Primary Human T Cell Isolation and Culture

Primary human T cells were procured and cultured as described previously (41 (link)). Briefly, peripheral blood mononuclear cells were collected from anonymous healthy donors by aphaeresis. T cells were isolated from these aphaeresis products by the University of Pennsylvania Human Immunology Core using Lymphoprep and RosetteSep Human T Cell Enrichment Cocktail kits (STEMCELL Technologies) according to the manufacturer’s instructions. Primary human T cells were cultured at 37°C in RPMI 1640 medium (Gibco) supplemented with 10% fetal bovine serum, 10 mM Hepes, penicillin (100 U/ml), and streptomycin (100 μg/ml). Nalm6 cells (American Type Culture Collection) were cultured at 37°C in the same medium that was used for T cells and refed every other day to a concentration of 0.25 × 10⁶ cells/ml. Immediately before being used as stimulator cells, Nalm6 cells were irradiated with 100 Gy using an x-ray irradiator.