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

IF 4.1 3区医学 Q2 IMMUNOLOGY

Clinical immunology Pub Date : 2026-02-01 Epub Date: 2025-12-02 DOI:10.1016/j.clim.2025.110654

Birthe Michiels , Rudi Beyaert , Jens Staal , Doreen Dillaerts , Maaike Cockx , Glynis Frans , Birgit Timmermans , Natalie Lorent , Isabelle Meyts , Xavier Bossuyt

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引用次数: 0

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.

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基于流式细胞术的ISG15诱导试验的开发和验证，以监测I型干扰素反应。

I型IFN反应在抵抗入侵病原体和某些自身炎症性疾病的免疫防御中起重要作用。在这项研究中，我们开发了一种基于流式细胞术的方法来测量ISG15在各种TLR配体和IFNα刺激下的产生。用TLR7/8、TLR3、TLR9配体和IFNα2刺激健康对照或患者外周血，测定ISG15的表达。通过TBK1/IKKε抑制剂和JAK1抑制剂分别抑制ISG15诱导，验证了该试验分析tlr依赖性I型IFN诱导和I型IFN依赖性JAK/STAT激活的能力。在AR IRF7缺乏的患者中，TLR7/8配体和TLR9配体诱导的ISG15产生被消除，但ifn α2诱导的ISG15产生未被消除。含有中和性抗ifn α2自身抗体的血清可消除ifn α2诱导的ISG15表达。基于流式细胞术的ISG15诱导试验可用于筛选I型IFN反应中的缺陷。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Clinical immunology 医学-免疫学

CiteScore

12.30

自引率

1.20%

发文量

212

审稿时长

34 days

期刊介绍： Clinical Immunology publishes original research delving into the molecular and cellular foundations of immunological diseases. Additionally, the journal includes reviews covering timely subjects in basic immunology, along with case reports and letters to the editor.