Anti cd25 fitc clone 7d4

Cell sorting was performed on a FACSAria W or L (BD Biosciences) cell sorter. For IL-2Rα and CD28 level sorting, cells were labelled with anti-CD28-PECy7 (clone 37.51, eBioscience) or anti-CD25-FITC (clone 7D4, BD).
Flow cytometry was performed on a FACSCanto II or LSRFortessa X-20 cytometer (both BD Biosciences). Data were analysed using FlowJo software (Treestar).
A known number of beads (Rainbow calibration particles, BD Biosciences) and propidium iodide (0.2 μg ml⁻¹, Sigma) was added to samples immediately prior to analysis. The ratio of beads to live cells was used to estimate the absolute cell number. The following monoclonal antibodies were used for the detection of cell surface markers: anti-CD25 -PECy7, or—APC (clone PC61, BD Biosciences) anti-CD28-PECy7 (clone 37.51, eBioscience). Staining was performed in PBS containing 0.1% BSA and 0.1% sodium azide (Sigma). In Supplementary Fig. 8, activated cells were defined as the 50% of cells with the highest FSC fluorescence. Spearman's correlation was calculated using Matlab 2011a's corr function.

Single cell suspensions from ILN were stimulated with 20 ng/ml PMA (Sigma-Aldrich) and 1 μg/ml ionomycin (Sigma-Aldrich) in the presence of 10 μg/ml brefeldin A (Sigma-Aldrich) for 4 hours at 37 °C, 5 % CO₂. Cells were stained with anti-CD4-PerCP antibody (clone RM4-5, BD Biosciences, San Diego, CA, USA), permeabilized with 0.5 % saponin and stained with anti-IFNγ-fluorescein isothiocyanate (FITC) (clone XMG1.2, BD Biosciences) and anti-IL17-PE (clone TC11-18H10, MiltenyiBiotec, Bergisch Gladbach, Germany) antibodies.
To detect Treg, single cell suspensions from ILN were stained with anti-CD4-PerCP and anti-CD25-FITC (clone 7D4, BD Biosciences) antibodies, fixed, permeabilized and stained with anti-Foxp3-PE antibody (cloneMF-23, BD Biosciences) following the manufacturer’s instructions. Isotype controls were included in all experiments to adjust the background signal. Flow cytometer analysis was performed in a FACSCalibur instrument and analyzed with Cell Quest Pro software (BD Biosciences).