Gentlemacs tube

Animals were sacrificed at the given time points. All organs were immediately frozen in −50 °C isopentane and transferred to −80 °C for long-term storage. Osseous organs were pulverized using pestle and mortar under continuous cooling with liquid nitrogen (particle size 1–2 mm). Powder and soft tissue organs were transferred to individual gentle macs tubes (Miltenyi Biotec, Bergisch Gladbach, Germany) and homogenized using Xiril Dispomix tissue tearer (Miltenyi Biotec, Bergisch Gladbach, Germany) at 400 rpms for 15 s twice. Lysates were constantly kept on ice and consequently centrifuged at 1300× g for 5 min at 4 °C (Heraeus Multifuge, Thermo Fisher Scientific, Waltham, MA, USA). Supernatant was transferred and utilized for in vitro luminometry.

The collected spleen pieces were transferred into gentleMACS tubes (Miltenyi Biotec, Bergisch-Gladbach, Germany) with 6 ml PBS solution each and disrupted using gentleMACS dissociator (Miltenyi Biotec). Subsequently, the cell suspension obtained was filtered through a cell strainer (70 μm) with 18 ml PBS (phosphate-buffered saline, Sigma-Aldrich/Merck, Darmstadt, Germany), the PBMCs were separated by density gradient centrifugation, and the erythrocytes were lysed [1 ml H₂O for 15 s, osmolarity reinstalled with 108 μl NaCl solution (8.8 %)]. The cell suspension was then filtered again through a cell strainer (50 μm). The cells were counted using a cell counter (Multisizer™ 3 Coulter Counter, Beckmann Coulter, Krefeld, Germany) and the cell number was adjusted to 2 × 10⁶ cells/ml in culture medium (RPMI-1640, PAN-Biotech, Aidenbach, Germany; 10% FBS, Biochrom, Berlin, Germany; 2 mM glutamine, 50 μg/ml gentamycin, 0.05 mM mercaptoethanol, Sigma-Aldrich/Merck) for the proliferation assay.

Total RNA was isolated from the placental tissue using the Gene MATRIX Universal RNA Purification Kit (EurX, Gdańsk, Poland) and supplemented with TRI-reagent (phenol equilibrated, stabilized: chloroform: isoamyl alcohol 25 : 24 : 1) (AppliChem GmbH, Darmstadt, Germany) and β-mercaptoethanol (Acros Organics, New Jersey, USA). The procedure was conducted according to the manufacturer's protocol. Three milligrams of each sample was homogenized using gentleMACS Tubes (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) and gentleMACS Dissociator (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany). Total RNA quantity and concentration were measured with Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific, MA, USA) and stored at −86°C. The RNA quantity included in the study ranged between 1.9 and 2.0, and the median RNA concentration for samples was 1000 ng/μl.

To remove epithelial cells, the small intestine was incubated in HBSS (Gibco, Carlsbad, CA) containing 2 mM EDTA, 2% FCS (PAA Laboratories, Pasching, Austria), and 1 mM DTT (Sigma, St. Louis, MO). This treatment was repeated twice for 20 min at 37°C with gentle agitation. The tissue was washed once with HBSS to remove EDTA and then transferred to a digestion solution containing 10% FCS, 60 CDU/ml collagenase D (Roche), 60 kunitz units/ml DNase I (Roche, Basel, Switzerland), 0.28 CDU/ml Dispase (Roche), and 1 mM CaCl₂ in HBSS for 20 min at 37°C with gentle agitation. The contents were transferred to gentleMACS tubes (Miltenyi Biotech, Bergisch Gladbach, Germany) and disrupted on a gentleMACS dissociator (Miltenyi Biotech). After filtration, single cell suspensions were obtained. MLN and PP were transferred to 5 ml of the digestion solution, incubated at 37°C for 45 min with agitation and the digested organs were pipetted into single cell suspensions. Spleens were incubated with 1 ml of digestion solution for 30 min at 37°C and then pressed through a nylon mesh to obtain single cells. Erythrocytes were lysed with 2 ml of a hypotonic solution of NH₄Cl. All cell suspensions were washed once with HBSS and resuspended in sterile PBS. Cells were counted using a Sysmex KX-21N cell counter (Sysmex Corporation, Kobe, Japan).

For in vitro analysis, cells were washed with phosphate-buffered saline and dissociated from the plate with accutase (Gibco) for 5–10 min at 37 °C to generate single cell suspensions. For in vivo studies, tumors were excised post-mortem, mechanically digested using gentle MACS tubes (Miltenyi) and enzymatically digested using a mixture of 0.5 mg/ml collagenase type III (Sigma-Aldrich), 0.01 mg/mL dispase, and 0.125 mg/ml DNAase (Sigma-Aldrich) with antibiotic, 30-min at 37 °C. Tissue dissociates were passed through a 70-µm filter to collect a single cell suspension. Single cell suspensions were washed once in flow staining buffer and incubated with respective flow antibodies at 4 °C for 30 min in the dark. DAPI was used to discriminate viable and dead cells. Tumor cells were gated on EpCAM-positive cells. Flow cytometry was performed using the following antibodies: HLA-A,B,C/Alexa Fluor488 (Biolegend, clone W6/32, 1:200), HLA-DR/PE-Cy7 (Biolegend, clone L243, 1:200), mouse MHC-I (H-2Kd/H-2Dd)/PE (Biolegend, clone 34-1-2S, 1:200), mouse MHC-II (I-A/I-E)/Alexa Fluor488 (Biolegend, clone M5/114.15.2, 1:200), mouse CD274(PD-L1)/APC (Biolegend, clone 10F.9G2, 1:200), and mouse EpCAM/PE-Cy7 (Biolegend, clone G8.8, 1:350). Samples were analyzed on an Attune NxT system (Life Technologies).

Madin-Darby Bovine Kidney cells (MDBK) were cultured in a rich glucose medium (BI medium) supplemented with 5% of fetal bovine serum (FBS) in a 150 cm² flask at 37 °C with 5% CO₂.
Several bovine lungs were collected from healthy animals (Centre de Recherche de Saint Vulbas, BI France). Several pieces were placed in a GentleMACS tubes (Miltenyi Biotec) for grinding. The lysates were then filtered on a 40 µm filter and centrifugated at 700 g during 10 min. The cell pellets were resuspended in a rich glucose medium (BI medium) supplemented with 10% of FBS. The obtained bovine lung primary cells (BPC) were pooled from 4 different lungs then cultured and conserved in nitrogen liquid with 10% DMSO.
The strain BVDV-1 NADL cp (AJ133738) were amplified internally on bovine cells. Viral titers in fluorescent antibodies infectious dose 50% (FAID50/mL) were determined on BPC or MDBK after 3 days with a pool of anti-Pestivirus monoclonal antibodies (BI collection) and Goat anti-Mouse IgG Secondary Antibody, Alexa Fluor 488 (Invitrogen). The CPE were observed on BPC or MDBK with ZOE Fluorescent Cell Imager (Biorad).