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: Stem cells (Dayton, Ohio)

Article Title: CD200 Expression Marks a Population of Quiescent Limbal Epithelial Stem Cells with Holoclone Forming Ability.

doi: 10.1002/stem.2903

Figure Lengend Snippet: Figure 2. CD200 expression in human and mouse cornea in vivo and during ex vivo expansion of human limbal epithelial cells. (A): Quantification of CD200 expression through different passages of limbal epithelial cells by flow cytometry. Values represent mean SEM, n = 3–10 (n, number of biological replicates), *, p < .05. (B): Quantification of CD200 expression during calcium induced differentiation of limbal epithelial cells by flow cytometry. Values represent mean SEM, n = 3, *, p < .05. (C): Immunohistochemical staining of human corneal tissue paraffin sections for ΔNp63 and CD200 within the central cornea and limbus. Nuclei are shown by Hoechst counter stain- ing. Scale bars 20 μm. (D): Immunohistochemical staining of murine corneal tissue cryosections for CK15, ΔNp63, and CD200 within the central cornea and limbus. Nuclei are shown by DAPI counter staining. The dashed line indicates the stromal-epithelial junction. Red arrows point at limbal region. Scale bars 20 μm. (E): Immunohistochemical staining of limbal epithelial cell colonies in vitro for CD200 and ΔNp63. Blue arrow points CD200+ cells. Nuclei are shown by Hoechst counter staining. Scale bar 50 μm. (F): Immunohistochemical stain- ing of limbal epithelial cell colonies in vitro for CD200 and Ki67. Red arrows point to CD200+ Ki67+ cells; orange arrows point to CD200+Ki67−cells. Nuclei are shown by Hoechst counter staining. Scale bar 50 μm. Abbreviations: ep, epithelium; st, stroma.

Article Snippet: The following primary antibodies were used at the indicated dilutions: anti CD109 (sc-271085, Santa Cruz, USA, 1:200), anti-human CD200 (329201, BioLegend, USA, 1:200), anti-mouse CD200 (AF3355, Novus Biologicals, USA, 1:100), anti p63 delta (NBP2-29467, Novus Biologicals, USA, 1:200), anti-cytokeratin 15 (ab52816, Abcam, Cambridge, UK, 1:100), and anti Ki67 antibody (ab15580, Abcam, UK, 1:100).

Techniques: Expressing, In Vivo, Ex Vivo, Cytometry, Immunohistochemical staining, Staining, In Vitro

Journal: Stem cells (Dayton, Ohio)

Article Title: CD200 Expression Marks a Population of Quiescent Limbal Epithelial Stem Cells with Holoclone Forming Ability.

doi: 10.1002/stem.2903

Figure Lengend Snippet: Figure 3. Colony forming efficiency and proliferative potential of sorted CD200 positive and negative population. (A): Pie chart showing the distribution of formed and aborted colonies in CD200+ population. (B): Comparison of colony forming efficiencies of CD200+ and CD200−cells. Values represent mean SEM, n = 3 (n, number of biological replicates). (C): Pie chart showing the distribution of formed and aborted colonies in CD200−population. Values represent mean SEM, n = 3. (D): Microscopic and macroscopic appearances of colo- nies formed by CD200+ cells. Scale bars 100 μm. (E): Microscopic and macroscopic appearances of colonies formed by CD200−cells. Scale bars 100 μm. (F): BrdU cell proliferation assay of CD200 negative and positive limbal epithelial cell population after 1- and 8-hours incuba- tion with BrdU. Values represent mean SEM, n = 3.(G): Quantification of cells in the S phase of the cell cycle in CD200+ and CD200−

Article Snippet: The following primary antibodies were used at the indicated dilutions: anti CD109 (sc-271085, Santa Cruz, USA, 1:200), anti-human CD200 (329201, BioLegend, USA, 1:200), anti-mouse CD200 (AF3355, Novus Biologicals, USA, 1:100), anti p63 delta (NBP2-29467, Novus Biologicals, USA, 1:200), anti-cytokeratin 15 (ab52816, Abcam, Cambridge, UK, 1:100), and anti Ki67 antibody (ab15580, Abcam, UK, 1:100).

Techniques: Comparison, BrdU Cell Proliferation Assay

Journal: Stem cells (Dayton, Ohio)

Article Title: CD200 Expression Marks a Population of Quiescent Limbal Epithelial Stem Cells with Holoclone Forming Ability.

doi: 10.1002/stem.2903

Figure Lengend Snippet: Figure 5. CD200 knockdown and its effect on clonal ability of limbal epithelial cells. (A): Quantitative reverse transcriptase poly- merase chain reaction expression data for control siRNA versus CD200 siRNA treated limbal epithelial cells. Values represent mean SEM, n = 3 (n, number of biological replicates), *, p < .05. (B): Pie chart showing distribution of paraclones, meroclones, and holoclones formed by control siRNA treated cells and (C) CD200 siRNA treated cells. (D): Representative images of colonies formed in control and CD200 siRNA group, with 500 or 1,000 cells seeded per well. Abbreviation: siRNA, small interfering RNA.

Article Snippet: The following primary antibodies were used at the indicated dilutions: anti CD109 (sc-271085, Santa Cruz, USA, 1:200), anti-human CD200 (329201, BioLegend, USA, 1:200), anti-mouse CD200 (AF3355, Novus Biologicals, USA, 1:100), anti p63 delta (NBP2-29467, Novus Biologicals, USA, 1:200), anti-cytokeratin 15 (ab52816, Abcam, Cambridge, UK, 1:100), and anti Ki67 antibody (ab15580, Abcam, UK, 1:100).

Techniques: Knockdown, Reverse Transcription, Expressing, Control, Small Interfering RNA

Journal: Stem cells (Dayton, Ohio)

Article Title: CD200 Expression Marks a Population of Quiescent Limbal Epithelial Stem Cells with Holoclone Forming Ability.

doi: 10.1002/stem.2903

Figure Lengend Snippet: Figure 4. Expression of putative limbal stem cell and corneal epi- thelial cell markers in the sorted CD109 and CD200 positive and negative cell populations. (A): Quantitative reverse transcriptase polymerase chain reaction expression data for CD109+ limbal epi- thelial cell population versus CD109−limbal epithelial cell popula- tion represented by the red line (value 1). Values represent mean SEM, n = 3 (n, number of biological replicates), *, p < .05; **, p < .01; ***, p < .001. (B): Quantitative reverse transcriptase polymerase chain reaction expression data for CD200+ limbal epi- thelial cell population versus CD200−limbal epithelial cell popula- tion represented by the red line (value 1). Values represent mean SEM, n = 3, *, p < .05; **, p < .01; ***, p < .001.

Article Snippet: The following primary antibodies were used at the indicated dilutions: anti CD109 (sc-271085, Santa Cruz, USA, 1:200), anti-human CD200 (329201, BioLegend, USA, 1:200), anti-mouse CD200 (AF3355, Novus Biologicals, USA, 1:100), anti p63 delta (NBP2-29467, Novus Biologicals, USA, 1:200), anti-cytokeratin 15 (ab52816, Abcam, Cambridge, UK, 1:100), and anti Ki67 antibody (ab15580, Abcam, UK, 1:100).

Techniques: Expressing, Reverse Transcription, Polymerase Chain Reaction

Journal: American journal of clinical pathology

Article Title: Diagnostic Utility of CD200 Immunohistochemistry in Distinguishing EBV-Positive Large B-Cell Lymphoma From Classic Hodgkin Lymphoma.

doi: 10.1093/ajcp/aqad053

Figure Lengend Snippet: FIGURE 2 Representative CD200 staining in Hodgkin and large B-cell lymphoma (LBCL). Representative images of H&E-stained sections and immunohistochemical stains for cases in each diagnostic category. All images ×400. CHL, classic Hodgkin lymphoma; DLBCL, diffuse large B-cell lymphoma; EBV, Epstein-Barr virus; NOS, not otherwise specified.

