Journal:

Article Title: The immunosuppressive surface ligand CD200 augments the metastatic capacity of squamous cell carcinoma

doi: 10.1158/0008-5472.CAN-09-4380

Figure Lengend Snippet: CD200R1+ stromal cells are CD11b+/Gr-1+ MDSCs. A, CD200R1 immunofluorescence in WD SCC, LN Met and Lung Met (white arrows). DAPI counterstain was conducted to visualize nuclei. Dashed line demarcates SCC keratinocytes in LN Met. B, For CD200R1 FACS analysis of CD200R1, CD11b, and Gr-1 or MHC II expression. The total population of CD200R1+ cells were gated and subsequently analyzed for expression of CD11b and Gr-1 or MHC class II. The percentage of the total CD200R1 pool for a single experiment is shown. Far right panel: murine SCC were stained with antibodies against CD200R1 (red) and CD11b (green) and counterstained with DAPI (blue). Arrows point to CD200R1+/CD11b+ (yellow) stromal MDSCs. C, Left: Bar graph showing the densitometric units representing GM-CSF and G-CSF levels in pRS-NS Lung Met/CD200R1+ co-cultures versus pRS-CD200 Lung Met/CD200R1+ co-cultures. *- statistically significant difference (GM-CSF, p = 0.049; G-CSF, p = 0.002). Right: H&E staining (top left) and G-CSF (red) and CD200 (green) immunofluorescence in murine PD SCC. Nuclei were delineated with DAPI (blue). Scale bars mark 50µm.

Article Snippet: Antibodies Antibodies were used against human CD200, mouse CD200, CD3ε, CD86, α6 integrin-FITC (BD Biosciences); NK1.1-488, c-kit, CD123, MHC II, CD11b-FITC, CD11b-PE, Gr-1-FITC, Gr-1-PE, CD11c (BioLegend); Langerin, Foxp3 (eBioscience); Keratin 14 (Covance), CD200R1 (R&D Systems), CD4-APC (Abcam), G-CSF (Santa Cruz), anti-rabbit HRP (Jackson Immunoresearch); species-specific Alexa Fluor-488, -594, -647, and -680 (Invitrogen); and human α6 integrin (kindly provided by Fiona Watt).

Techniques: Immunofluorescence, Expressing, Staining

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: Journal of neuroinflammation

Article Title: The regulation of NFKB1 on CD200R1 expression and their potential roles in Parkinson's disease.

doi: 10.1186/s12974-024-03231-3

Figure Lengend Snippet: Fig. 2 NFKB1 is a potential transcription factor of CD200R1 and regulates its transcriptional activity. (A) Schematic representation of the CD200R1 core promoter sequence. The region covers the nucleotides from − 482 to -146 based on the CD200R1 translation initiation site. (B) In silico prediction of the possible transcription factors. (C) Dual-luciferase assay results in human HEK293T cells transfected with the reporter construct containing the core pro moter and overexpressing indicated transcription factors. (D) Dual-luciferase assay results in human U937 cells after overexpressing the CD200R1 core promoter and RELA, CREB1 and NFKB1, respectively. (E) Dual-luciferase assay results in microglia cell line BV2 transfected with the core promoter vector and NFKB1. One-way ANOVA was used for comparison. **P < 0.01, **** P < 0.0001

Article Snippet: The validity of the antibody for ELISA assay was confirmed by the manufacturer with recombinant CD200R1 protein (https://www.rndsystems.com/cn/ products/human-cd200r1-antibody_af3414).

Techniques: Activity Assay, Sequencing, In Silico, Luciferase, Transfection, Construct, Plasmid Preparation, Comparison

Journal: Journal of neuroinflammation

Article Title: The regulation of NFKB1 on CD200R1 expression and their potential roles in Parkinson's disease.

doi: 10.1186/s12974-024-03231-3

Figure Lengend Snippet: Fig. 4 Expression levels of NFKB1 and CD200R1 in PD patients. (A, B) CD200R1 (A) and NFKB1 (B) mRNA levels were assessed in PBMCs from PD patients (n = 44) with age- and sex-matched healthy controls (n = 45) by RT-PCR. Non-parameter analysis was used for between-group comparison. (C) Spearman correlation analysis between CD200R1 and NFKB1 mRNA expressions in PBMCs from PD population

Article Snippet: The validity of the antibody for ELISA assay was confirmed by the manufacturer with recombinant CD200R1 protein (https://www.rndsystems.com/cn/ products/human-cd200r1-antibody_af3414).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Comparison

Journal: Journal of Cellular and Molecular Medicine

Article Title: CD200 expression in human cultured bone marrow mesenchymal stem cells is induced by pro‐osteogenic and pro‐inflammatory cues

doi: 10.1111/jcmm.12752

Figure Lengend Snippet: Primers used for (A) quantitative RT‐PCR, (B) classical RT‐PCR

Article Snippet: The primary unconjugated anti‐human CD200 mAb (BD Biosiences) was also used in some experiments, along with an isotype‐matched mouse anti‐human IgG1 control mAb (BD Biosciences).

