Boston Children's Hospital Newton, Massachusetts, United States
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Background/Purpose: Type I interferons (IFN-I) and type II interferon (IFN-g) are essential to host defense but dysregulated production of these cytokines is increasingly recognized in inflammatory diseases. Aberrant IFN-I production is associated with autoimmunity and excess IFN-g is linked to cytokine storm syndromes with T lymphocyte activation. Assays to measure IFN-I and IFN-g are not available in most institutions. We aimed to develop a clinical flow cytometry assay that rapidly captures the activity of IFN-I and IFN-g. Methods: We compared bulk RNA sequencing data from healthy controls and patients with macrophage activation syndrome (MAS), multisystem inflammatory syndrome in children (MIS-C), and systemic lupus erythematosus (SLE) to identify biomarkers of IFN-I and IFN-g signaling. We developed a flow cytometry assay to measure IFN-inducible markers on peripheral blood monocytes and acquired data across different pediatric inflammatory diseases. The findings were verified by a clinical laboratory that independently developed and validated this assay for clinical use. Results: Consistent with previous studies, RNA sequencing data from peripheral blood mononuclear cells identified a strong IFN-I signature in patients with SLE and a strong IFN-g signature in patients with MAS and MIS-C. We found that CD169 (SIGLEC-1) transcript levels positively correlated with the IFN-I signature while CD274 (PD-L1) expression reflected the IFN-g signature (Figure 1). Flow cytometry confirmed that surface expression of CD169 and CD274 on CD14+ monocytes are specific readouts of IFN-I and IFN-g activity, respectively (Figure 2). Based on these findings, we developed a flow cytometry assay to capture CD169 and CD274 expression, which requires 50 µL of whole blood and 30 minutes of hands-on time. We demonstrated the utility of this method for rapid screening of IFN dysregulation in >200 patients with a variety of pediatric inflammatory diseases including MAS, MIS-C, SLE, juvenile idiopathic arthritis, juvenile dermatomyositis, and monogenic autoinflammatory diseases (Figure 3A-D). We further illustrated the use of this test for therapeutic monitoring of recently approved medications that target the IFN pathways including Janus kinase inhibitors, emapalumab (anti-IFN-g), and anifrolumab (anti-IFN α/β receptor; Figure 3E-G). Finally, we outlined the steps of validating this assay for clinical use and summarize the findings during test validation and post-clinical implementation. Conclusion: We developed a clinically validated flow cytometry test to rapidly assess type I IFN and IFN-g activity. This test is useful for evaluation and monitoring of pediatric inflammatory diseases and can be readily implemented in clinical laboratories.
