Journal: Frontiers in Immunology

Article Title: Monocytes Differentiate to Immune Suppressive Precursors of Metastasis-Associated Macrophages in Mouse Models of Metastatic Breast Cancer

doi: 10.3389/fimmu.2017.02004

Figure Lengend Snippet: Metastasis-associated macrophage (MAM) precursor cells (MAMPCs) suppress cytotoxicity of CD8 + T cells through a reactive oxygen species (ROS)-mediated mechanism. (A,B) Effects of myeloid cells on the CD8 + T cell-induced tumor cell apoptosis. Splenic CD8 + T cells from normal C57BL/6 mice were cultured with anti-CD3/CD28 antibodies (effector; E) in the absence or presence of MAMPCs, MAMs, or resident macrophages (RMACs) from the metastatic lung of E0771-LG-injected mice (MAMPC/E, MAM/E, RMAC/E, respectively). The preincubated T cells were then isolated and cultured with E0771-LG cells expressing red fluorescent protein in the nuclei (target; T) at the indicated E:T ratio in the presence of green fluorogenic caspase-3 substrate. After 36 h, the number of apoptotic cancer cells indicated by red/green double positive nuclei was counted. (A) Representative images of cells cultured with the caspase-3 substrate for 36 h (E:T = 4:1). Scale bar; 50 µm, arrowhead; apoptotic cancer cell. (B) Number of apoptotic cancer cells cultured with preactivated CD8 + T cells ( n = 3, two independent experiments). Data are mean ± SEM, * P < 0.01 vs. E + T (4:1), # P < 0.01 vs. MAMPC/E + T. (C) Fold-change of genes encoding checkpoint T cell receptor ligands in MAMPCs and MAMs compared with classical monocytes (C-MOs) determined by microarray analyses in Figure (logFC > 1, FDR < 0.05). Data on expression values are presented as mean ± SEM. Note that the scale is exponential. (D) Mean fluorescence intensity of checkpoint T cell receptor ligands assessed by flow cytometry in C-MOs, MAMPCs, and MAMs ( n = 3, two independent experiments). Blood (for C-MOs) and lung digestion (for MAMPCs and MAMs) were prepared from E0771-LG-injected C57BL/6 mice at 14 days posttumor injection and stained with antibodies for indicated markers or isotype IgG. Data are mean ± SEM, * P < 0.01 vs. IM, # P < 0.01 vs. MAMPC. (E) Effects of checkpoint inhibitors on the suppressive activity of MAMPCs and MAMs ( n = 6, two independent experiments). CD8 + T cells were cultured with anti-CD3/CD28 antibodies and neutralizing antibodies for PD1 or CTLA4, or isotype IgG in the absence (effector; E) or presence of MAMPCs (MAMPC/E) or MAMs (MAM/E). Cytotoxicity of the precultured CD8 + T cells against E0771-LG cells at 4:1 E/T ratio were assessed as described above. Data are mean ± SEM that represent the ratio in number of apoptotic cancer cells relative to that induced by CD8 + T cells cultured with IgG in the absence of MAMPCs or MAMs (control). * P < 0.01 vs. control, # P < 0.01 vs. IgG. (F) Effects of inhibitors of nitric oxide or ROS production on the suppressive activity of MAMPCs and MAMs ( n = 6, two independent experiments). CD8 + T cells were cultured with anti-CD3/CD28 antibodies and L-NMMA, nor-NOHA, catalase and superoxide dismutase (SOD) (Cat/SOD), or vehicle (–) in the absence (E) or presence of MAMPCs (MAMPC/E) or MAMs (MAM/E). Cytotoxicity of the precultured CD8 + T cells against E0771-LG cells at 4:1 E/T ratio were assessed as described above. Data are mean ± SEM that represent the ratio of apoptotic cancer cells relative to that induced by CD8 + T cells cultured with PBS in the absence of MAMPCs or MAMs (control). * P < 0.01 vs. control, # P < 0.01 vs. PBS.

Article Snippet: We intraperitoneally injected d -luciferin in PBS (GoldBio, 1.5 mg/100 μL/20 g mouse) into anesthetized E0771-LG:Fl tumor-bearing mice.

