Journal: Frontiers in Immunology

Article Title: Enhanced Expression of CD47 Is Associated With Off-Target Resistance to Tyrosine Kinase Inhibitor Gefitinib in NSCLC

doi: 10.3389/fimmu.2019.03135

Figure Lengend Snippet: Surface CD47 and CRT expression in EGFR wild-type and mutant NSCLC cells. Surface CD47 (A) and ecto-CRT protein expression (B) shown as geometric MFI in a panel of six different NSCLC cell lines. Each histogram represents the mean (± SD) of three to five independent experiments. Comparisons made by ANOVA with Fisher's post hoc multiple comparison analysis. ### p < 0.03, ## p < 0.01, # p < 0.0005. Below each histogram, a matrix table where all p values resulting from post hoc analysis are reported. Expression levels of CD47 (C) and CRT mRNA (D) in 226 untreated primary NSCL adenocarcinomas (GEO accession number GSE31210 ). Middle lines in box plots represent the medians and whiskers represent 5–95% CI ( ### p < 0.03, Kruskal-Wallis test).

Article Snippet: Anti-mouse/human/rat CD47 mAb or mouse IgG isotype control were purchased from Bio X Cell.

Techniques: Expressing, Mutagenesis, Comparison

Journal: Frontiers in Immunology

Article Title: Enhanced Expression of CD47 Is Associated With Off-Target Resistance to Tyrosine Kinase Inhibitor Gefitinib in NSCLC

doi: 10.3389/fimmu.2019.03135

Figure Lengend Snippet: Modulation by gefitinib of surface CD47 and CRT expression in EGFR wild-type and mutant NSCLC cells. Flow cytometric profiles of surface CD47 (A) and ecto-CRT expression (B) on DMSO-treated (CTRL, gray lines) and gefitinib-treated (GEF, red lines) NSCLC cells. Histograms show the mean (± SD) of fold changes of CD47 (C) and ecto-CRT (D) geometric MFI, relative to DMSO-treated controls ( N = 3–5, * p < 0.05, ** p < 0.01 paired two-tailed Student's t -test).

Article Snippet: Anti-mouse/human/rat CD47 mAb or mouse IgG isotype control were purchased from Bio X Cell.

Techniques: Expressing, Mutagenesis, Two Tailed Test

Journal: Frontiers in Immunology

Article Title: Enhanced Expression of CD47 Is Associated With Off-Target Resistance to Tyrosine Kinase Inhibitor Gefitinib in NSCLC

doi: 10.3389/fimmu.2019.03135

Figure Lengend Snippet: Gefitinib-induced CD47 down-regulation promotes tumor cell phagocytosis by dendritic cells. Representative flow cytometric analyses and mean ± SD ( N = 4 independent healthy donors) of phagocytic activity of monocyte-derived dendritic cells (see Methods) against PC9 (A,B) , HCC827 (C,D) , and H1975 cells (E,F) treated with DMSO (CTRL) or gefitinib (GEF) as indicated. Cancer cells exposed to the drug for 48 h were labeled with DiO tracer and then co-cultured with dendritic cells for 2 h at a 1:1 ratio. Phagocytosis assays were also run at 4°C as controls. Histograms represent the percentages of positive cells for both CD11c and DiO tracer relative to total dendritic cells (* p < 0.05, n.s., not significant, paired two-tailed Student's t -test).

Article Snippet: Anti-mouse/human/rat CD47 mAb or mouse IgG isotype control were purchased from Bio X Cell.

Techniques: Activity Assay, Derivative Assay, Labeling, Cell Culture, Two Tailed Test

Journal: Frontiers in Immunology

Article Title: Enhanced Expression of CD47 Is Associated With Off-Target Resistance to Tyrosine Kinase Inhibitor Gefitinib in NSCLC

doi: 10.3389/fimmu.2019.03135

Figure Lengend Snippet: Blocking of CD47 on tumor cells induces phagocytosis by dendritic cells. Dendritic cells were co-cultured with DiO tracer-labeled HCC827 (A) and H1975 (B) cancer cells in the presence of IgG isotype control or anti-CD47 mAb as indicated. Shown is the mean (± SD, N = 3 independent healthy donors) percentage increase of CD11c/DiO tracer double positive cells, relative to dendritic cells co-cultured with DMSO-treated tumor cells ( # p < 0.05, ## p < 0.01, ANOVA with Fisher's post hoc analysis).

Article Snippet: Anti-mouse/human/rat CD47 mAb or mouse IgG isotype control were purchased from Bio X Cell.

