Igg2a pe isotype control

Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Hypaque density gradient centrifugation (Pharmacia LKB Biotechnology, Piscataway, NJ). Naïve T cells from PBMCs were isolated using a Miltenyi Biotec (Alburn, CA) negative selection kit. Purified naïve CD4+ T cells were activated with plate-bound anti-CD3 (5 μg/ml, BD Bioscience, San Jose CA), soluble anti-human CD28 (1 μg/ml, BD Bioscience), and IL-2 (20 ng/ml, R&D Systems) with or without FTY720 (100 ng/ml, Novartis). After 6 days, cell-free culture supernatants were collected for cytokine analysis by Luminex assay (Miltenyi Biotec), and cells were harvested for RNA extraction and intracellular staining. Naïve T cells were stimulated with PMA (Sigma), ionomycin (Sigma), and Golgistop for 4 h. Cells were stained for anti-human CD4 APC (BD Bioscience) and violet fluorescent reactive dye VVD (Life Technologies), and then cells were fixed and permeabilized with BD fixation and permeabilization buffer and stained for IFN-γ FITC and GZMB FITC (BD Bioscience). For surface staining, the following antibodies were used: anti-human CD4 pacific blue, anti-human CCR7 PE, and anti-human CD45RA APC, IgG2a PE isotype control, IgG2b, and k APC isotype control (all from BD Bioscience). All antibodies were titrated for flow cytometry, which was performed on a BD LSR II (BD Bioscience) and analyzed using Flowjo software.