Article Snippet: Staining was performed on a Leica Bond-Max (Leica Biosystems) following antigen retrieval (EDTA pH 9.0, 20 minutes) with a goat anti-human CD200 polyclonal antibody (AF2724; R&D Systems) at a 1:150 dilution.

Techniques: Staining, Immunohistochemical staining, Diagnostic Assay, Virus

Journal: American journal of clinical pathology

Article Title: Diagnostic Utility of CD200 Immunohistochemistry in Distinguishing EBV-Positive Large B-Cell Lymphoma From Classic Hodgkin Lymphoma.

doi: 10.1093/ajcp/aqad053

Figure Lengend Snippet: FIGURE 3 CD200 can help differentiate Epstein-Barr virus−positive (EBV+) classic Hodgkin lymphoma (CHL) with frequent Reed-Sternberg (RS) cells and EBV+ large B-cell lymphoma (LBCL). Representative images of H&E-stained sections and immunohistochemical stains for a case of EBV+ (CHL) with frequent RS cells and a case of EBV+ LBCL. In both cases, lesional cells were positive for CD30 and EBER and showed variable positivity for CD20. However, CD200 was positive in the case of EBV+ CHL and negative in the case of EBV+ LBCL. All images ×400.

Article Snippet: Staining was performed on a Leica Bond-Max (Leica Biosystems) following antigen retrieval (EDTA pH 9.0, 20 minutes) with a goat anti-human CD200 polyclonal antibody (AF2724; R&D Systems) at a 1:150 dilution.

Techniques: Virus, Staining, Immunohistochemical staining

Journal: American journal of clinical pathology

Article Title: Diagnostic Utility of CD200 Immunohistochemistry in Distinguishing EBV-Positive Large B-Cell Lymphoma From Classic Hodgkin Lymphoma.

doi: 10.1093/ajcp/aqad053

Figure Lengend Snippet: FIGURE 4 CD200 can help differentiate Epstein-Barr virus−positive (EBV+) large B-cell lymphoma (LBCL) with rare neoplastic cells from EBV+ classic Hodgkin lymphoma (CHL). Representative images of H&E-stained sections and immunohistochemical stains for a case of EBV+ CHL and a case of EBV+ LBCL with rare neoplastic cells and CHL-like morphology. In both cases, lesional cells were weakly positive for CD20 and CD30. However, CD200 was positive in the case of EBV+ CHL and negative in the case of EBV+ LBCL (arrows). All images ×600.

Article Snippet: Staining was performed on a Leica Bond-Max (Leica Biosystems) following antigen retrieval (EDTA pH 9.0, 20 minutes) with a goat anti-human CD200 polyclonal antibody (AF2724; R&D Systems) at a 1:150 dilution.

Techniques: Virus, Staining, Immunohistochemical staining

Journal: American journal of clinical pathology

Article Title: Diagnostic Utility of CD200 Immunohistochemistry in Distinguishing EBV-Positive Large B-Cell Lymphoma From Classic Hodgkin Lymphoma.

doi: 10.1093/ajcp/aqad053

Figure Lengend Snippet: FIGURE 5 A single case of CD200-negative classic Hodgkin lymphoma (CHL). The single case of CHL in which Reed-Sternberg cells were negative for CD200 was Epstein-Barr virus positive (EBV+), HIV associated, and refractory to CHL-directed therapy, raising the possibility that this may in fact have represented EBV+ large B-cell lymphoma. Arrows highlight large abnormal cells that show weak positivity for PAX5 (B) and variable positivity for CD20 (C) and are negative for CD200 (F). A, H&E. D, CD30. E, EBER. All images ×400.

Article Snippet: Staining was performed on a Leica Bond-Max (Leica Biosystems) following antigen retrieval (EDTA pH 9.0, 20 minutes) with a goat anti-human CD200 polyclonal antibody (AF2724; R&D Systems) at a 1:150 dilution.

Techniques: Virus

Journal: International Journal of Nanomedicine

Article Title: Preparation, characterization, and transfection efficiency of low molecular weight polyethylenimine-based nanoparticles for delivery of the plasmid encoding CD200 gene

doi: 10.2147/ijn.s140734

Figure Lengend Snippet: Figure 6 Cellular toxicity and transfection efficiency. Notes: Viability and gene transfer ability of unmodified and modified PEI derivatives complexed with pDNA encoding CD200 gene at polymer:plasmid (weight:weight) ratios of 0.25, 4, and 8 determined in triplicate in SH-SY5Y cell cultures in 96-well plates. (A) Cell survival was assayed by the MTT method, and expressed as the percentages of cell viability. (B) CD200 expression level measured by flow cytometry. *P,0.05, PEI derivative compared to unmodified parent polymer at the same C/P ratio. (n=3; error bars represent mean ± standard deviation). Abbreviations: C/P, carrier to plasmid ratio; pDNA, plasmid DNA; PEI, polyethylenimine; PEI-SUC, PEI-succinate conjugate; PEI-SUC-PEI, PEI-succinate-PEI conjugate.

Article Snippet: Human CD200 plasmid (pCMV-XL5-hCD200) was obtained from OriGene (Rockville, MD, USA).

Techniques: Transfection, Modification, Polymer, Plasmid Preparation, Expressing, Flow Cytometry, Standard Deviation

Journal: International Journal of Nanomedicine

Article Title: Preparation, characterization, and transfection efficiency of low molecular weight polyethylenimine-based nanoparticles for delivery of the plasmid encoding CD200 gene

doi: 10.2147/ijn.s140734

Figure Lengend Snippet: Figure 7 In vivo imaging of MS induced mice using fluorescently labeled plasmid encoding CD200 at C/P ratio 8. Notes: Mice were injected with the final volume of 100 μL of the polyplex formulation. (A) The control mice received 100 μL of HBG buffer. (B–I) Mice were injected with the polyplexes and imaging was performed at the time points of 0, 15, 30, 60, 120, 180 min and 24 h post injection. Magnification ×10. Abbreviations: C/P, carrier to plasmid ratio; MS, multiple sclerosis; HBG, HEPES buffered glucose.

Article Snippet: Human CD200 plasmid (pCMV-XL5-hCD200) was obtained from OriGene (Rockville, MD, USA).

Techniques: In Vivo Imaging, Labeling, Plasmid Preparation, Injection, Formulation, Control, Imaging

Journal: Neoplasia (New York, N.Y.)

Article Title: A Truncated form of CD200 (CD200S) Expressed on Glioma Cells Prolonged Survival in a Rat Glioma Model by Induction of a Dendritic Cell-Like Phenotype in Tumor-Associated Macrophages.

doi: 10.1016/j.neo.2016.02.006

Figure Lengend Snippet: Figure 3. Characterization of established cell lines expressing CD200L and CD200S. (A–C) Immunocytochemical confirmation of CD200 expression with antibodies recognizing N- (OX2) or C-termini of CD200. The C6 cell line transfected with an empty vector (A, C6-e) did not display positive immunostaining against either antibody. (B) The C6-L cell line was positive for both antibodies. (C) The C6-S line was only reactive to the C-terminal antibody. (D) qPCR confirmed the specific expression of CD200L or CD200S. A primer set amplifying cDNA derived from both CD200L- and CD200S-mRNA was called common ‘Com’ (see Supplementary Table 1). The other primer set ‘L’ amplified cDNA only derived from CD200L-mRNA. C6-e cells expressed none of the cDNA, C6-L cells expressed both, and C6-S cells expressed cDNA amplified only by the Com primer set. (E) Growth curves of each cell line. Data from four independent cultures are expressed as means ± SD except for the data of Empty at 5 days, which is expressed as mean −SD.