Techniques: Quantitative RT-PCR, Sequencing

Journal: Journal of Cellular and Molecular Medicine

Article Title: CD200 expression in human cultured bone marrow mesenchymal stem cells is induced by pro‐osteogenic and pro‐inflammatory cues

doi: 10.1111/jcmm.12752

Figure Lengend Snippet: Impact of BMP4 on ALPL expression by CD200 + cells

Article Snippet: The primary unconjugated anti‐human CD200 mAb (BD Biosiences) was also used in some experiments, along with an isotype‐matched mouse anti‐human IgG1 control mAb (BD Biosciences).

Techniques: Expressing

Journal: Journal of Cellular and Molecular Medicine

Article Title: CD200 expression in human cultured bone marrow mesenchymal stem cells is induced by pro‐osteogenic and pro‐inflammatory cues

doi: 10.1111/jcmm.12752

Figure Lengend Snippet: Impact of BMP2,4,7 on CD200 expression by BM MSCs

Article Snippet: The primary unconjugated anti‐human CD200 mAb (BD Biosiences) was also used in some experiments, along with an isotype‐matched mouse anti‐human IgG1 control mAb (BD Biosciences).

Techniques: Expressing

Journal: Journal of Cellular and Molecular Medicine

Article Title: CD200 expression in human cultured bone marrow mesenchymal stem cells is induced by pro‐osteogenic and pro‐inflammatory cues

doi: 10.1111/jcmm.12752

Figure Lengend Snippet: Impact of IL‐1β, ΤΝF‐α and PDTC on CD200 expression by BM MSCs ( n = 8)

Article Snippet: The primary unconjugated anti‐human CD200 mAb (BD Biosiences) was also used in some experiments, along with an isotype‐matched mouse anti‐human IgG1 control mAb (BD Biosciences).

Techniques: Expressing

Journal: Journal of Cellular and Molecular Medicine

Article Title: CD200 expression in human cultured bone marrow mesenchymal stem cells is induced by pro‐osteogenic and pro‐inflammatory cues

doi: 10.1111/jcmm.12752

Figure Lengend Snippet: Impact of dexamethasone on ALPL and CD200 expression

Article Snippet: The primary unconjugated anti‐human CD200 mAb (BD Biosiences) was also used in some experiments, along with an isotype‐matched mouse anti‐human IgG1 control mAb (BD Biosciences).

Techniques:

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: Aging cell

Article Title: Hedgehog signaling maintains hair follicle stem cell phenotype in young and aged human skin.

doi: 10.1111/j.1474-9726.2009.00526.x

Figure Lengend Snippet: Fig. 1 Characterization of human bulge cell markers. (A) CD200+ cells were stained in red by immunohistochemistry (magnification = 100·). Black rectangle identifies boundaries of 400· magnification shown in left inset. Positive cells are located in the outer root sheath of the hair follicle (HF), between insertions of sebaceous glands (SG) and arrector pili muscle (AP). Asterisk shows lumen of the HF (hair shaft was lost during staining). (B) Consecutive sections of human scalp skin biopsies from young individuals were stained for KRT15, CD200, and KRT19 by immunohistochemistry. Upper and lower limits of CD200 positive staining superimpose with those of KRT19 and KRT15 respectively (highlighted by black lines). Daggers indicate hair shafts remnants. (C) Schematic representation of localization of protein used as human bulge cell markers. Bulge cells are CD200+ ⁄ KRT15+ ⁄ KRT19+.

Article Snippet: Total HF cell suspensions were incubated with human IgGs (Sigma, St Louis, MO, USA) for 15 min at 4 C, and labeled with a mouse anti-CD200 antibody (AbD Serotec, Raleigh, NC, USA) at 4 C for 15 min. After three washes with MACS buffer, cells were incubated with microbeads coupled with rat antimouse IgG1 antibodies (Miltenyi Biotec) at 4 C for 15 min. After three washes with MACS buffer, cell suspension was loaded onto a column containing a ferromagnetic matrix exposed to the magnetic field of a permanent magnet.

