Cells were phenotypically analyzed using multi-color flow cytometry (Beckman-Coulter, Brea, CA, USA). Antibodies used for flow cytometry include: anti-CD3-PeCy5 (UCHT1), anti-CD4-PC5.5 (13B8.2), anti-CD27-PC5.5 (1A4CD27) (all from Beckman-Coulter), anti-CD25-PeCy7 (M-A25, BD). Anti-FOXP3-eFluo660 (PCH101) or -eFluo450 (PCH101), anti-IL-17A (eBio64DEC17)-AlexFluo88 and anti-IFNγ(4S.B3)-Pcy7 (all from eBioscience, San Diego, USA), anti-Helios-AlexFluo647 (22F6, Biolegend), anti-CTLA4-PE (BNI3) and anti-active caspase 3-PE (C92–605) (both from BD) were used after Fix-Perm-treatment (eBiosciences). Appropriate isotype mAbs were used to define marker settings. Fixable viability dye eFluor®780 (eBioscience) was used in some experiments. Data were analyzed using Kaluza software (Beckman-Coulter).
To measure the intracellular cytokine producing potential, the cells were stimulated with PMA (12.5 ng/mL) plus ionomycin (500 ng/mL) in the presence of Brefeldin-A (5 μg/mL) for 4 hours before analysis. To analyze the TCR-Vβ repertoire by flow cytometry we used a TCR-Vβ kit (Betamark, Beckman-Coulter) according to the manufacturer’s instructions. To monitor cell division by flow cytometry, cells were labeled with 0.5 μM CFSE (Invitrogen) and analyzed by flow cytometry as described previously45 .
To measure the intracellular cytokine producing potential, the cells were stimulated with PMA (12.5 ng/mL) plus ionomycin (500 ng/mL) in the presence of Brefeldin-A (5 μg/mL) for 4 hours before analysis. To analyze the TCR-Vβ repertoire by flow cytometry we used a TCR-Vβ kit (Betamark, Beckman-Coulter) according to the manufacturer’s instructions. To monitor cell division by flow cytometry, cells were labeled with 0.5 μM CFSE (Invitrogen) and analyzed by flow cytometry as described previously45 .