Techniques: Cell Culture, Injection, Isolation, Expressing, Microarray, Fluorescence, Flow Cytometry, Staining, Activity Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: TSLP Interferes with Airway Tolerance by Suppressing the Generation of Antigen-specific Regulatory T cells 1

doi: 10.4049/jimmunol.1002503

Figure Lengend Snippet: A, TSLP suppressed murine iTreg differentiation in the presence of TGF-β1. Naïve CD4+CD62L+ T cells were isolated from spleens and lymph nodes, cultured with 10 μg/ml plate-bound anti-CD3 and 2 μg/ml anti-CD28 in the presence of 3 ng/ml TGF-β1 for 5 days. TSLP was used at 10 ng/ml. Data represent mean ± SEM (n = 3). B, TSLP suppressed human iTreg differentiation in the presence of TGF-β1. Naïve CD4+ T cells were isolated from PBMC and cultured in the same condition as (A). Human T cells showed considerable variability in responding to TSLP. A responder is shown on top, a non-responder on the bottom. The percentage inhibition of iTreg differentiation by TSLP from all 20 donor samples is indicated in the graph on the right. C, Purity of naïve CD4+ T cells isolated from human PBMC. Samples of PBMC and purified T cells from the same donor were stained with Blood Dendritic Cell Enumeration Kit (Miltenyi Biotec) and analyzed by flow cytometry. Data represent mean ± SEM (n = 6). D, Quantitative PCR analysis of the expression of human TSLP receptor TSLPR in activated CD4+ T cells, presented relative to naïve cells. Including of TSLP in culture (+TSLP) further enhanced TSLPR expression. Naïve CD4+ T cells were purified from PBMC and cultured in the same condition as (A). Data represent mean ± SEM (n = 6). *: p < 0.05; **: p < 0.01 One way analysis of variance.

Article Snippet: Since it is reported that human TSLP mainly exerts its effect through DCs and had little effect on CD4 + T cells ( 32 ), we examined DC contamination in our purified human naïve CD4+ T cells by Blood Dendritic Cell Enumeration Kit (Miltenyi Biotec).

Techniques: Isolation, Cell Culture, Inhibition, Purification, Staining, Flow Cytometry, Real-time Polymerase Chain Reaction, Expressing

Journal: Journal of Virology

Article Title: Interferon-Induced Cell Membrane Proteins, IFITM3 and Tetherin, Inhibit Vesicular Stomatitis Virus Infection via Distinct Mechanisms ▿

doi: 10.1128/JVI.01328-10

Figure Lengend Snippet: Antiviral activities of IFITMs and tetherin against VSV in FLP-IN T Rex cells, expressing CAT or the desired ISGs, left untreated or treated with 1 μg/ml of tetracycline for 24 h. (A and C) Cells were harvested, and induction levels of IFITMs and tetherin expression were confirmed by Western blot analyses with antibody against Flag tag or tetherin protein. (B and D) Alternatively, cells were infected with VSV at an MOI of 0.001 and culturing continued in medium without or with tetracycline for an additional 16 h. VSV titers in the supernatants of the infected cells were determined by a plaque assay with Vero cells. Error bars indicate standard deviations of the means from three experiments. Statistical analysis was performed using Student's t test.

Article Snippet: Membranes were blocked with PBS containing 5% nonfat dry milk and probed with antibodies against Flag tag (Sigma), tetherin (Proteintech Group, Inc.), IFITM3 (Proteintech Group, Inc.), VSV G protein (Rockland, Inc.), or β-actin (Chemicon International).