Techniques: Blocking Assay, Cell Culture, Labeling, Control

Journal: Frontiers in Immunology

Article Title: Enhanced Expression of CD47 Is Associated With Off-Target Resistance to Tyrosine Kinase Inhibitor Gefitinib in NSCLC

doi: 10.3389/fimmu.2019.03135

Figure Lengend Snippet: Expression levels of surface CD47 increase in cancer cells acquiring resistance to gefitinib and inhibit tumor cell phagocytosis by dendritic cells. Surface CD47 (A) and ecto-CRT expression (B) in gefitinib-sensitive PC9 and HCC827 (gray lines) and resistant PC9GR and HCC827GR (green lines) cell lines. Representative flow cytometric histograms (left) and mean (± SD, N = 3–5) fold changes of treatment-resistant over sensitive cells (right). (C) Representative flow cytometric histogram plots (left) and mean (± SD) fold changes (right) of surface CD47 levels in resistant cell lines treated with DMSO (CTRL) or gefitinib (GEF) as indicated. Acquisition of resistance to gefitinib abolished drug-induced CD47 down-regulation in PC9GR (* p < 0.05, ** p < 0.01, n.s., not significant, paired two-tailed Student's t -test). (D) Mean ± SD ( N = 3 independent healthy donors) of phagocytic activity of monocyte-derived dendritic cells against PC9GR cells in the absence or presence of gefitinib treatment, performed at 4°C as control and at 37°C. Histograms represent the percentages of positive cells for both CD11c and DiO tracer relative to total dendritic cells (paired two-tailed Student's t -test. n.s., not significant). (E) Dendritic cells were co-cultured with gefitinib-treated, DiO tracer-labeled PC9GR cells in the presence of IgG isotype control or anti-CD47 mAb. Shown is the mean ± SD ( N = 3 independent healthy donors) percent change of CD11c + /DiO + tracer double positive dendritic cells, relative to dendritic cells co-cultured with DMSO-treated tumor cells ( ## p < 0.01, ANOVA with Fisher's post hoc analysis).

Article Snippet: Anti-mouse/human/rat CD47 mAb or mouse IgG isotype control were purchased from Bio X Cell.

Techniques: Expressing, Two Tailed Test, Activity Assay, Derivative Assay, Control, Cell Culture, Labeling

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: The pivotal role of cytotoxic NK cells in mediating the therapeutic effect of anti-CD47 therapy in mycosis fungoides

doi: 10.1007/s00262-021-03051-x

Figure Lengend Snippet: CD47 is overexpressed on malignant lymphocytes in mycosis fungoides (MF) tumors. a A representative image of a skin involved by MF demonstrates intense CD47 staining on atypical TOX + malignant cells. HE, hematoxylin and eosin (20x). b Targeted single-cell RNA transcriptomics as tSNE plots of concatenated tumors from three patients with MF tumors (n = 287 cells total). Clusters called by recursive dendrogram split and annotated from preferentially expressed genes. Tcm, T cell central memory; Tem, T cell effecor memory; DC, dendritic cells. c The intensity of CD47 expression (anti-CD47 antibody-oligo conjugate; AbSeq) over the various cell population defined in Fig. 1b. d Statistical analysis of expression of CD47 (molecules per cells) in different cell populations. *, p < 0.05; ***, p < 0.001 e CD47 expression on CD3 + TOX + MBL2 cells. Flow cytometry of a cell suspension from a primary cell culture. Grey tinted area, an isotype control. Red tinted area, anti-CD47 antibody. f Tumor growth curves of CD47hi WT MBL2 (WT) and CD47 KO MBL2 (CD47 KO) after implantation in B6.SJL mice. n = 5 mice in each group. g Representative imaging of mice 10 days after implantation of CD47hi WT MBL2 (WT) or CD47 KO MBL2 (KO) cells demonstrating large ulcerated tumor in WT, while KO mouse exhibited medium-size tumor without ulceration

Article Snippet: Antibody Anti-mouse CD47 antibody (MIAP301, rat IgG2aκ; RRID:AB_2687793) was obtained from BioXcell (West Lebanon, NH), stored at 4 °C in the dark, and diluted to 2 mg/mL immediately before use.

Techniques: Staining, Expressing, Flow Cytometry, Suspension, Cell Culture, Control, Imaging

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: The pivotal role of cytotoxic NK cells in mediating the therapeutic effect of anti-CD47 therapy in mycosis fungoides

