Journal: ERJ Open Research

Article Title: Distinct immune regulatory receptor profiles linked to altered monocyte subsets in sarcoidosis

doi: 10.1183/23120541.00804-2020

Figure Lengend Snippet: Distribution of the regulatory receptor CD200R and its ligand CD200L in sarcoidosis granulomas: transbronchial lung biopsy samples from two patients with sarcoidosis stained for a) CD200L and b) CD200R, with respective isotype control antibody staining in c) and d). f: fibroblasts; h: histiocytes (macrophages). Original magnification ×100.

Article Snippet: Antibodies were a polyclonal goat CD200L antibody (AF2724; R&D Systems, Abingdon, UK), normal goat IgG control (AB-108-C; R&D Systems), mouse monoclonal IgG1 anti-human CD200R (OX108, MCA 2282T; AbD Serotec/Bio-Rad, Kidlington, UK) and mouse monoclonal control IgG1 antibody (MOPC-21, 400101; Biolegend, London, UK).

Techniques: Staining, Control

Journal: ERJ Open Research

Article Title: Distinct immune regulatory receptor profiles linked to altered monocyte subsets in sarcoidosis

doi: 10.1183/23120541.00804-2020

Figure Lengend Snippet: Nonclassical monocytes express low levels of CD200R and expression of CD200R is lower on all monocyte subsets in patients with sarcoidosis compared with healthy controls. GMFI: geometric mean fluorescence intensity. a) Representative flow cytometry histograms from a healthy control subject comparing CD200R expression on classical, intermediate and nonclassical monocytes compared with isotype control antibody. b, c) Representative heat-map dot plots from b) a healthy control subject demonstrating low CD200R on nonclassical monocytes and c) a patient with sarcoidosis showing reduced CD200R expression on sarcoidosis monocyte subsets compared with the healthy control. On these pseudo-colour plots, orange indicates higher CD200R expression and green indicates lower CD200R expression. d–f) Expression of CD200R on d) classical, e) intermediate and f) nonclassical monocyte subsets in sarcoidosis patients and controls. Note the small y -axis scale for nonclassical monocytes. Individual data points are presented along with median and interquartile range. *: p<0.05; **: p<0.01 (Mann–Whitney U-test).

Article Snippet: Antibodies were a polyclonal goat CD200L antibody (AF2724; R&D Systems, Abingdon, UK), normal goat IgG control (AB-108-C; R&D Systems), mouse monoclonal IgG1 anti-human CD200R (OX108, MCA 2282T; AbD Serotec/Bio-Rad, Kidlington, UK) and mouse monoclonal control IgG1 antibody (MOPC-21, 400101; Biolegend, London, UK).

Techniques: Expressing, Fluorescence, Flow Cytometry, MANN-WHITNEY

Journal: ERJ Open Research

Article Title: Distinct immune regulatory receptor profiles linked to altered monocyte subsets in sarcoidosis

doi: 10.1183/23120541.00804-2020

Figure Lengend Snippet: CD200R expression on sarcoidosis classical and intermediate monocytes is dichotomous and consistent over time. GMFI: geometric mean fluorescence intensity. Patients with sarcoidosis in a longitudinal cohort (n=19) underwent serial measurements of CD200R expression on a) classical, b) intermediate and c) nonclassical monocyte subsets. Each patient is presented by a different coloured line.

Article Snippet: Antibodies were a polyclonal goat CD200L antibody (AF2724; R&D Systems, Abingdon, UK), normal goat IgG control (AB-108-C; R&D Systems), mouse monoclonal IgG1 anti-human CD200R (OX108, MCA 2282T; AbD Serotec/Bio-Rad, Kidlington, UK) and mouse monoclonal control IgG1 antibody (MOPC-21, 400101; Biolegend, London, UK).

Techniques: Expressing, Fluorescence

Journal: ERJ Open Research

Article Title: Distinct immune regulatory receptor profiles linked to altered monocyte subsets in sarcoidosis

doi: 10.1183/23120541.00804-2020

Figure Lengend Snippet: Characteristics of sarcoidosis patients stratified by monocyte CD200R phenotype

Article Snippet: Antibodies were a polyclonal goat CD200L antibody (AF2724; R&D Systems, Abingdon, UK), normal goat IgG control (AB-108-C; R&D Systems), mouse monoclonal IgG1 anti-human CD200R (OX108, MCA 2282T; AbD Serotec/Bio-Rad, Kidlington, UK) and mouse monoclonal control IgG1 antibody (MOPC-21, 400101; Biolegend, London, UK).

