Research Papers: Immunology:

Validation of a multicolor staining to monitor phosphoSTAT5 levels in regulatory T-cell subsets

Grégory Ehx _, Muriel Hannon, Yves Beguin, Stéphanie Humblet-Baron and Frédéric Baron

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Oncotarget. 2015; 6:43255-43266. https://doi.org/10.18632/oncotarget.6486

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Grégory Ehx1,*, Muriel Hannon1,*, Yves Beguin1,2, Stéphanie Humblet-Baron1,3,** and Frédéric Baron1,2,**

1 Hematology Research Unit, Groupe Interdisciplinaire de Génoprotéomique Appliquée (GIGA)-I³, University of Liège, Liège, Belgium

2 Department of Medicine, Division of Hematology, CHU of Liège, Liège, Belgium

3 Autoimmune Genetics Laboratory, University of Leuven, Leuven, Belgium

* These authors are co-first authors

** These authors are co-senior authors

Correspondence to:

Grégory Ehx, email:

Keywords: Treg, regulatory T cells, FOXP3, STAT5, IL-2, Immunology and Microbiology Section, Immune response, Immunity

Received: September 14, 2015 Accepted: November 26, 2015 Published: December 07, 2015


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.

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