CD3, CD25 (SK7 and M-A251 respectively; BD Biosciences) and CD127 (eBioRDR5; eBiosciences Hatfield, UK) antibodies were used for cell surface phenotyping. Cells were then further stained for intranuclear Foxp3 (PCH101; eBiosciences) using the Foxp3 staining kit as per the manufacturer's instructions (eBiosciences).
For intracellular cytokine staining on day 7, cells were restimulated for 4 hr with 5 ng/ml PMA and 500 ng/ml ionomycin, with 2 μm monensin (Sigma-Aldrich, St Louis, MO) added for the final 2 hr. Cells were washed, fixed and permeabilized using Cytofix/Cytoperm kit (BD Biosciences) and then stained with fluorescently labelled monoclonal antibodies to IL-2 (MQ1-17H12; eBiosciences). Dead cells (7-aminoactinomycin D-positive; Sigma-Aldrich, Gillingham, UK) were gated out.
For phospho-STAT5 staining two different buffer kits were used with the same antibody clone: 47/Stat5[pY694] obtained from BD Biosciences. The buffer set used for phospho-STAT5 staining on total CD4+ cell populations was PerFix EXPOSE Kit (Beckman Coulter, High Wycombe, UK). The buffer set used for phopspho-STAT5 staining on sorted Treg cell and effector T cell populations was CytoFix/CytoPerm Buffer and Perm Buffer III (BD Biosciences). Both buffer sets were used as per the manufacturer's instructions.
For intracellular cytokine staining on day 7, cells were restimulated for 4 hr with 5 ng/ml PMA and 500 ng/ml ionomycin, with 2 μ
For phospho-STAT5 staining two different buffer kits were used with the same antibody clone: 47/Stat5[pY694] obtained from BD Biosciences. The buffer set used for phospho-STAT5 staining on total CD4+ cell populations was PerFix EXPOSE Kit (Beckman Coulter, High Wycombe, UK). The buffer set used for phopspho-STAT5 staining on sorted Treg cell and effector T cell populations was CytoFix/CytoPerm Buffer and Perm Buffer III (BD Biosciences). Both buffer sets were used as per the manufacturer's instructions.