Techniques: Cell Counting

Journal: ERJ Open Research

Article Title: Distinct immune regulatory receptor profiles linked to altered monocyte subsets in sarcoidosis

doi: 10.1183/23120541.00804-2020

Figure Lengend Snippet: Oral corticosteroid therapy for sarcoidosis normalises monocyte subsets but has no effect on monocyte CD200R expression. GMFI: geometric mean fluorescence intensity. Pre- and 2-month post-steroid treatment data for three sarcoidosis patients who were treated with oral prednisolone 20 mg·day −1 . a, c, e) Percentages of classical, intermediate and nonclassical monocytes, respectively. b, d, f) CD200R expression on the respective monocyte subsets.

Article Snippet: Antibodies were a polyclonal goat CD200L antibody (AF2724; R&D Systems, Abingdon, UK), normal goat IgG control (AB-108-C; R&D Systems), mouse monoclonal IgG1 anti-human CD200R (OX108, MCA 2282T; AbD Serotec/Bio-Rad, Kidlington, UK) and mouse monoclonal control IgG1 antibody (MOPC-21, 400101; Biolegend, London, UK).

Techniques: Expressing, Fluorescence

Journal: ERJ Open Research

Article Title: Distinct immune regulatory receptor profiles linked to altered monocyte subsets in sarcoidosis

doi: 10.1183/23120541.00804-2020

Figure Lengend Snippet: Distribution of the regulatory receptor CD200R and its ligand CD200L in sarcoidosis granulomas: transbronchial lung biopsy samples from two patients with sarcoidosis stained for a) CD200L and b) CD200R, with respective isotype control antibody staining in c) and d). f: fibroblasts; h: histiocytes (macrophages). Original magnification ×100.

Article Snippet: Antibodies were a polyclonal goat CD200L antibody (AF2724; R&D Systems, Abingdon, UK), normal goat IgG control (AB-108-C; R&D Systems), mouse monoclonal IgG1 anti-human CD200R (OX108, MCA 2282T; AbD Serotec/Bio-Rad, Kidlington, UK) and mouse monoclonal control IgG1 antibody (MOPC-21, 400101; Biolegend, London, UK).

Techniques: Staining

Journal: The Journal of Neuroscience

Article Title: Newly Formed Endothelial Cells Regulate Myeloid Cell Activity Following Spinal Cord Injury via Expression of CD200 Ligand

doi: 10.1523/jneurosci.2199-16.2017

Figure Lengend Snippet: Figure1. SpatialdistributionofCD200LintheCNScompartmentsinhomeostasis.A,ImmunostainingforIII-tubulin,NeuN,andCD200Lofneuronslocatedinthehippocampus,cerebellum,and cortexofbrainsderivedfromnoninjuredWTmice.Scalebar,500m.Dataarerepresentativeoftwodifferentexperiments;n2miceineachgroup,oneachexperiment.B,Immunostainingfor III-tubulinandNeuNofneuronsexpressingCD200LintheDRGs,butnotinthespinalcordparenchymaofnoninjuredWTmice.Scalebar,100m.C,Whole-mountimmunostainingforCD31and CD200L in the choroid plexus derived from noninjured WT mice. D, Immunostaining for CD200L and vimentin ependymal cells in longitudinal sections of the central canal in the spinal cord of noninjuredWTmice.Inset,Immunostainingofcoronalsectionofthecentralcanal.E,F,Whole-mountimmunostainingforCD31andCD200Linthedura(E)andpia(F)materofmeningesderived fromnoninjuredWTmice.G,ImmunostainingforCD31andCD200LinspinalcordparenchymaofnoninjuredWTmice.H,Whole-mountimmunostainingforCD31andCD200Linthechoroidplexus derived from noninjured CD200L/ mice. C–H, Scale bar, 50 m. Data are representative of 2–5 independent experiments; n 3–5 mice in each group.