Article Snippet: RNA splicing generates CD200S with a shorter C–C′ loop or CDR2, all of which are responsible for the binding to a cells to generate C6-L or C6-S cell lines. fragments of CD200L or CD200S were amplified by PCR using CD200 cDNA purchased from Origene (Rockville, MD) with the following cloning primers containing BamHI and NotI respectively (see Figure 1): CD200L; forward 5′-GGA TCC ATG GGC AGT CCG GTA TTC AG-3′ and reverse 5′-ATG CGG CCG CTT ATT TCA TTC TTT GCA TCC CCT G-3′, CD200S; forward 5′-ACG GAT CCA TGA TCA CTT ACA GCA AAG CC-3′ and the same reverse primer as for CD200L.

Techniques: Expressing, Transfection, Plasmid Preparation, Immunostaining, Derivative Assay, Amplification

Journal: Neoplasia (New York, N.Y.)

Article Title: A Truncated form of CD200 (CD200S) Expressed on Glioma Cells Prolonged Survival in a Rat Glioma Model by Induction of a Dendritic Cell-Like Phenotype in Tumor-Associated Macrophages.

doi: 10.1016/j.neo.2016.02.006

Figure Lengend Snippet: Figure 7. Expression levels in tumors and isolated TAMs of mRNA encoding molecules related to CD200, DCs, lymphocytes and T cells, as revealed by qPCR. Data are shown in an alphabetical order. (A) cDNA was prepared from tumor masses dissected out 21 days after transplantation. CD200-mRNA amplified with ‘CD200 com’ primers was highly expressed by C6-L and C6-S tumors, whereas the CD200L-mRNA level was high only in the C6-L-tumors. The C6-S-tumors expressed DC markers, MHC class II α, CD11c, and CD80 at significantly higher levels than other tumors. Expression of mRNA encoding lymphocyte markers CD8α and β, CTL-related apoptosis-inducing molecules, granzyme, and perforin was significantly higher in the C6-S tumors. Despite not showing statistical significance, CD4 and IFNγ-mRNA were also highly expressed in the C6-S tumors. CLEC9A, which is expressed by myeloid DCs and responsible for cross-presentation, and chemokines (CCL12, CXCL10 and CXCL16), which are invovlved in recruitment of monocytes and lymphocytes, are also highly expressed in the C6-S tumor. (B) Shortly after isolation from the tumor mass, TAMs were subjected to qPCR analysis. TAMs from the C6-S-tumors expressed MHC class II α-, and CD11c-mRNA was expressed at significantly higher levels than in TAMs from other tumors. CD86-mRNA expression in TAMs from the C6-S tumors was also high. Data from 3 tumors of each cell lines are expressed as means ± SEM. *P b .05, **P b .01, ***P b .001 versus CD200S; #P b .05, ##P b .01 versus CD200L.

Article Snippet: RNA splicing generates CD200S with a shorter C–C′ loop or CDR2, all of which are responsible for the binding to a cells to generate C6-L or C6-S cell lines. fragments of CD200L or CD200S were amplified by PCR using CD200 cDNA purchased from Origene (Rockville, MD) with the following cloning primers containing BamHI and NotI respectively (see Figure 1): CD200L; forward 5′-GGA TCC ATG GGC AGT CCG GTA TTC AG-3′ and reverse 5′-ATG CGG CCG CTT ATT TCA TTC TTT GCA TCC CCT G-3′, CD200S; forward 5′-ACG GAT CCA TGA TCA CTT ACA GCA AAG CC-3′ and the same reverse primer as for CD200L.

Techniques: Expressing, Isolation, Transplantation Assay, Amplification

Journal:

Article Title: Elevated Neuronal Expression of CD200 Protects Wld s Mice from Inflammation-Mediated Neurodegeneration

doi: 10.2353/ajpath.2007.060677

Figure Lengend Snippet: Treatment of Wlds mice with blocking anti-CD200 antibody results in worsened EAE with increased macrophage/microglia infiltrates in the CNS. After the induction of EAE, Wlds and WT mice were treated with 200 μg/100 μl of blocking anti-CD200 antibody injected intravenously every other day from days 10 to 20. Control WT and Wlds mice were treated with PBS alone. Eight mice per treatment group were evaluated. a: Wlds mice treated with anti-CD200 antibody experienced a more severe disease course than untreated Wlds mice (P < 0.05, Student’s t-test—area under the curve). In comparison, disease in WT mice was similar even after treatment with anti-CD200 antibody (P = NS, Student’s t-test). b: Spinal cord sections harvested at day 20 from treated and control mice demonstrate enhanced immunofluorescence staining of macrophages/microglia (white arrows) in the CNS of anti-CD200-treated Wlds mice compared with Wlds controls. Macrophage/microglia staining was similar in treated and untreated WT mice. Immunofluorescence staining demonstrates more SMI-32-positive axonal ovoids (white arrows) in the spinal cord white matter of treated Wlds mice, compared with untreated controls. c and d: We performed flow cytometric analysis of immune cell populations in the spinal cords isolated from WT and Wlds mice treated with anti-CD200 antibody or rat IgG control antibody (days 10 to 20) on day 20 after immunization. The results from three to four mice per group were averaged and are shown in table form in d. Also shown is a representative FACS analysis of spinal cords from WT and Wlds mice treated with control Ig or anti-CD200 antibody and stained with CD11b-phycoerythrin and CD45-allophycocyanine (APC) antibodies (c). Original magnifications, ×10.

Article Snippet: The following antibodies were used: CD200 (clone 3B6, isotype rat IgM, 1:200; Cedarlane Laboratories, Hornby, ON, Canada); secondary: Alexa 488-conjugated goat anti-rat IgM (Molecular Probes); CD200R (anti-313015 CD200R peptide, 26 clone R252, isotype rabbit IgG, 1:200; Trillium Therapeutics Inc.); secondary: Alexa 594-conjugated goat anti-rabbit IgG (Molecular Probes); CD200R (clone OX-110, isotype rat IgG2a, 1:100; Serotec, Oxford, UK); secondary: Alexa 488-conjugated rabbit anti-rat IgG (Molecular Probes); mitogen-activated protein 2 (MAP-2) (clone HM-2: mouse anti-mouse IgG1, 1:100; Sigma); secondary: Alexa 594-conjugated goat anti-mouse IgG (Molecular Probes); NeuN (clone A60, isotype mouse IgG1, 1:100; Chemicon/Millipore, Temecula, CA); secondary: Alexa 594-conjugated goat anti-mouse IgG (Molecular Probes); GFAP cocktail (clones 4A11, 1B4, 2E1, isotype mouse IgG2b, 1:100; BD Pharmingen, Palo Alto, CA); secondary: Alexa 488- or Alexa 594-conjugated goat anti-mouse IgG (Molecular Probes); CNPase (clone 11-5B, isotype mouse IgG1, 1:100; Chemicon/Millipore); secondary: Alexa 488- or Alexa 594-conjugated goat anti-mouse IgG (Molecular Probes); β-tubulin (clone TUJ1, isotype mouse IgG2a; 1:100; Covance, Berkeley, CA); secondary: Alexa 488- or Alexa 594-conjugated goat anti-mouse IgG (Molecular Probes); CD4 (clone H29.129; isotype rat IgG2a; 1:50; BD Pharmingen); secondary: Alexa-488-conjugated goat anti-rat IgG (Molecular Probes); and CD8 (clone 53-6.7; isotype rat IgG2a, 1:50; BD Pharmingen); secondary: Alexa-488-conjugated goat anti-rat IgG (Molecular Probes).

