FIG. 1.

Gating strategy for the analysis of STAT5 phosphorylation within CD4⁺ T-cell subsets. PBMC from a healthy blood donor were stimulated or not stimulated with IL-7 for 15 min, permeabilized by methanol treatment, and labeled intracellularly with antibodies to CD3, CD4, CD45RA, CD25, Foxp3, and pSTAT5 (Y694). The gating strategy depicted allowed the characterization of pSTAT5 induction in four CD4⁺ T-cell subsets: conventional naive CD4⁺ T cells (Nv), naive Tregs (NTreg), conventional memory CD4⁺ T cells (Mem), and memory Tregs (MTreg). pSTAT5 inductions in response to high-dose IL-7 stimulation (purple histograms) did not differ markedly between subsets. In contrast, responses to low-dose IL-7 stimulation (orange lines) were more efficient in the Mem and Nv subsets than in the two Treg subsets. Basal pSTAT5 values are represented by dark shaded histograms (unstimulated samples). Negative isotypic control values are represented by light shaded histograms.