an unfavorable interaction with negatively charged posttranslational modifications in CCR7. Here, we confirm this using a CCL19-variant lacking the basic C-terminus. This variant displays a 22-fold higher potency at CCR7 compared to WT CCL19 and is highly unaffected by the presence of C21TP. WT CCL19 has a short basic C-terminus, CCL21 a longer one. Here, we propose a way to differentially boost CCL19 and CCL21 activity as short and long versions of C21TP boost CCL19 activity, whereas only a long C21TP version can boost chemokines with a full-length CCL21 C-terminus.

Keywords: CCL19; CCL21; CCR7; basic peptide; biased signaling.

Figure 1

Signaling profiles of CCL19^{CCL21N-term|C-term}, CCL21, and CCL19 in transfected cell lines and primary cells. (A) Alignment of CCL19, CCL21, and the chimeric chemokine CCL19^{CCL21N-term|C-term} encompassing the core domain of CCL19 (aa 17–77), the N-terminus (aa 1–16), and the C-terminus of CCL21 (aa 78–111). Basic residues in the C-terminal region of all three chemokines are written in bold and marked with blue, and the BBx(x)B motifs within CCL21 (3 motifs) and CCL19 (1 motif) are identified. As can be readily observed, the C-terminus of CCL19^{CCL21N-term|C-term} contains the 3 BBx(x)B motifs from CCL21, together with 1 BBx(x)B motif from CCL19, thus 4 BBx(x)B motifs in all. The signaling properties of CCL19^{CCL21N-term|C-term} in (B) G_αi signaling and (C) β-arrestin recruitment assay. G_αi signaling and β-arrestin recruitment were assessed using BRET-based assays. Statistical significance between dose–response curves was calculated using two-way ANOVA with Tukey’s multiple comparisons tests (n = 3–6). Calcium signal in DCs stimulated with (D) CCL21 or (E) CCL19^{CCL21N-term|C-term}. Changes in intracellular calcium are measured by measuring the fluorescence intensity of the dye Fluo-4, which increases fluorescence emission upon calcium-binding. Data are background subtracted to show relative changes in calcium flux (n = 5). (F) Dose–response curve of calcium signal in figure (D,E), where the maximal fluorescence value for each chemokine concentration is plotted. Statistical significance between dose–response curves was calculated using two-way ANOVA with Sidak’s multiple comparisons tests. (G) moDC chemotaxis and spider diagrams depicting the DC migration pattern towards the chemokine gradients; (H) CCL21 100 nM and (I) CCL19^{CCL21N-term|C-term} 100 nM. DC migration was assessed using time-lapse recordings (12 h) of moDCs. Statistical significance between bar graphs (CI values) was calculated using one-way ANOVA with Tukey’s correction for multiple tests. (n = 3). * p < 0.05, **** p < 0.0001, ns not significant.

Figure 2

Alanine mutation of R209 in CCR7 does not affect CCL19^{CCL21N-term|C-term} potency in G_αi signaling. Dose–response curves of CCR7_WT and CCR7_R209A stimulated with (A) CCL19, (B) CCL21, and (C) CCL19^{CCL21N-term|C-term} in a cAMP accumulation assay. Each set of data has been normalized to the WT dose–response curve within each individual experiment before the collection of data. Significant differences between CCR7_WT and CCR7_R209A have been analyzed using two-way ANOVA with Šídák’s multiple comparisons test. ** p < 0.01, **** p < 0.0001, ns not significant. Data are presented as mean values ± SEM (n = 3). (D) Serpentine structure of CCR7 displaying the location of residue R209 in the ECL2.

Figure 3

Long but not short C21TPs boost CCL19^{CCL21N-term|C-term} potency in G_αi signaling. Areas under the curve (AUCs, arbitrary units) of cAMP signaling are shown as bar graphs and AUC curves. The length and location of BBx(x)B motifs in the short and long C21TPs are shown as graphical bars. The effect of short (89–106) and long (81–111) C21TPs on G_αi signaling in response to (A) CCL19, (B) CCL21, and (C) CCL19^{CCL21N-term|C-term} was measured using BRET-based assays. C21TPs were added for a final concentration of 10 μM. Statistical significances were determined using one-way ANOVA with Tukey’s multiple comparisons test (n = 3). * p < 0.05, *** p < 0.001, **** p < 0.0001, ns non significant.

Figure 4

Long and short C21TP variants boost CCL21^1−91trunc activity in G_αi signaling equally. Areas under the curve (AUCs, arbitrary units) of cAMP signaling are shown as bar graphs and AUC curves. The length and location of BBx(x)B motifs in the short and long C21TPs are shown as graphical bars. (A) The effect of short (89–106) and long (81–111) C21TPs on G_αi signaling in response to CCL21^1−91trunc was measured using BRET-based assays. C21TPs were added to a final concentration of 10 μM. Statistical significances were determined using one-way ANOVA with Tukey’s multiple comparisons test (n = 3). *** p < 0.001, **** p < 0.0001, ns non significant. (B) Alignments of chemokine sequences; CCL19, CCL21, and CCL21^1−91trunc. Basic residues in the C-terminal region of the chemokines are written in bold and marked with blue. The alanine substitution (C80A) is written in bold and marked with red.

Figure 5

Removing C-terminal basic residues in CCL19 improves basal activity but does not boost activity in G_αi signaling. (A) Areas under the curve (AUCs, arbitrary units) of cAMP signaling are shown as bar graphs and AUC curves. The length and location of BBx(x)B motifs in the short and long C21TPs are shown as graphical bars. The effect of short (89–106) and long (81–111) C21TPs on G_αi signaling in response to CCL19 AMAAA⁷¹⁻⁷⁵ was measured using BRET-based assays. C21TPs were added for a final concentration of 10 μM. Statistical significances were determined using one-way ANOVA with Tukey’s multiple comparisons test (n = 3). * p < 0.05, ** p < 0.01, ns non significant. (B) Alignments of chemokine sequences; CCL19, CCL21, and CCL19 AMAAA⁷¹⁻⁷⁵. Basic residues in the C-terminal region of the chemokines are written in bold and marked with blue. The alanine substitutions K71A, K73A, R74A, and R75A are written in bold and marked with red.

Figure 6

The human HDP histatin-1-like short C21TP displays differential boosting of CCL19 and CCL21. Areas under the curve (AUCs, arbitrary units) of cAMP signaling are shown as bar graphs and AUC curves. The effect of histatin-1 on G_αi signaling in response to (A) CCL19 and (B) CCL21 was measured using BRET-based assays. Histatin-1 was added for a final concentration of 10 μM. The length and location of BBx(x)B motifs in histatin-1 and the short and long versions of C21TP are shown as graphical bars. Statistical significances were determined using one-way ANOVA with Tukey’s multiple comparisons test (n = 4). **** p < 0.0001. moDC chemotaxis in response to (C) 10 and 100 nM CCL21 in presence of 0.1, 1, 10, and 100 μM histatine-1. DC migration was assessed using time-lapse recordings (12 h) of moDCs. Statistical significance between bar graphs (CI values) was calculated using one-way ANOVA with Tukey’s correction for multiple tests. (n = 3). * p < 0.05, **** p < 0.0001, ns non significant.

Figure 7

The addition of free CCL21 C-terminal peptides affects chemokine–receptor interaction.