doi: 10.1007/s00262-021-03051-x

Figure Lengend Snippet: Anti-CD47 therapy is efficient in controlling the malignant lymphoma growth in a murine model of mycosis fungoides (MF). a Design of anti-CD47 experiments (I.P., intraperitoneal). b Tumor thickness after MBL2 implantation during treatment with anti-CD47 antibody or irrelevant IgG control (n = 5 mice per group). c Percentage of TOX + tumor cells in the inflammatory infiltrate of auricular skin at day 24 after implantation. The percentage of caspase-3 + cells is indicated in black (n = 5 mice per group) (Tx, treatment). d Percentage of F4/80 + cells in the inflammatory infiltrate of auricular skin at day 24 after implantation (n = 5 mice per group). e Percentage of MHC class II + cells among F4/80 + cells in the inflammatory infiltrate of auricular skin at day 24 after implantation (n = 5 mice per group). f Percentage of TNF-α + cells among F4/80 + cells in the inflammatory infiltrate of auricular skin at day 24 after implantation (n = 5 mice per group). g Percentage of NK1.1 + cells in the inflammatory infiltrate of auricular skin at day 24 after implantation (n = 5 mice per group). h Percentage of IFN-γ + cells among NK1.1 + cells in the inflammatory infiltrate of auricular skin at day 24 after implantation (n = 5 mice per group). i Percentage of CD62L + NKG2A-cells among NK1.1. + cells in the inflammatory infiltrate of auricular skin at day 24 after implantation (n = 5 mice per group). j Schematic of experimental design. k Representative images of mice with tumors after treatment with IFN-α, anti-CD47, or combination anti-CD47 + IFN-α. l The percentage of malignant cells (TOX +) in the TME on day 24 after implantation of MBL2 (n = 5 mice per group). m Representative flow cytometry of CD107a and IFN-γ NK cells (gated on NK1.1 + cells) during therapy with anti-CD47 antibody. n Quantification of CD107a + IFN-γ + NK cells and CD107a + IFN-γ-NK cells (n = 5 mice per group). o The cytotoxic assay of NK cells derived from splenocytes of treated mice co-cultured with MBL2 cells at the indicated effector to target cell ratios

Article Snippet: Antibody Anti-mouse CD47 antibody (MIAP301, rat IgG2aκ; RRID:AB_2687793) was obtained from BioXcell (West Lebanon, NH), stored at 4 °C in the dark, and diluted to 2 mg/mL immediately before use.

Techniques: Control, Flow Cytometry, Derivative Assay, Cell Culture

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: The pivotal role of cytotoxic NK cells in mediating the therapeutic effect of anti-CD47 therapy in mycosis fungoides

doi: 10.1007/s00262-021-03051-x

Figure Lengend Snippet: The effect of anti-CD47 therapy is mediated by cytotoxic NK cells and does not depend on IFN-γ. a Schematic of NK1.1 depletion experiment. b A representative flow showing the percentage of NK cells in non-depleted and NK-depleted mice prior to therapy. c Representative images of mice with tumors 14 days after MBL2 implantation with and without NK1.1 depletion prior to therapy initiation. D The percentage of malignant cells (CD3 + TOX + cells) per ear in non-depleted and NK-depleted mice 24 days after MBL2 implantation. N = 5 mice per group. ***, p < 0.001 e Tumor thickness 24 days after MBL2 implantation in mice treated with irrelevant IgG or anti-CD47 antibody (n = 5 mice per group). f Schematic of experimental design for IFN-γ KO mice. g The volume of lymph nodes 24 days after MBL2 implantation in mice treated with anti-CD47 antibody (n = 3 mice per group). *,p < 0.05; **,p < 0.01; ****,p < 0.0001; ns, non-significant

Article Snippet: Antibody Anti-mouse CD47 antibody (MIAP301, rat IgG2aκ; RRID:AB_2687793) was obtained from BioXcell (West Lebanon, NH), stored at 4 °C in the dark, and diluted to 2 mg/mL immediately before use.

Techniques:

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: The pivotal role of cytotoxic NK cells in mediating the therapeutic effect of anti-CD47 therapy in mycosis fungoides

doi: 10.1007/s00262-021-03051-x

Figure Lengend Snippet: The anti-CD47 therapy is accompanied by an influx of NK cells in the TME in patients with relapsed/refractory mycosis fungoides (MF). a Representative images of high and low CD56 number in the dermal infiltrate of tumor MF. b Inverse correlation of the number of CD56 cells per 100 dermal lymphocytes and histoscore of CD47 on the epidermotropic malignant lymphocytes (n = 17, r = – 0.48, p < 0.05). c Representative images of a patient treated with six intra-tumoral injections of 10 mg TTI-621 demonstrate a significant reduction of all tumors and plaques after treatment (I, injected tumor; C, control non-injected tumor). d Multispectral fluorescent immunohistochemistry of CD3, CD56, TOX, and DAPI staining of representative tumors before and after treatment with TTI-621 (60X). e Percentage of malignant cells (CD3 + CD4 + TOX +) and NK cells (CD3-CD56 +) in the TME (n = 11 patients; 4 responders and 7 non-responders). f tSNE analysis of cellular composition in patients with cutaneous T cell lymphoma who had responded to therapy with TTI-621 (n = 4 patients), showing eight distinct clusters of NK cells. g Phenotypic characterization of eight distinct clusters of NK cells depending on the percentage of markers. h Grouping of eight NK cell clusters in two groups based on high vs. dim CD56 expression. i Changes in CD56high NK cells vs. CD56dim NK cells after intra-tumoral injection of TTI-621 (2 weeks of therapy) in patients who responded to TTI-621 (n = 4). j Percentage of NK cells before and after six intra-tumoral injections of TTI-621 (2 weeks of therapy) in patients who responded to TTI-621 (n = 4).. *,p < 0.05; **,p < 0.01

Article Snippet: Antibody Anti-mouse CD47 antibody (MIAP301, rat IgG2aκ; RRID:AB_2687793) was obtained from BioXcell (West Lebanon, NH), stored at 4 °C in the dark, and diluted to 2 mg/mL immediately before use.

