Journal: Journal of leukocyte biology

Article Title: CD40 signaling restricts RNA virus replication in macrophages, leading to rapid innate immune control of acute virus infection

doi: 10.1002/JLB.4HI0420-285RR

Figure Lengend Snippet: A) Peritoneal cells from WT or CD40−/− male mice were infected with the indicated viruses (MOI=1 for all viruses except VSV where MOI = 0.5). Twenty-four hours following infection, RNA was extracted and viral load was quantified by qRT-PCR. B) Peritoneal cells from WT male mice were stimulated with an agonistic CD40 antibody, FGK4.5/FGK45, for 24 hours prior to infection and quantitation. C) Peritoneal cells from WT or CD40−/− mice were infected with EBOV (Mayinga) (MOI = 0.0015) and evaluated for virus load at 24 hours by qRT-PCR. D) Peritoneal cells from WT or CD40−/− mice were infected with by EBOV (Mayinga) (MOI = 0.0015) and number of infected cells were assessed at 24 hours by fixation followed by staining with anti-VP40 antibody and Hoescht dye. Infected cells were quantified by microscopy. E-F) Peritoneal cells from WT mice (E) or 6 day matured human MDMs (hMDMs) (F) were stimulated with an agonistic CD40 antibody (mouse CD40 antibody, FGK4.5/FGK45, or human CD40 antibody, G28.5) or left untreated (MOI = 0.0015 pmacs; MOI = 0.1 MDMs). After 24 hours, non-adherent cells were removed from the cultures and enriched pmacs or hMDMs were infected with EBOV(Mayinga). At 24 hours, cells were processed and stained as described in D. In parallel, supernatants from infected cells were titered on Vero cells and quantified using the same microscopic method. All experiments were performed at least three independent times. For all experiments, * indicates p<0.05. All qRT-PCR is quantified by delta Ct method comparing viral RNA to HPRT.

Article Snippet: In vivo ligation of CD40 was performed by administering a single 250 μg dose of an agonistic rat anti-mouse CD40 mAb (Clone FGK4.5, BioXCell, West Lebanon, NH) or rat IgG2a control (Clone 2A3, BioXCell) 24 hours prior to viral challenge.

Techniques: Infection, Quantitative RT-PCR, Quantitation Assay, Virus, Staining, Microscopy

Journal: Journal of leukocyte biology

Article Title: CD40 signaling restricts RNA virus replication in macrophages, leading to rapid innate immune control of acute virus infection

doi: 10.1002/JLB.4HI0420-285RR

Figure Lengend Snippet: A) WT or CD40−/− female mice were injected i.p. with 200 μg of agonistic CD40 antibody, blocking CD154 or IgG control as noted. Twenty-four hours later, mice were infected i.p. with a lethal dose of rVSV/EBOV GP. Peritoneal cells were isolated, RNA was harvested, and viral RNA was quantified by qRT-PCR at 24 hours of infection. B) Female Ifnar−/− and Ifnar/CD40−/− mice were infected i.p. with a dose of rVSV/EBOV GP that was lethal to Ifnar−/− mice. Twenty-four hours after infection, peritoneal cells were harvested, RNA was isolated and qRT-PCR analysis of viral RNA was performed. C-D) Enriched pmacs from male Ifnar−/− mice were incubated for 24 hours with agonistic CD40-specific mAb (C and D) or insect cell membranes expressing CD154 (C only). Treatments were removed and cells infected with rVSV/EBOV GP (MOI=1). Infection was quantified 24 hours following infection by flow cytometry and data are expressed as a percent change in the number of GFP+ cells relative to the unstimulated controls (C). Supernatants were titered on Vero cells (D). All experiments were performed three times. For all experiments, * indicates p<0.05.

Article Snippet: In vivo ligation of CD40 was performed by administering a single 250 μg dose of an agonistic rat anti-mouse CD40 mAb (Clone FGK4.5, BioXCell, West Lebanon, NH) or rat IgG2a control (Clone 2A3, BioXCell) 24 hours prior to viral challenge.

