Ruxolitinib

CD11b⁺-enriched cells (10,000–20,000 cells/well) from 27 samples were cultured in RPMI-1640 media supplemented with 10% fetal bovine serum, 10,000 units/mL of pen/strep, 1% GlutaMAX (Thermo Fisher Scientific, Waltham, MA), and 5 ng/mL GM-CSF. Six different experimental conditions were tested per sample: (1) unstimulated cells; (2) stimulated with 10 μg/mL LPS; (3) 1 μM^{11 (link),54 (link)} ruxolitinib (InvivoGen, San Diego, CA); (4) 1 μM tofacitinib (Invivogen); (5) 10 μg/mL LPS 1 mM ruxolitinib; and (6) 10 μg/mL LPS + 1 μM tofacitinib. After overnight incubation, supernatants were collected for cytokine quantification by Luminex using a Human High Sensitivity Cytokine customized premixed magnetic bead panel (R&D Systems, Minneapolis, MN) for the following: IL-1 β/IL-1F2, IL-6, IL-8/CXCL8, and TNF. The median fluorescence intensity data were analyzed using MILLIPLEX Analyst Software V.3.5 (MilliporeSigma), and concentrations were calculated in pg/mL in relation to an R&D standard.

On day 0, GPL cells were pre-treated with 100 IU/ml or 1000 IU/ml universal human IFN-I (PBL Assay Science). The next day, IFN pre-treated and control untreated cells were infected separately with either WT GPCMV or vIET mutant virus (0.1 MOI) and incubated at 37°C with 5% CO₂. After 72 hrs, supernatants and monolayers were harvested and titrated in duplicate on GPL cells as previously described. IFN-I IU/ml were confirmed by VSV growth experiments in the presence or absence of different concentrations of IFN-I on GPL cells prior to reported studies (data not shown). GPL cells were pre-treated with 10 μM of the JAK1/JAK2 inhibitor ruxolitinib (Invivogen) for 1–6 hrs. Depending on the experimental condition, some cells were treated with 100 IU/ml of universal human IFN-I (PBL Assay Science). Cells were subsequently infected with vIET (0.1 MOI) for 1 hr. Cells were washed and fresh F-12 medium supplemented with 10% FCS plus 10 μM ruxolitinib were added then incubated at 37°C with 5% CO₂ for 4 days. Cells and supernatant were harvested and virus titrated on GPL cells.