Pe conjugated anti ctla4

Naïve CD4+ T cells were cultured for 3 days under the appropriate conditions for each subset. Next, cells were stimulated for 4 h with PMA (50 ng/mL) and ionomycin (1 μM, Sigma, St. Louis, MO, USA) in the presence of Brefeldin A (BioLegend, San Diego, CA, USA). After stimulation, cells were fixed in BD Cytofix/Cytoperm^TM buffer or FOXP3 Fix/Perm buffer and then permeabilized with BD Perm/Wash^TM or FOXP3 Perm Buffer. The following antibodies were used for staining: PE-conjugated anti-IFN-γ (Biolegend, San Diego, CA, USA), PE-conjugated anti-IL-1 (BioLegend, San Diego, CA, USA), PE-conjugated anti-IL-17 (BioLegend, San Diego, CA, USA), APC-conjugated anti-FOXP3 (eBioscience, San Diego, CA, USA), PE-conjugated anti-CD25 (BioLegend, San Diego, CA, USA), PE-conjugated anti-ICOS (BioLegend, San Diego, CA, USA), PE-conjugated anti-GITR (BioLegend), PE-conjugated anti-CTLA4 (BioLegend, San Diego, CA, USA), PE-conjugated anti-ERK1/2 phospho (T302/Y204) (BioLegend, San Diego, CA, USA), PE-conjugated anti-AKT phospho (T308) (BD Biosciences, Franklin Lakes, NJ, USA), APC-conjugated anti-S6 phospho (S235/236) (Invitrogen, Carlsbad, CA, USA), and PercP-conjugated anti-STAT5 (Y694) (BD Biosciences, Franklin Lakes, NJ, USA). Cells were analyzed using a BD FACSCalibur or a BD Accuri C6 Plus cytometer. FlowJo software was used for data analysis and to draw graphs.