Techniques: Expressing, Western Blot, FLAG-tag, Infection, Plaque Assay

Journal: Journal of Virology

Article Title: Interferon-Induced Cell Membrane Proteins, IFITM3 and Tetherin, Inhibit Vesicular Stomatitis Virus Infection via Distinct Mechanisms ▿

doi: 10.1128/JVI.01328-10

Figure Lengend Snippet: IFITM and tetherin restrict VSV infection. (A) HeLa cells were transfected with SMARTpool siRNA targeting IFITM2 and IFITM3 or nontargeting siRNA (control), following the directions of the manufacturer (Dharmacon). Forty-eight hours posttransfection, cells were left untreated or were treated with the indicated concentrations of IFN-α for 24 h. Cells were harvested for Western blot analysis of IFITM protein in the cell lysates with an antibody recognizing IFITM2 and -3 (Proteintech). β-Actin served as the loading control. (B) Following IFN-α treatment, HeLa cells were infected with VSV at an MOI of 0.01. Virus yields in culture medium samples harvested at 24 h postinfection were determined with a plaque assay and are expressed as means ± standard errors (n = 3). *, P < 0.001. (C) HeLa cells were transfected with SMARTpool siRNA targeting tetherin or nontargeting siRNA (control), following the directions of the manufacturer (Dharmacon). Six hours posttransfection, cells were left untreated or treated with the indicated concentrations of IFN-α for 24 h. Cells were harvested for Western blot analysis of tetherin expression in the cell lysates or, alternatively, infected with VSV at an MOI of 0.01. (D) Virus yields in culture medium samples harvested at 24 h postinfection were determined with a plaque assay and are expressed as means ± standard errors (n = 3). Statistical analysis was performed using Student's t test. **, P < 0.0001; #, P < 0.05.

Article Snippet: Membranes were blocked with PBS containing 5% nonfat dry milk and probed with antibodies against Flag tag (Sigma), tetherin (Proteintech Group, Inc.), IFITM3 (Proteintech Group, Inc.), VSV G protein (Rockland, Inc.), or β-actin (Chemicon International).

Techniques: Infection, Transfection, Control, Western Blot, Virus, Plaque Assay, Expressing

Journal: Journal of Virology

Article Title: Interferon-Induced Cell Membrane Proteins, IFITM3 and Tetherin, Inhibit Vesicular Stomatitis Virus Infection via Distinct Mechanisms ▿

doi: 10.1128/JVI.01328-10

Figure Lengend Snippet: Expression and membrane topology of IFITM3 and tetherin in HEK293 cells. FLP-IN T Rex cell lines expressing either IFITM3 or tetherin were left untreated or treated with 1 μg/ml of tetracycline for 24 h. (A and B) Expression levels of IFITM3 (A) and tetherin (B) were visualized by immunofluorescent staining. Cell nuclei were visualized by using 4′,6-diamidino-2-phenylindole staining. (C and D) For FACS analysis, after induction of ISG expression, cells were either left untreated (intact) or permeabilized by incubation with buffer containing 0.1% Triton X-100. Expression of the Flag tag on the surface of intact cells (blue) or in the permeabilized cells (green) was revealed by flow cytometry. Histograms were gated to analyze the population expressing the Flag tag. Diagrams to depict the putative membrane topologies of IFITM3 and tetherin are also presented.

Article Snippet: Membranes were blocked with PBS containing 5% nonfat dry milk and probed with antibodies against Flag tag (Sigma), tetherin (Proteintech Group, Inc.), IFITM3 (Proteintech Group, Inc.), VSV G protein (Rockland, Inc.), or β-actin (Chemicon International).

Techniques: Expressing, Membrane, Staining, Incubation, FLAG-tag, Flow Cytometry

Journal: Journal of Virology

Article Title: Interferon-Induced Cell Membrane Proteins, IFITM3 and Tetherin, Inhibit Vesicular Stomatitis Virus Infection via Distinct Mechanisms ▿

doi: 10.1128/JVI.01328-10

Figure Lengend Snippet: Identification of the VSV replication step(s) targeted by IFITM3 and tetherin. FLP-IN T Rex cell lines expressing the control protein CAT or the antiviral proteins IFITM3 or tetherin were left untreated or treated with 1 μg/ml of tetracycline for 24 h prior to infection with VSV at an MOI of 5. At the indicated time points after infection, culture supernatants and cells were harvested. (A) The levels of cell-associated viral RNA were determined in a real-time RT-PCR assay, and the results are expressed as copies per 100 ng of total cellular RNA. (B) Virus yields were measured in a plaque assay. Error bars indicate standard deviations of the means (n = 3). (C) Accumulation of viral G protein was determined by Western blot assay. (D) Cells were fixed with 2% paraformaldehyde and photographed with a Nikon microscope.