Techniques: Injection, Control, Immunohistochemistry, Staining, Expressing

Journal: Mucosal immunology

Article Title: Neutrophil expressed CD47 regulates CD11b/CD18-dependent neutrophil transepithelial migration in the intestine in vivo .

doi: 10.1038/s41385-020-0316-4

Figure Lengend Snippet: a Schematic presentation of different fractions collected for neutrophil (PMN) quantification (left) and model of CD47 regulation of CD11b/CD18-dependent PMN transepithelial migration (TEpM) across intestinal mucosa (right). b Analysis of PMN TEpM in vivo using an ileal loop model reveals that Cd47 −/− mice have significantly reduced numbers of PMN that migrated to the intestinal lumen 1 hr after intraluminal instillation of leukotriene B 4 (LTB 4 ; 1 nM) compared with WT mice. Dots represent individual mice. Data are Means ± SEM of three independent experiments, 14 mice/group (*p ≤ 0.05) as determined by Mann-Whitney U test. c, d After luminal content collection, sections of intestinal loop were enzymatically digested to obtain a Lamina propria (LP)-enriched fraction or fixed and paraffin-embedded for histological analysis. c Quantification of leukocytes by flow cytometry shows comparable numbers of PMN within the LP from WT and Cd47 −/− mice. Data are Means ± SEM of 2 independent experiments, 8 mice/group. d Representative images of immunohistochemical staining depicting Ly6G + PMN. 20x (top) and 40x (bottom) objectives. Scale bars: 100 μm and 50 μm, respectively.

Article Snippet: Recombinant murine E-selectin-Fc, VCAM-1-Fc and ICAM-1-Fc chimera molecules, and the polyclonal goat anti-mouse CD47 (cat#AF1866) and goat anti-human JAM-A (cat#AF1077) antibodies were from R&D Systems (Minneapolis, MN).

Techniques: Migration, In Vivo, MANN-WHITNEY, Flow Cytometry, Immunohistochemical staining, Staining

Journal: Mucosal immunology

Article Title: Neutrophil expressed CD47 regulates CD11b/CD18-dependent neutrophil transepithelial migration in the intestine in vivo .

doi: 10.1038/s41385-020-0316-4

Figure Lengend Snippet: a CD47 expression on bone marrow neutrophils (BMN) by western blotting and b densitometry analysis of three experiments reveal that nearly 80% of CD47 is selectively depleted on PMN from mice with specific deletion of CD47 on PMN ( MRP8-Cre;Cd47 fl/fl ) compared to Cd47 fl/fl control mice. c Reduced surface expression of CD47 was corroborated by flow cytometry on BMN from MRP8-Cre;Cd47 fl/fl compared to Cd47 fl/fl mice. d Analysis of PMN TEpM in vivo using a murine ileal loop model reveals significant reduction of transmigrated PMN into the intestinal lumen 1 hr after intraluminal instillation of LTB 4 (1 nM) in MRP8-Cre;Cd47 fl/fl compared with Cd47 fl/fl mice. Data are Means ± SEM of at least three independent experiments, 16-18 mice/group (**p≤0.01) as determined by Mann-Whitney U test. e PMN TEpM in vivo in mice with specific deletion of CD47 on IECs ( Villin-Cre;Cd47 fl/fl ) revealed similar numbers of PMN in the intestinal lumen 1 hr after intraluminal instillation of LTB 4 (1 nM) compared with Cd47 fl/fl control littermates. Data are Means ± SEM of 2 independent experiments, 6-9 mice/group.

Article Snippet: Recombinant murine E-selectin-Fc, VCAM-1-Fc and ICAM-1-Fc chimera molecules, and the polyclonal goat anti-mouse CD47 (cat#AF1866) and goat anti-human JAM-A (cat#AF1077) antibodies were from R&D Systems (Minneapolis, MN).

Techniques: Expressing, Western Blot, Control, Flow Cytometry, In Vivo, MANN-WHITNEY

Journal: Mucosal immunology

Article Title: Neutrophil expressed CD47 regulates CD11b/CD18-dependent neutrophil transepithelial migration in the intestine in vivo .