Techniques: Blocking Assay, Injection, Control, Comparison, Immunofluorescence, Staining, Isolation

Journal:

Article Title: Elevated Neuronal Expression of CD200 Protects Wld s Mice from Inflammation-Mediated Neurodegeneration

doi: 10.2353/ajpath.2007.060677

Figure Lengend Snippet: Decreased ubiquitination of CD200 in spinal cord lysates of Wlds mice. a: Representative immunoblot of spinal cord lysates from naïve WT mice (lanes 1 and 2), naïve Wlds mice (lanes 3 and 4), WT mice day 22 after immunization (lanes 5 and 6), Wlds mice day 22 after immunization (lanes 7 and 8), WT mice day 60 after immunization (lanes 9 and 10), and Wlds mice day 60 after immunization (lanes 11 and 12) shows increased expression of CD200 in Wlds spinal cord lysates at all time points from compared with those from WT mice. β-Actin control immunoblot shows similar protein amounts in all samples. b: Densitometric quantification of immunoblots demonstrates increased expression of CD200 during the course of EAE in Wlds mice but not WT mice. c: Immunoprecipitation of CD200, with immunoblotting (IB) of ubiquitin and CD200. Sample numbers are the same as in a, except sample 10 was omitted. There was decreased expression ubiquitination of CD200 in Wlds mice samples at d0 and d22 compared with WT samples. At d60, the expression of ubiquitin was increased in Wlds samples and was comparable with the WT sample.

Article Snippet: The following antibodies were used: CD200 (clone 3B6, isotype rat IgM, 1:200; Cedarlane Laboratories, Hornby, ON, Canada); secondary: Alexa 488-conjugated goat anti-rat IgM (Molecular Probes); CD200R (anti-313015 CD200R peptide, 26 clone R252, isotype rabbit IgG, 1:200; Trillium Therapeutics Inc.); secondary: Alexa 594-conjugated goat anti-rabbit IgG (Molecular Probes); CD200R (clone OX-110, isotype rat IgG2a, 1:100; Serotec, Oxford, UK); secondary: Alexa 488-conjugated rabbit anti-rat IgG (Molecular Probes); mitogen-activated protein 2 (MAP-2) (clone HM-2: mouse anti-mouse IgG1, 1:100; Sigma); secondary: Alexa 594-conjugated goat anti-mouse IgG (Molecular Probes); NeuN (clone A60, isotype mouse IgG1, 1:100; Chemicon/Millipore, Temecula, CA); secondary: Alexa 594-conjugated goat anti-mouse IgG (Molecular Probes); GFAP cocktail (clones 4A11, 1B4, 2E1, isotype mouse IgG2b, 1:100; BD Pharmingen, Palo Alto, CA); secondary: Alexa 488- or Alexa 594-conjugated goat anti-mouse IgG (Molecular Probes); CNPase (clone 11-5B, isotype mouse IgG1, 1:100; Chemicon/Millipore); secondary: Alexa 488- or Alexa 594-conjugated goat anti-mouse IgG (Molecular Probes); β-tubulin (clone TUJ1, isotype mouse IgG2a; 1:100; Covance, Berkeley, CA); secondary: Alexa 488- or Alexa 594-conjugated goat anti-mouse IgG (Molecular Probes); CD4 (clone H29.129; isotype rat IgG2a; 1:50; BD Pharmingen); secondary: Alexa-488-conjugated goat anti-rat IgG (Molecular Probes); and CD8 (clone 53-6.7; isotype rat IgG2a, 1:50; BD Pharmingen); secondary: Alexa-488-conjugated goat anti-rat IgG (Molecular Probes).

Techniques: Ubiquitin Proteomics, Western Blot, Expressing, Control, Immunoprecipitation

Journal:

Article Title: Elevated Neuronal Expression of CD200 Protects Wld s Mice from Inflammation-Mediated Neurodegeneration

doi: 10.2353/ajpath.2007.060677

Figure Lengend Snippet: Increased expression of CD200 in the spinal cord of Wlds mice. Spinal cord sections from WT and Wlds mice on days 0, 22, and 60 after immunization were double-stained with CD200 (green) and NeuN (red) marker for neurons. a: Shown are representative merged confocal images. CD200 expression is markedly increased in Wlds sections compared with WT sections, with increasing expression after the induction of EAE. b and c: Splitway confocal images showing co-localization of CD200 and NeuN staining in WT (b) and Wlds (c) sections. CD200 expression is increased on Wlds neuronal bodies and processes. d: Confocal merge profiles and intensity profile shows co-localization of CD200 (green) and NeuN (red) in the surface and cytoplasm of cells and processes but not the nucleus. e: Confocal intensity profile of CD200 staining shows a punctate pattern of staining consistent with surface staining of the molecule. f: Confocal reconstruction (2.5-dimension) of Z-stacked images demonstrates punctate areas of high-intensity staining (red > yellow > green), consistent with surface staining (red), as well as medium intensity staining in cytoplasmic regions (yellow). Original magnifications, ×63.

Article Snippet: The following antibodies were used: CD200 (clone 3B6, isotype rat IgM, 1:200; Cedarlane Laboratories, Hornby, ON, Canada); secondary: Alexa 488-conjugated goat anti-rat IgM (Molecular Probes); CD200R (anti-313015 CD200R peptide, 26 clone R252, isotype rabbit IgG, 1:200; Trillium Therapeutics Inc.); secondary: Alexa 594-conjugated goat anti-rabbit IgG (Molecular Probes); CD200R (clone OX-110, isotype rat IgG2a, 1:100; Serotec, Oxford, UK); secondary: Alexa 488-conjugated rabbit anti-rat IgG (Molecular Probes); mitogen-activated protein 2 (MAP-2) (clone HM-2: mouse anti-mouse IgG1, 1:100; Sigma); secondary: Alexa 594-conjugated goat anti-mouse IgG (Molecular Probes); NeuN (clone A60, isotype mouse IgG1, 1:100; Chemicon/Millipore, Temecula, CA); secondary: Alexa 594-conjugated goat anti-mouse IgG (Molecular Probes); GFAP cocktail (clones 4A11, 1B4, 2E1, isotype mouse IgG2b, 1:100; BD Pharmingen, Palo Alto, CA); secondary: Alexa 488- or Alexa 594-conjugated goat anti-mouse IgG (Molecular Probes); CNPase (clone 11-5B, isotype mouse IgG1, 1:100; Chemicon/Millipore); secondary: Alexa 488- or Alexa 594-conjugated goat anti-mouse IgG (Molecular Probes); β-tubulin (clone TUJ1, isotype mouse IgG2a; 1:100; Covance, Berkeley, CA); secondary: Alexa 488- or Alexa 594-conjugated goat anti-mouse IgG (Molecular Probes); CD4 (clone H29.129; isotype rat IgG2a; 1:50; BD Pharmingen); secondary: Alexa-488-conjugated goat anti-rat IgG (Molecular Probes); and CD8 (clone 53-6.7; isotype rat IgG2a, 1:50; BD Pharmingen); secondary: Alexa-488-conjugated goat anti-rat IgG (Molecular Probes).

