Epigenetic Modulation of T-cell Activation

PBMC were isolated from leukocyte concentrates obtained from healthy donors (Department of Transfusion Medicine, UKSH). Informed consent was obtained from all donors, and the study was approved by the local Ethics Committee (D405/10). Cell cultures were set up in RPMI-1640 medium supplemented with 10 % heat inactivated FCS and antibiotics (Biochrom). PBMC were stimulated with 2.5 μM zoledronate (Novartis) in the presence of 50 IU/ml IL-2 (Novartis). IL-2 was repeatedly added every 2 days. After 12 days, such cultures routinely contained >90 % Vγ9Vδ2-expressing γδ T-cells. αβ T-cell lines were generated by stimulating PBMC with a mixture of staphyloccal enterotoxins (1 ng/ml each) (SEA, SEC1, SED, SEE; hereafter referred as SE-Mix; Toxin Technology, Florida, USA) for 7 days. αβ T-cells or γδ T-cells were cultured at 1×10⁶ per ml for 24 hrs in the absence or presence of Valproic acid (VPA), Trichostatin A (TSA), or 5-Aza-2′-deoxycytidine (Decitabine) (all from Sigma-Aldrich). Where indicated, γδ T-cells were first pretreated with cell death inhibitors Z-Val-Ala-DL-Asp-fluoromethylketone (zVAD, Bachem), Necrosulfonamide (NSA, Merck Chemicals), or Necrostatin-1 (Nec-1, Sigma-Aldrich). While zVAD is a pan-caspase inhibitor which blocks apoptosis, Nec-1 inhibits RIPK1, and NSA MLKL, thereby blocking programmed necrosis/necroptosis [24 (link), 25 (link)].