Validation of a multicolor staining to monitor (phospho)STAT5 levels in regulatory T-cell subsets

BACKGROUND: Regulatory T cells (T(regs)) 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 T(reg) 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 (phospho)STAT5 staining. RESULTS: In human PBMCs, the PFE kit allowed the detection of surface antigens, FOXP3, KI67 and (phospho)STAT5 in similar proportions to what was observed without permeabilization (for surface antigens), or with PFA or methanol permeabilizations for FOXP3/KI67 and (phospho)STAT5, respectively. Comparable observations were made with murine splenocytes. Further, the PFE kit allowed determining the response of different human and murine T(reg) subsets to IL-2. It also allowed demonstrating that human T(reg) subsets with the highest levels of (phospho)STAT5 had also the highest suppressive activity in vitro, and that anti-thymocyte glogulin (ATG) induced T(reg) independently of the STAT5 pathway, both in vitro and in vivo. CONCLUSIONS: We have validated a multicolor staining method that allows monitoring (phospho)STAT5 levels in T(reg) subsets. This staining could be useful to monitor responses of various T(reg) subsets to IL-2 therapy.