Detection of autoAbs against type I IFNs in AIRE ^C337F individuals. (A) Neutralization of type I IFNs determined by a Renilla luciferase reporter assay using transfected HEK-293T cells treated with IFN-α2 (10 ng/ml or 100 pg/ml), IFN-β (10 ng/ml), or IFN-ω (10 ng/ml or 100 pg/ml) in the absence or presence of plasma from healthy donors (HD) (n = 48, blue circles), individuals with the heterozygous AIRE^C337F variant (n = 3, red circles), or patients with autoAbs against type I IFNs (n = 15, purple circles; positive controls). Data are expressed as relative luciferase activity (ISRE dual luciferase activity, with normalization against Renilla luciferase activity) (24, 25). (B and C) Multiplex particle-based assay to detect autoAbs against (B) type I IFNs (IFNα2, IFNβ, and IFNω) and IFNγ or (C) IL-1, IL-3, IL-4, IL-6, IL-7, IL-10, IL-12, IL-17A, IL-17F, IL-21, IL-22, IL-23, IL-27, MCP-1, TGF β, TNF, and GM-CSF present in plasma from healthy donors (n = 4, blue circles), individuals with the heterozygous AIRE^C337F variant (n = 3, red circles), a patient with AR APECED (orange circles), or serum samples obtained from individuals with autoAbs against IFNα2, IFNβ, IFNω, IFNγ, IL-6, IL-10, IL-12, IL-17A, IL-17F, IL-22, IL-23, TNF, and GM-CSF (purple circles; positive controls). NB: Serum containing autoAbs against IL-β, IL-3, IL-4, IL-7, IL-21, IL-27, MCP-1, and TGFβ were not available for testing as positive controls at the time of testing the AD AIRE-deficient individuals (24, 25).