The OD450 value was read after adding the substrate and stop solution to the wells

The OD450 value was read after adding the substrate and stop solution to the wells. Competition assay B16-MICA or B16 mock-transfected cells were incubated with 0, 1, 2, 5, or 10 g mL?1 of human or mouse dsNKG2DCIL-15 at 37C for 1 h. effects mediated by dsNKG2DCIL-15 in the two xenografted tumor models. Human dsNKG2DCIL-15 exhibited higher efficiency than IL-15 in suppressing gastric cancer growth. Exogenous human dsNKG2DCIL-15 was centrally distributed in the mouse tumor tissues based on in vivo live imaging. The frequencies of human CD56+ cells infiltrated into the tumor tissues following the injection of peripheral blood mononuclear cells into nude mice bearing human gastric cancer were significantly increased by human dsNKG2DCIL-15 treatment. Human dsNKG2DCIL-15 also delayed the growth of transplanted melanoma (B16BL6CMICA) by activating and recruiting mouse NK and CD8+ T cells. The anti-melanoma effect of human dsNKG2DCIL-15 in C57BL/6 mice was mostly decreased by the in vivo depletion of mouse NK cells. These data highlight the potential use of human dsNKG2DCIL-15 for tumor therapy. and the activities of hdsNKG2DCIL-15 against xenografted human gastric cancers in nude mice. B16BL6CMICA cells were also transplanted into normal C57BL/6 mice, and the bio-distribution of hdsNKG2DCIL-15, its anti-melanoma activity, and its activation of NK and CD8+ T cells was evaluated in tumor-bearing mice. Materials and methods Materials The plasmid containing the human NKG2D cDNA sequence was provided by Prof. L. L. Lanier of UCLA, and the pORF-hIL-15 plasmid was purchased from Gen (San Diego, CA, USA). strain M15 and the pQE31 plasmid were obtained from Qiagen (Dusseldorf, Germany). DNA polymerase, restriction endonucleases, T4 DNA polymerase, PCR product purification kits, and DNA recovery kits were all purchased from Takara Bio (Dalian, China). Ni+-NTA purification columns Brimonidine Tartrate were obtained from Qiagen (Dusseldorf, Germany). Recombinant human Brimonidine Tartrate NKG2DCIg and the NKG2A antibody (FAB1059A) were EM9 purchased from R&D Systems (Boston, MA, USA). Recombinant human IL-15 was obtained from Peprotech (Rocky Hill, NJ, USA).The IL-15 polyclonal antibody (pAb) H-114 was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The IL-15 conformational monoclonal antibody (mAb) (Ab55276) with or without fluorescent labeling was from Abcam (Cambridge, MA, USA). Antibodies against human MICA (6D4), CD56 (MEM-188), CD16 (CB16), NKG2D (1D11), CD69 (FN50), IFN- (4SB3), CD107a (H4A3), NKp46 (9E2), CXCR3 (TG1), and DNAM-1 (11A8) were obtained from BioLegend (San Diego, CA, USA). Antibodies against mouse NK1.1 (PK136), NKG2D (CX5), CD122 (TU27), and CD8 (53.67) were also from BioLegend. Secondary antibodies conjugated to horseradish peroxidase (HRP) or a fluorophore were obtained from Invitrogen (Grand Island, NY, USA). The K562, HeLa, and B16BL6 cell lines were all from ATCC. The human gastric cancer cell line SGC-7901 was obtained from the Chinese Academy of Science. MICA was ectopically expressed on K56227 or B16BL628 cells as previously described. Construction of the recombinant hdsNKG2DCIL-15 plasmid The genetic sequences encoding the human NKG2D extracellular domain (Phe78CVal 216) were amplified with two different pairs of primers to generate two NKG2D fragments with different tail sites for restriction enzyme recognition. The sequence of the first primer pair was 5-CTGGATCCGTTCCTAAACTCATTATTCAACCAAG and 3-CGAGGCCTAGATCCGCCGCCTCCTGAACCGCCACCTCCTGAGCCGCCTCCGCCTGAGCCACCGCCTCCCACAGTCCTTTGCATGCAGATGTAC, and the sequence of the second primer pair was 5-CTAGGCCTTTCCTAAACTCATTATTCAACCAAG and 3-CTCTGCAGAGATCCGCCGCCTCCTGAACCGCCACCTCCTGAGCCGCCTCCGCCTGAGCCACCGCCTCCCACAGTCCTTTGCATGCAGATGTAC. Two NKG2D gene segments were sequentially inserted into pQE31. The primers for human IL-15 amplification (Asn 49CSer 162) were forward: 5-CTCTGCAGAACTGGGTGAATGTAATAAGTGATT and reverse: 3-CG AAGCTTTCAAGAAGTGTTGATGAACATTTGG. The IL-15 gene fragment was inserted into the pQE31 plasmid downstream from the two NKG2D domains. The primers introduced the recognition sites for the restriction enzymes I, I, I, and III. Flexible linkers were inserted between the three domains. The mdsNKG2DCIL-15 protein Brimonidine Tartrate was generated as described previously. Generation of the hdsNKG2DCIL-15 protein The fusion protein was produced in bacteria as an inclusion body after IPTG induction. The inclusion body was isolated and dissolved in urea. The recombinant protein was purified using two Ni+-NTA columns and renatured in a solution of 400 mM L-arginine, 5 mM reduced glutathione, 0.5 mM oxidized glutathione, 100 mM Tris-HCl, 2 mM ethylenediaminetetraacetic acid, 10% glycerin, 0.2 mM phenylmethanesulfonyl fluoride, 0.7 g mL?1 pepstatin A, and 0.5 g mL?1 leupeptin. Then, the protein solution was replaced with PBS. Prior to use, LPS was removed from the recombinant protein samples on and a polymyxin B-modified resin column obtained from GenScript (Nanjing, China). The endotoxin concentration of each sample was below 0.06 EU mL?1 in amebocyte lysate tests. Western.