Techniques: Staining, Immunohistochemistry

Journal: Aging cell

Article Title: Hedgehog signaling maintains hair follicle stem cell phenotype in young and aged human skin.

doi: 10.1111/j.1474-9726.2009.00526.x

Figure Lengend Snippet: Fig. 2 Characterization of isolated human bulge cells from young human skin. (A) CD200, KRT15 and KRT19 mRNA levels were measured by Q-PCR on CD200+ (bulge) and CD200) (differentiated) cells of the hair follicle (HF) in young (< 40 years) individuals. N = 6; *P < 0.05. (B) Transglutaminase 1 (TGM1) mRNA levels in same cell populations. N = 3; P = 0.022.

Article Snippet: Total HF cell suspensions were incubated with human IgGs (Sigma, St Louis, MO, USA) for 15 min at 4 C, and labeled with a mouse anti-CD200 antibody (AbD Serotec, Raleigh, NC, USA) at 4 C for 15 min. After three washes with MACS buffer, cells were incubated with microbeads coupled with rat antimouse IgG1 antibodies (Miltenyi Biotec) at 4 C for 15 min. After three washes with MACS buffer, cell suspension was loaded onto a column containing a ferromagnetic matrix exposed to the magnetic field of a permanent magnet.

Techniques: Isolation

Journal: Aging cell

Article Title: Hedgehog signaling maintains hair follicle stem cell phenotype in young and aged human skin.

doi: 10.1111/j.1474-9726.2009.00526.x

Figure Lengend Snippet: Fig. 3 Additional markers of young human bulge keratinocytes. (A, B) Frozen scalp skin sections from young individuals were immunostained for CD34 (A) and follistatin (B). Original magnification = 100·. Black rectangle indicates location of left inset (magnification 400·). Black arrows represent upper and lower limits of bulge area. Representative of at least four individuals. (C) Quantitative expression of gene transcripts for various potential hair follicle (HF) stem cell markers in bulge (CD200+) and differentiated (CD200)) cells of the HF in young human skin. N = 6; *P < 0.05; NS = not significant vs. bulge cells. Gas1, EGFR, CCN2 and S100A4 are represented in separate bar graphs to account for higher basal expression levels.

Article Snippet: Total HF cell suspensions were incubated with human IgGs (Sigma, St Louis, MO, USA) for 15 min at 4 C, and labeled with a mouse anti-CD200 antibody (AbD Serotec, Raleigh, NC, USA) at 4 C for 15 min. After three washes with MACS buffer, cells were incubated with microbeads coupled with rat antimouse IgG1 antibodies (Miltenyi Biotec) at 4 C for 15 min. After three washes with MACS buffer, cell suspension was loaded onto a column containing a ferromagnetic matrix exposed to the magnetic field of a permanent magnet.

Techniques: Expressing

Journal: Aging cell

Article Title: Hedgehog signaling maintains hair follicle stem cell phenotype in young and aged human skin.

doi: 10.1111/j.1474-9726.2009.00526.x

Figure Lengend Snippet: Fig. 5 Aging does not alter bulge marker expression in human skin. Total RNA from bulge (CD200+) and differentiated (CD200)) cells of the hair follicle (HF) were isolated from scalp skin biopsies of young (< 40 years) and aged (> 70 years) individuals, and analyzed by Q-PCR for human HF stem cell markers. (A) Quantitative expression of CD200, KRT15 and KRT19 human bulge cell markers. (B) Direct comparison of gene transcript levels of various potential additional bulge cell markers between young and aged individuals. Gas1, EGFR and CCN2 are represented in separate bar graphs to account for higher expression levels. N = 6; *P<0.05; NS = not significant vs. young counterpart.

Article Snippet: Total HF cell suspensions were incubated with human IgGs (Sigma, St Louis, MO, USA) for 15 min at 4 C, and labeled with a mouse anti-CD200 antibody (AbD Serotec, Raleigh, NC, USA) at 4 C for 15 min. After three washes with MACS buffer, cells were incubated with microbeads coupled with rat antimouse IgG1 antibodies (Miltenyi Biotec) at 4 C for 15 min. After three washes with MACS buffer, cell suspension was loaded onto a column containing a ferromagnetic matrix exposed to the magnetic field of a permanent magnet.

Techniques: Marker, Expressing, Isolation, Comparison

Journal: Aging cell

Article Title: Hedgehog signaling maintains hair follicle stem cell phenotype in young and aged human skin.

doi: 10.1111/j.1474-9726.2009.00526.x

Figure Lengend Snippet: Fig. 6 Hedgehog (Hh) pathway components are selectively expressed in human bulge cells vs. differentiated hair follicle (HF) keratinocytes (KCs). Transcript levels of Hh pathway components and target genes were quantified by Q-PCR in bulge (CD200+) and differentiated (CD200)) cells of human HFs. N = 6; (A) young and (B) aged individuals. *P < 0.05; NS = not significant vs. differentiated HF KCs. (C) Quantitative expression of levels of Hh pathway components and target genes transcripts in bulge cells of young and aged individuals. NS = not significant vs. young counterpart.

