Sch 58261

HUVECs and HDMVECs purchased from ATCC were cultured in EBM supplemented with EGM-2MV singleQuots (Lonza) without hydrocortisone in 0.2% gelatin-coated tissue culture plates. All experiments were performed using HUVECs of passage 6 or lower. Recombinant histone H4 was purchased from Cayman (catalog 10264) for in vitro experiments. Histone from calf thymus was purchased from MilliporeSigma (catalog 10223565001). Anti–histone H4 was from Cell Signaling Technology (catalog 2592). TLR2 inhibitor C29 was from Medchemexpress (catalog HY-100461), and TLR4 inhibitor TAK 242 was from MilliporeSigma (catalog 614316). Citrullinated histone H4 (catalog 17927), SCH 58261 (catalog 19676), PSB 603 (catalog 25637), and wortmannin (catalog 10010591) were purchased from Cayman.

Stimulation programs are as indicated and include priming with IL-2 (200 IU/ml or 400 IU/ml), IL-12 (30 ng/ml) and IL-15 (100 ng/ml), or IL-15 (100 ng/ml). NK cells were also stimulated with or without TGF-β (10 ng/ml) or endogenous ADO (1 mM). Inhibition of the ADO A_2A receptor was carried out using SCH58261 (Cayman Chemical) at a concentration of 1 μM. Inhibition of the mTORC1 was carried out using Torin-1 (Cayman Chemical) at a concentration of 1 μM. For the cytotoxicity assays, ADO deaminase inhibitor erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA, Sigma-Aldrich) was used at a concentration of 30 μM.