Techniques: Expressing, Staining, Marker

Journal:

Article Title: Elevated Neuronal Expression of CD200 Protects Wld s Mice from Inflammation-Mediated Neurodegeneration

doi: 10.2353/ajpath.2007.060677

Figure Lengend Snippet: Increased expression of CD200 during EAE co-localizes with CNPase and GFAP marker. a: Splitway confocal images show partial co-localization of CD200 and CNPase markers in Wlds and WT spinal cord sections. Expression in both strains is enhanced at day 22 after immunization compared with naïve spinal cords. b: Splitway confocal images show partial co-localization of CD200 and GFAP markers in Wlds and WT spinal cord sections. Expression is enhanced particularly in Wlds sections at day 22 after immunization compared with naïve spinal cords. Original magnifications, ×63.

Article Snippet: The following antibodies were used: CD200 (clone 3B6, isotype rat IgM, 1:200; Cedarlane Laboratories, Hornby, ON, Canada); secondary: Alexa 488-conjugated goat anti-rat IgM (Molecular Probes); CD200R (anti-313015 CD200R peptide, 26 clone R252, isotype rabbit IgG, 1:200; Trillium Therapeutics Inc.); secondary: Alexa 594-conjugated goat anti-rabbit IgG (Molecular Probes); CD200R (clone OX-110, isotype rat IgG2a, 1:100; Serotec, Oxford, UK); secondary: Alexa 488-conjugated rabbit anti-rat IgG (Molecular Probes); mitogen-activated protein 2 (MAP-2) (clone HM-2: mouse anti-mouse IgG1, 1:100; Sigma); secondary: Alexa 594-conjugated goat anti-mouse IgG (Molecular Probes); NeuN (clone A60, isotype mouse IgG1, 1:100; Chemicon/Millipore, Temecula, CA); secondary: Alexa 594-conjugated goat anti-mouse IgG (Molecular Probes); GFAP cocktail (clones 4A11, 1B4, 2E1, isotype mouse IgG2b, 1:100; BD Pharmingen, Palo Alto, CA); secondary: Alexa 488- or Alexa 594-conjugated goat anti-mouse IgG (Molecular Probes); CNPase (clone 11-5B, isotype mouse IgG1, 1:100; Chemicon/Millipore); secondary: Alexa 488- or Alexa 594-conjugated goat anti-mouse IgG (Molecular Probes); β-tubulin (clone TUJ1, isotype mouse IgG2a; 1:100; Covance, Berkeley, CA); secondary: Alexa 488- or Alexa 594-conjugated goat anti-mouse IgG (Molecular Probes); CD4 (clone H29.129; isotype rat IgG2a; 1:50; BD Pharmingen); secondary: Alexa-488-conjugated goat anti-rat IgG (Molecular Probes); and CD8 (clone 53-6.7; isotype rat IgG2a, 1:50; BD Pharmingen); secondary: Alexa-488-conjugated goat anti-rat IgG (Molecular Probes).

Techniques: Expressing, Marker

Journal:

Article Title: Elevated Neuronal Expression of CD200 Protects Wld s Mice from Inflammation-Mediated Neurodegeneration

doi: 10.2353/ajpath.2007.060677

Figure Lengend Snippet: Expression of CD200 in Splenocytes from Wld s Mice

Article Snippet: The following antibodies were used: CD200 (clone 3B6, isotype rat IgM, 1:200; Cedarlane Laboratories, Hornby, ON, Canada); secondary: Alexa 488-conjugated goat anti-rat IgM (Molecular Probes); CD200R (anti-313015 CD200R peptide, 26 clone R252, isotype rabbit IgG, 1:200; Trillium Therapeutics Inc.); secondary: Alexa 594-conjugated goat anti-rabbit IgG (Molecular Probes); CD200R (clone OX-110, isotype rat IgG2a, 1:100; Serotec, Oxford, UK); secondary: Alexa 488-conjugated rabbit anti-rat IgG (Molecular Probes); mitogen-activated protein 2 (MAP-2) (clone HM-2: mouse anti-mouse IgG1, 1:100; Sigma); secondary: Alexa 594-conjugated goat anti-mouse IgG (Molecular Probes); NeuN (clone A60, isotype mouse IgG1, 1:100; Chemicon/Millipore, Temecula, CA); secondary: Alexa 594-conjugated goat anti-mouse IgG (Molecular Probes); GFAP cocktail (clones 4A11, 1B4, 2E1, isotype mouse IgG2b, 1:100; BD Pharmingen, Palo Alto, CA); secondary: Alexa 488- or Alexa 594-conjugated goat anti-mouse IgG (Molecular Probes); CNPase (clone 11-5B, isotype mouse IgG1, 1:100; Chemicon/Millipore); secondary: Alexa 488- or Alexa 594-conjugated goat anti-mouse IgG (Molecular Probes); β-tubulin (clone TUJ1, isotype mouse IgG2a; 1:100; Covance, Berkeley, CA); secondary: Alexa 488- or Alexa 594-conjugated goat anti-mouse IgG (Molecular Probes); CD4 (clone H29.129; isotype rat IgG2a; 1:50; BD Pharmingen); secondary: Alexa-488-conjugated goat anti-rat IgG (Molecular Probes); and CD8 (clone 53-6.7; isotype rat IgG2a, 1:50; BD Pharmingen); secondary: Alexa-488-conjugated goat anti-rat IgG (Molecular Probes).

Techniques: Expressing

Journal:

Article Title: Elevated Neuronal Expression of CD200 Protects Wld s Mice from Inflammation-Mediated Neurodegeneration

doi: 10.2353/ajpath.2007.060677

Figure Lengend Snippet: Neuronal cultures from Wlds E16 embryos are protected from LPS-activated microglial-induced toxicity. Cortical neuronal cultures were derived from WT and Wlds E16 embryos and plated at a high-density concentration of 200,000 cells/well/0.5 ml in 24-well plates. a: Representative fluorescence microscopy photomicrographs of MAP-2 (red), CD200 (green), and merged images from cortical cultures. CD200 expression is increased in Wlds cultures compared with WT cultures, and co-localizes with MAP-2-positive cells (white arrows). In some cases, CD200 expression does not co-localize with MAP-2 (arrowheads). Controls are stained with isotype control antibody and secondary antibodies. b and c: Shown are representative photomicrographs of WT and Wlds neuronal cultures with LPS-activated (b) or IFN-γ-activated (c) primary microglia. Cultures were immunostained with anti-MAP-2 antibody (red) and LB4 (green). Wlds axons and neurons remain intact after co-culture with activated microglia; however, there is significant increase in axonal beading in WT co-cultures. Percentage of beaded axons/total number of axons in 10 fields was quantified for each condition. Protection of Wlds neurons from neurotoxicity induced by activated microglia is ameliorated after the addition of a blocking anti-CD200 antibody or anti-CD200 F(Ab′)2 fragment (both conditions, P < 0.0001; Student’s t-test). Original magnifications, ×40 (a); ×63 (b, c).

