Supplementary Figure 4: Lower proportions of CCR7⁺CD86⁺ DCs in skin-draining LNs of CCRL1-deficient mice.

(a) Gating strategy to identify “migratory” CD86^pos CCR7^pos DC in inguinal LNs of wild type (WT)and CCRL1 deficient (KO) mice. After single cell gate and exclusion of dead cells, total CD45⁺ cells were analysed for CD11c and CD11b expression as displayed for representative wild type and CCRL1 deficient LNs. Populations of CD11b^high (blue gate) and CD11b^low (green gate) DCs were further analysed for CCR7 and CD86 expression to identify CCR7⁺CD86⁺ “migratory” DCs. To identify LDCs total DCs (red gate) were analysed for langerin against CCR7. This gate encompasses both epidermal-derived Langerhans cells and langerin^pos dermal DCs. (b) Proportions of both CD11b^highCD11c⁺CD86^highCCR7⁺ and CD11b^lowCD11c⁺CD86^highCCR7⁺ DCs are significantly lower in CCRL1 deficient mice (KO, white box) compared to wild-type (WT grey box), Significance indicated: *, P < 0.00. Data are from four litters of 8 week old F2 female mice. n=9 per group. (c) Representative histogram of CCR7 expression in WT (blue) versus CCRL1 ko (red) gated on all DCs, representative of 3 independent experiments. (e) Geometric mean fluorescent intensity of CCR7 in gated CD86^high cells from WT (grey box) and KO (white box). F2 littermates, n=4 and 5 for CCRL1 ko and WT mice, respectively. Box-plot graphs show median values with quartiles and minimum/maximum.