Novel assay to detect increased level of neutralizing anti-interferon gamma autoantibodies in non-tuberculous mycobacterial patients

Subjects exposed to non-tuberculous mycobacterium (NTM) species do not always develop an active disease, which likely reflects underlying host susceptibility factors. Recent reports have shown that anti interferon gamma (IFN-γ) neutralizing autoantibodies (IFN-γ Ab) are associated with the development of disseminated NTM in patients without known evidence of immunodeficiency. The purpose of this study is to establish the screening method if subjects have IFN-γ Ab. Whole blood was obtained from patients with disseminated NTM, those with pulmonary NTM, and healthy controls. The neutralizing capacity to IFN-γ activity was assessed as an inhibition of Signal Transducer and Activation of Transcription 1 (STAT-1) phosphorylation in leukocyte after stimulation with exogenous IFN-γ by flow cytometer. The strength of phosphorylation was described as STAT1 phosphorylation index. Antigen capture assay was performed to measure the relative titer of Immunoglobulin-G fraction of IFN-γ Ab. STAT1 phosphorylation by IFN-γ was significantly inhibited in the leukocytes from patients with disseminated NTM compared to that in healthy subjects, while this inhibition was not observed in patients with pulmonary NTM. All subjects with inhibited STAT1 phosphorylation had high titer of Immunoglobulin-G that reacted with IFN-γ in the antigen capture assay. The measurement of STAT1 phosphorylation index in whole blood leukocytes and antigen capture assay are simple and useful method for detection of anti-IFN-γ neutralizing autoantibodies, and is valuable in the pathophysiological diagnosis of disseminated NTM patients without obvious immunodeficiency.