Recombinant murine ifn γ

Mouse glucagonoma αTC1.6 cells were purchased from the American Type Culture Collection (ATCC). Cells were maintained in Dulbecco's Modified Eagle Medium (DMEM—Sigma-Aldrich, Saint Louis, MO, USA) containing 10% fetal bovine serum (FBS), 2 mM L-glutamine, 0.15% 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) 15 mM, 1% non-essential amino acids (NEAA), 0.02% bovine serum albumins (BSA, Sigma-Aldrich), 25 mM d-glucose (Sigma-Aldrich), 100 U/ml penicillin, and 100 μg/ml streptomycin, at 37°C, with 5%-CO₂ humidified incubator. Mouse insulinoma βTC1 cells were also from ATCC; cells were cultured in DMEM with 25 mM glucose (Sigma Aldrich), supplemented with 2 mM L-Glutamine, 15% horse serum (HS), 2.5% FBS, 1% penicillin/streptomycin, in 95% humidified air, with 5% CO₂ at 37°C. Cells were passaged once a week after trypsinization and replaced with new medium twice weekly. Both cell lines were treated with a cytokine cocktail (recombinant murine IL-1β, specific activity 0.1 U/ml, Peprotech, London, UK, UE; recombinant murine IFN-γ, specific activity 25 U/ml, Peprotech; recombinant murine TNF-α, specific activity 25 U/ml, Peprotech), as previously described (22 (link)).

Cells at 5 × 10⁵.ml⁻¹ were treated with B. abortus RNA, other prokaryotic or eukaryotic RNAs, E. coli RNase I (Life Technologies)-treated B. abortus RNA or B. abortus lipoproteins in the presence of 150 U.ml⁻¹ IFN-γ for 48 h as it was formerly described (14 (link)). MHC-II, MHC-I, CD40, CD86, or CD80 expressions were assessed by flow cytometry. In the experiments that involved murine macrophages, BMDM were treated with different doses of B. abortus RNA in presence of 10 ng.ml⁻¹ recombinant murine IFN-γ (PeproTech) for 48 h. Murine MHC-II expression was assessed by flow cytometry.

B. subtilis were grown on LB-chloramphenicol with 1 mM IPTG
plates and processed into LB chloramphenicol as described above. Once the
bacteria were normalized to OD₆₀₀ = 1 in LB chloramphenicol, an MOI
of 100 was added to 0.5 × 10⁶ primary murine macrophages from WT
C57BL/6 or C57BL/6 deficient for phox (gp91^phox-/-)
mice (The Jackson Laboratory, stock # 002365) that have been activated using 100
ng/well recombinant murine IFN-γ (Peprotech, #315–05) for 18 hr. The cells were
spinfected at 200 x g for 5 min. At 1.5 hr, cells were washed 2x with sterile
PBS and gentamicin was added to the cells. pBMMs were lysed in 500 µL cold water
at hours 2, 6, 7, and 8 and plated for CFU. Colonies were enumerated after
overnight growth at 30°C on LB plates.

To determine the expression of PD-L1 by various immune cells and mature/immature BMM-derived DCs or M0/M1 BMM-derived macrophages, these cells were obtained, cultured, and harvested as described previously. To evaluate the effect of IFN-γ on PD-L1 expression of macrophages, BMM-derived M0 macrophages were treated with 500 U/mL recombinant murine IFN-γ (PeproTech) in a 5% CO₂ atmosphere at 37 °C for 24 h. To visualize surface PD-L1 expression on T cells, NK cells, NKT cells, MDSCs, and B cells, splenocytes were stained with FITC-conjugated anti-CD3 Ab (BioLegend), PE-conjugated anti-NK1.1 (BioLegend), PE-conjugated anti-CD11b (BioLegend), FITC-conjugated anti-Gr-1 (BioLegend), PE-conjugated anti-CD19 (BioLegend), and PerCP-eFluor 710-conjugated anti-CD274 (PD-L1) (eBioscience), respectively. To visualize the surface expression of PD-L1 on DCs and macrophages, BMM-derived DCs or BMM-derived macrophages were stained with PE-conjugated anti-CD11c Abor anti-F4/80 Ab (BioLegend), FITC-conjugated anti-PD-L1 Ab (BioLegend), and PE-Cy5-conjugated anti-CD80 Ab (BioLegend) or PE-Cy5-conjugated anti-CD86 or anti-MHC-II Ab (BioLegend), respectively. To visualize surface PD-L1 expression on TC-1 tumor cells, cells were stained with PE-conjugated anti-PD-L1 Ab (BioLegend). Flow cytometric analysis was performed using a FACSCalibur flow cytometer with CELLQuest software (BD Biosciences).

BM cell differentiation was done according to [24 (link)] with little modifications. Briefly, single cell suspensions were generated from wildtype and Bcl3^-/- BM cells. The cells were suspended in DMEM medium supplemented with 10% FCS, 1% L-glutamine, 1% sodium pyruvate, 1% MEM-NEAA and 1% penicillin/streptomycin, 55 mM 2-mercaptoethanol and Flt3L (100 ng/ml) (Cat no. RP-8665, Invitrogen, Thermo Fischer Scientific) then were cultured at 37°C in a humidified atmosphere at 5% CO2. On day 3, the cells were transferred to a single well containing a monolayer of mitomycin C (Cat no. 50-07-7, Millipore Sigma, Merck, Darmstadt, Germany)-treated OP9 or OP9-DL1 cells, or else were kept unaltered. At Day 7, the cells were supplemented with murine rIFN-γ (40 μg/ml) (Recombinant Murine IFN-γ, Catalog Number:315–05, Peprotech, East Windsor, NJ, USA) or were kept unaltered. At Day 9, all the cells were harvested and used for DC phenotyping by flow cytometry or were used further in the antigen presentation assay described above.