Nature Immunology: doi: /ni Supplementary Figure 1

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1 Supplementary Figure 1 BALB/c LYVE1-deficient mice exhibited reduced lymphatic trafficking of all DC subsets after oxazolone-induced sensitization. (a) Schematic overview of the mouse skin oxazolone contact hypersensitivity model, with tracking of endogenous DCs (1) labeled by fluorescein skin painting, which was carried out at the same time as topical application of oxazolone. Alternatively, mice were pre-sensitized to oxazolone, prior to intradermal injection of CMFDA-labeled BMDCs (2) and further topical application of oxazolone. (b-c) Inguinal and axillary draining LNs from sensitized mice were harvested 6-48 h following application of oxazolone and FITC and analyzed by flow cytometry, with plots showing gating strategy for CD45 + CD11c + FITC + live cells (b), measuring numbers of CD45 + leukocytes and CD45 + CD11c + FITC + MHCclII + DC subsets (c). Data represent the mean ± s.e. (n = 5) from one experiment of three. n.s., not significant; *P < 0.05, **P < 0.01, Mann-Whitney U test.

2 Supplementary Figure 2 No overt differences were observed in lymphoid DC populations in naive Lyve / and Lyve +/+ BALB/c mice. (a-d) DC populations in inguinal LNs of naïve BALB/c Lyve -/- and Lyve +/+ littermates as assessed by flow cytometry, by total number of live cells (a), number of CD11c + MHCclII + DCs shown by representative density plots (b) and bar-and-whiskers graphs (c), or numbers of CD11c + MHCclII + subsets (d). Data are the mean ± s.e. from one representative experiment of three.

3 Supplementary Figure 3 C57BL/6 Lyve1 / mice exhibited a transient reduction in lymphatic trafficking of DCs after oxazolone-induced sensitization. Inguinal and axillary draining LNs from C57BL/6 Lyve1 -/- and Lyve1 +/+ mice were harvested at 6 h (a), 24 h (b) and 48 h (c) following skin sensitization with oxazolone and FITC and analyzed by flow cytometry, measuring numbers of CD45 + leukocytes and CD45 + CD11c + FITC + MHCclII + DC subsets. Data represent the mean ± s.e. (n = 5) from one experiment of three, * p < 0.05, ** p < 0.01, Mann-Whitney U test.

4 Supplementary Figure 4 Receptor-binding characteristics of LYVE-1 HA-blocking mabs, and effects on DC trafficking in BALB/c mice. (a, b) Binding affinity of rat anti-mouse LYVE-1 mabs B1/10, C1/8 and mab2125 to mouse LYVE-1 (a) or Jurkat cells transfectants (b), assessed by ELISA and flow cytometry respectively. (c) Relative potency of LYVE-1 mabs as inhibitors of bha binding to mouse LYVE-1 measured using transfected Jurkat cells and flow cytometry. Data are the mean fluorescence intensity (MFI) ± s.e. (n = 3), one experiment of three. (d) Partial epitope mapping of the mouse HABD, using single residue mutagenesis of recombinant receptor in transfected Jurkat cells. Location of residues contributing to mab epitopes (as identified by a reduction in mab binding of > 25% relative to wild-type receptor) plotted on individual space-filling models of the LYVE-1 HABD and colored red, blue or green. Residues forming the epitope for the human LYVE-1 HA blocking mab 3A are shown in a corresponding model of human LYVE-1 HABD, colored magenta. The conserved Cys84-Cys105 disulfide is highlighted in yellow and mutation of a predicted N-glycosylation site in tan. (e-i) Numbers of CD45 + leukocytes and CD45 + CD11c + FITC + MHCclII + DC subsets recovered from draining LNs of BALB/c mice immunized with anti-lyve-1 mabs, 24 h after oxazolone/fitc painting, assessed by flow cytometry. Data represent the mean ± s.e. (n = 5), one representative experiment of two. n.s., not significant; *P < 0.05; **P < 0.01, Mann-Whitney U test.

