Ifn α 2b

All cell lines in this study (Table 1) were incubated at 37°C and 5% CO₂ and cultured in Dulbecco's modified Eagle medium, high glucose, GlutaMAX, 25 mM HEPES (Thermo Fisher Scientific) supplemented with 10% fetal calf serum (FCS; Thermo Fisher Scientific) and 50 μg/ml gentamicin (PAN-Biotech) (D10) except for HUVEC, which were maintained in standard endothelial cell growth medium 2 (PromoCell), and iSLK cells (61 (link)), which were maintained in D10 supplemented with 2.5 μg/ml puromycin (InvivoGen) and 250 μg/ml G418 (Carl Roth). IFN-α treatment was performed by supplementing the respective culture medium with IFN-α 2b (Sigma; 5,000 U/ml). For seeding and subculturing of cells, the medium was removed, and the cells were washed with phosphate-buffered saline (PBS; PAN-Biotech) and detached with trypsin (PAN-Biotech). All transfections were performed using polyethylenimine (PEI; Polysciences) at a 1:3 ratio (mg DNA/mg PEI) mixed in Opti-MEM (Thermo Fisher Scientific). Cytotoxicity was measured using the CellTiter-Glo luminescent cell viability assay (Promega) according to the manufacturer’s instructions.

All cell lines in this study (Table 2) were incubated at 37 °C and 5% CO₂ and cultured in Dulbecco’s modified Eagle medium, high glucose, GlutaMAX, 25 mM HEPES (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal calf serum (FCS; Thermo Fisher Scientific, Waltham, MA, USA) and 50 μg/mL gentamicin (PAN-Biotech, Aidenbach, Germany) (D10) except for HUVEC, which were maintained in standard Endothelial Cell Growth Medium 2 (PromoCell, Heidelberg, Germany), and BJAB and MDA-MB-231 cells, which were maintained in RPMI (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% FCS and 50 μg/mL gentamicin. Interferon-alpha (IFN-α) treatment was performed by supplementing the respective culture medium with IFN-α 2b (Sigma; 5000 U/mL). For seeding and subculturing of cells, the medium was removed, the cells were washed with phosphate-buffered saline (PBS; PAN-Biotech, Aidenbach, Germany) and detached with trypsin (PAN-Biotech, Aidenbach, Germany). All transfections were performed using polyethylenimine (PEI; Polysciences) at a 1:3 ratio (µg DNA/µg PEI) mixed in Opti-MEM (Thermo Fisher Scientific, Waltham, MA, USA).

Separately, depending on availability of PBMC, RNA was extracted from cells using Picopure RNA isolation (Thermofisher, KIT0214) immediately upon thawing, or after 20 h stimulation with IFNα2b (Sigma, SRP4595) at 1000 IU/ml. RNA gene expression was quantified using Nanostring Plexset technology, whereby digital barcodes hybridize to oligonucleotide probes to assess for expression of pre-chosen genes, after correction to house-keeping genes (POL2RA, G6PD, SDHA) and inbuilt positive and negative controls (27 (link)). A panel of genes was chosen (Supplementary Table 6) and analyzed by linear regression, applying the Benjamini-Hochberg method of correction for multiple testing for the significance value of each coefficient.

LLPCs were differentiated in vitro using a multi-step culture system as previously described^{41 (link)}. All cultures were performed in IMDM supplemented with 10% FBS, 1% penicillin-streptomycin, and 1% ITS Liquid Media Supplement. At the first step, isolated PBMCs were activated as outlined above and expanded for 5 days with the same cytokine cocktail. At the second step, PBs were generated by seeding cells in medium with IL-2 (20 U/ml), IL-6 (50 ng/ml, PeproTech), IL-10 (50 ng/ml), and IL-15 (10 ng/ml) for 3 days. At the third step, PBs differentiated into early PCs by adding hIL-6 (50 ng/ml), hIL-15 (10 ng/ml), and IFN-α-2b (500 U/ml, Merck) for 3 days. At the last step, early PCs differentiated into LLPCs through co-culturing with irradiated 293T-APRIL feeder cells and adding hIL-6 (10 ng/ml). Fresh medium and cytokines were renewed twice a week and cultures were maintained until day 30.