Development and validation of a flow cytometry-based ISG15 induction assay to monitor the type I interferon response

The type I IFN response plays an important role in the immune defence against invading pathogens and in certain autoinflammatory diseases. In this study, a flow cytometry-based assay to measure ISG15 production in response to stimulation with various TLR ligands and IFNα was developed. PBMCs of healthy controls or patients were stimulated with TLR7/8, TLR3 and TLR9 ligands and IFNα2 whereafter ISG15 expression was measured. The ability of the assay to analyse TLR-dependent type I IFN induction and type I IFN-dependent JAK/STAT activation was verified by inhibition of ISG15 induction by a TBK1/IKKε inhibitor and a JAK1 inhibitor, respectively. TLR7/8 ligand- and TLR9 ligand-induced ISG15 production but not IFNα2-induced ISG15 production was abolished in a patient with AR IRF7 deficiency. IFNα2-induced ISG15 expression was abolished by serum containing neutralizing anti-IFNα2 autoantibodies. The flow cytometry-based ISG15 induction assay can be applied to screen for defects in the type I IFN response.