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:

Article Title: Targeted disruption of the interferon-? receptor 2 gene results in severe immune defects in mice

doi:

Figure Lengend Snippet: The IFN-γR2 chain disruption results in defective JAK-STAT activation by IFN-γ. (A) IFN-γR2 is required for activation of Jak1 and Jak2 by IFN-γ. Embryonic fibroblasts from both wild-type mice (lanes 1 and 2) and IFN-γR2 −/− mice (lanes 3 and 4) were cultured with IFN-γ (lanes 2 and 4) for 10 min. The extracts from these cells were immunoprecipitated with anti-Jak1 or anti-Jak2 antisera and blotted with either the 4G10 antibody and anti-Jak1 or anti-Jak2 antisera. (B) IFN-γR2 is required for Stat1 activation by IFN-γ. Splenocytes from wild-type mice (lanes 1 and 2) and IFN-γR2 −/− mice (lanes 3 and 4) were either cultured without (lanes 1 and 3) or with IFN-γ (lanes 2 and 4) for 15 min. (C) Total splenocytes were isolated from mice and cultured with IFN-γ for 2 hr. The total RNA was analyzed by Northern blotting with specific IRF-1, GBP-1, and glyceraldehyde-3-phosphate dehydrogenase probes.

Article Snippet: Various cytokines and antibodies were added to these cultures: murine IL-2 at 1 ng/ml, murine IL-4 at 10 ng/ml, murine IFN-γ (Genzyme) at 10 ng/ml, anti-IL-4 antibodies at 10 μg/ml, and anti-IFN-γ antibody at 15 μg/ml.

Techniques: Activation Assay, Cell Culture, Immunoprecipitation, Isolation, Northern Blot

Journal:

Article Title: Targeted disruption of the interferon-? receptor 2 gene results in severe immune defects in mice

doi:

Figure Lengend Snippet: Defect in class switching in B cells isolated from IFN-γR2 −/− mice. IgM+ B cells were isolated from total splenocytes and then cultured for 6 days in either LPS, or LPS plus IL-4, or LPS plus IFN-γ, or LPS plus IL-4 plus IFN-γ. The culture supernatants were then collected and assayed for IgG2a (A), IgE (B), and IgG1 (C) production by ELISA.

Article Snippet: Various cytokines and antibodies were added to these cultures: murine IL-2 at 1 ng/ml, murine IL-4 at 10 ng/ml, murine IFN-γ (Genzyme) at 10 ng/ml, anti-IL-4 antibodies at 10 μg/ml, and anti-IFN-γ antibody at 15 μg/ml.

Techniques: Isolation, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal:

Article Title: Targeted disruption of the interferon-? receptor 2 gene results in severe immune defects in mice

doi:

Figure Lengend Snippet: Altered Th1 responses in IFN-γR2 −/− mice. +/+, wild-type mice. −/−, IFN-γR2-deficient mice. (A) IFN-γ production of in vitro differentiated CD4 T cells. Naive (Mel14+) CD4+ T cells were cultured for 7 days in anti-CD3 antibody-coated wells with IL-2 (bar set 1), or IL-2, IL-4, and anti-IFN-γ antibody (bar set 2); or IL-2, IFN-γ, and anti-IL-4 antibody (bar set 3); or IL-12 and anti-IL-4 antibody (bar set 4). The results represent the average of three independent experiments. (B) IL-4 production of the in vitro differentiated CD4+ T cell cultures described above in A. The results represent the average of three independent experiments. (C and D) Antigen-specific T cell cytokine production in local LNCs isolated from KLH-immunized mice. Both wild-type mice (bars 1 and 2) and IFN-γR2 −/− mice (bars 3 and 4) were immunized with KLH in complete Freund’s adjuvant. The LNCs were isolated on day 10 and cultured with (bars 2 and 4) or without KLH (at 100 μg/ml) for 2 days. The supernatants were then assayed for IFN-γ production (C). The duplicated wells of cells were pulsed with 3H on day 2 for 8 hr and proliferation was assayed on day 3 (D). The results represent the average of four independent experiments.

