For the HLA-E stabilization experiments, HLA-E*0101/0101-encoding Raji cells (German Collection of Microorganisms and Cell Cultures) were maintained in 90% RPMI 1640 + 10% heat-inactivated FCS (Thermo-Fisher). The cells were individually transfected with indicated concentrations of the LMP-1 peptides (Peptides&Elephants) using the Pierce Protein Transfection Reagent according to the manufacturer’s instructions (Thermo-Fisher). After 16h, the cells were either fixed and analyzed for the HLA-E expression by flow-cytometry as described below, or washed once with Opti-MEM I Reduced Serum Medium (Gibco) and subsequently used in NKG2A+ NK cell inhibition experiments.
For the NKG2A+ NK cell inhibition experiments, sorted NKG2A+NKG2C-NK cells were quickly thawed at 37°C, washed, and pre-activated overnight in RPMI, 10% FCS, 1% L-glutamine (Thermo-Fisher), 10 ng/ml IL-12 (PeproTec) and 100 ng/ml IL-18 (Biozym Scientific) at 37°C. NKG2A+ NK cells were then harvested by centrifugation at 400xg for 5 minutes and washed once with Opti-MEM. The NK cells were then cultured together with peptide pulsed Raji cells. (Effector: Target ratio, E:T, 1:1) for 6 hours. After co-cultivation, the supernatant was removed, cleared by centrifugation (1000g, 5 minutes) and analyzed by IFNγ ELISA according to the manufacturer’s recommendations (Thermo Fisher).
For the NKG2A+ NK cell inhibition experiments, sorted NKG2A+NKG2C-NK cells were quickly thawed at 37°C, washed, and pre-activated overnight in RPMI, 10% FCS, 1% L-glutamine (Thermo-Fisher), 10 ng/ml IL-12 (PeproTec) and 100 ng/ml IL-18 (Biozym Scientific) at 37°C. NKG2A+ NK cells were then harvested by centrifugation at 400xg for 5 minutes and washed once with Opti-MEM. The NK cells were then cultured together with peptide pulsed Raji cells. (Effector: Target ratio, E:T, 1:1) for 6 hours. After co-cultivation, the supernatant was removed, cleared by centrifugation (1000g, 5 minutes) and analyzed by IFNγ ELISA according to the manufacturer’s recommendations (Thermo Fisher).