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

  • Clin Immunol. 2025 Dec 2:283:110654. doi: 10.1016/j.clim.2025.110654.
Birthe Michiels  1 ,  Rudi Beyaert  2 ,  Jens Staal  3 ,  Doreen Dillaerts  4 ,  Maaike Cockx  5 ,  Glynis Frans  6 ,  Birgit Timmermans  7 ,  Natalie Lorent  8 ,  Isabelle Meyts  9 ,  Xavier Bossuyt  10
Affiliations
  • 1. Department of Laboratory Medicine, University Hospitals Leuven, Leuven, Belgium; Department of Microbiology, Immunology and Transplantation, Clinical and Diagnostic Immunology, KU Leuven, Leuven, Belgium. Electronic address: [email protected].
  • 2. Unit of Molecular Signal Transduction in Inflammation, VIB-UGent Center for Inflammation Research, Belgium; Department of Biomedical Molecular Biology, Ghent University, Belgium. Electronic address: [email protected].
  • 3. Unit of Molecular Signal Transduction in Inflammation, VIB-UGent Center for Inflammation Research, Belgium; Department of Biomedical Molecular Biology, Ghent University, Belgium; Department of Biochemistry and Microbiology, Ghent University, Belgium. Electronic address: [email protected].
  • 4. Department of Microbiology, Immunology and Transplantation, Clinical and Diagnostic Immunology, KU Leuven, Leuven, Belgium. Electronic address: [email protected].
  • 5. Department of Microbiology, Immunology and Transplantation, Clinical and Diagnostic Immunology, KU Leuven, Leuven, Belgium; PharmAbs, The KU Leuven Antibody Center, University of Leuven, Leuven, Belgium. Electronic address: [email protected].
  • 6. Department of Laboratory Medicine, University Hospitals Leuven, Leuven, Belgium; Department of Microbiology, Immunology and Transplantation, Clinical and Diagnostic Immunology, KU Leuven, Leuven, Belgium. Electronic address: [email protected].
  • 7. Department of Laboratory Medicine, University Hospitals Leuven, Leuven, Belgium. Electronic address: [email protected].
  • 8. Department of Respiratory Diseases, University Hospitals Leuven, Leuven, Belgium; Department of Chronic Diseases, Metabolism and Aging, BREATHE laboratory, KU Leuven, Leuven, Belgium. Electronic address: [email protected].
  • 9. Department of Pediatrics, University Hospitals Leuven, Leuven, Belgium; Department of Microbiology, Immunology and Transplantation, Laboratory for Inborn Errors of Immunity, KU Leuven, Leuven, Belgium. Electronic address: [email protected].
  • 10. Department of Laboratory Medicine, University Hospitals Leuven, Leuven, Belgium; Department of Microbiology, Immunology and Transplantation, Clinical and Diagnostic Immunology, KU Leuven, Leuven, Belgium. Electronic address: [email protected].
PMID: 41344526 DOI: 10.1016/j.clim.2025.110654
Abstract

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.

Keywords
Anti-IFNα2 autoantibodies; Flow cytometry; IRF7; ISG15; TLR; Type I IFN response.
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