Article Snippet: Various cytokines and antibodies were added to these cultures: murine IL-2 at 1 ng/ml, murine IL-4 at 10 ng/ml, murine IFN-γ (Genzyme) at 10 ng/ml, anti-IL-4 antibodies at 10 μg/ml, and anti-IFN-γ antibody at 15 μg/ml.

Techniques: In Vitro, Cell Culture, Isolation

Journal: The Journal of Experimental Medicine

Article Title: T-bet regulates Th1 responses through essential effects on GATA-3 function rather than on IFNG gene acetylation and transcription

doi: 10.1084/jem.20052165

Figure Lengend Snippet: Elevated IL-4 and GATA-3 and committed Th2 cells in T-bet −/− mice. (A) Naive CD4 + T cells and whole CD4 + T cells from T-bet −/− , T-bet +/+ , and wild-type mice were stimulated with anti-CD3/anti-CD28 and maintained under Th1 conditions (IL-12 only) and expanded with IL-2. On day 6, the cells were subjected to intracellular cytokine staining after restimulation with PMA/ionomycin. These data are representative of results obtained from three independent experiments. (B) Naive CD4 + T cells from T-bet −/− and wild-type mice were stimulated with anti-CD3/anti-CD28 under Th1 conditions (IL-12 only) or neutral (no cytokines or antibodies added) conditions for 48 h, and then culture supernatants were harvested for IFN-γ and IL-4 determination by ELISA. The data shown are averages ± SE derived from three independent experiments. (C) Naive CD4 + T cells from T-bet −/− and wild-type mice were stimulated with anti-CD3/ anti-CD28 under Th1 conditions (IL-12 only) or neutral conditions and maintained for the time periods shown in the figure. At each time point, cells were harvested and total RNAs were isolated and subjected to real-time PCR analysis for GATA-3 mRNA quantification. Independently, total RNAs from freshly isolated naive and memory CD4 + T cells were prepared for day 9 samples. The data shown are averages ± SE derived from three independent experiments.

Article Snippet: Murine recombinant IL-12, IL-4, anti–mouse IL-12, and anti–mouse IFN-γ antibody were purchased from PeproTech.

Techniques: Staining, Enzyme-linked Immunosorbent Assay, Derivative Assay, Isolation, Real-time Polymerase Chain Reaction

Journal: The Journal of Experimental Medicine

Article Title: T-bet regulates Th1 responses through essential effects on GATA-3 function rather than on IFNG gene acetylation and transcription

doi: 10.1084/jem.20052165

Figure Lengend Snippet: T-bet–independent Th1 development and IL-12Rβ2 chain expression. (A) Naive CD4 + T cells from T-bet −/− and wild-type mice (purified by flow cytometric sorting) were stimulated with Con A/APC (30 Gy-irradiated splenocytes from wild-type mice) under optimal Th1 conditions (IL-12 + anti–IL-4), suboptimal Th1 conditions (IL-12 only or anti–IL-4 only), neutral conditions, or suboptimal Th2 (IL-4 only) conditions. The cells were then expanded with IL-2 as well as cytokines and antibodies on days 2 and 4. On day 8, the cells were restimulated by PMA/ionomycin and subjected to intracellular cytokine staining for IL-4 and IFN-γ. See Materials and methods for further details. (B) Experimental design was the same as in A, except that cells subject to two kinds of stimulation (Con A/APC and anti-CD3/anti-CD28) were compared. These data are representative of results obtained from three independent experiments. (C) Naive CD4 + T cells from T-bet −/− and wild-type mice (purified by flow cytometric sorting) were stimulated with anti-CD3/anti-CD28 in the presence of IL-12 and 20 μg/ml anti–IL-4 (for optimal Th1 conditions) or with 5 ng/ml IL-12 (for suboptimal Th1 conditions). The cells were expanded by the addition of IL-2 on day 4 of culture and then washed and restimulated with PMA/ionomycin or plate-bound (pb) anti-CD3 on day 6, and culture supernatants were collected after 6 and 24 h, respectively, for assay of IFN-γ by ELISA (See Materials and methods).

