Journal: Oncogene

Article Title: miR-21 Depletion in Macrophages Promotes Tumoricidal Polarization and Enhances PD-1 Immunotherapy

doi: 10.1038/s41388-018-0178-3

Figure Lengend Snippet: (a, b) qPCR measurement of (a) Tnf , Il6 , and Nos2 and (b) Argc1, Mrc1 , and Il4ra gene transcripts in WT and miR-21 −/− bone marrow-derived macrophages at resting conditions, co-cultured with B16 cells, or co-cultured with B16 cells in addition to IFN-γ and LPS or IL-4 treatment. Y axis denotes log 2 values of the relative expression levels of genes (except for Il4ra , which was the relative level of Il4ra ) normalized to Gapdh . NOS2 was undetectable (N.D.) without stimulation. (c–f) Mature miR-21 was measured by qPCR in WT BMDMs upon stimulation with IFN-γ, IFN-γ and LPS, IL-4, or co-culture with B16 cells. Y axis denotes the relative expression levels of miR-21 using snoRNA-153 as a reference. Values are mean ± s.e.m.; * P ≤ 0.05, * * P ≤ 0.01, *** P ≤ 0.001.

Article Snippet: To stimulate MEFs and BMDMs, LPS was purchased from Sigma-Aldrich, recombinant murine IFN-γ was from Gold Biotechnology (St. Louis, MO), and recombinant murine IL-4 and recombinant M-CSF were from Peprotech (Rocky Hill, NJ).

Techniques: Derivative Assay, Cell Culture, Expressing, Co-Culture Assay

Journal: Oncogene

Article Title: miR-21 Depletion in Macrophages Promotes Tumoricidal Polarization and Enhances PD-1 Immunotherapy

doi: 10.1038/s41388-018-0178-3

Figure Lengend Snippet: (a) Putative miR-21 binding sites within the 3′UTRs of STAT1 and JAK2. The matching sites are indicated by the vertical lines. (b) Activity of luciferase reporters containing wild type (WT) or mutant (Mut) miR-21 target sites in the STAT1 3′UTR. (c) mRNA levels of Stat1 and Jak2 in WT and miR-21 −/− BMDMs. *P ≤ 0.05, * * P ≤ 0.01, n.s., not significant. (d and e) Immunoblotting analysis of protein levels of JAK1, JAK2, phosphorylation of STAT1 on tyrosine 701 (pSTAT1), STAT1, PDCD4, and actin in BMDMs (d) and MEFs (e). (f and g) Immunoblotting analysis of protein levels of pSTAT1, STAT1, PDCD4 and actin in miR-21 −/− MEFs transfected with miR-21 (f) or in WT MEFs transfected with a locked nucleic acid miR-21 inhibitor (g) and treated with IFN-γ (20 ng/mL) for up to 60 min. (h) Immunoblotting analysis of protein levels of phospho-IKKα/β, phospho-p65, phospho-IκB, and total IκB in WT and miR-21 −/− BMDMs upon LPS (50 ng/mL) stimulation for the indicated times. (i) Immunoblotting analysis of protein levels of pSTAT6, STAT6, PDCD4, and actin in WT and miR-21 −/− BMDMs upon stimulation with IL-4 (10 ng/mL) for indicated times.

Article Snippet: To stimulate MEFs and BMDMs, LPS was purchased from Sigma-Aldrich, recombinant murine IFN-γ was from Gold Biotechnology (St. Louis, MO), and recombinant murine IL-4 and recombinant M-CSF were from Peprotech (Rocky Hill, NJ).

Techniques: Binding Assay, Activity Assay, Luciferase, Mutagenesis, Western Blot, Transfection

Journal: Oncogene

Article Title: miR-21 Depletion in Macrophages Promotes Tumoricidal Polarization and Enhances PD-1 Immunotherapy

doi: 10.1038/s41388-018-0178-3

Figure Lengend Snippet: (a, b) Flow cytometry analysis of PD-L1 expression in TAMs isolated from tumors as described in . (a) The portion of PD-L1-positive TAMs in total macrophages. (b) Mean fluorescence intensity (MFI) of the PD-L1 signal from TAMs. (c) Immunoblotting analysis of the protein levels of JAK2, pSTAT1, and PD-L1 in immortalized WT and miR-21 −/− BMDMs treated with IFN-γ. (d–i) Mice implanted with B16 and macrophages as described in were treated with IgG or antibodies against PD-1. N = 6–7 per group. (d) Representative images of tumors at 16 days post inoculation. (e) Tumor weights. (f) Tumor volumes. Cell populations within tumors were analyzed by flow cytometry analysis; (g) M1 and M2 TAMs; (h) M1:M2 ratio; and (i) CD8 + T cells within tumors. (j) The role of miR-21 in macrophage polarization of TAMs. miR-21 suppresses the expression of STAT1, JAK2, and PDCD4 to inhibit STAT1 and NF-κB activation and prevent TAMs towards M1 polarization. Yet elevated STAT1 activation mediated by miR-21 deficiency promotes PD-L1 expression in TAMs, which can be mitigated by PD-1 antibody blockade. * P ≤ 0.05, *** P ≤ 0.001; n.s., not significant.

