Journal: Nature Communications

Article Title: GPR182 limits antitumor immunity via chemokine scavenging in mouse melanoma models

doi: 10.1038/s41467-021-27658-x

Figure Lengend Snippet: a , b Two published single-cell RNA sequencing datasets of human melanoma were queried for GPR182 expression based on cell type ( a ). b Analysis of ECs in human melanoma further revealed that GPR182 is primarily expressed in LECs. c Human melanoma tissues were stained for GPR182 (green) together with EC markers, including CD31, podoplanin (PDPN), or Prox-1. d A lymphatic score was generated using 288 metastatic melanoma samples from the TCGA database. The lymphatic score was calculated based on relative expression levels of PDPN , LYVE1 , and VEGFC in each sample. Lymphatic score was plotted against GPR182 mRNA expression level (RSEM, log2 normalized). Pearson’s correlation coefficient, r , and p -values shown from two-sided test. e , f Human melanoma tissues with adjacent normal skin were stained for PDPN (red), CD31(blue), and GPR182 (green) (E); S-100 staining (brown) was used to identify melanocytes and tumor cells. f Quantification of GPR182 median fluorescent intensity (MFI) in PDPN+ lymphatic vessels from paired tumor and adjacent normal tissue. n = 15, P -value from two-sided paired t -test. Error bars represent SEM.

Article Snippet: After blocking with 2.5% normal horse serum, the slides were then stained overnight at 4 ° C with the following primary antibodies: anti-mouse-GPR182 (1:500, A14854, ABclonal) and anti-mouse-LYVE1–β-gal (1:500, AF2125-SP, R&D).

Techniques: RNA Sequencing Assay, Expressing, Staining, Generated

Journal: PLoS Pathogens

Article Title: Rapid Lymphatic Dissemination of Encapsulated Group A Streptococci via Lymphatic Vessel Endothelial Receptor-1 Interaction

doi: 10.1371/journal.ppat.1005137

Figure Lengend Snippet: A-D: Adhesion of encapsulated M18 GAS untransfected and LYVE-1 lentivirus-transfected HDLECs as indicated above each panel. (A and B) Left to right; numbers of adherent GAS were determined by quantitative culture and representative fluorescence microscopy of adherent GAS (30 min incubation) in the presence of control mAb or LYVE-1 blocking mAb. Scale bars (20 μm). (C) Dose-dependent inhibition of GAS adhesion to HDLEC by a soluble human LYVE-1 ectodomain fragment. (D) Dose-dependent inhibition of GAS adhesion to lentivirus transfected HDLECs in the presence of high (HMWHA, solid line) and low (LMWHA, dashed line) molecular weight HA. (A-D: n = 4, data represent mean+/-SD) (Mann Whitney U;* = p<0.05). (E) High resolution confocal microscopy of M18 GAS following infection of HDLEC. Scale bars (10 μm). Red = LYVE-1, green = GAS, blue = nuclei. Arrows indicate encapsulated GAS adhering to clusters of LYVE-1.

Article Snippet: For LYVE-1 blocking experiments mice were injected intra-peritoneally with 0.5 mg anti mLYVE-1 mAb2125 (R&D) or control rat IgG (isotype control or polyclonal) (R&D) 24 hours prior to infection.

Techniques: Transfection, Fluorescence, Microscopy, Incubation, Blocking Assay, Inhibition, Molecular Weight, MANN-WHITNEY, Confocal Microscopy, Infection

Journal: PLoS Pathogens

Article Title: Rapid Lymphatic Dissemination of Encapsulated Group A Streptococci via Lymphatic Vessel Endothelial Receptor-1 Interaction

doi: 10.1371/journal.ppat.1005137

Figure Lengend Snippet: Adhesion of M18 GAS to MDLECs. Left to right; numbers of adherent GAS were determined by quantitative culture (n = 4; Data represent mean+/-SD) (Mann Whitney U;* = p<0.05) and representative fluorescence microscopy of adherent GAS (30 min incubation) in the presence of control mAb or LYVE-1 blocking mAb. Scale bars (20 μm). Red = LYVE-1, green = GAS, blue = nuclei. Arrows indicate encapsulated GAS adhering to clusters of LYVE-1.