Article Snippet: Murine recombinant IL-12, IL-4, anti–mouse IL-12, and anti–mouse IFN-γ antibody were purchased from PeproTech.

Techniques: Expressing, Purification, Irradiation, Staining, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Experimental Medicine

Article Title: T-bet regulates Th1 responses through essential effects on GATA-3 function rather than on IFNG gene acetylation and transcription

doi: 10.1084/jem.20052165

Figure Lengend Snippet: STAT4 signaling is impaired in T-bet −/− CD4 + T cells. (A) Naive CD4 + T cells from T-bet −/− and wild-type mice were stimulated with anti-CD3/anti-CD28 under either suboptimal Th1 conditions (IL-12 only) or neutral conditions. After maintenance for the time periods shown in the figure, whole cell lysates were subjected to Western blot analysis. Independently, lysates from freshly isolated naive CD4 + T cells were prepared for day 0 samples. Similar results were obtained in three other independent experiments. (B) Naive CD4 + T cells from T-bet −/− and wild-type mice were stimulated with anti-CD3/anti-CD28 under either optimal Th1 conditions (IL-12 + anti–IL-4) or suboptimal Th1 conditions (IL-12 only), and then expanded with IL-12 as well as cytokines and antibodies on days 2 and 4. On day 7, the cells were restimulated with fresh IL-12 for 30 min and lysed for Western blot analysis. Recombinant GATA-3 protein was prepared from GATA-3–expressing retrovirus-transfected Phoenix-Eco packaging cell line. Similar results were obtained in three other independent experiments. (C) Naive CD4 + T cells from T-bet −/− mice were stimulated and maintained as described in B. On day 2, the cells were infected with either control retrovirus (GFP-RV) or STAT4-expressing retrovirus (STAT4-RV). On day 7, the cells were restimulated by PMA/ionomycin and subjected to intracellular cytokine staining for IL-4 and IFN-γ. (D) The cells used in C were sorted by a flow cytometer according to GFP expression on day 6 and then lysed for Western blot analysis. The lysate of the Th2 line from T-bet −/− mice was also prepared as a positive control for GATA-3 protein. These data are representative of those obtained in three independent experiments.

Article Snippet: Murine recombinant IL-12, IL-4, anti–mouse IL-12, and anti–mouse IFN-γ antibody were purchased from PeproTech.

Techniques: Western Blot, Isolation, Recombinant, Expressing, Transfection, Infection, Control, Staining, Flow Cytometry, Positive Control

Journal: The Journal of Experimental Medicine

Article Title: T-bet regulates Th1 responses through essential effects on GATA-3 function rather than on IFNG gene acetylation and transcription