Article Snippet: The following antibodies were used: CD200 (clone 3B6, isotype rat IgM, 1:200; Cedarlane Laboratories, Hornby, ON, Canada); secondary: Alexa 488-conjugated goat anti-rat IgM (Molecular Probes); CD200R (anti-313015 CD200R peptide, 26 clone R252, isotype rabbit IgG, 1:200; Trillium Therapeutics Inc.); secondary: Alexa 594-conjugated goat anti-rabbit IgG (Molecular Probes); CD200R (clone OX-110, isotype rat IgG2a, 1:100; Serotec, Oxford, UK); secondary: Alexa 488-conjugated rabbit anti-rat IgG (Molecular Probes); mitogen-activated protein 2 (MAP-2) (clone HM-2: mouse anti-mouse IgG1, 1:100; Sigma); secondary: Alexa 594-conjugated goat anti-mouse IgG (Molecular Probes); NeuN (clone A60, isotype mouse IgG1, 1:100; Chemicon/Millipore, Temecula, CA); secondary: Alexa 594-conjugated goat anti-mouse IgG (Molecular Probes); GFAP cocktail (clones 4A11, 1B4, 2E1, isotype mouse IgG2b, 1:100; BD Pharmingen, Palo Alto, CA); secondary: Alexa 488- or Alexa 594-conjugated goat anti-mouse IgG (Molecular Probes); CNPase (clone 11-5B, isotype mouse IgG1, 1:100; Chemicon/Millipore); secondary: Alexa 488- or Alexa 594-conjugated goat anti-mouse IgG (Molecular Probes); β-tubulin (clone TUJ1, isotype mouse IgG2a; 1:100; Covance, Berkeley, CA); secondary: Alexa 488- or Alexa 594-conjugated goat anti-mouse IgG (Molecular Probes); CD4 (clone H29.129; isotype rat IgG2a; 1:50; BD Pharmingen); secondary: Alexa-488-conjugated goat anti-rat IgG (Molecular Probes); and CD8 (clone 53-6.7; isotype rat IgG2a, 1:50; BD Pharmingen); secondary: Alexa-488-conjugated goat anti-rat IgG (Molecular Probes).

Techniques: Derivative Assay, Concentration Assay, Fluorescence, Microscopy, Expressing, Staining, Control, Co-Culture Assay, Blocking Assay

Journal: Nature Communications

Article Title: CD200R1-CD200 checkpoint inhibits phagocytosis differently from SIRPα-CD47 to suppress tumor growth

doi: 10.1038/s41467-025-60456-3

Figure Lengend Snippet: a Mass spectrometry analysis of CD11b immunoprecipitates from BMDMs. Left: experimental workflow for isolating the CD11b-interacting proteins. Right: relative abundance of CD11b-interacting proteins, with a focus on cell surface receptors with inhibitory potential, indicated by a color gradient. ITIM, immunoreceptor tyrosine-based inhibitory motif; ITSM, immunoreceptor tyrosine-based switch motif; NPXY, asparagine, proline, any residue, tyrosine. IP, immunoprecipitation. b Immunoblot analysis of CD11b and CD200R1 interaction in immunoprecipitates from WT, Itgb2 −/− and Cd200r1 −/− BMDMs. Relative abundance is shown at the bottom of each panel. c Flow cytometry analysis of CD200 expression on parental (top) and Tac expression on Tac + (bottom) mouse tumor cell lines. Red curves represent staining with CD200 or Tac mAbs. Filled curves, control (Ctrl) mAbs. d Microscopy-based phagocytosis assay of non-opsonized (−) or IgG-opsonized (+) Tac + WEHI-231, Tac + A20, Tac + J558 and Tac + TUBO cells by WT BMDMs, in presence of CD200 mAb OX-90 (rat IgG2a) or Ctrl mAb 2A3 (rat IgG2a). Tumor cells were opsonized with Tac mAb 7G7 (mouse IgG2a), ( n = 3). e Time-course pHrodo-based phagocytosis assay using IgG-opsonized Tac + WEHI-231 cells and WT BMDMs. WEHI-231 cells were labeled with pHrodo red dye, and BMDMs were labeled with CSFE. Phagocytosis over time (0-4 h) was analyzed using an IncuCyte Live Cell Analyzer. Left: representative images at 2 h (scale bar, 100 μm; arrows, BMDMs with engulfed tumor cells). Right: quantification of cumulative phagocytosis (top) and time-specific increase in phagocytosis (Δ phagocytosis; bottom) over 0-4 h, ( n = 3). f Confocal microscope-based conjugate formation and actin polarization assay of IgG-opsonized WEHI-231 cells labeled with CSFE (green) and co-incubated with WT BMDMs labeled with Cell Trace Violet (CTV; blue), in presence of CD200 mAb or Ctrl mAb. Actin (red) was detected by β-actin mAb. Left: representative images (scale bar, 10 μm; arrows, BMDMs with fully polarized actin). Scale, 10 μm. Right: quantification of conjugate formation (top) and of conjugates with fully polarized actin (bottom), ( n = 3). g As per Fig. 1d, except that phagocytosis of complement (C3bi)-opsonized WEHI-231 cells by WT BMDMs in the presence of blocking CD200 mAb OX-90, blocking CD11b mAb 5C6 (rat IgG2b), Ctrl mAb 2A3 or Ctrl mAb LTF-2 (rat IgG2b), ( n = 3). h As per Fig. 1d, expect that phagocytosis of IgG-opsonized WEHI-231 cells by WT BMDMs in the presence of CD200R1 mAb OX-131 (mouse IgG1, Fc-silent) or Ctrl mAb (mouse IgG1, Fc-silent), ( n = 3). Data are from three ( a – h ) independent experiments. Each symbol represents one mouse. Data are presented as mean ± s.e.m. Statistical analysis: two-tailed t -test ( d – f , h ) with multiple comparisons ( e ); One-way ANOVA test with multiple comparisons ( g ); ns, not significant. See also Supplementary Fig. and Supplementary Table .

Article Snippet: For phagocytosis assays and in vivo assays, the following mAbs were used: CD200 mAb OX-90 (BioXCell, Lebanon, NH); CD200R1 mAb OX-131 (Absolute Antibody, Boston, MA), SIRPα mAb no. 27 ; rat IgG2b isotype Ctrl mAb LTF-2 (BioXCell); rat IgG2a isotype Ctrl mAb 2A3 (BioXCell); Ctrl mAb MOPC-21 (in-house generated); and blocking CD11b mAb 5C6 (BioXCell).

Techniques: Mass Spectrometry, Residue, Immunoprecipitation, Western Blot, Flow Cytometry, Expressing, Staining, Control, Microscopy, Phagocytosis Assay, Labeling, Incubation, Blocking Assay, Two Tailed Test

Journal: Nature Communications

Article Title: CD200R1-CD200 checkpoint inhibits phagocytosis differently from SIRPα-CD47 to suppress tumor growth

doi: 10.1038/s41467-025-60456-3

Figure Lengend Snippet: a Flow cytometry analysis of CD200R1 expression (left) on WT (top) or Cd200r1 −/− (bottom) BMDMs, and CD200 expression (right) on Cd200 +/+ (top) or Cd200 −/− (bottom) WEHI-231 cells, as measured by flow cytometry. Red curves, CD200R1 or CD200 mAbs. Filled curves, Ctrl mAb. b As per Fig. , except that phagocytosis of IgG-opsonized WEHI-231 cells in the presence of WT or Cd200r1 −/− BMDMs was studied, ( n = 3). c As per Fig. 2b, except that phagocytosis of IgG-opsonized Cd200 +/+ and Cd200 −/− WEHI-231 cells, in the presence of WT or Cd200r1 −/− BMDMs, ( n = 3). Data are from three independent experiments ( a – c ). Each symbol represents one mouse. Data are mean ± s.e.m. Statistical analysis: two-way ANOVA test, with multiple comparisons ( b , c ), ns not significant. See also Supplementary Fig. .

Article Snippet: For phagocytosis assays and in vivo assays, the following mAbs were used: CD200 mAb OX-90 (BioXCell, Lebanon, NH); CD200R1 mAb OX-131 (Absolute Antibody, Boston, MA), SIRPα mAb no. 27 ; rat IgG2b isotype Ctrl mAb LTF-2 (BioXCell); rat IgG2a isotype Ctrl mAb 2A3 (BioXCell); Ctrl mAb MOPC-21 (in-house generated); and blocking CD11b mAb 5C6 (BioXCell).