Article Snippet: Membranes were blocked with PBS containing 5% nonfat dry milk and probed with antibodies against Flag tag (Sigma), tetherin (Proteintech Group, Inc.), IFITM3 (Proteintech Group, Inc.), VSV G protein (Rockland, Inc.), or β-actin (Chemicon International).

Techniques: Expressing, Control, Infection, Quantitative RT-PCR, Virus, Plaque Assay, Western Blot, Microscopy

Journal: Journal of Virology

Article Title: Interferon-Induced Cell Membrane Proteins, IFITM3 and Tetherin, Inhibit Vesicular Stomatitis Virus Infection via Distinct Mechanisms ▿

doi: 10.1128/JVI.01328-10

Figure Lengend Snippet: IFITM3, but not tetherin, inhibits the synchronized infection of a recombinant VSV that expresses GFP. FLP-IN T Rex cell lines expressing the control protein CAT (A) or the antiviral proteins IFITM3 (B) or tetherin (C) were left untreated (red) or treated with 1 μg/ml of tetracycline (blue) for 24 h prior to infection with VSV-GFP at an MOI of 5. At 4 h postinfection, cells were fixed and the levels of GFP expression were analyzed by flow cytometry. (D) Ratio of the MFI in cells treated with tetracycline versus that in cells left untreated, plotted as the relative MFI. Error bars indicate standard deviations of the means (n = 3).

Article Snippet: Membranes were blocked with PBS containing 5% nonfat dry milk and probed with antibodies against Flag tag (Sigma), tetherin (Proteintech Group, Inc.), IFITM3 (Proteintech Group, Inc.), VSV G protein (Rockland, Inc.), or β-actin (Chemicon International).

Techniques: Infection, Recombinant, Expressing, Control, Flow Cytometry

Journal: Journal of Virology

Article Title: Interferon-Induced Cell Membrane Proteins, IFITM3 and Tetherin, Inhibit Vesicular Stomatitis Virus Infection via Distinct Mechanisms ▿

doi: 10.1128/JVI.01328-10

Figure Lengend Snippet: IFITM3, but not tetherin, inhibits VSV-G protein-mediated virus entry. (A) FLP-IN T Rex cells expressing the control protein CAT or the indicated ISGs were left untreated or treated with 1 μg/ml of tetracycline for 24 h. Cells were then infected with VSV-G-pseudotyped lentiviral particles at an MOI of 1. Forty-eight hours postinfection, intracellular levels of firefly luciferase expressed by the recombinant lentiviral vector were determined. Results represent the means ± standard deviations (n = 6) of the ratios of light units obtained from wells cultured in the presence of tetracycline versus those obtained from wells that were cultured in the absence of tetracycline. (B) FLP-IN T Rex cells induced to express CAT, IFITM3, or tetherin were cultured in the absence or presence of tetracycline for 36 h. Cells were infected with VSV at an MOI of 5 for 1 h on ice to allow attachment. After three washes with PBS, RNA was extracted to measure the amount of cell-bound virus. To quantify virus entry, virus inocula were removed after 1 h of binding on ice, and cells were either directly subjected to trypsin treatment or incubated for another 10 min at 37°C, followed by trypsin treatment to remove any cell-associated virus that had not entered the cytoplasm. Intracellular viral RNA was measured in a real-time RT-PCR assay, and the results are expressed as copies per 100 ng of total cellular RNA. Error bars indicate standard deviations of the means (n = 3).

Article Snippet: Membranes were blocked with PBS containing 5% nonfat dry milk and probed with antibodies against Flag tag (Sigma), tetherin (Proteintech Group, Inc.), IFITM3 (Proteintech Group, Inc.), VSV G protein (Rockland, Inc.), or β-actin (Chemicon International).

Techniques: Virus, Expressing, Control, Infection, Luciferase, Recombinant, Plasmid Preparation, Cell Culture, Binding Assay, Incubation, Quantitative RT-PCR