title: Immunogenic effects of recombinant interferon-beta therapy disrupt the JAK/STAT pathway in primary immune cells from patients with multiple sclerosis.
abstract: BACKGROUND
  Immunogenicity of recombinant interferon-β (IFN-β) 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-β responsive cells and signalling pathways in peripheral blood mononuclear cells (PBMCs) with phospho-specific flow cytometry.
  
  
  METHODS
  PBMCs from 10 IFN-β-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-β (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-naïve 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%.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Neurology, Haukeland University Hospital, Bergen, Norway. gavasso@yahoo.com
authors.forename: S
authors.initials: S
authors.lastname: Gavasso
authors.forename: Bt
authors.initials: B
authors.lastname: Gjertsen
authors.forename: E
authors.initials: E
authors.lastname: Anderssen
authors.forename: Km
authors.initials: K
authors.lastname: Myhr
authors.forename: C
authors.initials: C
authors.lastname: Vedeler
mesh_terms: D000328:Adult; D000368:Aged; D057134:Antibodies, Neutralizing / Q000097:blood*; D016022:Case-Control Studies; D005260:Female; D005434:Flow Cytometry; D006801:Humans; D007155:Immunologic Factors / Q000276:immunology / Q000627:therapeutic use*; D016899:Interferon-beta / Q000276:immunology / Q000627:therapeutic use*; D053612:Janus Kinases / Q000378:metabolism*; D016018:Least-Squares Analysis; D007963:Leukocytes, Mononuclear / Q000187:drug effects* / Q000201:enzymology / Q000276:immunology; D008297:Male; D008875:Middle Aged; D020529:Multiple Sclerosis, Relapsing-Remitting / Q000097:blood / Q000188:drug therapy* / Q000201:enzymology / Q000276:immunology; D009664:Norway; D010766:Phosphorylation; D025341:Principal Component Analysis; D011994:Recombinant Proteins / Q000627:therapeutic use; D050791:STAT Transcription Factors / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects*; D016896:Treatment Outcome
chemical_list: D057134:Antibodies, Neutralizing; D007155:Immunologic Factors; D011994:Recombinant Proteins; D050791:STAT Transcription Factors; D016899:Interferon-beta; D053612:Janus Kinases