Article Snippet: To stimulate MEFs and BMDMs, LPS was purchased from Sigma-Aldrich, recombinant murine IFN-γ was from Gold Biotechnology (St. Louis, MO), and recombinant murine IL-4 and recombinant M-CSF were from Peprotech (Rocky Hill, NJ).

Techniques: Flow Cytometry, Expressing, Isolation, Fluorescence, Western Blot, Activation Assay

Journal: PLoS ONE

Article Title: Selective Blockade of Interferon-α and -β Reveals Their Non-Redundant Functions in a Mouse Model of West Nile Virus Infection

doi: 10.1371/journal.pone.0128636

Figure Lengend Snippet: Type I IFNs (IFN-β, 800 pg; IFN-α1, 40,000 pg; IFN-α4, 4000 pg; IFN-α5, 400 pg; IFN-α11, 1000 pg; or IFN-α13, 1000 pg) were preincubated with 1 to 10 μg of HDβ-4A7 ( A, B ) or 0.1 to 10 μg of TIF-3C5 ( C, D ), MAR1-5A3 (10 μg) or control hamster IgG (10 μg) for 30 min then added to fibrosarcoma cells for 72 h and stained for H2-K b class I MHC antigens by flow cytometry. Plots and histograms are representative of three or more independent assays using either F510 ( A, B ) or 1969 ( C, D ) fibrosarcoma cells.

Article Snippet: Recombinant murine IFN-α1, -α4, -α11 and-α13 were provided by Anthony Coyle and Ricardo Cibotti (MedImmune, Inc., Gaithersburg, MD).

Techniques: Control, Staining, Flow Cytometry

Journal: PLoS ONE

Article Title: Selective Blockade of Interferon-α and -β Reveals Their Non-Redundant Functions in a Mouse Model of West Nile Virus Infection

doi: 10.1371/journal.pone.0128636

Figure Lengend Snippet: Ten units IFN-β ( A ) or IFN-α ( B ) were preincubated for 1 h with indicated mAbs and then added to L929 cells overnight. Ten units/well type I IFN activity used in these assays are as follows: IFN-β, 16 pg; IFN-αA, 22 pg; IFN-α1, 15 pg; IFN-α4, 70 pg; IFN-α5, 20 pg; IFN-α13, 13 pg. IFN-treated cells subsequently were infected with VSV for 48 h and cell viability determined by crystal violet staining and optical density measurements. Results shown are representative of three or more independent experiments.

Article Snippet: Recombinant murine IFN-α1, -α4, -α11 and-α13 were provided by Anthony Coyle and Ricardo Cibotti (MedImmune, Inc., Gaithersburg, MD).

Techniques: Activity Assay, Infection, Staining

Journal: PLoS ONE

Article Title: Selective Blockade of Interferon-α and -β Reveals Their Non-Redundant Functions in a Mouse Model of West Nile Virus Infection

doi: 10.1371/journal.pone.0128636

Figure Lengend Snippet: Approximately 20 units of recominant IFN-β ( A ) or IFN-α1 ( B ), IFN-α4 ( C ) or IFN-α13 ( D ) were preincubated for 1 h with indicated mAbs and then added to L929 cells overnight. Constant levels of Type I IFN used in these assays are as follows: IFN-β, 4.4 ng; IFN-α1, 0.15 ng; IFN-α4, 0.05 ng; IFN-α13, 34 ng. IFN-treated cells subsequently were infected with VSV for 48 h and cell viability determined by crystal violet staining and optical density measurements. Results shown are representative of two independent experiments.

Article Snippet: Recombinant murine IFN-α1, -α4, -α11 and-α13 were provided by Anthony Coyle and Ricardo Cibotti (MedImmune, Inc., Gaithersburg, MD).

Techniques: Infection, Staining

Journal:

Article Title: Dendritic cell maturation enhances CD8 + T-cell responses to exogenous antigen via a proteasome-independent mechanism of major histocompatibility complex class I loading

doi: 10.1046/j.1365-2567.2003.01664.x

Figure Lengend Snippet: Antigen dose dependence of CD8+ T-cell responses to ISCOMS-associated antigen. DC were pulsed with 0–10 μg/ml OVA ISCOMS with or without LPS activation, washed and co-cultured with OT-1 lymphocytes. (a,b) The expression of CD69 and CD25 on CD8+ Vα2+ T cells was assessed after 24 hr in culture, and (c) IFN-γ production was assessed after 48 hr in culture. Results shown are mean ±1 SD for triplicate cultures. (*P < 0·02; **P < 0·01 versus OVA ISCOMS pulse only).

Article Snippet: As described previously, 13 interferon-γ (IFN-γ) levels were determined using a sandwich enzyme-linked immunosorbent assay, with a pair of IFN-γ-specific antibodies (PharMingen) and standardized with known concentrations of recombinant murine IFN-γ (PharMingen).

Techniques: Activation Assay, Cell Culture, Expressing