Ifn β

Human Primary Proximal Tubule (HPPT) Cells (ATCC^® PCS-400–010^™) were cultured in low-serum medium consisting of Renal Epithelial Cell Basal Medium (ATCC^® PCS-400–030^®) with Renal Epithelial Cell Growth Kit (ATCC^® PCS-400–040^®), Penicillin-Streptomycin-Amphotericin B Solution (ATCC^® PCS-999–002^®) and Phenol Red (ATCC^® PCS-999–001^®) added. Cells were obtained at passage 2, cultured according to manufacturer’s instructions and used between passages 4 and 6. In addition to characteristic cobblestone growth pattern when confluent, cells were confirmed to express several proximal tubule markers including y-glutamyltransferase-1 and HAVCR1 (KIM1) as assessed with RNA-seq. Cells were stimulated with IFNα, IFNβ, IFNγ, TNFα, IL-6 and IL-1β (all obtained from Peprotech) for 12 hours in concentration of 10 ng/ml. Cells were treated with ruxolitinib (Peprotech) at 10 μM for 12 hours together with IFNβ. At least three biological replicates were prepared for all experiments.

In total, 1 × 10⁶ CD4⁺ T cells were activated for 24 hours with 1 μg/mL of anti-CD3 (clone OKT3, eBioscience) and 1 μg/mL of anti-CD28 Abs (clone CD28.2, eBioscience). To assess TLR7 agonist effect on CD4⁺ T cells, 1 × 10⁶ cells/mL were stimulated with 5 μg/mL of IMQ (Invivogen) for 30 hours. For IFN-β stimulation, CD4⁺ T cells were cultured with 10 ng/mL IFN-β (Peprotech) for 6 hours and washed 3 times with RPMI. For some assays, 1 μg/mL of Brefeldin A (BD Biosciences) was added to cell cultures for 6 hours to evaluate the intracellular accumulation of inflammatory cytokines.
To investigate pathways promoting TLR7 expression in CD4⁺ T cells, we pretreated 1 × 10⁶ HIV^free CD4⁺ T cells with 10 ng/mL IFN-β for 6 hours in the presence or absence of conditioned medium with 10% v/v of DAMPs containing supernatant, obtained from PBMCs treated with staurosporine for 24 hours, before TCR activation or TLR7 stimulation. The supernatant was prepared as follows: 1 × 10⁶/mL PBMC from HIV^free donors were cultured in vitro in the presence of 2 μM staurosporine for 2 hours at 37°C; cells were then washed 3 times with PBS and left in culture for a further 4 hours at 37°C, before collecting the supernatant.

Human hepatoma cell lines HepG2 (DSMZ-No: ACC-180) and Hep3B (DSMZ-No: ACC-93) were purchased from German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany). PLC/PRF/5 human hepatoma cells were received from the European Collection of Cell Cultures (ECACC, Salisbury, UK; catalogue no: 85061113). VERO-B4 cells (African green monkey; DSMZ-No: ACC-33) were also obtained from DSMZ. Hep3B and PLC/PRF/5 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Sigma Aldrich; Munich, Germany) supplemented with 10% fetal bovine serum (FBS, Biowest, Nuaillé, France). HepG2 cells were cultured using a minimum glucose DMEM (Sigma Aldrich) supplemented with 10% FBS and l-glutamine (10 ml/l). Hepatoma cells were stored in an incubator at 37 °C in a humidified atmosphere enriched with 5% CO₂. Stimulation with human IFN-β (IFN-β; Pepro-Tech, Rocky Hill, NJ) was achieved by adding 1,000 U/ml IFN-β to the culture medium.