Techniques: Injection, Blocking Assay, Control, Infection, Isolation, Quantitative RT-PCR, Incubation, Expressing, Flow Cytometry

Journal: Journal of leukocyte biology

Article Title: CD40 signaling restricts RNA virus replication in macrophages, leading to rapid innate immune control of acute virus infection

doi: 10.1002/JLB.4HI0420-285RR

Figure Lengend Snippet: A) Peritoneal cells from male Ifnar−/− and Ifnar/CD40−/− mice were isolated. Forty-eight hours after isolation, cells were either washed to remove non-adherent cells, or incubated in the presence of non-adherent cells and infected with rVSV/EBOV GP (MOI=0.1). RNA was isolated 24 hours following infection and viral RNA was quantified by qRT-PCR. B) Peritoneal cells were isolated from male Ifnar−/− mice and cells expressing the indicated surface protein were removed via magnetic bead separation. The remaining cells were infected with rVSV/EBOV GP (MOI=0.1). RNA was isolated at 24 hours and quantified by qRT-PCR. C-D) Female C57BL/6 Ifnar−/− mice were injected i.p. with antibodies against CD4 and CD8 (200 μg each) or appropriate IgG controls. Twenty-four hours later, mice were infected i.p. with a sublethal dose of rVSV/EBOV GP. At 24 hours, serum was collected and titers were quantified by endpoint dilution on Vero cells (C) or peritoneal cell RNA was harvested, and viral RNA was quantified by qRT-PCR (D). All experiments were performed three times. All qRT-PCR is quantified by delta Ct method comparing VSV-M gene to HPRT. * indicates p<0.05.

Article Snippet: In vivo ligation of CD40 was performed by administering a single 250 μg dose of an agonistic rat anti-mouse CD40 mAb (Clone FGK4.5, BioXCell, West Lebanon, NH) or rat IgG2a control (Clone 2A3, BioXCell) 24 hours prior to viral challenge.

Techniques: Isolation, Incubation, Infection, Quantitative RT-PCR, Expressing, Injection

Journal: Journal of leukocyte biology

Article Title: CD40 signaling restricts RNA virus replication in macrophages, leading to rapid innate immune control of acute virus infection

doi: 10.1002/JLB.4HI0420-285RR

Figure Lengend Snippet: A-B) Pmacs were harvested from male Ifnar−/− mice and stimulated with agonistic CD40 antibody in the presence of varying concentrations of a TRAF6 inhibitor (7651589) for 7 hours. Following stimulation, RNA was isolated and IL-12p35 mRNA was quantified by qRT-PCR (A). In parallel, cells were stimulated for 24 hours with CD40 agonist (+/− 10 μM TRAF6 inhibitor 7651589) and subsequently challenged with rVSV/EBOV GP (MOI=1). RNA was harvested and viral RNA was assessed by qRT-PCR (B). C) Peritoneal cells were harvested from male Ifnar−/− mice and transfected with 3 TRAF6 siRNAs (25 nM each). At 48 hours, cells were stimulated with agonistic CD40 antibody or IL-12. After 24 hours cells were washed to remove non-adherent cells, media was refreshed, and cells were infected with rVSV/EBOV GP (MOI=1). RNA was harvested 24hpi and virus was quantified by qRT-PCR.

Article Snippet: In vivo ligation of CD40 was performed by administering a single 250 μg dose of an agonistic rat anti-mouse CD40 mAb (Clone FGK4.5, BioXCell, West Lebanon, NH) or rat IgG2a control (Clone 2A3, BioXCell) 24 hours prior to viral challenge.

Techniques: Isolation, Quantitative RT-PCR, Transfection, Infection, Virus

Journal: Journal of leukocyte biology

Article Title: CD40 signaling restricts RNA virus replication in macrophages, leading to rapid innate immune control of acute virus infection