Article Snippet: For LYVE-1 blocking experiments mice were injected intra-peritoneally with 0.5 mg anti mLYVE-1 mAb2125 (R&D) or control rat IgG (isotype control or polyclonal) (R&D) 24 hours prior to infection.

Techniques: MANN-WHITNEY, Fluorescence, Microscopy, Incubation, Blocking Assay

Journal: PLoS Pathogens

Article Title: Rapid Lymphatic Dissemination of Encapsulated Group A Streptococci via Lymphatic Vessel Endothelial Receptor-1 Interaction

doi: 10.1371/journal.ppat.1005137

Figure Lengend Snippet: A-C: Confocal microscopic images of murine ear skin 6–24 hours after local intradermal inoculation. (A) Low magnification images of lymphatic vessels in skin and surrounding infected tissue 24 hours post infection (scale bar 20 μm). (B) 3D rendered images and (C) orthogonal views of the same z-stacks at 6 hours post infection. Arrows indicate individual streptococci interacting with LYVE-1 dense regions of lymphatic vessel endothelium (scale bar 5 μm). D-E: Epifluorescence microscopic images of frozen sections of draining cervical nodes 6 hours post infection at low power (100X magnification)(D) and high power (400X)(E) (Scale bar 50 μm and 20 μm respectively). Red = LYVE-1, green = GAS, blue = nuclei. Arrows indicate encapsulated GAS adhering to clusters of LYVE-1.

Article Snippet: For LYVE-1 blocking experiments mice were injected intra-peritoneally with 0.5 mg anti mLYVE-1 mAb2125 (R&D) or control rat IgG (isotype control or polyclonal) (R&D) 24 hours prior to infection.

Techniques: Infection

Journal: PLoS Pathogens

Article Title: Rapid Lymphatic Dissemination of Encapsulated Group A Streptococci via Lymphatic Vessel Endothelial Receptor-1 Interaction

doi: 10.1371/journal.ppat.1005137

Figure Lengend Snippet: Dissemination of M18 GAS in murine soft-tissue infection in constitutive LYVE-1 -/- mice (n = 7/group). Numbers of GAS recovered at site of infection (A), ipsilateral draining LN (B), spleen (C) and blood (D) 3 hours post-infection. Lines depict median values in each case (Mann Whitney U;* = p<0.05, ** = p<0.01). (E-I) Confocal micrographs of frozen sections of representative draining inguinal lymph nodes from wildtype (n = 5) (E-G) and LYVE-1 -/- (n = 4) (H and I) mice that were resected 3 hours post infection. Original magnification was 100X (E and H) or 400X (F, G and I). Scale bars are 100, 50, 20, 100 and 50 μm for E, F, G, H and I respectively.

Article Snippet: For LYVE-1 blocking experiments mice were injected intra-peritoneally with 0.5 mg anti mLYVE-1 mAb2125 (R&D) or control rat IgG (isotype control or polyclonal) (R&D) 24 hours prior to infection.

Techniques: Infection, MANN-WHITNEY

Journal: PLoS Pathogens

Article Title: Rapid Lymphatic Dissemination of Encapsulated Group A Streptococci via Lymphatic Vessel Endothelial Receptor-1 Interaction

doi: 10.1371/journal.ppat.1005137

Figure Lengend Snippet: Dissemination of M18 GAS in murine soft-tissue infection following LYVE-1 mAb blockade (n = 22/group). Quantitative culture of GAS at site of infection (A), ipsilateral draining LN (B), spleen (C) and blood (D) 3 hours post-infection. Lines depict median values in each case (Mann Whitney U;* = p<0.05, ** = p<0.01). (Control antibody group: circles indicate isotype control antibody and triangles indicate polyclonal control IgG).

Article Snippet: For LYVE-1 blocking experiments mice were injected intra-peritoneally with 0.5 mg anti mLYVE-1 mAb2125 (R&D) or control rat IgG (isotype control or polyclonal) (R&D) 24 hours prior to infection.

