Journal: Frontiers in Oncology

Article Title: YB-1-based oncolytic virotherapy in combination with CD47 blockade enhances phagocytosis of pediatric sarcoma cells

doi: 10.3389/fonc.2024.1304374

Figure Lengend Snippet: Infection with XVir-N-31 (XVir) increases both calreticulin (CALR) and CD47 surface expression on pediatric sarcoma cell lines. (A) Analysis of CALR (‘eat-me’) and CD47 surface expression (‘don’t-eat-me’) surface expression of pediatric sarcoma cell lines A673, SKNMC, and U2OS 48 hours post infection (hpi) at indicated multiplicity of infection (MOI) assessed by FACs analysis after dead cell exclusion via DAPI. Y-axis depicts the fold change of expression compared to controls (ctrl) using frequency of parent minus isotype (IT). (B) Analysis MOI/dose-dependency of CD47 surface expression at 48hpi using indicated MOI. Statistical analysis was performed using the unpaired student’s t-test in (A) and one way ANOVA with multiple comparison and Tukey correction in (B) . Plotted is the mean with SD. Each dot represents one biological replicate. Experiments were repeated at least twice to ensure reproducibility. Levels of significance are indicated as asterisks *p<0,0332; **p<0,0021; ***p<0,0002; ****p<0,0001.

Article Snippet: Following antibodies were used: Human CD47 Antibody, (#AF4670, R&D Systems, 1:500), Adenovirus-Hexon (#AB1056, Merck Millipore, 1:1000), GAPDH (#2118S, Cell Signaling Technology, 1:2000), Mouse anti-rabbit IgG HRP (#Sc-2357, Santa Cruz Biotech, 1:1000), anti-sheep IgG HRP (#HAF016, R&D Systems, 1:1000).

Techniques: Infection, Expressing, Comparison

Journal: Frontiers in Oncology

Article Title: YB-1-based oncolytic virotherapy in combination with CD47 blockade enhances phagocytosis of pediatric sarcoma cells

doi: 10.3389/fonc.2024.1304374

Figure Lengend Snippet: The combination (combo) of XVir-N-31 (XVir) and the CD47-inhbitor (CD47i) B6H12.2 shows the highest levels of phagocytosis for all tested phagocytes and cell lines. Phagocytosis by THP-1 macrophages (A) , THP-1 imDCs (B) , and healthy donor-derived monocytic imDCs (C) was assessed at indicated MOI (48hpi) and time (y-axis) for A673 (left panels) and U2OS (right panels). CD47i was added when starting phagocytosis. Each dot represents one biological replicate. Experiments were repeated at least three times. Plotted is the normalized phagocytosis compared to ctrl as mean and SD. One way ANOVA with multiple comparison and Tukey correction was used for statistical analysis. Levels of significance are indicated as asterisks *p<0,0332; **p<0,0021; ***p<0,0002; ****p<0,0001.

Article Snippet: Following antibodies were used: Human CD47 Antibody, (#AF4670, R&D Systems, 1:500), Adenovirus-Hexon (#AB1056, Merck Millipore, 1:1000), GAPDH (#2118S, Cell Signaling Technology, 1:2000), Mouse anti-rabbit IgG HRP (#Sc-2357, Santa Cruz Biotech, 1:1000), anti-sheep IgG HRP (#HAF016, R&D Systems, 1:1000).

Techniques: Derivative Assay, Comparison

Journal: Frontiers in Immunology

Article Title: IL-27 Derived From Macrophages Facilitates IL-15 Production and T Cell Maintenance Following Allergic Hypersensitivity Responses

doi: 10.3389/fimmu.2021.713304

Figure Lengend Snippet: CD3, CD47, and CD172a expression in human ACD clusters. (A–C) Immunofluorescence staining of CD3 (green), CD47 (red), CD172a (purple), CD14 [green, a serial slide section with the staining of CD172a (purple)], and Hoechst (blue) in human donor-matched patch-test negative control and patch-test (+) ACD skin. White dashed lines mark the epidermal-dermal junction. Data are representative of 3 patient samples per tested condition. (A) Scale bars are 200 µm (left) and 100 µm (right). (B, C) Scale bars are 20 µm.

