Extended Data Fig. 4: Analysis of CCR8 ligand binding, internalization and migration capacities.
(a, b) Interactions between mouse CCL17 or CCL1 and CCR4, CCR5 or CCR8 were assessed on the surface of adherent cDCs isolated from LNs of Apoe−/− mice using Duolink proximity-ligation assay after incubation with recombinant mouse CCL17, CCL1 (100 ng/ml) or PBS (control) and respective antibodies to CCR4, CCR5 and CCR8, as indicated. (a) Shown are representative images recorded with a Leica SP8 confocal microscope for anti-CCR8 and anti-CCL17 after PBS and CCL17 treatment (scale bar = 10 µm); (b) Signals generated by interactions between ligands and receptors on the cDC surface were quantified and normalized to untreated controls (dotted line); number of independent experiments per bar from left to right: n = 5, 4, 4, 4, 3, 5; (c) Representative histograms displaying CCR8 expression in HEK293 CCR8-transfectants and controls; (d,e) CCR4 internalization in CCR8-deficient or CCR8-competent CD4+ T cells from thymus (d, number of independent experiments per bar from left to right: n = 3, 4, 3, 4) and LNs (e, number of independent experiments per bar from left to right: n = 3, 4, 3, 4) stimulated with CCL17 (100 ng/ml) or vehicle and analyzed by flow cytometry; (f) Dose-response curve to compare CCR8 internalization by CCL1 and CCL17 at indicated concentrations in primary CCR8-expressing human T cells; (g) Representative histograms displaying CCR4 expression in HEK293 CCR4-transfectants and controls; (h) Area under the curve (AUC) for Rluc in Glosensor assays monitoring cAMP levels in HEK293 CCR8-transfectants to obtain dose-response curves for CCL1, CCL17, CCL18 or irrelevant CCL20 using indicated concentrations (CCL1, CCL18, CCL20, n = 6 independent experiments; CCL17, n = 3 independent experiments); (i) Transwell migration assay with human CD4+ T cells towards recombinant human CCL17 (100 ng/ml) or CCL1 (50 ng/ml) in the presence or absence of a blocking antibody to CCR8 (2 µg/ml). Migrated cells were quantified by flow cytometry; number of replicates in parentheses over number of independent experiments per bar from left to right: n = (40)8, (16)5, (46)8, (21)5, (18)5, (18)5; (j-l) Transwell migration assays depicted as dose-response curves of Apoe−/−CD4+ T cell migration induced by CCL1 (j), CCL17 (k) or CCL22 (l) at indicated concentrations in the presence or absence of anti-CCR8 antibody (2 µg/ml) or C021 (0.5 µM) (j-l, n = 4 independent experiments; CCL17+anti-CCR8, n = 3 independent experiments). (a-l) Data represent mean ± SEM. Two-sided P values as indicated and analyzed by Mann-Whitney U-test or unpaired Student’s t-test (b), two-way ANOVA (d,e) or nested ANOVA (i) with Holm-Šídák’s post hoc test (d,e), generalized linear model (f).