Techniques: Flow Cytometry, Expressing

Journal: Nature Communications

Article Title: CD200R1-CD200 checkpoint inhibits phagocytosis differently from SIRPα-CD47 to suppress tumor growth

doi: 10.1038/s41467-025-60456-3

Figure Lengend Snippet: a – g Tac + WEHI-231 tumor (with or without luciferase expression) were injected intravenously into Rag1 −/− mice, followed by intraperitoneal injection of Tac mAb combined with CD200 mAb or Ctrl mAb every 2 days. Mice were euthanized on day 15 ( b – d ) or monitored over time using luminescence, if cells were expressing luciferase ( e – g ), ( n = 12, b , c , n = 10, e – g ). a Schematic representation of the experimental workflow. I.V., intravenously; I.P., intraperitoneally. b Representative photographs of mice euthanized on day 15, with or without tumor injection, and treated with the indicated mAbs. Scale bar, 1 cm. c Liver weight of mice injected or not with tumor cells. d Hematoxylin and eosin staining of liver sections, showing blood vessels (blue asterisks) and tumor cell aggregates adjacent to blood vessels (outlined in white). Scale bars, 100 µm (10× magnification), and 20 µm (40× magnification). e Representative luminescence images on day 12. f Tumor progression over time as measured by luminescence. sec, second, st, steradian. g Kaplan–Meier analysis of survival. h – j Tac + A20 cells were injected subcutaneously in Rag1 −/− mice followed by intraperitoneal injection of Tac mAb combined with CD200 mAb or Ctrl mAb, every 2 days, ( n = 8). h Schematic representation of the experimental workflow. S.C, subcutaneously. i Tumor volume over time. j Tumor weight. Data are from three c or two d – j independent experiments, respectively. Each symbol represents one mouse. Data are presented as mean ± s.e.m. Statistical analysis: one-way ANOVA test, with multiple comparisons ( c ); two-tailed t-test ( f , i , j ). log-rank (Mantel–Cox) test ( g ). ns, not significant. See also Supplementary Figs. and .

Article Snippet: For phagocytosis assays and in vivo assays, the following mAbs were used: CD200 mAb OX-90 (BioXCell, Lebanon, NH); CD200R1 mAb OX-131 (Absolute Antibody, Boston, MA), SIRPα mAb no. 27 ; rat IgG2b isotype Ctrl mAb LTF-2 (BioXCell); rat IgG2a isotype Ctrl mAb 2A3 (BioXCell); Ctrl mAb MOPC-21 (in-house generated); and blocking CD11b mAb 5C6 (BioXCell).

Techniques: Luciferase, Expressing, Injection, IF-cells, Staining, Two Tailed Test

Journal: Nature Communications

Article Title: CD200R1-CD200 checkpoint inhibits phagocytosis differently from SIRPα-CD47 to suppress tumor growth

doi: 10.1038/s41467-025-60456-3

Figure Lengend Snippet: a Flow cytometry analysis of CD200R1 expression on human blood monocyte-derived macrophages, either unprimed or primed for 1 day with the indicated stimuli. Red curves, CD200R1 mAb. Filled curves, Ctrl mAb. Number in histograms indicated mean fluorescence intensity (MFI). b Fold changes of RNA expression of human blood monocyte-derived macrophages treated with or without IL-4. CD200R1 is highlighted in red. Fold changes (log 2 ; x -axis) and adjusted p -values (log 10 ; y-axis) are shown. The red line represents a 4-fold change threshold. Data are from dataset GSE195440 . c Frequency and relative expression levels of CD200R1 RNA across various human TAM subtypes, distinguished by gene expression profiles , . Data are from single-cell RNA sequencing (scRNA-seq) datasets GSE154763 and GSE146771 of TAMs from patients with the cancers indicated in Supplementary Fig. , and colon carcinoma. d RNA expression profiles of human hematological malignancies, determined by microarray analysis. Left: UMAP plots of samples with tumor diagnosis are shown and colored by cluster identity. Middle: CD200 and CD47 RNA expression levels overlaid onto the UMAP (color gradient shows relative expression). Right: mean expression of CD200 and CD47 RNA in common subtypes of hematological malignancies and normal immune cells (color gradient). Data are from published dataset HEMAP ( n = 7092). e Single-cell RNA sequencing analysis of human melanoma. Left: t-SNE profiles of melanoma malignant or non-malignant cells. Malignant melanoma cells are clustered by patient sample (top), while infiltrating immune cells are clustered by cell type (bottom). The middle and right: normalized expression of selected markers overlaid onto the t-SNE space, with a color gradient reflecting relative expression. Macrophages are outlined by a blue line. Data are from published dataset GSE115978 . f Same as d , except that expression of CD200 and CD47 RNA was analyzed in normal hematopoietic cells. g Flow cytometry analysis of expression of CD200 and CD47 on resting or activated human B cells, CD4 + T cells, and CD8 + T cells. Data are from three independent experiments ( a, g ). Each symbol represents one gene ( b ), one cell ( c ), one healthy donor or patient ( d ). Statistical analysis: Wald test followed by multiple comparisons ( b ); Kruskal–Wallis H test followed by Dunn’s post-hoc test with multiple comparisons ( c , left); two-tailed Mann–Whitney U test ( c , right panels). See also Supplementary Fig. .

Article Snippet: For phagocytosis assays and in vivo assays, the following mAbs were used: CD200 mAb OX-90 (BioXCell, Lebanon, NH); CD200R1 mAb OX-131 (Absolute Antibody, Boston, MA), SIRPα mAb no. 27 ; rat IgG2b isotype Ctrl mAb LTF-2 (BioXCell); rat IgG2a isotype Ctrl mAb 2A3 (BioXCell); Ctrl mAb MOPC-21 (in-house generated); and blocking CD11b mAb 5C6 (BioXCell).

Techniques: Flow Cytometry, Expressing, Derivative Assay, Fluorescence, RNA Expression, Gene Expression, RNA Sequencing, Microarray, Biomarker Discovery, Two Tailed Test, MANN-WHITNEY

Journal: Nature Communications

Article Title: CD200R1-CD200 checkpoint inhibits phagocytosis differently from SIRPα-CD47 to suppress tumor growth

doi: 10.1038/s41467-025-60456-3

Figure Lengend Snippet: a Flow cytometry analysis of CD200 (top) or tumor antigens (CD20, CD38, CD123, DLL3 and SLAMF7; bottom) on various human tumor cell lines. Red curves, CD200 mAb or tumor antigen-specific mAbs. Filled curves, Ctrl mAbs. b As per Fig. , except using IL-4-primed human blood monocyte-derived macrophages and human tumor cells in the presence of samalizumab (human IgG1, Fc-silent) or Ctrl mAb MOPC21 (human IgG1, Fc-silent). IgG opsonization was performed using CD20 mAb rituximab (SLVL, 721.221), CD38 mAb daratumumab (NCI-H929), CD123 mAb talacotuzumab (KG-1a), DLL3 mAb rovalpituzumab (NCI-H209), or SLAMF7 mAb elotuzumab (SK-MEL-28) ( n = 3 or 4). c As per Fig. 5b, except using the indicated cell lines and samalizumab (human IgG1, Fc-silent), CD47 mAb B6H12 (human IgG1, Fc-silent), or Ctrl mAb MOPC21 (human IgG1, Fc-silent). Tumor cells were opsonized as detailed for Fig. 5b, ( n = 3). d –f Subcutaneous injection of SLVL cells in NSG mice, followed by intraperitoneal injection of rituximab (mouse IgG2a version) combined with samalizumab or Ctrl mAb every 2 days, ( n = 8). d Schematic representation of the experimental workflow. e Tumor volume over time. f Tumor weight. g –i Subcutaneous injection of 721.221 cells in NSG mice, followed by intraperitoneal injection of rituximab (mouse IgG2a version) combined with samalizumab or Ctrl mAbs every 2 days, ( n = 10). g Schematic representation of the experimental workflow. h Tumor volume over time. i Tumor weight. Data are from three to four ( a, b ), three ( c ), or two ( d-i ) independent experiments. Each symbol represents one human sample or one mouse. Data are presented as mean ± s.e.m. Statistical analysis: two-tailed t-test ( f , i ), with multiple comparisons ( b, c , e , h ). ns, not significant. See also Supplementary Fig. .