Article Snippet: Mouse IgG1 isotype control (MOPC-21) (Tonbo Biosciences), Goat IgG isotype control (R&D Systems), Sheep IgG isotype control (R&D Systems), Rabbit isotype control (Southern Biotech, Birmingham, AL), anti-human CD14 (61D3, Tonbo Biosciences), anti-human iNOS (polyclonal, Thermo Fisher Scientific), anti-human CD8 (MCD8, Santa Cruz Biotechnology, Dallas, TX), and IL27R (polyclonal, R&D Systems), anti-human IL-27 (polyclonal, R&D Systems), anti-human CD86 (IT2.2, Biolegend), anti-human CD3 (SP7, Abcam, Cambridge, England), anti-human CD47 (polyclonal, R&D Systems), anti-human SIRP alpha (CD172a) (OTI7B3, Origene), anti-human IL-15 (polyclonal, R&D systems), anti-human BCL2 (clone 100, BioLegend), anti-mouse CD3 (17A2, Tonbo Biosciences), and anti-mouse CD8 (YTS 105.18, Novus Biologicals, Littleton, CO) followed by reaction with Cy3, Alexa Fluor 555, Alexa Fluor 647, Alexa Fluor 488, or FITC-conjugated secondary antibodies (Thermo Fisher Scientific).

Techniques: Expressing, Immunofluorescence, Staining, Negative Control

Journal: Blood

Article Title: Targeted inhibition of CD47-SIRPα requires Fc-FcγR interactions to maximize activity in T-cell lymphomas

doi: 10.1182/blood.2019001744

Figure Lengend Snippet: Human CD47 protein expression is heterogeneous, whereas MHC class I expression is homogeneous across TCL. (A) CD47 expression on normal peripheral blood-derived CD3+ T cells from healthy donors and TCL lines was determined by flow cytometry. CD47 surface density was plotted after mean fluorescence intensity was normalized for cell size. Each point represents a different healthy donor (n = 10) or TCL line (ALCL = 9 [DL-40, FEPD, KI-JK, L82, MAC2A, Karpas 299, SR-786, SU-DHL-1, SUP-M2], CTCL = 4 [HH, HuT-78, MJ, Myla], NKTCL = 3 [MTA, KHYG-1, SNK6], γδTCL [Karpas 384], HSTCL [DERL-2], PTCL-NOS = 2 [OCI-Ly12, SMZ-1], ATLL = 1 [HuT-102], and NKLGL = 1 [NKL]) or BCL line (Burkitt [Raji, Daudi], DLBCL [SU-DHL-2, SU-DHL-5, and Mantle cell [JVM-2]). **P = .0021 by 2-sided Welch t test. Normalized mean expression for each population was: CD3+ T cells 69.85 (range, 47.1-93.5) and TCL lines 176.9 (range, 20.9-694.8). (B) Resting CD3+ T cells from 2 additional donors (D#1, D#2) and TCL whole cell lysates were immunoblotted with the indicated antibodies. (C) Differential CD47 expression measured by RNA-seq was batch corrected and plotted. Three healthy donors were used for naïve, effector, and follicular helper T-cell groups. Cell lines: ALK+ ALCL = 3 (KI-JK, L82, SR-786), ALK- ALCL = 3 (MAC2A, OCI-Ly13.2, FEPD), PTCL-NOS = 2 (OCI-Ly12, SMZ-1), CTCL = 3 (HH, HuT-78, Myla), NK/TCL = 2 (KHYG-1, MTA). PDXs: ALK+ ALCL = 2 (WCTL-91953, WCTL-81162), AITL = 3 (DFTL-94393, DFTL-78024, DFTL-47880), HSTL = 1 (DFTL-81777), ATLL = 1 (DFTL-69579), T-PLL = 1 (DFTL-28776), and NK/TCL = 1 (DFTL-85005). (D) Representative images of patient lymph nodes stained for H&E, CD47 showing low and high H-scores and for MHC class I. Scale bar: 0.05 mm. (E) Histological analysis of CD47 expression in TCL patient-derived primary samples in the TMA (other: CTCL = 3, ATLL = 5, NK/TCL = 3, T-PLL = 1, subcutaneous panniculitis-like TCL = 2, γ/Δ TCL = 2, TCL unclassifiable = 3, and T-cell lymphoblastic lymphoma = 2). (F) PFS comparison between quartile groups by H-score (log-rank, P = .43). (G) PFS comparison between quartile groups by percent CD47+ lymphoma cells (log-rank, P = .38). H&E, hematoxylin and eosin.