doi: 10.1038/s41385-020-0316-4

Figure Lengend Snippet: a PMN TEpM in vivo in response to LTB 4 (1 nM) was determined in ileal loops of WT and Cd47 −/− mice after intraluminal instillation of blocking mAb against CD11b (clone M1/70; 20 μg/mL), or rat IgG 2b isotype control (20 μg/mL). WT PMN migration was significantly reduced by blocking CD11b in the intestinal lumen compared to mice treated with isotype control. CD47 deficiency also resulted in a significant reduced PMN migration in Cd47 −/− mice compared with WT mice. Importantly, blocking CD11b on Cd47 −/− mice did not further decrease PMN migration compared to Cd47 −/− mice treated with isotype control. Data are Means ± SEM of 3 independent experiments, 12-14 mice/group (**p≤0.01) as determined by two-way ANOVA analysis. b After luminal content collection, intestinal sections were enzymatically digested for quantification of PMN in LP-enriched fraction by flow cytometry, or fixed and paraffin-embedded for histological analysis. Numbers of PMN within the LP fraction were similar between different treatments. Data are Means ± SEM of 2 independent experiments, 8-14 mice/group. c Representative images of immunohistochemical staining including higher magnification insets, depicting Ly6G+ PMN. 40x objective. Scale bars: 50 μm.

Article Snippet: Recombinant murine E-selectin-Fc, VCAM-1-Fc and ICAM-1-Fc chimera molecules, and the polyclonal goat anti-mouse CD47 (cat#AF1866) and goat anti-human JAM-A (cat#AF1077) antibodies were from R&D Systems (Minneapolis, MN).

Techniques: In Vivo, Blocking Assay, Control, Migration, Flow Cytometry, Immunohistochemical staining, Staining

Journal: Mucosal immunology

Article Title: Neutrophil expressed CD47 regulates CD11b/CD18-dependent neutrophil transepithelial migration in the intestine in vivo .

doi: 10.1038/s41385-020-0316-4

Figure Lengend Snippet: a Schematic representation of BMN migration in vitro in response to a chemoattractant gradient of LTB 4 (10 nM) for 2 hr at 37°C. b CD47 cell surface expression on BMN isolated from WT and Cd47 −/− mice by flow cytometry. c WT and Cd47 −/− BMN migration in response to LTB 4 in presence of blocking mAb against CD11b, CD47 or isotype control (20 μg/mL in each case). WT BMN migration was significantly reduced by addition of inhibitory CD11b or CD47 mAb. Loss of CD47 on Cd47 −/− BMN resulted in a significant reduced migration compared to WT BMN. Addition of blocking mAbs against CD11b or CD47 did not further reduce Cd47 −/− BMN migration in comparison to Cd47 −/− BMN migration in the presence of isotype mAb. Data are Means ± SEM of three independent experiments. *p ≤ 0.01 as determined by two-way ANOVA analysis.

Article Snippet: Recombinant murine E-selectin-Fc, VCAM-1-Fc and ICAM-1-Fc chimera molecules, and the polyclonal goat anti-mouse CD47 (cat#AF1866) and goat anti-human JAM-A (cat#AF1077) antibodies were from R&D Systems (Minneapolis, MN).

Techniques: Migration, In Vitro, Expressing, Isolation, Flow Cytometry, Blocking Assay, Control, Comparison

Journal: Mucosal immunology

Article Title: Neutrophil expressed CD47 regulates CD11b/CD18-dependent neutrophil transepithelial migration in the intestine in vivo .

doi: 10.1038/s41385-020-0316-4

Figure Lengend Snippet: a, b Murine CD18, CD11a, CD11b and CD47 were immunoprecipitated from non-stimulated WT and Cd47 −/− BMN lysates and immunoblotted for CD47 as detailed in the methods. a Representative western blot confirming the expression of CD47 in total lysates from WT but CD47 loss in Cd47 −/− BMN. b CD47 was co-immunoprecipitated with CD11b and CD18, and, to a lesser extent, with CD11a on WT BMN. Cd47 −/− BMN were used as negative control. c The association between CD47 and CD11b on unstimulated, non-permeabilized BMN was confirmed by in situ proximal ligation assay (PLA) using a combination of CD47 and CD11b Abs as detailed in the methods. The positive fluorescent staining (white dots) indicates close proximity (<40 nm) between CD47 and CD11b targets. Positive controls include use of CD11b-CD18 Abs as a known heterodimeric interaction. Furthermore, as a negative control, CD47-CD11b interaction was probed on BMN from Cd47 −/− mice and no signal was detected. Nuclei were counter-stained with Hoechst 33342. 100x objective. Scale bars: 50 μm.

Article Snippet: Recombinant murine E-selectin-Fc, VCAM-1-Fc and ICAM-1-Fc chimera molecules, and the polyclonal goat anti-mouse CD47 (cat#AF1866) and goat anti-human JAM-A (cat#AF1077) antibodies were from R&D Systems (Minneapolis, MN).

