Fig. 2

GARP specifically defines suppressive activated Tregs. A Gating strategy and representative flow cytometry plots of GARP expression are shown. GARP is selectively expressed in CD4⁺CD25⁺ and CD4⁺Fop3⁺ cells after TCR stimulation. CD25⁺GARP⁺ and CD25⁺GARP⁻ subpopulations can be sorted for in vitro suppressive function assay. B Induction of GARP on CD4⁺Fop3⁺ cells after TCR stimulation (anti-CD3 and anti-CD28 beads). C In vitro suppressive function assay of the CD25⁺GARP⁺ and CD25⁺GARP⁻ subsets. The evaluation of the suppressive function of each subset was performed by CFSE staining of the responder cells. Percentages of dividing responder cells per well were indicated. Data are representative of five separate experiments. D The percentages of CD25⁺GARP⁻ and CD25⁺GARP⁺ cells prohibiting proliferation in each group are shown in a summary graph. EFlow cytometry of the secretion of IFN-γ vs. IL-17 by GARP⁻ and GARP⁺ subsets after stimulation with PMA + ionomycin for 5 h. Summary graph showing the percentages of IFN-ɣ and IL-17 secreting cells in each subset, respectively (F, G). One-way ANOVA with post hoc pairwise multiple comparisons using the Tukey’s method. For D, F and G, data are from n = 5 independent experiments using T cells from different healthy donors (n = 5 subjects)