Article Snippet: Transduced cell lines were cultured for 5 to 7 days in RPMI1640 supplemented with 20% fetal calf serum and l -glutamine before depletion of CD47-expressing cells using an anti-CD47 mAb (B6H12.2, ATCC) and magnetic beads coated with sheep anti-mouse IgG (Dynabeads, Life Technologies, Carlsbad, CA).

Techniques: Expressing, Derivative Assay, Flow Cytometry, Fluorescence, RNA Sequencing, Staining, Comparison

Journal: Blood

Article Title: Targeted inhibition of CD47-SIRPα requires Fc-FcγR interactions to maximize activity in T-cell lymphomas

doi: 10.1182/blood.2019001744

Figure Lengend Snippet: Therapy with anti-CD47 antibody SRF231 induces durable responses in TCL PDX and murine models in vivo. (A-F) Tumor burden and organ weights of mice engrafted with the HSTCL PDX DFTL-81777 and treated with hIgG4 isotype control or anti-CD47 antibody. (G-N) Tumor burden in blood and BM (n = 3 per antibody group). *P < .05; **P < .01; ***P < .001, ****P < .0001 by 2-sided Welch t test. Kaplan-Meier survival analyses (n = 3-6 per antibody group) by log-rank test. Arrows indicate start (day 0) and stop (day 10 for models 81777 and 22685) and start (day 0) and stop (day 4 for model 85005) of treatment. (O) Kaplan-Meier survival analysis of ITK-SYK xenograft model treated with murine anti-CD47 antibody, MIAP410, or with mIgG1κ isotype control, MOPC-21 (n = 3 per antibody group) log-rank test. BM, bone marrow.

Article Snippet: Transduced cell lines were cultured for 5 to 7 days in RPMI1640 supplemented with 20% fetal calf serum and l -glutamine before depletion of CD47-expressing cells using an anti-CD47 mAb (B6H12.2, ATCC) and magnetic beads coated with sheep anti-mouse IgG (Dynabeads, Life Technologies, Carlsbad, CA).

Techniques: In Vivo, Control

Journal: Blood

Article Title: Targeted inhibition of CD47-SIRPα requires Fc-FcγR interactions to maximize activity in T-cell lymphomas

doi: 10.1182/blood.2019001744

Figure Lengend Snippet: Anti-CD47 antibody efficacy is macrophage-mediated in TCL PDX. (A) Burden at involved sites for each mouse. P values by 2-sided Welch t test. (B) Flow plots of Gr-1+ neutrophils in the peripheral blood of mice before and after treatment with anti-Ly6G Ab. (C) Flow plots of F4/80+ macrophages in the peripheral blood of mice before and after treatment with clodronate. PBS, phosphate-buffered saline.

Article Snippet: Transduced cell lines were cultured for 5 to 7 days in RPMI1640 supplemented with 20% fetal calf serum and l -glutamine before depletion of CD47-expressing cells using an anti-CD47 mAb (B6H12.2, ATCC) and magnetic beads coated with sheep anti-mouse IgG (Dynabeads, Life Technologies, Carlsbad, CA).

