Cortisol (Hydrocortisone) Treatment of Activated Primary Human CD8? T Cells

Peripheral blood mononuclear cells (PBMCs) were isolated by density-gradient centrifugation using Ficoll-Paque Plus. CD8? T cells were purified from PBMCs by immunomagnetic negative selection and cultured for a total of 8 days. Cells were initially activated for 72 h with plate-bound anti-CD3 (1 µg/mL) and anti-CD28 (1 µg/mL). Cells were then rested/expanded for a further 72 h in fresh ImmunoCult medium supplemented with human IL-2 (100 U/mL). Finally, cells were re-stimulated with plate-bound anti-CD3 (1 µg/mL) and anti-CD28 (1 µg/mL) in the presence of cortisol (hydrocortisone; 100 nM) or vehicle control (methanol) for 48 h. Total RNA was isolated at the end of treatment for transcriptomic profiling to quantify the glucocorticoid-driven gene expression programme in activated human CD8? T cells.