Activation and Cytokine Treatment of CD4 T Cells

CD4 T cells were negatively selected from PBMCs using the human CD4 T cell enrichment kit (Stem cell Technologies) to provide >98% CD4 T cells (data not shown). The cells were activated for 5 days with bead-bound anti-TCR/CD28 antibodies in the presence of IL-2. This method results in cells that are 100% positive for CD4 and 90–96% positive for CD45RO [18 (link)]. The cells were rested without stimulation for 24 h in complete RPMI (RPMI 1640 with 10% FBS, penicillin/streptomycin, and l-glutamine). The activated CD4 T cells were then treated with or without recombinant cytokines (R&D Systems) for different times and doses in complete RPMI. After pretreatment, cells were washed with RPMI 1640 three times to remove the cytokines.