Article Snippet: Total HF cell suspensions were incubated with human IgGs (Sigma, St Louis, MO, USA) for 15 min at 4 C, and labeled with a mouse anti-CD200 antibody (AbD Serotec, Raleigh, NC, USA) at 4 C for 15 min. After three washes with MACS buffer, cells were incubated with microbeads coupled with rat antimouse IgG1 antibodies (Miltenyi Biotec) at 4 C for 15 min. After three washes with MACS buffer, cell suspension was loaded onto a column containing a ferromagnetic matrix exposed to the magnetic field of a permanent magnet.

Techniques: Expressing

Journal: Aging cell

Article Title: Hedgehog signaling maintains hair follicle stem cell phenotype in young and aged human skin.

doi: 10.1111/j.1474-9726.2009.00526.x

Figure Lengend Snippet: Fig. 7 The hedgehog transmembrane receptor and target gene product Ptch is expressed in CD200+ cells of the bulge area of human hair follicles. (A, B) Ptch protein expression was stained by immunohistochemistry 100· magnification (A). Black rectangle in A identifies boundaries of 200· magnification shown in B. (C) Double immunofluorescence staining of human bulge area. Human scalp sections were stained with CD200 (green) and Ptch (red). Overlay shows that Ptch is expressed by CD200+ cells of the human bulge area. Magnification = 400·. Representative of at least three individuals.

Article Snippet: Total HF cell suspensions were incubated with human IgGs (Sigma, St Louis, MO, USA) for 15 min at 4 C, and labeled with a mouse anti-CD200 antibody (AbD Serotec, Raleigh, NC, USA) at 4 C for 15 min. After three washes with MACS buffer, cells were incubated with microbeads coupled with rat antimouse IgG1 antibodies (Miltenyi Biotec) at 4 C for 15 min. After three washes with MACS buffer, cell suspension was loaded onto a column containing a ferromagnetic matrix exposed to the magnetic field of a permanent magnet.

Techniques: Expressing, Staining, Immunohistochemistry

Journal: Cell Journal (Yakhteh)

Article Title: Hair Follicle Generation by Injections of Adult Human Follicular Epithelial and Dermal Papilla Cells into Nude Mice

doi: 10.22074/cellj.2016.3916

Figure Lengend Snippet: Presence of human hair stem cells in human scalp samples. A. Longitudinal section of human anagen hair showed CD200 and K15 expressions in the outermost (ORS) layer of human hair. CD200 expressed between the insertion point of the arrector pili muscle (APM) and the sebaceous gland (SG). K15 expressed in the bulge and inferior of the bulge area (sub bulge). White arrows show APM, SG, B. CD200 expressed between the APM and SG. Dotted square represents CD200 expression, C, D. Magnified CD200 expression area, E. K15 expressed in the bulge and inferior of the bulge area (sub bulge). Dotted square represents K15 expression, F and G. Magnified K15 expression area. Nuclei were stained with DAPI (scale bar: 50 μm).

Article Snippet: Slides were washed and treated with blocking reagent followed by overnight incubation with the primary antibodies KRT15 (Abcam1385 LHKRT15, 1:100) and goat anti-human CD200 (R&D Systems, 1:50).

Techniques: Expressing, Staining

Journal: Cell Journal (Yakhteh)

Article Title: Hair Follicle Generation by Injections of Adult Human Follicular Epithelial and Dermal Papilla Cells into Nude Mice

doi: 10.22074/cellj.2016.3916

Figure Lengend Snippet: Human dermal papilla and epithelial cell characterization and culture. A. Dermal papilla produced a sunflower colony in culture (dotted circle), B. Upper panel showed magnified colony, C, D. Dermal papilla colony tested strongly positive for alkaline phosphatase and toluidine blue staining (dotted circles) (scale bar: 200 μm), E. Epithelial cells began to proliferate after four days (scale bar: 100 μm), F. Epithelial cells became confluent after 14 days, G and H. CD200 expression detected after culture (Red). Nuclei were stained with DAPI (blue) (scale bar: 200 μm).

Article Snippet: Slides were washed and treated with blocking reagent followed by overnight incubation with the primary antibodies KRT15 (Abcam1385 LHKRT15, 1:100) and goat anti-human CD200 (R&D Systems, 1:50).

Techniques: Produced, Staining, 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 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