Article Snippet: For phagocytosis assays and in vivo assays, the following mAbs were used: CD200 mAb OX-90 (BioXCell, Lebanon, NH); CD200R1 mAb OX-131 (Absolute Antibody, Boston, MA), SIRPα mAb no. 27 ; rat IgG2b isotype Ctrl mAb LTF-2 (BioXCell); rat IgG2a isotype Ctrl mAb 2A3 (BioXCell); Ctrl mAb MOPC-21 (in-house generated); and blocking CD11b mAb 5C6 (BioXCell).

Techniques: Flow Cytometry, Derivative Assay, Injection, Two Tailed Test

Journal: Nature Communications

Article Title: CD200R1-CD200 checkpoint inhibits phagocytosis differently from SIRPα-CD47 to suppress tumor growth

doi: 10.1038/s41467-025-60456-3

Figure Lengend Snippet: a Partial sequences of the cytoplasmic domain of CD200R1 from different species. The conserved tyrosines (Y 286 , Y 289 , and Y 297 ; based on mouse amino acid numbering) are in red, whereas the conserved NPxY motif is boxed. Identical residues are depicted by asterisks (*), while conserved and semi-conserved amino acids are highlighted by colons (:) and periods (.), respectively. b Phagocytosis of IgG-opsonized WEHI-231 cells by WT BMDMs expressing GFP alone, or Cd200r1 −/− BMDMs expressing GFP alone or CD200R1 variants, in the presence of CD200 mAb or Ctrl mAb, ( n = 3). c Mass spectrometry analyzes of cytoplasmic proteins with inhibitory potential interacting with synthetic biotinylated CD200R1 peptides, with or without phosphorylation at Y 286 or Y 297 in pull-down assays. Peptides are depicted at the top. Interactors identified by phosphorylated peptides are shown below. Negative regulators of immune cell activation, either adaptors, kinases, phosphatases or Ras-GAP, are indicated. d Immunoblot analysis of Dok-1 and Dok-2 expression (left) and phagocytosis of IgG-opsonized WEHI-231 cells (right) by BMDMs from WT or Dok1 −/− Dok2 −/− mice. β-actin as loading Ctrl (left). Normalized protein abundance (in %) relative to actin is shown below the top panel, ( n = 4). e Same as Fig. 6d except that WT BMDMs transduced with Ctrl or Csk-specific siRNAs [ Csk knockdown ( Csk KD )] were used. Two different Csk-specific siRNAs were studied. Csk expression (left) and phagocytosis (right) were studied, ( n = 4). f Summary of fold-changes in phagocytosis for the various genetically deficient BMDMs in response to CD200 mAb, compared to WT BMDMs, ( n = 3 or 4). g WT or Dok1 −/− Dok2 −/− BMDMs were stimulated or not for 1 min with biotinylated CD200R1 mAb OX-110 and streptavidin. Cell lysates were immunoprecipitated with α-Dok-1, α-Dok-2, α-Csk, or normal rabbit serum (NRS), and probed by immunoblotting with antibodies targeting phosphotyrosine (pTyr), Dok-1, Dok-2 or Csk. h WT or Dok1 −/− Dok2 −/− BMDMs were stimulated or not for 30 s with biotinylated CD200R1 mAb OX-110 and streptavidin. Cell lysates were probed with α-pLyn (Tyr 507) or α-Lyn Abs (top). A quantification of multiple independent experiments is shown at the bottom, ( n = 3). Data are from three ( b , e , g , h ) or four d independent experiments, two (pY 297 peptide) and three (pY 286 peptide) ( c ) independent experiments. Each symbol represents one mouse ( b , d , e ). Data are mean ± s.e.m. Statistical analysis: two-way ANOVA test, with multiple comparisons ( b , d , e , h ); One-way ANOVA test, with multiple comparisons ( f ). ns not significant. See also Supplementary Figs. , and Supplementary Table .

Article Snippet: For phagocytosis assays and in vivo assays, the following mAbs were used: CD200 mAb OX-90 (BioXCell, Lebanon, NH); CD200R1 mAb OX-131 (Absolute Antibody, Boston, MA), SIRPα mAb no. 27 ; rat IgG2b isotype Ctrl mAb LTF-2 (BioXCell); rat IgG2a isotype Ctrl mAb 2A3 (BioXCell); Ctrl mAb MOPC-21 (in-house generated); and blocking CD11b mAb 5C6 (BioXCell).

Techniques: Expressing, Mass Spectrometry, Phospho-proteomics, Activation Assay, Western Blot, Quantitative Proteomics, Transduction, Knockdown, Immunoprecipitation

Journal: Nature Communications

Article Title: CD200R1-CD200 checkpoint inhibits phagocytosis differently from SIRPα-CD47 to suppress tumor growth

doi: 10.1038/s41467-025-60456-3

Figure Lengend Snippet: a Flow cytometry analysis of CD200 (red curves; top) and CD47 (lavender curves; bottom) expression on J558, A20 and WEHI-231 cells (left). Filled curves, Ctrl mAbs. The right panel shows relative expression levels of CD200 and CD47. b As per Fig. , except that mAbs were used in combination: CD200 mAb OX-90 (rat IgG2a), SIRPα mAb 27 (mouse IgG2a, Fc-silent), Ctrl mAb 2A3 (rat IgG2a), and Ctrl mAb MOPC21 (mouse IgG2a, Fc-silent), ( n = 3). c – f Luciferase + Tac + GFP + WEHI-231 cells were injected intravenously into Rag1 −/− mice, followed by intraperitoneal injection of Tac mAb combined with the indicated mAbs every 2 days starting from day 4, ( n = 5). c Schematic representation of the experimental workflow. Tumor progression was measured over time using luminescence. Representative photographs of mice ( d ) and quantification ( e ). f Kaplan – Meier curves of survival. g Phagocytosis of normal activated human T cells or B cells by autologous human macrophages, in the presence of samalizumab (human IgG1, Fc-silent), CD47 mAb B6H12 (human IgG1, Fc-silent) or Ctrl mAb (human IgG1, Fc-silent). T cells were not opsonized (implying phagocytosis was mediated by SLAMF7), whereas B cells were opsonized with rituximab (human IgG1, Fc-active) ( n = 3). Data are from three ( a , b , g ) or two ( c – f ) independent experiments. Each symbol represents one mouse or donor. Data are mean ± s.e.m. Statistical analysis: two-way ANOVA test, with multiple comparisons ( b , e ); log-rank (Mantel-Cox) test ( f ); one-way ANOVA test, with multiple comparisons ( g ). ns, not significant.

Article Snippet: For phagocytosis assays and in vivo assays, the following mAbs were used: CD200 mAb OX-90 (BioXCell, Lebanon, NH); CD200R1 mAb OX-131 (Absolute Antibody, Boston, MA), SIRPα mAb no. 27 ; rat IgG2b isotype Ctrl mAb LTF-2 (BioXCell); rat IgG2a isotype Ctrl mAb 2A3 (BioXCell); Ctrl mAb MOPC-21 (in-house generated); and blocking CD11b mAb 5C6 (BioXCell).

Techniques: Flow Cytometry, Expressing, Luciferase, Injection