Techniques: Infection, MANN-WHITNEY

Journal: Nature immunology

Article Title: Dendritic cells enter lymph vessels by hyaluronan-mediated docking to the endothelial receptor LYVE-1.

doi: 10.1038/ni.3750

Figure Lengend Snippet: Figure 3 LYVE-1-specific mAbs inhibit in vivo trafficking of DCs through dermal lymphatics. (a,b) The recovery of endogenous DCs in draining LNs in BALB/c mice after injection of rat IgG or anti-LYVE-1 mAb B1/10, C1/8 or mAb2125, during either sensitization (a) or challenge (b) by topical application of oxazolone and FITC. Data show numbers of CD45+FITC+CD11c+ DCs (n = 5). (c) The effect of anti-LYVE-1 mAbs on DC trafficking in C57BL/6 mice 24 h after skin painting with oxazolone and FITC. The anti-ICAM-1 mAb YN1-1 was used as a positive control (n = 4). (d) The recovery of DCs from draining LNs 6–48 h after topical application of oxazolone and FITC in BALB/c mice injected with rat IgG2a or C1/8 (n = 4). (e,f) The entry of CMFDA-labeled BMDCs into dermal afferent lymphatics immunostained with rabbit anti-LYVE-1 after oxazolone skin painting. Magnification, 630×; scale bars, 20 µm (rat IgG and B1/10) or 50 µm (C1/8 and mAb2125) (e). Arrows indicate BMDCs within lymphatic vessel lumens; arrowheads indicate BMDCs restricted to basolateral surfaces of vessels. Data in f are expressed as a percentage of lymphatic-vessel-associated BMDCs (n = 5). (g) The recovery of BMDCs from draining LNs 24 h after rat IgG or mAb injection. (h,i) Results of ex vivo skin crawl-out assays from ear tissue, showing numbers of egressed DCs (h) and remaining dermal DCs (i) (n = 5). Data in plots represent the mean ± s.e.m. n.s., not significant; *P < 0.05, **P < 0.01, Mann-Whitney U-test. Data are from one experiment representative of three (a–c,e–i) or two (d) different experiments.

Article Snippet: Rat anti-mouse LYVE-1 (mAb2125) was from R&D Systems; mAbs B1/10 and C1/8 were generated previously, with mouse LYVE-1 Fc used as the immunogen28,29, as was polyclonal rabbit anti-LYVE-1 (ref. 20).

Techniques: In Vivo, Injection, Positive Control, Labeling, Ex Vivo, MANN-WHITNEY

Journal: Nature immunology

Article Title: Dendritic cells enter lymph vessels by hyaluronan-mediated docking to the endothelial receptor LYVE-1.

doi: 10.1038/ni.3750

Figure Lengend Snippet: Figure 7 DCs adhere to LECs in an HA- and LYVE-1-dependent manner via LYVE-1-enriched transmigratory cups. (a,b) Adhesion of LPS-matured, CMFDA-labeled BMDCs to primary mLEC monolayers after 3 h of incubation in the presence of rat IgG or anti-LYVE-1 mAbs (a), or after 2 h of preincubation with HAase (b), as assessed by fluorescence plate reader. Data are the mean and s.e.m.; n = 4. (c–e) Basolateral-to-luminal transmigration of LPS-matured, fluorescently labeled BMDCs across mLEC monolayers grown on the undersurface of Transwell filters, quantitated over time by fluorescence plate reader in the presence of rat IgG, B1/10 (c), C1/8 (d) or mAb2125 (e). Data are the mean ± s.e.m.; n = 4. (f–h) Confocal microscopy images of cultured primary mLEC monolayers immunostained with rabbit anti-LYVE-1 (f), viewed 3 h after the addition of fluorescently labeled BMDCs (CMFDA), and counterstained with DAPI (g,h). LYVE-1-enriched cups surrounding individual DCs (asterisks) are indicated by arrows (g). A digital zoomed-in view of an orthogonal view is shown in h (magnification, 630×; scale bars, 20 µm). *P < 0.05, Mann-Whitney U-test. Data and images are from one experiment representative of three separate experiments (a–h).

Article Snippet: Rat anti-mouse LYVE-1 (mAb2125) was from R&D Systems; mAbs B1/10 and C1/8 were generated previously, with mouse LYVE-1 Fc used as the immunogen28,29, as was polyclonal rabbit anti-LYVE-1 (ref. 20).

Techniques: Labeling, Incubation, Fluorescence, Transmigration Assay, Confocal Microscopy, Cell Culture, MANN-WHITNEY