Techniques: Immunoprecipitation, Western Blot, Expressing, Negative Control, In Situ, Ligation, Staining

Journal: Mucosal immunology

Article Title: Neutrophil expressed CD47 regulates CD11b/CD18-dependent neutrophil transepithelial migration in the intestine in vivo .

doi: 10.1038/s41385-020-0316-4

Figure Lengend Snippet: a WT and Cd47 −/− BMN express comparable surface levels of CD11a and CD11b integrins. b-d BMN were drawn across coverslips coated with immobilized ICAM-1-Fc, VCAM-1-Fc or E-selectin-Fc adhesion molecules at decreasing levels of shear stress ranging from 1.5 to 0.75 dynes/cm 2 , and BMN adhesion was measured as described in methods. In some experiments, BMN were pre-treated with function blocking mAbs anti-CD11a (M1/70; 20 μg/mL) or anti-CD11b (M1/70; 20 μg/mL). Data are Means ± SEM of at least three independent experiments. *p≤0.05, **p≤0.01, ***p≤0.001 as measured by ANOVA analysis followed by Tukey’s multiple comparison test.

Article Snippet: Recombinant murine E-selectin-Fc, VCAM-1-Fc and ICAM-1-Fc chimera molecules, and the polyclonal goat anti-mouse CD47 (cat#AF1866) and goat anti-human JAM-A (cat#AF1077) antibodies were from R&D Systems (Minneapolis, MN).

Techniques: Shear, Blocking Assay, Comparison

Journal: Mucosal immunology

Article Title: Neutrophil expressed CD47 regulates CD11b/CD18-dependent neutrophil transepithelial migration in the intestine in vivo .

doi: 10.1038/s41385-020-0316-4

Figure Lengend Snippet: CD47 was knocked down by CRISPR/Cas9 in the human promyelocitic cell line (HL60). HL60 and CD47-null HL60 (HL60/E2) were then differentiated into a neutrophil-like phenotype as described in the methods. a, b CD11b expression on resting, differentiated HL60 was not significantly altered by CD47 knockdown: a cell surface expression of CD11b on differentiated PMN was quantified by flow cytometry and b total CD11b content was determined by western blotting. Image shows a representative western blot and graph represents a densitometry analysis of three different assays. c CD47 and CD11b association in HL60 was assessed by PLA. Positive fluorescent signals were elicited between CD47 and CD11b Abs, as well as between CD11b and CD18 Abs indicating close association of these proteins. CD47 and CD11b Abs did not elicit fluorescent signals on HL60/E2 (CD47 null). Nuclei were counterstained with Hoechst 33342. 100x objective. Scale bars: 50 μm. d Differentiated HL60 and HL60/E2 were stimulated with fMLF (1 μM) for different times and CD11b surface expression was determined by flow cytometry. Increased surface expression of total CD11b after fMLF stimulation was not different between HL60 and HL60/E2. e, f CD11b and CD18 activation on HL60 was determined by using activation reporter mAbs, CBRM1/5 and m24 respectively. fMLF stimulation resulted in activation of CD11b and CD18 on HL60 control cells, but the magnitude was significantly reduced in HL60/E2. Data are Means ± SEM of at least three independent experiments. *p≤0.05, **p≤0.01 as determined by Two-way ANOVA analysis. No Tx (no fMLF treatment).

Article Snippet: Recombinant murine E-selectin-Fc, VCAM-1-Fc and ICAM-1-Fc chimera molecules, and the polyclonal goat anti-mouse CD47 (cat#AF1866) and goat anti-human JAM-A (cat#AF1077) antibodies were from R&D Systems (Minneapolis, MN).

Techniques: CRISPR, Expressing, Knockdown, Flow Cytometry, Western Blot, Activation Assay, Control

Journal: Journal for Immunotherapy of Cancer

Article Title: Differential phagocytosis induces diverse macrophage activation states in malignant gliomas

doi: 10.1136/jitc-2025-012211

Figure Lengend Snippet: Combining radiation therapy with anti-CD47 mAb treatment enhances in vitro phagocytosis of human DIPG and mouse glioma cell lines. Human patient-derived DMG cell lines (BT-245, SU-DIPGXVII, and SU-DIPGXXV) were exposed to either 0 or 4 Gy×3 and incubated with human peripheral blood-derived macrophages in the presence of anti-CD47 mAb, HU5F9-G4. Flow cytometry ( A , C , and E ) as well as histogram ( B , D , and F ) plots show that combining fractionated irradiation and anti-CD47 antibody treatment increases the phagocytosis of DIPG/DMG cells by macrophages compared with individual treatments alone. ( G–J ) Mouse glioblastoma cell lines (SB28 and CT2A) were exposed to either 0 or 4 Gy×3 and incubated with mouse bone marrow-derived macrophages in the presence of anti-CD47 mAb, MIAP301. Data shown are consistent with two independent experiments (n=3) and are shown as mean+SD. Unpaired Student’s t-test. *p<0.05, **p<0.01 and ***p<0.0001. One-way analysis of variance, ****p<0.0001 (BT245), ***p<0.001 (DIPGXVII) and ****p<0.0001 (DIPGXXV). ***p<0.001 (CT2A) and **p<0.001 (SB28). DMG, diffuse midline glioma; mAb, monoclonal antibody.