Techniques: Saline

Journal: Blood

Article Title: Targeted inhibition of CD47-SIRPα requires Fc-FcγR interactions to maximize activity in T-cell lymphomas

doi: 10.1182/blood.2019001744

Figure Lengend Snippet: Anti-human CD47 monoclonal antibodies, B6H12 and SRF231 induce phagocytosis of TCL cells over nonmalignant T cells in vitro. (A) B6H12 induced phagocytosis of peripheral blood CD3+ T cells from 4 healthy donors using mBMDM and hMDM in comparison with isotype control (mIgG1κ) are presented. The human anti-CD20 mAb rituximab (Rit) and anti-CD52 mAb alemtuzumab (Alem) both with hIgG1 isotype were used as negative and positive controls, respectively. (B, C) B6H12-mIgG1κ and SRF231 mediated phagocytosis of HuT-78, Myla, and HH (cutaneous T-cell lymphoma) and SUP-M2, MAC2A, Karpas 299 (K-299) (peripheral T-cell lymphoma) cells compared with isotype controls (mIgG1κ and hIgG4). Fold change in CFSE+ of CD11b+ mBMDMs and of CD14+ hMDMs relative to the isotype control (mIgG1κ for B6H12-mIgG1κ, hIgG4 for SRF231, and hIgG1 for B6H12-hIgG1) for that cell line. Alemtuzumab was used as a positive control for induction of ADCP for CD52-expressing HuT-78 cell line and as a negative control for other lines. B6H12-mIgG1κ and B6H12-hIgG1 mediated phagocytosis of Myla and MAC2A relative to isotype controls is plotted for direct comparison between antibody isotypes. (D) Flow plots of mBMDM-engulfed CFSE+ tumor cells with SRF231 and hIgG4 isotype control upon incubation of tumor cells ex vivo harvested from PDXs (ALK+ ALCL [WCTL-81162], T-PLL [DFTL-28776], AITL [DFTL-47880], and PTCL-NOS [DFTL-84867]).

Article Snippet: Transduced cell lines were cultured for 5 to 7 days in RPMI1640 supplemented with 20% fetal calf serum and l -glutamine before depletion of CD47-expressing cells using an anti-CD47 mAb (B6H12.2, ATCC) and magnetic beads coated with sheep anti-mouse IgG (Dynabeads, Life Technologies, Carlsbad, CA).

Techniques: Bioprocessing, In Vitro, Comparison, Control, Positive Control, Expressing, Negative Control, Incubation, Ex Vivo

Journal: Blood

Article Title: Targeted inhibition of CD47-SIRPα requires Fc-FcγR interactions to maximize activity in T-cell lymphomas

doi: 10.1182/blood.2019001744

Figure Lengend Snippet: Anti-CD47 monoclonal antibodies exert minimal apoptosis, no CDC, but affect ADCC. (A) TCL cells were incubated with the indicated antibodies at 10 µg/mL or 10 μM staurosporine (positive control) for 2 h and the percentage of live cells was quantified by flow cytometry. (B, C) CDC assay with 30% murine and human complement was performed in triplicate and % PI+ cells are reported. Rituximab and Raji cells were used as positive controls, whereas trastuzumab was used as negative control. (D) ADCC reporter assay measuring luminescence with murine FcγRIIIA-expressing Jurkat cells was performed in triplicate at an effector:target ratio of 25:1 with indicated antibodies. Human anti-CD3 mAb (OKT3) was used as positive control along with its isotype control, mIgG2aκ. Fold induction is relative to isotype control (mIgG1κ for B6H12 and mIgG2aκ for OKT3). Shown are mean values of technical triplicates with error bars representing SEM. *P < .05, **P < .01, ***P < .001, ****P < .0001 by 2-sided Welch t-test. (E) CFSE-labeled TCL cells were incubated with human-derived NK cells in the presence of indicated antibodies for 4 hours. Percentage of propidium iodide positive (% PI pos) (of CFSE+) cells was determined by flow cytometry and plotted. Rituximab and Raji cells were used as positive controls whereas trastuzumab was used as negative control. Shown are mean values of technical triplicates with error bars representing SEM. *P < .05, **P < .01, ***P < .001, ****P < .0001 by 2-sided Welch t test. SEM, standard error of the mean.