doi: 10.1084/jem.20052165

Figure Lengend Snippet: T-bet overexpression suppresses GATA-3 and Th2 cytokines. (A) Naive CD4 + T cells from T-bet −/− and wild-type mice were stimulated with anti-CD3/anti-CD28 under strict Th2 conditions (IL-4 + anti–IFN-γ + anti–IL-12). On day 2, the cells were infected with control (mock) or T-bet–expressing retrovirus (T-bet-RV). Finally, on day 7, GFP + cells were sorted by flow cytometry and lysed for Western blot studies. These data are representative of those obtained in three independent experiments. (B) Naive CD4 + T cells from T-bet −/− and wild-type mice were stimulated with anti-CD3/anti-CD28 under optimal Th1 conditions. On day 2, the cells were infected with control (mock) or STAT4-, IL-12Rβ2 chain–, or T-bet–expressing retroviruses. Finally, on day 7, cells were restimulated by PMA/ionomycin and subjected to intracellular cytokine staining for IL-4. The values shown were the percentages of IL-4 + cells in the GFP + population. These data are representative of those obtained from two independent experiments. (C) An established murine Th2 cell line (D10 cells) was stimulated with conalbumin/APCs and then infected with a T-bet–expressing retrovirus on days 2, 3, 4, and 5. On day 14, GFP + cells were enriched by flow cytometric sorting and restimulated with antigen/APC. Finally, on day 21, GFP + and GFP − cells were separated by sorting (purities were >92%) and lysed for Western blot analysis. (D) T-bet–expressing and nonexpressing D10 cells were obtained and cultured as described in C. On day 21, the cells were restimulated with either high (2 μg/ml) or low (0.2 μg/ml) concentrations of plate-bound anti-CD3 and/or conalbumin/APCs. 48 h after such restimulation, culture supernatants were collected and IL-4, IL-5, and IFN-γ were measured by ELISA. Similar results were obtained in three independent experiments. (E) T-bet–expressing and nonexpressing D10 cells were obtained and cultured as described in C. On day 21, 2 μg/ml actinomycin D was added to the cultures, and cells were harvested every 30 min for extraction of total RNA and subsequent real-time PCR analysis for GATA-3 mRNA. The left panel depicts the relative amount of GATA-3 mRNA normalized by 18S rRNA, and the right panel depicts the percent decreases from the nontreated sample. Similar results were obtained in two independent experiments.

Article Snippet: Murine recombinant IL-12, IL-4, anti–mouse IL-12, and anti–mouse IFN-γ antibody were purchased from PeproTech.

Techniques: Over Expression, Infection, Control, Expressing, Flow Cytometry, Western Blot, Staining, Cell Culture, Enzyme-linked Immunosorbent Assay, Extraction, Real-time Polymerase Chain Reaction

Journal: The Journal of Experimental Medicine

Article Title: T-bet regulates Th1 responses through essential effects on GATA-3 function rather than on IFNG gene acetylation and transcription

doi: 10.1084/jem.20052165

Figure Lengend Snippet: STAT4 signal induces T-bet in the absence of STAT1. (A) Naive CD4 + T cells from BALB/c and STAT1 −/− mice were stimulated with anti-CD3/anti-CD28 under Th2 conditions for 4 d. 24 h after stimulation, the cells were infected with mock-RV or STAT4-RV. On day 4, the cells were washed extensively and maintained under Th1 conditions (IL-12 and anti–IL-4 antibody) for another 3 d. Finally, the cells were restimulated by PMA/ionomycin and subjected to intracellular cytokine staining for IL-4 and IFN-γ. Similar results were obtained in three independent experiments. (B) The same cells used in A were initially cultured under Th2 conditions for 4 d and then either cultured under Th1 conditions or continued under Th2 conditions for 3 d. The cells were then sorted on day 7 according to GFP expression to obtain cells expressing STAT4-RV and then lysed for Western blot analysis. Similar results were obtained in three independent experiments. (C) The same cells used in A were initially cultured under Th2 conditions for 4 d and then either cultured under Th1 conditions with two concentrations of IL-12 or continued under Th2 conditions for 3 d. The cells were then sorted on day 7 according to GFP expression to obtain cells expressing STAT4-RV or GFP-RV (mock) and then lysed for Western blot analysis. Similar results were obtained in three independent experiments. (D) Naive CD4 + T cells from C57BL/6, STAT1 −/− , IFN-γ −/− , and IFN-γR −/− mice were stimulated with anti-CD3/anti-CD28 either under strict Th1 conditions (IL-12 + anti–IL-4 antibody) or suboptimal Th1 conditions (IL-12 only) and expanded by hu-IL-2. On day 7, these cell lines were lysed and subjected to Western blot analysis. Similar results were obtained in two independent experiments.

Article Snippet: Murine recombinant IL-12, IL-4, anti–mouse IL-12, and anti–mouse IFN-γ antibody were purchased from PeproTech.

