Fluorofix

For each participant, venous blood samples were collected in EDTA vacutainers (Becton Dickinson, Franklin Lakes, NJ). Blood was processed by Ficoll- Hypaque density gradient to isolate PBLs. Samples were stored in liquid nitrogen and shipped on dry ice to University of Colorado for further processing.
PBLs were thawed and washed with complete RPMI 1640 medium prior to staining. After washing, cells were resuspended in flow buffer containing phosphate buffered saline, 1% BSA, and 0.1% sodium azide, and counted using a hemocytometer; one million viable cells were stained for flow cytometry. Cells were first stained for viability using Live/Dead fixable blue dye (Thermo Fisher Scientific, Waltham, MA) as per manufacturer’s instructions. Cells were then blocked for 10 min at 4°C with 100 μL of a 1:100 diluted Fc blocking reagent (eBioscience, San Diego, CA) in 30 μL flow buffer. After that, cells were incubated with an antibody cocktail containing CD19 Spark NIR 685 clone: HIB19 (Biolegend) and CD14 Spark Blue 550 clone: 63D3 (Biolegend) antibodies. Incubation was performed for 30 min in the dark at 4°C. Cells were then fixed in 500 μL of Fluorofix (Biolegend, San Diego, CA). Samples were run on a Cytek^® Aurora (Cytek Bioscience, Fremont, CA) equipped with 5 lasers. Data were analyzed using FlowJo version 9.5.2 (Tress Star, Ashland, OR).

Splenocytes were harvested and processed into single cell suspensions and RBC lysed. Cells were then stained for surface markers (NK1.1, NKp46, CD3ε, CD19, Ly6C, KLRG1, Ly49I, and Ly49H) and viability (Zombie Red, BioLegend), and then rested for 1 hour at 37° C in 75 μL of RPMI-1640 (RPMI) supplemented with 2 mM glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, 50 μg/ml gentamicin, 110 μg/ml sodium pyruvate, 50 μM 2-mercaptoethanol, 10 mM HEPES, and 10% FCS. Splenocytes were then stimulated with IFN-α1 (BioLegend) or IL-18 (BioLegend) in 2-fold dilution series in 75μL of supplemented RPMI, starting at 50 ng/mL or 10 ng/mL, respectively. Cells were incubated at 37° C for 15 minutes, and then fixed using 150 μL of FluoroFix (BioLegend), followed by permeabilizing using True-Phos Perm buffer as recommended by manufacturer (BioLegend). Cells were then intracellularly stained with antibodies against phospho-STAT1^Ser727 (S727) (clone A15158B, BioLegend), and phospho-p38 (clone 36/p48 (pT180/pY182), BD Biosciences. Cells were then analyzed using a flow cytometer.