Article Snippet: Mouse monoclonal anti-CD47 antibody (clone B6H12) was purchased from Bio X Cell (West Lebanon, New Hampshire, USA) and diluted in PBS (phosphate buffered saline) before use.

Techniques: In Vitro, Derivative Assay, Incubation, Flow Cytometry, Irradiation

Journal: Journal for Immunotherapy of Cancer

Article Title: Differential phagocytosis induces diverse macrophage activation states in malignant gliomas

doi: 10.1136/jitc-2025-012211

Figure Lengend Snippet: Single-cell RNA sequencing reveals the enrichment of distinct macrophage subsets following co-culture with diffuse midline glioma cells pretreated with either control, RT, anti-CD47 therapy, or combination of RT and anti-CD47 therapy. ( A ) Schematic diagram of the workflow used for in vitro phagocytosis and single-cell RNA sequencing. Briefly, BT245 cells were irradiated (4Gy×3) treated for three consecutive days and treated with either PBS or anti-CD47 mAb for 30 min at 37°C and co-cultured with PBMC-derived macrophages for 24 hours. BT245 cells not exposed to radiation and treated with PBS served as controls. Macrophages that either phagocytose (“eaters”) or do not phagocytose (“non-eaters”) tumor cells were sorted using flow cytometry and subjected to single-cell RNA-sequencing. ( B ) UMAP projection displaying 11 distinct cell clusters from the eater’s cohort. Each dotted line and arrow indicate the identity of that specific cell cluster. ( C–D ) UMAP plots and proportion of cells in each cluster from four treatment groups: control, 4 Gy×3, anti-CD47, or 4 Gy×3+anti-CD47. Dotted lines indicate the expansion/enrichment of distinct macrophage clusters in that treatment condition. Note the expansion/enrichment of two distinct cell clusters (1 and 3) in the combination treatment. ( E ) Heatmap of marker genes in each cell cluster. Representative genes with higher gene expression for each cluster are outlined on the left. Bubble plots demonstrating expression of marker genes associated with antigen presenting ( F ), inflammatory ( G ), M1-like ( H ), M2-like ( I ), proliferation ( J ), and tumor cell signature ( K ) by the various cell clusters. The dotted box indicates the cell clusters with higher average marker gene expression. The size of the bubble dot is proportional to the percentage of cells in a cluster expressing the marker gene and the color intensity is proportional to average scaled marker gene expression within a cluster. mAb, monoclonal antibody; PBMC, peripheral blood mononuclear cell; PBS, phosphate-buffered saline; RT, radiotherapy; UMAP, Uniform Manifold Approximation and Projection.

Article Snippet: Mouse monoclonal anti-CD47 antibody (clone B6H12) was purchased from Bio X Cell (West Lebanon, New Hampshire, USA) and diluted in PBS (phosphate buffered saline) before use.

Techniques: RNA Sequencing, Co-Culture Assay, Control, In Vitro, Irradiation, Cell Culture, Derivative Assay, Flow Cytometry, Marker, Gene Expression, Expressing, Saline

Journal: Journal for Immunotherapy of Cancer

Article Title: Differential phagocytosis induces diverse macrophage activation states in malignant gliomas

doi: 10.1136/jitc-2025-012211

Figure Lengend Snippet: Characterization of macrophages that are enriched following phagocytosis of diffuse midline glioma cells pretreated with Control, anti-CD47 therapy or RT. ( A ) Volcano plot showing the top differentially upregulated and downregulated genes in the macrophages that are enriched after co-culture with control treated BT245 cells (cell Cluster 0) compared with macrophages that were enriched after co-culture with either anti-CD47 (cell Cluster 1) or RT (cell Cluster 3) treated BT245 cells. ( B ) Gene Ontology enrichment analysis of biological process for significantly upregulated genes between control-enriched macrophages versus anti-CD47-enriched and RT-enriched macrophages. Note only the top 15 biological processes are shown. ( C ) Volcano plot showing the top differentially upregulated and downregulated genes in the macrophages that are enriched after co-culture with anti-CD47 treated BT245 cells (cell Cluster 1) compared with macrophages that were enriched after co-culture with either control (cell Cluster 0) or RT (cell Cluster 3) treated BT245 cells. ( D ) Gene Ontology enrichment analysis of biological processes for significantly upregulated genes between anti-CD47-enriched macrophages versus control-enriched and RT-enriched macrophages. Note only the top 15 biological processes are shown. ( E ) Volcano plot showing the top differentially upregulated and downregulated genes in the macrophages that are enriched after co-culture with RT treated BT245 cells (cell Cluster 3) compared with macrophages that were enriched after co-culture with either control (cell Cluster 0) or anti-CD47 (cell Cluster 1) treated BT245 cells. ( F ) Gene Ontology enrichment analysis of biological processes for significantly upregulated genes between control-enriched macrophages versus anti-CD47-enriched and RT-enriched macrophages. Note only the top 15 biological processes are shown. BP, biological process; FC, fold change; NS, not significant; RT, radiotherapy.