doi: 10.1002/JLB.4HI0420-285RR

Figure Lengend Snippet: A/B) Peritoneal cells from male mice (A) or human monocyte derived macrophages (B) were untreated or treated with IL-12. After incubation, non-adherent cells were removed and enriched pmacs were infected for 24 hours with WT EBOV (MOI = 0.0015 pmacs; MOI = 0.1 MDMs) under BSL-4 containment. Cells were subsequently fixed, stained with anti-VP40 antibody and Hoescht dye, and infected cells were quantified by microscopy. Supernatants from infected cells were titered on Vero cells and quantified by the same microscopic method. C) Enriched pmacs from male Ifnar−/− mice were stimulated with IL-12 and at 24 hours infected with rVSV/EBOV GP (MOI=1) or VSV (MOI=0.5). Twenty-four hour following infection, supernatants were collected, filtered and TCID50s were determined on Vero cells. D/E) Pmacs were harvested from male Ifnar−/− mice and treated with agonistic CD40-specific mAb with or without the IL-12 inhibitor, apilimod, for 8 hours. RNA was harvested and qRT-PCR was performed to quantify IL-12p35 expression (D). In parallel, cells were stimulated with apilimod for 8 hours and infected with rVSV/EBOV GP (MOI=1). Twenty-four hours following infection, RNA was isolated and viral RNA was assessed by qRT-PCR (E). F) Enriched pmacs were obtained from Ifnar−/− and Ifnar/CD40−/− mice. Cells were stimulated with CD40 agonistic mAb or IL-12 and IL-12p35 gene expression was quantified by qRT-PCR. G) Pmacs from IL-12p40−/− mice were harvested and stimulated with agonistic CD40-specific antibody or IL-12 for 24h prior to infection with rVSV/EBOV GP (MOI=5). Twenty-four hours post infection, RNA was extracted and viral RNA was quantified by qRT-PCR. H) Enriched pmacs were obtained from Ifnar−/− and Ifnar/CD40−/− mice. Cells were stimulated with IL-12 and 24 hours later infected with rVSV/EBOV GP. Infections were evaluated for virus load by qRT-PCR at 24 hours. All experiments were performed three times. For all experiments, * indicates p<0.05.

Article Snippet: In vivo ligation of CD40 was performed by administering a single 250 μg dose of an agonistic rat anti-mouse CD40 mAb (Clone FGK4.5, BioXCell, West Lebanon, NH) or rat IgG2a control (Clone 2A3, BioXCell) 24 hours prior to viral challenge.

Techniques: Derivative Assay, Incubation, Infection, Staining, Microscopy, Quantitative RT-PCR, Expressing, Isolation, Gene Expression, Virus

Journal: Journal of leukocyte biology

Article Title: CD40 signaling restricts RNA virus replication in macrophages, leading to rapid innate immune control of acute virus infection

doi: 10.1002/JLB.4HI0420-285RR

Figure Lengend Snippet: A) Enriched pmacs were obtained from Ifnar−/− and Ifnar/CD40−/− mice. Cells were stimulated with CD40 agonist, IL-12 or IFN-γ and IRF-1 gene expression was quantified by qRT-PCR. B-C) Peritoneal cells from male WT (B) or CD40−/− (C) mice were incubated with agonistic CD40 mAb or IL-12 for 24 hours. Some cells were also treated with blocking anti-IFN-γ antibody as noted during antibody or cytokine treatment. After incubation, non-adherent cells were removed and enriched pmacs were infected with EBOV (Mayinga)(MOI = 0.0015) under BSL-4 conditions. At 24 hours of infection, cells were fixed, stained with anti-VP40 antibody and Hoescht dye, and infected cells were quantified by microscopy. In parallel, 24-hour supernatants from infected cells were titered on Vero cells and quantified by the same microscopic method. D-E) Enriched pmacs from male C57BL/6 Ifngr−/− mice were stimulated with the indicated stimuli. Twenty-four hours after stimulation, RNA was isolated from cells and IL-12p35 and IRF-1 mRNA was quantified by qRT-PCR (D) or infected with WT EBOV (MOI 0.0015) or rVSV/EBOV GP (MOI=5) and viral RNA was quantified 24 hours following infection by qRT-PCR (E). All experiments were performed three times. For all experiments, * indicates p<0.05.

Article Snippet: In vivo ligation of CD40 was performed by administering a single 250 μg dose of an agonistic rat anti-mouse CD40 mAb (Clone FGK4.5, BioXCell, West Lebanon, NH) or rat IgG2a control (Clone 2A3, BioXCell) 24 hours prior to viral challenge.

