CHICAGO — The use of a clinically validated flow cytometry test can assess type 1 and type 2 interferon activity in children with various pediatric inflammatory diseases to evaluate and monitor their disease activity and their responses to medications, according to research presented at American College of Rheumatology (ACR) 2025 Annual Meeting.

“Interferons are really important cytokines for immunity, but when you have too much of these cytokines, it can lead to development of rheumatic diseases,” Pui Y. Lee, MD, PhD, a rheumatologist at Boston Children’s Hospital and an assistant professor of pediatrics at Harvard Medical School, both in Boston, told attendees.

Type 1 interferons, which include interferon alpha and interferon beta among others, coordinate the body’s antiviral response but increased levels of them have been linked to systemic lupus erythematosus (SLE) and juvenile dermatomyositis (JDM). Type 2 interferons, which are interferon gamma, are key mediators in activating macrophages, but these are increased in cytokine storm syndromes such as macrophage activation syndrome (MAS), hemophagocytic lymphohistiocytosis (HLH), and multisystem inflammatory syndrome in children (MIS-C).

Despite long-time recognition that dysregulation of these interferons plays a role in inflammatory disease, most institutions lack the ability to assess these cytokines with existing approaches, such as quantifying interferon-stimulated genes (for type 1) and measuring CXCL9 (for type 2).

Development of the Assay

“It remains very challenging to measure these cytokines, in part because of limited sensitivity and specificity of traditional assays,” Lee said. His lab therefore worked on using bulk RNA sequencing of peripheral blood mononuclear cells to develop biomarkers of interferon signaling that could be used clinically for therapeutic monitoring and diagnostic evaluation.

They compared RNA sequencing data from healthy control individuals to those of children who had MAS, MIS-C, and SLE. The researchers identified a strong type 1 interferon signature in patients with SLE and a strong type 2 interferon signature in patients with MAS and MIS-C. Specifically, expression of the protein CD169 correlated with type 1 interferon and was elevated most prominently in children with SLE and JDM, although it was seen in other inflammatory diseases as well. Expression of the protein CD274 (PD-L1) correlated with type 2 interferon and CXCL9 levels and was elevated in those with MAS and MIS-C. CD274 expression also reflected interferon gamma upregulation in familial HLH.

“We’re focused on studying CD274 and CD169 because these proteins are expressed on the surface of monocytes and can be measured readily using flow cytometry,” Lee said.

The researchers confirmed these associations with flow cytometry and then developed a flow cytometry assay that assesses expression of both CD169 and CD274, using just 50 µL of whole blood and taking just 30 minutes “from start to finish,” Lee said.

The researchers used this assay to screen more than 200 patients with various pediatric inflammatory diseases — including MAS, MIS-C, SLE, juvenile idiopathic arthritis, JDM, and monogenic autoinflammatory diseases — for CD169 and CD274 expression. Lee showed how the assay might be used for longitudinal therapeutic monitoring of MAS, for example, where changes in CD274 expression preceded corresponding changes in ferritin levels. Similarly, CD169 expression can be used for monitoring therapy that targets type 1 interferon.

The researchers also assessed how the assay could be used to monitor recently approved medications that target both interferon pathways, such as JAK inhibitors, the anti-type 2 interferon (interferon gamma) monoclonal antibody emapalumab, and the anti-type 1 interferon receptor inhibitor anifrolumab.

The assay was already clinically implemented in October 2023, with more than 2000 tests completed for centers in North America.

Advantages and Limitations of the Assay

Advantages of this assay include its low cost, fast turnaround time, and easy implementation in labs with flow cytometry experience. It also does not have any special requirements for sample collection, and samples are stable for up to 72 hours at room temperature, Lee said.

The assay has some limitations, Lee said, because its results can be affected by cytopenia, laboratories that might perform the assay have their own reference range or different instruments for flow cytometry quantification, and interferon dysregulation can only indicate immune patterns and not a particular diagnosis.

‘Findings Are Very Exciting’

“These findings are very exciting as they suggest a relatively easy way to quantify interferon activity in rheumatic diseases that can be readily translated into clinical practice,” Grant Schulert, MD, PhD, an associate professor of pediatric rheumatology at Cincinnati Children’s Hospital, Cincinnati, told Medscape Medical News.

“There is significant interest in being able to measure type 1 and 2 interferons in a clinical setting, but it is challenging,” said Schulert, who was not involved with this research. “The cytokines themselves are hard to measure as they generally stay in tissue and have very low circulating levels, so instead we need to measure a proxy for this activity.”

He said that flow cytometry-based tests like the ones described here are already commonly used in molecular and diagnostic immunology labs, “so these results offer a roadmap” for clinical testing for interferon activity and support “its utility in both diagnosis and disease monitoring.”

Lee and his coauthors reported having no relevant disclosures, and no external funding was noted. Schulert reported consulting for SOBI and Novartis and receiving research support from IpiNovyx.

Tara Haelle is a science/health journalist based in Dallas.