When tumor size was >108 radiance and/or >500 mm3, tumors from 3 mice that escaped agonistic αCD40 (harvested at day 40–75), 2 tumors that escaped agonistic αCD40+αPD-L1 (harvested at day 80) ,and 2 tumors that escaped αPD-1+αPD-L1 (harvested at day 80) were expanded in vitro as described (20 ). Following in vitro establishment of cell lines (~1 week), 3×105KPC tumor epithelial cells per well were plated in 6-well plates in tumor media. After 24 hours, supernatant was removed and replaced with tumor media ± 50 ng/ml recombinant mouse IFNγ (R&D Systems). After ∼48 h, adherent tumor cells were lifted in 10 mM EDTA (Invitrogen), washed and stained with antibodies directly conjugated to PD-L1 (10F.9G2, BioLegend). Data were acquired on Fortessa flow cytometer (BD) using BD FACSDiva software and analyzed using FlowJo v10. Re-derived cell lines were also plated at equal numbers and cultured in vitro ± recombinant mouse IFNγ (100 ng/mL, R&D Systems) for 24 hours in 6-well plates. Cells were lifted in 10 mM EDTA (Invitrogen), RNA was extracted (QIAGEN RNeasy Mini Kit) and concentration/purity was assessed by Nanodrop. cDNA was generated using RT Buffer Mix and RT Enzyme Mix (Thermo Fisher). Real time PCR was performed in triplicate on a BioRad CFX96 Touch Real-Time PCR Detection System by measuring SYBR Green (BioRad) fluorescence for 40 cycles similar to as we described (20 ).
Recombinant mouse ifn γ
Manufactured by R&D Systems
Sourced in United States, France
Recombinant mouse IFN-γ is a recombinant protein that represents the mature form of mouse interferon gamma. Interferon gamma is a cytokine that plays a key role in the immune response.
Automatically generated - may contain errors
Lab products found in correlation
Ethylene diamine tetraacetic acid (edta), by Thermo Fisher Scientific (4 mentions)
Poly 1 c tlr3 ligand, by Merck Group (4 mentions)
Rneasy mini kit, by Qiagen (3 mentions)
Rt enzyme mix, by Thermo Fisher Scientific (3 mentions)
Sybr green, by Bio-Rad (3 mentions)
Cfx96 touch real time pcr detection system, by Bio-Rad (3 mentions)
Fortessa flow cytometer, by BD (3 mentions)
Rt buffer mix, by Thermo Fisher Scientific (3 mentions)
Ifn γ, by BioLegend (3 mentions)
M csf, by R&D Systems (3 mentions)
Lipopolysaccharides (lps), by Merck Group (3 mentions)
Tnf α, by R&D Systems (3 mentions)
Duoset elisa kit, by R&D Systems (3 mentions)
Recombinant human m csf, by BioLegend (2 mentions)
Lipopolysaccharides (lps), by Thermo Fisher Scientific (2 mentions)
Interleukin 4 (il 4), by BioLegend (2 mentions)
Gm csf, by R&D Systems (2 mentions)
Poly 1 c, by InvivoGen (2 mentions)
Facsdiva software, by BD (2 mentions)
Percp cy5, by BioLegend (2 mentions)
61 protocols using recombinant mouse ifn γ
1
Characterizing PD-L1 Expression in KPC Tumors
2
Evaluating IFNγ Response in KPC2a Tumors
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KPC2a tumors from 2 mice treated with αPD-L1 (harvested at day 45), 1 mouse treated with αPD-1 (harvested at day 45), and one mouse treated with dual blockade (harvested at day 63) were expanded in vitro using culture conditions described above. Parental, KPC2a, and re-derived cell lines were plated at equal numbers and cultured in vitro ± recombinant mouse IFNγ (100ng/mL, R&D Systems) for 24 hours in 6-well plates. Cells were lifted in 10mM EDTA (Invitrogen), RNA was extracted (QIAGEN RNeasy Mini Kit) and concentration/purity was assessed by Nanodrop. cDNA was generated using RT Buffer Mix and RT Enzyme Mix (Thermo Fisher). Real time PCR was performed in triplicate on a BioRad CFX96 Touch Real-Time PCR Detection System by measuring SYBR Green (BioRad) fluorescence for 40 cycles. MSLN was used as a negative control and ATP5b as a reference gene because they are unlikely to be influenced by IFNγ. Calculations were performed according to the geNorm method to calculate fold change and standard error (Vandesompele et al., 2002 ).
3
Isolation and Culture of Mouse Macrophages
4
Evaluating DSP-7888 and PD-1 Inhibition Synergy
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To evaluate potential synergy between DSP-7888 Emulsion and PD-1 inhibition, 8-week-old female HLA-A*02:01 transgenic mice received a single intradermal injection of either DSP-7888 Emulsion (n = 5) or nelatimotide-only emulsion (n = 5). After 7 days, mice were euthanized via carbon dioxide inhalation, and splenocytes from each group were harvested and pooled. A portion of splenocytes from each group were pulsed with 100 µg/mL WT1126-134 peptide for 1 h, washed twice, and used as antigen-presenting cells. LLC-HHD-WT1 cells were irradiated with X-ray, stimulated for 2 days with 100 ng/mL recombinant mouse IFN-γ (R&D Systems), and used as tumor cells. Splenocytes, antigen-presenting cells, and tumor cells were co-cultured (10:1:1) in 96-well U-bottom plates (“with tumor cells” culture). Control wells received culture medium instead of tumor cells (“without tumor cells” culture). Both the “with” and “without” tumor cell cultures were incubated with anti-PD-1 (29F.1A12, BioLegend) or an isotype control antibody (rat IgG2aκ, BD Biosciences) for 3 days. Each sample was plated in triplicate. Secreted IFN-γ was measured using the Mouse IFN-γ ELISA Set (R&D Systems).