Techniques: Gene Expression, Quantitative RT-PCR, Incubation, Blocking Assay, Infection, Staining, Microscopy, Isolation

Journal: Journal of leukocyte biology

Article Title: CD40 signaling restricts RNA virus replication in macrophages, leading to rapid innate immune control of acute virus infection

doi: 10.1002/JLB.4HI0420-285RR

Figure Lengend Snippet: A) Survival curves of female WT (n=6) and CD40−/− (n=4) mice treated with 1 μg anti-IFNAR (MAR-1) antibody one day prior to challenge with a sublethal dose of rVSV/EBOV GP. Mice were monitored daily. B) Survival curve of female Ifnar−/− (n=9) and Ifnar/CD40−/− (n=13) mice that were challenged with a dose of rVSV/EBOV GP which was sublethal to Ifnar−/− mice by i.p. injection. Mice were monitored daily (DPI=days post infection). C) Survival curve of female Ifnar−/− (n=6) and Ifnar/CD40−/−(n=6) mice infected with a dose of rVSV/EBOV GP that was sublethal to Ifnar−/− mice by retro-orbital injection. Mice were monitored daily. D) Female Ifnar−/− (n=8) and Ifnar/CD40−/− (n=8) mice were infected i.p. with a dose of rVSV/EBOV GP that is sublethal to Ifnar−/− mice. Serum was collected at 12-hour intervals and viremia was assessed by end point dilutions on Vero cells. E/F) Female Ifnar−/− (n=12) and Ifnar/CD40−/− (n=12) mice were infected i.p. with a dose of rVSV/EBOV GP that is sublethal to Ifnar−/− mice. At 24 or 48 hours following infection, mice were anesthetized, perfused through the heart with PBS, and euthanized prior to organ harvest. RNA was isolated and qRT-PCR analysis of viral mRNA was performed (E). In parallel organs were homogenized and titers were assessed on Vero cells (n=4 organs/group) (F). All experiments were performed three times. For all experiments, * indicates p<0.05.

Article Snippet: In vivo ligation of CD40 was performed by administering a single 250 μg dose of an agonistic rat anti-mouse CD40 mAb (Clone FGK4.5, BioXCell, West Lebanon, NH) or rat IgG2a control (Clone 2A3, BioXCell) 24 hours prior to viral challenge.

Techniques: Injection, Infection, Isolation, Quantitative RT-PCR

Journal: Cell reports

Article Title: Gain-of-function p53 R172H mutation drives accumulation of neutrophils in pancreatic tumors, promoting resistance to immunotherapy

doi: 10.1016/j.celrep.2021.109578

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: InVivoMAb CD40 rat anti-mouse IgG2a mAb (FGK45) , BioXCell , Cat# BE0016-2; RRID:AB_1107647.

Techniques: Blocking Assay, Marker, Recombinant, Mutagenesis, TA Cloning, SYBR Green Assay, Staining, Software, Expressing

Journal: Cell reports

Article Title: Gain-of-function p53 R172H mutation drives accumulation of neutrophils in pancreatic tumors, promoting resistance to immunotherapy

doi: 10.1016/j.celrep.2021.109578

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: InVivoMAb CD40 rat anti-mouse IgG2a mAb (FGK45) , BioXCell , Cat# BE0016-2; RRID:AB_1107647.

Techniques: Blocking Assay, Marker, Recombinant, Mutagenesis, TA Cloning, SYBR Green Assay, Staining, Software, Expressing

Journal: Cell reports

Article Title: Gain-of-function p53 R172H mutation drives accumulation of neutrophils in pancreatic tumors, promoting resistance to immunotherapy

doi: 10.1016/j.celrep.2021.109578

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: InVivoMAb CD40 rat anti-mouse IgG2a mAb (FGK45) , BioXCell , Cat# BE0016-2; RRID:AB_1107647.

Techniques: Blocking Assay, Control, Marker, Virus, Recombinant, Mutagenesis, TA Cloning, SYBR Green Assay, Reverse Transcription, Staining, Software, Gene Expression, Expressing

Journal: Cell reports

Article Title: Gain-of-function p53 R172H mutation drives accumulation of neutrophils in pancreatic tumors, promoting resistance to immunotherapy

doi: 10.1016/j.celrep.2021.109578

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: InVivoMAb CD40 rat anti-mouse IgG2a mAb (FGK45) , BioXCell , Cat# BE0016-2; RRID:AB_1107647.

Techniques: Blocking Assay, Control, Marker, Virus, Recombinant, Mutagenesis, TA Cloning, SYBR Green Assay, Reverse Transcription, Staining, Software, Gene Expression, Expressing