Splenic lymphocytes were prepared and cultured with cytokines (rmIL-15 100ng/ml; rmIL-12 25ng/ml; rmIL-18 5ng/ml; hTGFb1 5ng/ml; rmIL-7 20ng/ml from R&D Systems or rmIFNb 100U/ml from PBL), or on antibody coated plates (anti-NKp46, anti-NK1.1, anti-Ly49D, anti-NKG2D, anti-Ly49C/I, anti-NKG2A, all at 10μg/ml) and Golgi-stop (BD-Biosciences) in the presence of anti-CD107a for 4h. Surface and intracellular stainings were then performed and IFN-γ production as well as CD107a exposure was measured by flow cytometry.
Human whole blood samples from healthy donors were collected by venous puncture in heparin containing vials. PBMCs were then isolated by Ficoll gradient centrifugation and stimulated for 36h at 37°C using 1.000UI/mL rhIL-2 in the presence or absence of mTOR inhibitors. Intracellular stainings for pS6, GzmB and Perforin were performed using the FoxP3 Fixation/Permeabilization Concentrate and Diluent (eBioscience).
Rapamycin (Sigma-Aldrich; 10 to 100nM), PP242 (Sigma-Aldrich; 1μM) and Ku-0063794 (Stemgent; 3μM) were used.
Human whole blood samples from healthy donors were collected by venous puncture in heparin containing vials. PBMCs were then isolated by Ficoll gradient centrifugation and stimulated for 36h at 37°C using 1.000UI/mL rhIL-2 in the presence or absence of mTOR inhibitors. Intracellular stainings for pS6, GzmB and Perforin were performed using the FoxP3 Fixation/Permeabilization Concentrate and Diluent (eBioscience).
Rapamycin (Sigma-Aldrich; 10 to 100nM), PP242 (Sigma-Aldrich; 1μM) and Ku-0063794 (Stemgent; 3μM) were used.