5
Nitric Oxide Production in Macrophages Infected with Toxoplasma gondii
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The RAW 264.7 and J774-A1 cells were seeded at the density of 5 × 104 cells per well in 96-well plates, activated with 200 U/ml of recombinant mouse IFN-γ (R&D Systems, United States) and 0.2 μg/ml of LPS from Escherichia coli 0111:B4 (Sigma-Aldrich, United States) for 24 h prior of the T. gondii infection. After 24 h of activation, cells were washed twice with sterile PBS, DMEM supplemented with 3% FBS was added, cells were infected with a 5 T. gondii per macrophage (5:1) ratio and incubated at 37°C for 2 h. The T. gondii-macrophage ratio used was determined by previous experiments examining NO production and number of adhered macrophages after 24 h infection. After infection, cells were washed twice in sterile PBS, and DMEM supplemented with 10% FBS with IFN-γ and LPS or these activators and L -arginine (Sigma-Aldrich, United States) at different concentration, was added. Supernatants were collected at 2, 4, 6, and 24 h after infection for the nitrite assay.
6
Multiparametric Flow Cytometry Analysis
7
T Cell Activation and Regulation
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The CD8+ T cells and CD4+ T cells were isolated from the spleens or lymph nodes of BALB/c and DUC18 Tg mice using CD8a (Ly‐2) MicroBeads and CD4 (L3T4) MicroBeads according to manufacturer’s protocol (Miltenyi Biotec). The purity of isolated cells was greater than 95%. CD8+ T cells were stimulated by Dynabeads Mouse T‐Activator CD3/CD28 (Invitrogen) at several bead‐to‐cell ratios in the absence or presence of syngeneic CD4+ T cells (CD8 / CD4 ratio = 0.5). In some experiments, recombinant human IL‐2 (provided Takeda Pharmaceutical), recombinant mouse IL‐21 (R&D Systems), and/or recombinant mouse IFN‐γ (R&D Systems) were added. In some experiments, Transwell system (BD Biosciences) was used to separate CD8+ T cells from CD4+ T cells in the absence or presence of neutralizing mAb to cytokines, such as anti‐IL‐2 mAb (S4B6) (BD Biosciences), anti‐IL‐21 polyclonal Ab (BD Biosciences), or anti‐IFN‐γ mAb (H22) (kindly provided Dr RD Schreiber, Washington University School of Medicine).
8
Immunohistochemical and Western Blot Analyses
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The following mAbs were used for immunohistochemical and Western blot analyses: rat anti-mouse IDO mAb (Novus, Littleton, CO, USA), rabbit anti-mouse CD8 mAb (Epitomics, Burlingame, CA, USA), rat anti-mouse pan-NK mAb (Becton Dickinson, Franklin Lakes, NJ, USA), and α-SMA mAb (DakoCytomation, Copenhagen, Denmark). 1-Methyl-D-tryptophan was purchased from Sigma-Aldrich (St. Louis, MO, USA). Recombinant mouse IFN-γ, TGF-β1, and IL-10 were purchased from R&D Systems (Minneapolis, MN, USA).
9
Interferon-gamma Activation of MBECs
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MBECs were stimulated with 10 ng/ml recombinant mouse IFNγ (R&D Systems) 24 h before parasites (3 × 106 freeze-thawed PbA mature iRBCs unless otherwise stated) were added. After a further 24 h, the MBECs were washed and 6 × 104 reporter cells in 0.4 ml RPMI complete medium were added. Following overnight co-incubation, the reporter cells were resuspended and moved to a 96-well filter plate for X-gal staining.
10
Murine Macrophage Infection with Toxoplasma
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The murine leukemia monocyte/macrophage cell line RAW264.7 (TIB 71; ATCC, Rockville, MD, USA) was cultured in RPMI 1640 containing 4.5 g/l glucose, 10% FCS, 1 mM sodium pyruvate, 10 mM HEPES, 100 U/ml penicillin and 100 μg/ml streptomycin. Tachyzoites of the mouse-avirulent type II T. gondii strain NTE (62 (link)) were propagated in L929 fibroblasts as described previously (17 (link)). Prior to infection, parasites were isolated by differential centrifugation and thoroughly washed (39 (link)). Unless stated otherwise, host cells were infected at a parasite-to-host cell ratio of 6:1 for 24 h. Infected cells or non-infected controls were stimulated with 100 U/ml (all experiments except ChIP) or 300 U/ml (ChIP) of recombinant mouse IFN-γ (R&D Systems, Wiesbaden, Germany) starting at 3–23.5 h after infection as indicated. For some experiments, RAW264.7 cells were treated with 0.5 μM of 5-aza-2-deoxycytidine (AZA) for 7 days prior to infection with T. gondii and/or treatment with IFN-γ.
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