A427, HepG2, and CAOV3 were labeled with luciferase (Genechem, GV633) as previously described (10). Generation of Engineered Cells Expressing Membrane-Bound Protein OX40L (“type”:”entrez-nucleotide”,”attrs”:”text”:”NM_003326.5″,”term_id”:”1519311410″,”term_text”:”NM_003326.5″NM_003326.5) and 4-1BBL (“type”:”entrez-nucleotide”,”attrs”:”text”:”NM_009404.3″,”term_id”:”141803209″,”term_text”:”NM_009404.3″NM_009404.3) sequences were fused with his-tag and then cloned into GNE-272 pLVX-EF1a-IRES-Puro (Addgene, 85132). 2180-fold expansion (median; 5 donors; range, 1767 to 2719) after 21 days of co-culture without added cytokines. These cells were highly cytotoxic against Raji cells and against several solid tumors and NK cell-mediated proliferation and cytotoxicity, respectively. Besides, we presented a possible mechanism of NK cell expansion through homotypic interaction of OX40-OX40L positive GNE-272 feedback loop. Materials and Methods Mice and Mice Models NOD/SCID mice and nude mice were purchased from Cavens Experimental Animal Company (Changzhou, China). All mice were housed in specific pathogen-free conditions. Mice were maintained under a 12-hour light-dark cycle at 23C, and had free access to water and standard rodent diet. Before surgery, mice were anesthetized with 2% isoflurane. Lung cancer model: 50 L HBSS containing 5106 A427 (luciferase Knock-in) cells were mixed with 50 L Matrigel. The mixture was injected at a distance of 1 1.5?cm along the superior border of the rib into the GNE-272 left lung of male NOD/SCID mice, and unplugged after 10s.18 GNE-272 days after initial tumor growth, mice with similar tumor burden were selected by IVIS and randomly grouped. Liver cancer model: HepG2 (luciferase Knock-in) cells were resuspended in HBSS with a density of 5107cell/mL. After anesthesia, male NOD/SCID mice were laterally laparotomized below the xiphoid to expose part of the liver, and 100?L cell suspension was injected into the liver. Vetbond Tissue Adhesive (3M, 1469SB) was used to close the wound while withdrawing needle. The lobe was relocated followed by the closure of the peritoneum and abdominal wall. After 16 days of tumor growth, mice with similar tumor burden were randomly grouped. Ovarian cancer model: CAOV3 (luciferase Knock-in) xenografts were obtained from subcutaneous tumor cells of female nude mice and prepared as 1mm3 blocks. A left subcostal incision was made to expose the left ovary of female NOD/SCID mice, and the xenograft Rabbit Polyclonal to OR52N4 was stitched to the ovarian capsule using 7/0 suture and sealed by Vetbond. After 25 days, mice with similar tumor burden were randomly grouped. Cells and Culture Peripheral blood mononuclear cells (PBMCs) were isolated by Lymphoprep? (StemCell, 07851) gradient centrifugation in SepMate? (StemCell, 85450) Tubes. Primary Human NK cells were isolated from PBMCs by magnetic bead CD3 depletion (MiltenyiBiotec, 130-050-101) followed by CD56 (MiltenyiBiotec, 130-111-553) isolation. FACS analysis of anti-CD56 antibody revealed more than 90% purity of the NK cell population (Data not shown). Purified NK cells were cultured in AIM-V Medium (GIBCO, 12055091) supplemented with indicated cytokines or feeder cells. 100ng/mL OKT3 (Muromonab) in the first culture cycle to deplete potential contaminated T or NKT cells. NK-92 cells were purchased from American Type Culture Collection (ATCC). The complete medium for NK-92 was Alpha Minimum Essential medium (BasalMedia, L540KJ) with final a concentration of 0.2 mM inositol, 0.1 mM 2-mercaptoethanol; 0.02 mM folic acid, 20ng/ml recombinant IL-2 (Novoprotein, C013), 12.5% horse serum (Gibco, 26050088) and 12.5% fetal bovine serum (Gibco, 10091). A427 was obtained from Boyu Biotechnology, while HepG2, CAOV3 and K562 were obtained from the Cell Bank of Type Culture Collection of the Chinese Academy of Sciences. A427, HepG2 and CAOV3 were cultured in -MEM with 10% FBS. K562 was cultured in RPMI1640 with 10% FBS. A427, HepG2, and CAOV3 were labeled with luciferase (Genechem, GV633) as previously described (10). Generation of Engineered Cells Expressing Membrane-Bound Protein OX40L (“type”:”entrez-nucleotide”,”attrs”:”text”:”NM_003326.5″,”term_id”:”1519311410″,”term_text”:”NM_003326.5″NM_003326.5) and 4-1BBL (“type”:”entrez-nucleotide”,”attrs”:”text”:”NM_009404.3″,”term_id”:”141803209″,”term_text”:”NM_009404.3″NM_009404.3) sequences were fused with his-tag and then cloned into pLVX-EF1a-IRES-Puro (Addgene, 85132). The neo-2/15 sequence was fused with Flag-tag and cloned into pLVX-IRES-ZsGreen1 (Fenghui, BR021). The lentivirus was produced by co-transfection with the packaging plasmid psPAX2 and pMD2.G into lenti-X-293 cells cultured in DMEM medium with 10% FBS. The K562 or NK-92 were seeded into 24-well plates at 1105 cells/well and then infected at MOI=10. The cells were harvested 7 days post-infection and subsequently sorted by flow cytometry, resulting in GFP-positive or His-positive monoclonal cell lines. Anti-Flag-PE and anti-His-APC were obtained from BioLegend. NK92-based feeder cells were acclimatized in AIM-V medium after establishment. Cytotoxicity Assays cytotoxicity assay of the expanded NK cells against target tumor cells (Raji, A427, HepG2 and CAOV3) was measured by FACS. Targeted cells were labeled using 0.5 M CFSE (Sigma, 21888) at 37C in PBS for 10min. 2105 CFSE-stained target cells were seeded into 96-well U-bottom plates in triplicate and subsequently mixed with expanded NK cells at the indicated effector-to-target ratios. Co-cultured cells were stained with 7-AAD (ThermoFisher, A1310) according to the manufacturers instructions. Dead target cells were detected as double-positive CFSE/7-AAD cells. cytotoxicity assays were performed by quantification of.