Article Snippet: Transduced cell lines were cultured for 5 to 7 days in RPMI1640 supplemented with 20% fetal calf serum and l -glutamine before depletion of CD47-expressing cells using an anti-CD47 mAb (B6H12.2, ATCC) and magnetic beads coated with sheep anti-mouse IgG (Dynabeads, Life Technologies, Carlsbad, CA).

Techniques: Bioprocessing, Incubation, Positive Control, Flow Cytometry, CDC Assay, Negative Control, Reporter Assay, Expressing, Control, Labeling, Derivative Assay

Journal: Blood

Article Title: Targeted inhibition of CD47-SIRPα requires Fc-FcγR interactions to maximize activity in T-cell lymphomas

doi: 10.1182/blood.2019001744

Figure Lengend Snippet: Anti-CD47 antibody increases phagocytosis through FcγR-independent and FcγR-dependent mechanisms and is not Mac-1 dependent. (A) CFSE+ TCL cells were incubated in vitro with B6H12 and F(ab′)2 fragment of B6H12 or isotype control in the presence of mBMDMs and hMDMs. (B) Same as panel A, using full-length and F(ab′)2 fragments of 2D3 and MIAP410. (C) CFSE+ TCL cells were incubated with WT or Fcer1g−/− (KO) mBMDMs in the presence of the indicated antibodies. Shown are mean values of technical triplicates with error bars representing SEM. *P < .05, **P < .01, ***P < .001, ****P < .0001 by 2-sided Welch t test. (D-E) CFSE+ TCL cells were incubated with WT or Mac1−/− (KO) mBMDMs in the presence of the indicated antibodies or F(ab′)2 fragment of B6H12. A representative experiment performed in triplicate with error bars representing SEM is shown. (F) On the left, phagocytosis of CFSE-labeled CTCL cells, Myla and HH by hMDMs in the presence of various antibodies including Moga at concentrations of 5 µg/mL is plotted. On the right, percent PI positive of the CFSE-labeled Myla and HH cells is plotted after culture with human NK cells in the presence of various antibodies at concentrations of 10 µg/mL. Shown are mean values of technical triplicates with error bars representing SEM. *P < .05, **P < .01, ***P < .001, ****P < .0001 by 2-sided Welch t test. (G) Phagocytosis of Myla, HuT-78, MAC2A, and K-299 cells mediated by full-length B6H12-mIgG1κ, anti-HLA A,B,C mAb, W6/32 and their F(ab′)2 portions alone and in combination relative to isotype controls at 10 µg/mL (mIgG1κ for B6H12-mIgG1κ and mIgG2aκ for W6/32). Percentage of CFSE+ macrophages (of CD14+ hMDMs) is plotted. Shown are mean values of technical triplicates ± SEM. *P < .05; **P < .01; ***P < .001, ****P < .0001 by 2-sided Welch t test. KO, knockout; ns, not significant.

Article Snippet: Transduced cell lines were cultured for 5 to 7 days in RPMI1640 supplemented with 20% fetal calf serum and l -glutamine before depletion of CD47-expressing cells using an anti-CD47 mAb (B6H12.2, ATCC) and magnetic beads coated with sheep anti-mouse IgG (Dynabeads, Life Technologies, Carlsbad, CA).

