Pe conjugated anti icos

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.

For intracellular cytokine staining, cells were re-stimulated for 4 h before harvest with phorbol myristate acetate (PMA) (50 ng/ml), ionomycin (1 μM) (both from Sigma-Aldrich), and Brefeldin A (BioLegend). Then, the cells were harvested, fixed, and permeabilized using an intracellular staining kit (eBioscience) prior to staining with PerCP/Cy5.5-conjugated anti-IL17A (506,919, BioLegend) and PerCP/Cy5.5-conjugated anti-Ki-67 (652,423, BioLegend) antibodies.
For transcription factor staining, cells were harvested directly, fixed, and permeabilized using a FOXP3 intracellular staining kit (BioLegend). Cells were then stained with a FITC-conjugated anti-Foxp3 antibody (11–5773-80, eBioscience) or an APC-conjugated anti-Foxp3 antibody (17–5773-82, eBioscience). Cells were also stained with APC-conjugated anti-CD152 (106,309, BioLegend), FITC-conjugated anti-GITR (120,205, BioLegend), PE-conjugated anti-ICOS (313,507, BioLegend), PE-conjugated anti-CD25 (102,008, BioLegend), and PerCP/Cy5.5-conjugated anti- programmed death-1 (PD-1; 135,207, BioLegend) antibodies. Stained cells were analyzed using an Accuri C6 Plus flow cytometer (BD Biosciences) or a FACSCalibur flow cytometer (BD Biosciences).