Effect of APECED sera on ISG expression and STAT1 phosphorylation. (A) Expression of ISGs in control monocytes incubated in 20% autologous sera with the addition of 2% APECED sera-positive (Ab⁺) or -negative (Ab⁻) for IFN-α antibodies, or with control serum (Ctrl) for 18 hours. *P < .05 between IFN-α antibody–positive APECED patients and healthy controls. (B) U937 cells were treated with 1000 U/mL IFN-α for 15 minutes or with the same concentration of IFN-α preincubated with 2%, 5%, or 10% of APECED (A1-A5) or 10% of control (C1-C3) sera, stained for intacellular pSTAT1 and measured by flow cytometry. (C) Control PBMCs were incubated with 50% of serum samples from APECED patients positive (Ab⁺) or negative (Ab⁻) for IFN-α antibodies, healthy controls (Ctrl), or SLE patients for 15 minutes and stained for intracellular pSTAT1 to test for IFN activity in the sera (left panel). Control PBMCs were incubated with 50% of serum samples from an APECED patient negative for IFN-α autoantibodies (A3), an SLE patient with IFN activity, or two healthy controls for 15 minutes with or without the addition of neutralizing anti–IFN-α antibody or isotype control antibody as indicated, and stained for intracellular pSTAT1 (right panel).