title: Deficient phosphorylation of Stat1 in leukocytes identifies neutralizing antibodies in multiple sclerosis patients treated with interferon-beta.
abstract: BACKGROUND
  Anti interferon-beta (IFN-β) neutralizing antibodies (NAb) affect efficacy of treatment of multiple sclerosis patients, but exactly when the detrimental effects of NAbs offset therapeutic efficacy is debated. Quantification of intracellular pathway-specific phosphorylation by phospho-specific flow cytometry (phosphoflow) is a promising tool for evaluation of these effects in primary immune cells from treated patients at the single-cell level.
  
  
  METHODS
  Samples for phosphoflow and gene expression changes were collected before administration of IFN-β and at four, six, and eight hours thereafter. Patients were NAb negative (n = 3) or were NAb positive with low/medium (n = 1) or high (n = 2) NAb titers. Levels of phosphorylation of six Stat transcription factors (pStat) in seven cell subtypes and expression levels of 71 pathway-specific genes in whole blood were measured. The data was subjected to principal component analysis (PCA), fifty-fifty MANOVA, ANOVA, and partial least square regression (PLSR).
  
  
  RESULTS
  PCA of pStat levels clustered patients according to NAb class independently of time. PCA of gene expression data clustered patients according to NAb class but was affected by time and treatment. In the fifty-fifty MANOVA, NAb class was significant for both pStat levels and gene expression data. The ANOVA identified pStat1 protein in several cell subtypes as significantly affected by NAb class. The best fitting model for NAb prediction based on PLSR included pStat1 in monocytes, T cells, or lymphocytes and pStat3 in monocytes (r = 0.97). Gene expression data were slightly less predictive of NAb titers.
  
  
  CONCLUSIONS
  Based on this proof of concept study, we hypothesize that NAb effects can be monitored by evaluation of a single biomarker, pStat1, in either monocytes or T cells by phosphoflow directly after IFN-β administration. The method will significantly reduce cost relative to labor intensive in vitro methods and offers a patient-specific approach to NAb evaluation.
authors.affiliation: Department of Neurology, Haukeland University Hospital, Bergen, Norway ; The Norwegian Multiple Sclerosis Competence Centre, Department of Neurology, Haukeland University Hospital, Bergen, Norway ; Department of Clinical Medicine, University of Bergen, Bergen, Norway.
authors.forename: Sonia
authors.initials: S
authors.lastname: Gavasso
authors.affiliation: Nofima, Ås, Norway.
authors.forename: Ellen Faergestad
authors.initials: EF
authors.lastname: Mosleth
authors.affiliation: The Norwegian Multiple Sclerosis Competence Centre, Department of Neurology, Haukeland University Hospital, Bergen, Norway.
authors.forename: Tove
authors.initials: T
authors.lastname: Marøy
authors.affiliation: Department of Clinical Science, Hematology Section, University of Bergen, Bergen, Norway.
authors.forename: Katarina
authors.initials: K
authors.lastname: Jørgensen
authors.affiliation: Department of Clinical Medicine, University of Bergen, Bergen, Norway.
authors.forename: Hanne-Linda
authors.initials: HL
authors.lastname: Nakkestad
authors.affiliation: Department of Clinical Science, Hematology Section, University of Bergen, Bergen, Norway ; Department of Internal Medicine, Haukeland University Hospital, Bergen, Norway.
authors.forename: Bjørn-Tore
authors.initials: BT
authors.lastname: Gjertsen
authors.affiliation: Department of Neurology, Haukeland University Hospital, Bergen, Norway ; The Norwegian Multiple Sclerosis Competence Centre, Department of Neurology, Haukeland University Hospital, Bergen, Norway ; Department of Clinical Medicine, University of Bergen, Bergen, Norway ; The KG Jebsen Centre for MS Research, University of Bergen, Bergen, Norway.
authors.forename: Kjell-Morten
authors.initials: KM
authors.lastname: Myhr
authors.affiliation: Department of Neurology, Haukeland University Hospital, Bergen, Norway ; Department of Clinical Medicine, University of Bergen, Bergen, Norway ; The KG Jebsen Centre for MS Research, University of Bergen, Bergen, Norway.
authors.forename: Christian
authors.initials: C
authors.lastname: Vedeler
mesh_terms: D000328:Adult; D057134:Antibodies, Neutralizing / Q000276:immunology*; D005260:Female; D005786:Gene Expression Regulation / Q000187:drug effects / Q000276:immunology; D006801:Humans; D016899:Interferon-beta / Q000097:blood / Q000276:immunology* / Q000494:pharmacology / Q000627:therapeutic use*; D007962:Leukocytes / Q000187:drug effects / Q000378:metabolism*; D008297:Male; D008875:Middle Aged; D009103:Multiple Sclerosis / Q000188:drug therapy* / Q000276:immunology* / Q000473:pathology; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects / Q000276:immunology; D050794:STAT1 Transcription Factor / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects / Q000276:immunology; D059010:Single-Cell Analysis; D017211:Treatment Failure
chemical_list: D057134:Antibodies, Neutralizing; D010750:Phosphoproteins; D050794:STAT1 Transcription Factor; C494079:STAT1 protein, human; D016899:Interferon-beta