Techniques: Incubation, In Vitro, Control, Labeling, Knock-Out

Journal: Blood

Article Title: Targeted inhibition of CD47-SIRPα requires Fc-FcγR interactions to maximize activity in T-cell lymphomas

doi: 10.1182/blood.2019001744

Figure Lengend Snippet: Therapy with anti-CD47 antibody SRF231 induces durable responses in TCL PDX and murine models in vivo. (A-F) Tumor burden and organ weights of mice engrafted with the HSTCL PDX DFTL-81777 and treated with hIgG4 isotype control or anti-CD47 antibody. (G-N) Tumor burden in blood and BM (n = 3 per antibody group). *P < .05; **P < .01; ***P < .001, ****P < .0001 by 2-sided Welch t test. Kaplan-Meier survival analyses (n = 3-6 per antibody group) by log-rank test. Arrows indicate start (day 0) and stop (day 10 for models 81777 and 22685) and start (day 0) and stop (day 4 for model 85005) of treatment. (O) Kaplan-Meier survival analysis of ITK-SYK xenograft model treated with murine anti-CD47 antibody, MIAP410, or with mIgG1κ isotype control, MOPC-21 (n = 3 per antibody group) log-rank test. BM, bone marrow.

Article Snippet: Therapeutic agents and antibodies B6H12.2, MIAP410, and murine IgG1κ isotype controls were obtained from BioXCell.

Techniques: In Vivo, Control

Journal: Blood

Article Title: Targeted inhibition of CD47-SIRPα requires Fc-FcγR interactions to maximize activity in T-cell lymphomas

doi: 10.1182/blood.2019001744

Figure Lengend Snippet: Anti-CD47 antibody increases phagocytosis through FcγR-independent and FcγR-dependent mechanisms and is not Mac-1 dependent. (A) CFSE+ TCL cells were incubated in vitro with B6H12 and F(ab′)2 fragment of B6H12 or isotype control in the presence of mBMDMs and hMDMs. (B) Same as panel A, using full-length and F(ab′)2 fragments of 2D3 and MIAP410. (C) CFSE+ TCL cells were incubated with WT or Fcer1g−/− (KO) mBMDMs in the presence of the indicated antibodies. Shown are mean values of technical triplicates with error bars representing SEM. *P < .05, **P < .01, ***P < .001, ****P < .0001 by 2-sided Welch t test. (D-E) CFSE+ TCL cells were incubated with WT or Mac1−/− (KO) mBMDMs in the presence of the indicated antibodies or F(ab′)2 fragment of B6H12. A representative experiment performed in triplicate with error bars representing SEM is shown. (F) On the left, phagocytosis of CFSE-labeled CTCL cells, Myla and HH by hMDMs in the presence of various antibodies including Moga at concentrations of 5 µg/mL is plotted. On the right, percent PI positive of the CFSE-labeled Myla and HH cells is plotted after culture with human NK cells in the presence of various antibodies at concentrations of 10 µg/mL. Shown are mean values of technical triplicates with error bars representing SEM. *P < .05, **P < .01, ***P < .001, ****P < .0001 by 2-sided Welch t test. (G) Phagocytosis of Myla, HuT-78, MAC2A, and K-299 cells mediated by full-length B6H12-mIgG1κ, anti-HLA A,B,C mAb, W6/32 and their F(ab′)2 portions alone and in combination relative to isotype controls at 10 µg/mL (mIgG1κ for B6H12-mIgG1κ and mIgG2aκ for W6/32). Percentage of CFSE+ macrophages (of CD14+ hMDMs) is plotted. Shown are mean values of technical triplicates ± SEM. *P < .05; **P < .01; ***P < .001, ****P < .0001 by 2-sided Welch t test. KO, knockout; ns, not significant.

Article Snippet: Therapeutic agents and antibodies B6H12.2, MIAP410, and murine IgG1κ isotype controls were obtained from BioXCell.

Techniques: Incubation, In Vitro, Control, Labeling, Knock-Out