title: Validation of a multicolor staining to monitor phosphoSTAT5 levels in regulatory T-cell subsets.
abstract: BACKGROUND
  Regulatory T cells (Tregs) are key players in immune tolerance. They express the transcription factor FOXP3 and are dependent of the STAT5 signaling for their homeostasis. So far, the study of phosphorylated epitopes by flow cytometry required treating the cells with methanol, which is harmful for several epitopes.
  
  
  METHODS
  Here we assessed whether the PerFix EXPOSE reagent kit (PFE)(Beckman Coulter) allowed monitoring the phosphorylation level of STAT5 in Treg subpopulations together with complex immunophenotyping. Results observed with the PFE kit were compared to those observed without cell permeabilization for surface markers, with paraformaldehyde permeabilization for non-phosphorylated intracellular epitopes, and with methanol-based permeabilization for phosphoSTAT5 staining.
  
  
  RESULTS
  In human PBMCs, the PFE kit allowed the detection of surface antigens, FOXP3, KI67 and phosphoSTAT5 in similar proportions to what was observed without permeabilization (for surface antigens), or with PFA or methanol permeabilizations for FOXP3/KI67 and phosphoSTAT5, respectively. Comparable observations were made with murine splenocytes. Further, the PFE kit allowed determining the response of different human and murine Treg subsets to IL-2. It also allowed demonstrating that human Treg subsets with the highest levels of phosphoSTAT5 had also the highest suppressive activity in vitro, and that anti-thymocyte glogulin (ATG) induced Treg independently of the STAT5 pathway, both in vitro and in vivo.
  
  
  CONCLUSIONS
  We have validated a multicolor staining method that allows monitoring phosphoSTAT5 levels in Treg subsets. This staining could be useful to monitor responses of various Treg subsets to IL-2 therapy.
authors.affiliation: Hematology Research Unit, Groupe Interdisciplinaire de Génoprotéomique Appliquée (GIGA)-I³, University of Liège, Liège, Belgium.
authors.forename: Grégory
authors.initials: G
authors.lastname: Ehx
authors.affiliation: Hematology Research Unit, Groupe Interdisciplinaire de Génoprotéomique Appliquée (GIGA)-I³, University of Liège, Liège, Belgium.
authors.forename: Muriel
authors.initials: M
authors.lastname: Hannon
authors.affiliation: Hematology Research Unit, Groupe Interdisciplinaire de Génoprotéomique Appliquée (GIGA)-I³, University of Liège, Liège, Belgium.|Department of Medicine, Division of Hematology, CHU of Liège, Liège, Belgium.
authors.forename: Yves
authors.initials: Y
authors.lastname: Beguin
authors.affiliation: Hematology Research Unit, Groupe Interdisciplinaire de Génoprotéomique Appliquée (GIGA)-I³, University of Liège, Liège, Belgium.|Autoimmune Genetics Laboratory, University of Leuven, Leuven, Belgium.
authors.forename: Stéphanie
authors.initials: S
authors.lastname: Humblet-Baron
authors.affiliation: Hematology Research Unit, Groupe Interdisciplinaire de Génoprotéomique Appliquée (GIGA)-I³, University of Liège, Liège, Belgium.|Department of Medicine, Division of Hematology, CHU of Liège, Liège, Belgium.
authors.forename: Frédéric
authors.initials: F
authors.lastname: Baron
mesh_terms: D000328:Adult; D000818:Animals; D005260:Female; D005434:Flow Cytometry / Q000379:methods; D064593:Heterografts; D006801:Humans; D016130:Immunophenotyping / Q000379:methods; D008297:Male; D051379:Mice; D016688:Mice, Inbred NOD; D016513:Mice, SCID; D010766:Phosphorylation; D050799:STAT5 Transcription Factor / Q000032:analysis*; D013194:Staining and Labeling / Q000379:methods*; D016176:T-Lymphocyte Subsets / Q000276:immunology*; D050378:T-Lymphocytes, Regulatory / Q000276:immunology*; D055815:Young Adult
chemical_list: D050799:STAT5 Transcription Factor
keywords: FOXP3; IL-2; Immune response; Immunity; Immunology and Microbiology Section; STAT5; Treg; regulatory T cells