Ifn β

Mouse fetal livers were extracted from E14.5 embryos homozygous for the floxed LDB1 gene with or without CRE under control of the Mx1 gene promoter (Li et al. 2010 (link)). Cells were cultured as described (von Lindern et al. 2001 (link)). CRE expression was induced by 250 U/mL IFN-β (Millipore) in culture medium. Genotyping and Cre-mediated deletion were as described (Li et al. 2010 (link)).

A wild-type CD8⁺ dendritic cell line (MuTu1940) was established and cultured as described previously (22 (link),39 (link)). For ChIP-seq and ChIP-qPCR experiments, CD8⁺ dendritic cells (DCs) were treated for 90 min with 5 μg/ml of high molecular weight polyinosinic–polycytidylic acid (pIC, InvivoGen), 1 mM class B CpG oligonucleotide 1826 (CpG, InvivoGen) or 100 U/ml interferon-β (IFN-β, Millipore) or a combination of these ligands. The binding sites of IRF3, IRF5 and IRF9 were determined in DCs stimulated by pIC, CpG and IFN-β, respectively. The binding of cRel and Junb was determined in CpG-stimulated and unstimulated DCs. For gene expression qPCR experiments, CD8⁺ DCs were treated for 1.5, 3, 6 or 12 h with 5 μg/ml pIC, 1 mM CpG or 100 U/ml IFN-β.

At passage 6 and at ~80% confluency, cells were primed with 1500 UI/mL IFN-β (Merck Millipore, Ireland) for 1 hour in hPL-free DMEM as previously described.^{41 (link)} Primed with IFN-β and non-primed MSCs were washed with PBS and cultured for an additional 48 hours in media containing EVs-depleted hPL (hPL was EV depleted by ultracentrifugation at 120 000 × g for 18 hours at 4°C; Ti70 rotor, κ-factor 298.0, Beckman Coulter, Ireland), and the CM was collected. To remove cells, CM (100 mL) was centrifuged at 2000 × g for 10 minutes. To remove debris and large-sized EVs, supernatants were centrifuged at 10 000 × g for 45 minutes at 4°C in a Sorvall ST 8 small benchtop centrifuge using an HIGHConic III fixed angle rotor (κ-factor 2488, Thermo Fisher Scientific, Ireland), followed by filtration using a 0.2-µm pore filter membrane (Thermo Fisher Scientific). Next, small-sized EVs were isolated using the polyethylene glycol 6000 (PEG) precipitation protocol as previously described^{43-45 (link)} followed by ultracentrifugation at 100 000 × g for 91 minutes (Ti70 rotor, κ-factor 298.0, Beckman Coulter, Ireland) at 4°C. MSC-EV samples were resuspended in 0.5 mL of 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)/0.9% sodium chloride (NaCl, Thermo Fisher Scientific) and stored at−80°C.