Cd25 bv605

For surface staining, cells were incubated with CD25 BV605 (clone 2A3; BD), CD4 Alexa Fluor 700 (clone RPA--T4; BD), CD45RA PerCP-Cy5.5 (clone HI100; eBioscience) at 4°C for 30 minutes. For analysis of total CTLA--4 and Foxp3 expression, cells were fixed and permeabilized with Foxp3 staining buffer (eBioscience) and incubated with Foxp3 allophycocyanin (clone 236A--E7; eBioscience) and CTLA--4 phycoerythrin (clone BNI3; BD). Cells were acquired on a BD LSRII cytometer and the data analyzed using FlowJo software (TreeStar). In some experiments relative expression of CTLA--4 or CD25 was calculated based on: level of expression (MFI) in memory Treg (CD45RA--Foxp3+)/ level of expression (MFI) in naïve conventional CD4 cells (CD4+CD45RA+ Foxp3--) 2.5 T--cell stimulation CD4 T cells were resuspended at 1 × 10 6 /mL in RPMI 1640 with 10% fetal bovine serum, 2 mM l--glutamine, 1% penicillin, and 1% streptomycin. 96,000 Tells were stimulated with 0.5µg/ml anti--CD3 plus 72,000 CHO--cells expressing the CD80 ligand as previously described [24] . Cells were cultured in a 96--well round--bottomed plate at 37°C, 95% humidity, and 5% CO2.

Whole blood (100 μl) was immunolabeled for 15 minutes at room temperature using the following antibodies: FITC-CD3, APC-Cy7-CD4, PerCP-Cy5.5-CD8, PE-CD127, V500-CD19, PE-CF594-CD183, PE-Cy7-CD196, BV605-CD25, BV421-CD194, Alexa Fluor 700-CD14, and APC-CD56 (all from BD Biosciences, Mississauga, Canada). Red blood cells were lysed (0.06 M NH 4 Cl, 4 mM KHCO 3 , 0.052 mM EDTA), and peripheral blood mononuclear cells washed with PBS, PFA-fixed, and analyzed using a flow cytometer (LSRFortessa, BD Biosciences, Ontario, Canada). A minimum of 300,000 events were acquired per sample. Data analysis was performed using FlowJo (Tree Star Inc, Ashland, OR) (gating strategy is shown in Supplemental Figure 1).