Techniques: Infection, Staining, Cell Culture, Expressing, Western Blot

Journal: Stem Cell Research & Therapy

Article Title: Efficient co-expression of bicistronic proteins in mesenchymal stem cells by development and optimization of a multifunctional plasmid

doi: 10.1186/scrt56

Figure Lengend Snippet: Bioactivity of Mu-IFNαA translated from bicistronic vectors . In conditioned medium from mesenchymal stem cell (MSC) clones where both bioactivity and immunoreactivity were measured, the specific activity of interferon in each medium was calculated and each clone was separately color-coded. In all cases, the activity was determined by comparison with a standard murine interferon, produced in Escherichia coli and purified to homogeneity, and the mean of each group of values are shown with a horizontal line. (a) Left: conditioned medium from six monoclonal MSC lines stably transfected with (from left to right) pEF3-MuIFNαA, pEF3-MuIFNαAEMCV*ChFP and pEF3-MuIFNαAcmycChFP were assayed for bioactivity by the antiviral assay. Right: in a separate experiment, conditioned medium from 12 monoclonal MSC lines stably transfected with pEF3-MuIFNαA and from four monoclonal MSC lines stably transfected with pEF3-MuIFNαAEMCVChFP were assayed. (b) Conditioned medium from six monoclonal MSC lines stably transfected with pEF3-MuIFNαAEMCV*ChFP (left) and pEF3-MuIFNαAcmycChFP (right) were assayed for Mu-IFNα immunoconcentration by ELISA. (c) Specific activities of the Mu-IFNαA within each conditioned medium were calculated by dividing the bioactivity by the immunoconcentration. Bacterial recombinant Mu-IFNαA purified to homogeneity has a specific activity of 5 × 10 7 to 10 × 10 7 units/mg; this range is indicated with the brackets between the two datasets. ChFP, monomeric cherry fluorescent protein.

Article Snippet: Murine IFNαA (purified, 1,350 units/μl - also known as Mu-IFNα3; #12100-1) as well as rat anti-mouse monoclonal neutralizing antibodies raised against Mu-IFNαA (#22100-1) were gifts from PBL Interferon Source (Piscataway, NJ, USA).

Techniques: Clone Assay, Activity Assay, Comparison, Produced, Purification, Stable Transfection, Transfection, Antiviral Assay, Enzyme-linked Immunosorbent Assay, Recombinant

Journal:

Article Title: Coccidioides releases a soluble factor that suppresses nitric oxide production by murine primary macrophages

doi: 10.1016/j.micpath.2010.11.006

Figure Lengend Snippet: Spherule initials of Coccidioides suppress NO production by activated, primary macrophages isolated from C57BL/6 mice. Peritoneal macrophages were activated with IFN-γ + LPS, and NO concentration was measured by fluorescence using DAF-2DA and expressed as relative fluorescence units (RFU). Spherule initials of Coccidioides were co-cultured with macrophages to yield a multiplicity of infection (MOI) of 2:1, 5:1 or 10:1 (macrophages:fungal cells), and incubated 1, 3, 6 or 12 h in a 5% CO2 incubator at 37° C. All experiments were performed at least three times. Results of a representative experiment are shown. Error bars indicate standard errors of the mean (SEM). Statistically significant differences in NO production between test groups was determined using the Student t-test for the following comparisons: # vs non-activated macrophages = P<0.001, and + or ¥ vs activated, non-infected macrophages = P<0.001 & P<0.05, respectively. Non-ifx., non-infected macrophages; Ctl., non-activated, control macrophages.

Article Snippet: Murine IFN-γ was purchased from BD Biosciences/Pharmingen (San Jose, CA), and ultrapure E. coli -derived lipopolysaccharide (LPS) was obtained from InvivoGen (San Diego, CA).

Techniques: Isolation, Concentration Assay, Fluorescence, Cell Culture, Infection, Incubation