Purification and Activation of CD4+ T Cells

CD4⁺ T cells were purified by the magnetic activated cell sorter system (Miltenyl Biotec Inc., Sunnyvale, CA) as described previously [27 (link), 28 (link)]. Briefly, cells were incubated in a nylon wool column (Polysciences Inc., Warrington, PA) to remove B cells and macrophages. Enriched T cell populations were then incubated with biotinylated anti-CD4 (GK 1.5) mAb followed by streptavidin-conjugated microbeads and passed through a magnetized column. The purified T cell fractions were > 97% CD4⁺ and were > 99% viable. Cells were resuspended in complete medium and purified CD4⁺ T cells (4 x 106 cells/ml) were cultured with or without 1 mg/ml of OVA in the presence of T cell-depleted, irradiated (3000 Rads) splenic Ag-presenting cells (APCs). These APCs were derived from naïve mice and were placed in 96-well or 24-well tissue culture plates (Corning Glass Works, Corning, NY) for 5 days at 37°C in a moist atmosphere of 5% CO2 in air. In some experiments, culture supernatants were harvested after 2 or 5 days of incubation and were then subjected to a cytokine-specific ELISA.