Immunogenic effects of recombinant interferon-beta therapy disrupt the JAK/STAT pathway in primary immune cells from patients with multiple sclerosis

Background: Immunogenicity of recombinant interferon-beta (IFN-beta) is a known complication in the therapy of relapsing-remitting multiple sclerosis (RRMS). Neutralizing antibodies (NAbs) that can interfere with efficacy are quantified using in vitro bioassays; however, these assays do not reveal the immunogenic state of the patient and are not predictive of treatment outcome. Objective: Assessment of the impact of NAbs on IFN-beta responsive cells and signalling pathways in peripheral blood mononuclear cells (PBMCs) with phospho-specific flow cytometry. Method: PBMCs from 10 IFN-beta-treated patients with RRMS, two untreated patients, and two healthy controls were re-stimulated in autologous sera and media with a serial dilution of IFN-beta (0-8000 U/ml) and levels of phosphorylation of STAT1/3/4/5/6 transcription factors were quantified in PBMC subtypes (NAb titres 0 to > 6000 neutralizing units). Data was subjected to principal component analysis, Hotelling's T-2, and partial least squares analysis. Results: Three significantly distinct clusters of individuals were revealed in autologous sera: therapy-naive and healthy, treated NAb-negative, and treated NAb-positive. Compared with controls STATs signalling patterns were modulated in treated NAb-negative patients and inhibited in all treated NAb-positive patients independently of NAb titres. In media no clustering of patients could be found. The predictability of NAb titres based on the phospho-flow data was 74%. Conclusion: Phospho-specific flow cytometry can delineate subset-specific cell responses that can act as surrogates for NAb exposure in blood. Immunogenic effects alter the response in primary cells even at low NAb levels. Cell line-based immunogenicity testing is not readily transferable to the immunogenic response in patients.