5 Supplementary Figure 5 Characterization of mouse BMDCs. (a-b) Bone marrow cells were extracted from wild-type BALB/c mice and cultured for 7 days in the presence of IL-4 and GM-CSF, then inducing maturation by addition of LPS prior to analysis of nonadherent cells by flow cytometry. Plots show gating strategy (a) with almost 90% of cells CD11c + MHCclII +. (b) Detection of surface HA using bvg1 and streptavidin-647 (grey shaded histogram), with hyaluronidase-treated cells as a control for non-specific binding (dark grey), revealed that HA was present on CD11c + MHCclII + cells (i) but largely absent from the minor population of CD11c - MHCclII + cells (ii). (c) Assessment of BMDC viability using LiveDead780 dye exclusion and analysis by FACS, following treatment for 72 h with 4-MU ( mm). 0.1 mm 4-MU was used in subsequent experiments. (d-e) Expression of the activation marker CD86 following treatment of BMDCs with 4-MU for 72 h (d) and then again at 24 h following removal of 4-MU from the medium (e). Data represent the mean ± s.e. (BMDCs prepared from 5 mice) from one representative experiment of three, **** p < , Mann-Whitney U test.

6 Supplementary Figure 6 In situ detection of HA on dermal DCs. (a) Whole-mount preparations of mouse ear tissue from CD11c-GFP (green) reporter mice, freshly resected then incubated for 2 h at 37 o C in either the absence (mock) or presence of hyaluronidase (HAase) prior to immunostaining using anti-mhcclii mab and anti-rat AlexaFluor568 (red), and bvg1 with streptavidin647 (blue). Bar = 10 m in upper panels, bar = 20 m in lower panels. (b) In parallel, ex vivo culturing of mouse ear tissue from CD11c-GFP (green) reporter mice was carried out for 24 h, prior to incubation in either absence or presence of hyaluronidase, then whole-mount immunostaining as above. Bar = 10 m. (c-e) Cells that had egressed from the skin explants during culture were fixed to microscope slide by cytospin before staining for immunofluorescence microscopy (e) HAase-treated egressed cells were also stained for bvg1, to show specificity of binding. Images captured by confocal microscope at magnification: 250X, (c, e) and 630X (a, b, d). (c) Bar = 20 m; (d) bar = 5 m; (e) bar = 10 m. Representative images from three separate repeated experiments are shown.

7 Supplementary Figure 7 LYVE-1 HA adhesive interactions are critical for endothelial transmigratory cup formation. (a, b) Confocal imaging of LYVE-1 + cup formation (red, AlexaFluor546) around DCs (green, CMFDA, either mouse BMDCs (a) or human MDDCs (b)) adhering to mouse (a) or human (b) LEC monolayers, incubated (3 h) in the presence of either the indicated rat anti-mouse LYVE-1-HA blocking mabs or control rat IgG (blue, AlexaFluor594), (a), or the anti-human HA blocking mab891, with HA detected by bvg1 and streptavidin647, (blue), (b). LYVE-1-enriched cups, indicated by arrows, are absent around murine DCs adhering to mlecs treated with mabs C1/8, mab2125 or mab891. (c) Mouse BMDCs and/or mlec monolayers were treated with hyaluronidase (HAase) prior to co-incubation (3 h) and then imaging by confocal microscopy. Cells were immunostained with bvg1 (green), with anti-cd44 (blue, as a marker for the leukocyte cell surface) and anti-lyve-1 (red). Magnification: 630X. (a) Bar = 10 m in Rat IgG, B1/10 and C1/8 panels; Bar = 20 m in mab2125 panels; (b) bar = 10 m; (c) bar = 20 m. Representative images from three separate repeated experiments are shown.

8 Supplementary Figure 8 Detection of LYVE-1-enriched transmigratory cups within mouse lymphatic capillaries in vivo. Confocal imaging of whole mount sections of mouse dermis, immunostained with rabbit anti-lyve-1 and goat anti-rabbit AlexaFluor 546 (red), 24 h following topical application of oxazolone and intradermal injection of CMFDA-labeled BMDCs (green). Images show LYVE-1 + endothelial cup-like structures surrounding individual DCs (indicated by arrows) at the basolateral surface of lymphatic vessels, with digital zoom images of selected z-stacks in panels on right. Magnification: 630X, bar = 20 m.