Article Snippet: The tissues were blocked with M.O.M. immunodetection kit reagent (Vector Laboratories) containing 0.1% Triton X-100 (SigmaAldrich), and stained with the following primary antibodies: rabbit antiGlial fibrillary acidic protein (GFAP) (1:400; Dako), rabbit anti-Ionized calcium binding adapter molecule-1 (Iba-1) (1:300; Wako), goat anti-CD200L (1:40; R&D Systems), goat anti-CD200R1 (1:100; R&D Systems), rat anti-CD31 (1:40; BD Biosciences PharMingen), rabbit antiIII tubulin (1:5000; Covance), mouse anti-Neuronal Nuclei (NeuN) (1:150; Millipore Bioscience Research Reagents International), rabbit anti-Ki67 (1:100; Abcam), and chicken anti-vimentin (1:500; Millipore); Fluorescein isothiocyanate (FITC)-conjugated Bandeiraea simplicifolia isolectin B4 (1:40; Sigma-Aldrich) was added for 1 h to the secondary antibody solution.

Techniques: Derivative Assay, Immunostaining

Journal: The Journal of Neuroscience

Article Title: Newly Formed Endothelial Cells Regulate Myeloid Cell Activity Following Spinal Cord Injury via Expression of CD200 Ligand

doi: 10.1523/jneurosci.2199-16.2017

Figure Lengend Snippet: Figure2. CD200LisinvolvedinthefunctionalrecoveryfollowingSCIandisupregulatedbynewlyformedendothelialcellswithinthelesionsite.A,ImmunostainingforGFAP,todetectreactive astrocytes that surround the lesion site, together with CD200L on days 3, 7, and 14 following SCI. Scale bar, 500 m. Day 7: Enlarged image of CD200L Hoechst cells. Scale bar, 100 m. B, C, Immunostaining for CD200L and CD31 at the epicenter of the lesion site 7 d following SCI in WT (B) and CD200L/ (C) mice. D, Immunostaining for CD200L and CD31 distal to the lesion site 7 d followingSCIinWTmice.E,ImmunostainingforIII-tubulin,NeuN,andCD200Lshowingneuronsthatarelocatedadjacenttothelesionsite7dfollowingSCI.Scalebar,100m.F,Immunostaining for CD200LCD31 endothelium positive for Ki67 at the epicenter of the lesion site 7 d following SCI in WT mice. B–D, F, Scale bar, 50 m. A–F, Data are representative of 2–5 independent experiments;n3–5miceineachgroup.G,HindlimbmotorfunctionassessedaccordingtotheBMSforWT(filledblackcircles)andCD200L/(hollowedredcircles)mice.Left,Motorscoreas a function of time. FInteraction(1,152) 10.2; ***p 0.001 (repeated-measures two-way ANOVA). Right, Scores of individual mice (d20). ***p 0.001 (Student’s t test). H, Hindlimb motor function, assessed according to the BMS for [WT CD200L/] (hollowed orange squares) and [CD200L/ WT] (filled gray squares) BM chimeras. Left, Motor score as a function of time. FInteraction(1,60)4.94;***p 0.001(repeated-measurestwo-wayANOVA).Right,Scoresofindividualmice(d20).*p0.0252(Student’sttest).G,H,Dataarerepresentativeoftwodifferent experiments; n 10 – 11 mice in each group. *p 0.05. ***p 0.001. Data are mean SEM.

Article Snippet: The tissues were blocked with M.O.M. immunodetection kit reagent (Vector Laboratories) containing 0.1% Triton X-100 (SigmaAldrich), and stained with the following primary antibodies: rabbit antiGlial fibrillary acidic protein (GFAP) (1:400; Dako), rabbit anti-Ionized calcium binding adapter molecule-1 (Iba-1) (1:300; Wako), goat anti-CD200L (1:40; R&D Systems), goat anti-CD200R1 (1:100; R&D Systems), rat anti-CD31 (1:40; BD Biosciences PharMingen), rabbit antiIII tubulin (1:5000; Covance), mouse anti-Neuronal Nuclei (NeuN) (1:150; Millipore Bioscience Research Reagents International), rabbit anti-Ki67 (1:100; Abcam), and chicken anti-vimentin (1:500; Millipore); Fluorescein isothiocyanate (FITC)-conjugated Bandeiraea simplicifolia isolectin B4 (1:40; Sigma-Aldrich) was added for 1 h to the secondary antibody solution.

Techniques: Immunostaining