2. Academic Validation
  3. Development and validation of a flow cytometry-based ISG15 induction assay to monitor the type I interferon response

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

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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