IL-7 and IL-2 binding to receptors and association of CD25 and CD132
IL-7 and IL-2 binding to their respective receptors on the cell surface were measured using the IL-7 or the IL-2 Fluorokine kit according to manufacturer instructions (R&D System). Tregs were suspended in PBS at 4×106 cells/mL in the presence of rhIL-2 (Bio-Techne) or rhIL-7 (R&D Systems) at concentrations as indicated in figure 2 for 15 minutes at 37C and incubated 1µg/ml of biotinylated IL-7 or IL-2 for 20 minutes on ice. Avidin-FITC was then added for an additional 30 min. Finally, cells were washed in PBS and analyzed by FACS. To determine the association of IL-2 with CD25 and CD132 Treg were suspended in X-vivo 15 without serum and incubated with or without IL-7 at concentrations indicated in figure 2 for 15 min at 37 C. Treg were washed twice in PBS and cell pellets were frozen at −80 C, subsequently thawed and lysed in 50 mM Hepes, 150 mM NaCl, 15 mM MgCl2, 1 mM EDTA, 10% glycerol and 1% Triton X-100. For the detection of association of CD25 and CD132, cell lysates were incubated with anti-CD25 PE (mouse IgG1, clone M-A251) on ice for 20 min. Anti-mouse IgG1,j,-coupled beads (Comp beads, BD-Pharmingen) or uncoupled beads as control were added to the lysates for 20 min on ice to bind the anti-CD25 complex to the beads. Association of CD132 with CD25 was detected after washing beads in PBS containing BSA and incubation with biotinylated anti-CD132 (rat IgG2,j, clone TUGh4) for 20min on ice. Beads were then incubated with streptavidin-APC for 20 min at room temperature, washed and analyzed by flow cytometry.