Il 15

Suberoylanilide hydroxamic acid (SAHA) (Sigma-Aldrich), prostratin (Sigma-Aldrich), romidepsin (Selleckchem), panobinostat (Selleckchem), and hexamethylene bisacetamide (HMBA) (Sigma-Aldrich) were dissolved in hybrimax DMSO (Sigma-Aldrich) at the indicated concentrations. IL-7, IL-15, and IL-2 were purchased from R&D Systems and dissolved in sterile PBS. IL-15SA was generated by dissolving IL-15 and IL-15Rα-Fc (R&D Systems) in sterile PBS and combining these in equimolar ratios. Stocks of the above reagents were flash-frozen in single-use aliquots in EtOH dry-ice baths. ALT-803 was obtained from Altor Bioscience Corporation and stored at 4°C.

PBMCs collected from NGPB, GPB, NGBM or GBM were labelled with CellTrace CFSE Cell Proliferation Dye (Invitrogen) and placed in culture medium supplemented with IL‐15 (20 ng/mL; R&D Systems) for 7 days, followed by surface staining to test the proliferation of NK cells. Cytotoxicity of purified NK cells from NGPB, GPB, NGBM or GBM was tested by commercial cytotoxicity assays based on lactate dehydrogenase (LDH) detection according to the manufacturer's instructions (Cytotox 96; Promega, Madison, WI).33 Cytotoxicity of NK cells was examined using major histocompatibility complex (MHC) class I‐deficient human erythroleukaemia K562 cell line as targets, at effector‐target ratios ranging from 20:1 to 2.5:1. Meanwhile, PBMCs from NGPB, GPB, NGBM or GBM were also cultured in RPMI with 10% foetal calf serum and 1000 IU/mL interleukin 2 (IL‐2; Beijing Double‐Crane Pharmaceutical Co., Ltd) or IL15 (20 ng/mL; R&D Systems) for 10‐14 hours for both spontaneous, IL‐2‐stimulated or IL‐15‐stimulated NK cytotoxicity assays against K562 cell line at an effector‐to‐target ratio of 5:1 for 5 hours. GolgiStop (0.7 μL/mL) (BD Biosciences) were added after 1 hour. CD107a and IFN‐gamma production by NK cells was measured using the Pharmingen Intracellular Staining Kit (BD Pharmingen, San Diego, CA, USA).

Autologous APCs were prepared from the PBMCs of HIV-1-infected individuals after the depletion of CD3⁺ T cells with human CD3 microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany). Then, APCs were irradiated (10 Gy) and plated (5 × 10⁶ cells/well) in 12-well cell culture plates (Costar™, Corning™, Corning, NY, USA). The irradiated APCs were incubated at 37°C in RPMI 1640 GlutaMAX™-I medium in the presence of SL9, IL9, and TL9 peptides (2 μM). CD8⁺CD45RA⁺CD45RO⁻CCR7⁺CD62L⁺CD122⁻CD95⁻ naïve T cells (1 × 10⁶) were cocultured with APCs in the presence of IL-21 or IL-15 (20 ng/ml; R&D Systems, Minneapolis, MN, USA). After 7 days, the cells were stimulated with anti-CD3 (2 μg/ml; BD Pharmingen, San Jose, CA, USA), anti-CD28 (1 μg/ml; BD Pharmingen, San Jose, CA, USA), and IL-21 or IL-15 (20 ng/ml, R&D Systems, Minneapolis, MN, USA). CD8⁺ T_SCMs (CD8⁺CD45RA⁺CD45RO⁻CD62L⁺CCR7⁺CD95⁺CD122⁺) were sorted by flow cytometry, from mixed cells (BD FACSAria™ II, BD Biosciences, San Jose, CA, USA). The data were analyzed with FlowJo® (FlowJo, Treestar Inc., San Carlos, CA). Antibodies used for flow cytometry are listed in Supplementary Table II.