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

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.