Article Snippet: Mouse monoclonal anti-CD47 antibody (clone B6H12) was purchased from Bio X Cell (West Lebanon, New Hampshire, USA) and diluted in PBS (phosphate buffered saline) before use.

Techniques: Control, Co-Culture Assay

Journal: Journal for Immunotherapy of Cancer

Article Title: Differential phagocytosis induces diverse macrophage activation states in malignant gliomas

doi: 10.1136/jitc-2025-012211

Figure Lengend Snippet: Combination of RT and anti-CD47 treatment reduces tumor burden and prolongs the survival of mice bearing BT245 xenografts compared with monotherapy. ( A ) Schematic diagram showing the experimental treatment plan followed. ( B ) Quantification of total IVIS flux values over time course. ( C ) Kaplan-Meier survival analysis of BT245 xenografts with indicated treatments, control, n=10; RT, n=9; anti-CD47, n=10; and RT+anti-CD47, n=10. The log-rank test was used to calculate statistical significance. *p<0.05, **p<0.01. ( D–F ) Bar graphs demonstrating the relative percentages of F4/80 + , CD80 + (M1-like) and CD206 + (M2-like) tumor-associated macrophages in control, RT, anti-CD47, or RT+anti-CD47 treated mice bearing BT245 xenografts. Data shown are obtained from n=3 mice for each group and are represented as mean+SD. Unpaired Student’s t-test: *p<0.05, **p<0.01 and ***p<0.0001. IP, intraperitoneal; IVIS, In Vivo Imaging System; RT, radiotherapy.

Article Snippet: Mouse monoclonal anti-CD47 antibody (clone B6H12) was purchased from Bio X Cell (West Lebanon, New Hampshire, USA) and diluted in PBS (phosphate buffered saline) before use.

Techniques: Control, In Vivo Imaging

Journal: Journal for Immunotherapy of Cancer

Article Title: Differential phagocytosis induces diverse macrophage activation states in malignant gliomas

doi: 10.1136/jitc-2025-012211

Figure Lengend Snippet: Combination of RT and anti-CD47 treatment reduces tumor burden and prolongs the survival of mice-bearing CT-2A intracranial allografts compared with monotherapy. ( A ) Schematic diagram showing the experimental treatment plan followed. ( B ) Quantification of total IVIS flux values over time course. ( C ) Kaplan-Meier survival analysis of CT-2A allografts with indicated treatments, control, n=8; RT, n=8; anti-CD47, n=8; and RT+anti-CD47, n=8. The log-rank test was used to calculate statistical significance. *p<0.05, **p<0.01, ***p<0.001. ns, not significant. ( D–F ) Bar graphs demonstrating the relative percentages of F4/80 + , CD80 + (M1-like) and CD206 + (M2-like) tumor-associated macrophages in control, RT, anti-CD47, or RT+anti-CD47 treated mice bearing CT-2A intracranial allografts. Data shown are obtained from n=3 mice for each group and are represented as mean+SD. Unpaired Student’s t-test: *p<0.05, **p<0.01 and ***p<0.0001. IP, intraperitoneal; IVIS, In Vivo Imaging System; RT, radiotherapy.

Article Snippet: Mouse monoclonal anti-CD47 antibody (clone B6H12) was purchased from Bio X Cell (West Lebanon, New Hampshire, USA) and diluted in PBS (phosphate buffered saline) before use.

Techniques: Control, In Vivo Imaging

Journal: Journal for Immunotherapy of Cancer

Article Title: Differential phagocytosis induces diverse macrophage activation states in malignant gliomas

doi: 10.1136/jitc-2025-012211

Figure Lengend Snippet: Validation of marker genes identified from single-cell RNA-sequencing using tumor-associated macrophages obtained from murine diffuse midline gliomas and glioblastoma intracranial allografts treated with either control, RT, anti-CD47, or RT+anti-CD47. ( A ) Dot plot indicates the average expression of CLEC7A, CD44 ( D ), and ( G ) for each cell cluster identified from single-cell RNA-sequencing. ( B–C ) Representative overlay histograms and median fluorescence intensity (MFI) values of CLEC7A, CD44 ( E–F ), and CD63 ( H–I ), expression in gliomas isolated from mice treated with either phosphate-buffered saline (control), RT, anti-CD47 therapy, or RT with anti-CD47 combination therapy. Data shown are obtained from n=3 mice for each group and are represented as mean+SD. Unpaired Student’s t-test: *p<0.05, **p<0.01 and ***p<0.0001. RT, radiotherapy.

Article Snippet: Mouse monoclonal anti-CD47 antibody (clone B6H12) was purchased from Bio X Cell (West Lebanon, New Hampshire, USA) and diluted in PBS (phosphate buffered saline) before use.

Techniques: Biomarker Discovery, Marker, RNA Sequencing, Control, Expressing, Fluorescence, Isolation, Saline