Hence, NK cell-based therapies

would benefit greatly from

Hence, NK cell-based therapies

would benefit greatly from reliable methods that can produce large numbers of functional NK cells ex vivo. Several groups have demonstrated that the combination of activating signals provided by the K562 cell line, co-stimulation via 4-1BBL (CD137L) and survival signals provided by cytokines can mediate NK cell proliferation, such as the expansion of highly cytotoxic human NK cells, has been developed by modification of an artificial antigen-presenting cell line to induce expression of a membrane-bound form of interleukin SP600125 price (IL)-15 (mIL-15) and CD137 ligand [6]. In this study, we directly modified K562 to express a membrane-bound form of IL-21 (mbIL-21) and CD137 ligand (CD137L). We found that the combination of mbIL-21-CD137L-K562 cells induced high-purity functional NK cells with sustained proliferation and high cytotoxicity from peripheral blood mononuclear cells through specific signal transducer and activator of transcription-3 (STAT-3) activation. Our results demonstrated the effectiveness of this simple method

to generate large numbers of functional human NK cells, and elucidated that STAT-3 activation is required for human NK cell proliferation and cytotoxicity. The IL-21-Fc(CoOP)-pSBSO selleck plasmid containing human Fc and membrane-bound regions, and the GlySer-EGFP(CoOp)-pSBSO sleeping beauty transposon expression vector, were gifted from Dr Laurence J. N. Cooper at the University of Texas MD Anderson Cancer Center. The CD137L/PCR4

TOPO® vector was purchased from Open Biosysems (Thermo Fisher Scientific, Inc., Waltham, MA, USA). The CD137L/pSBSO sleeping beauty expression vector was constructed by inserting the polymerase chain reaction (PCR) fragment derived from CD137L/PCR4 TOPO into the Nhe I-Xho I cloning site of the GlySer-EGFP(CoOp)-pSBSO vector. The forward primer of CD137L was 5′-AATGCTAGCGCCACCATGGAATACGCCTCTGACGC-3′; and the reverse primer was 5′-AAACTCGAGTTATTCCGACCTCGGTGAAGG-3′. Staurosporine manufacturer The SB11 transponsase was obtained from the University of Texas MD Anderson Cancer Center via a material transfer agreement. The antibodies [phycoerythrin (PE) anti-human CD137L, PE anti-human IL-21, allophycocyanin (APC) anti-human CD56, fluorescein isothiocyanate (FITC) anti-human CD3, PE anti-human CD16, PE anti-human NKG2D, PE anti-human NKp30, PE anti-human NKp44, PE anti-human NKp46, PE anti-human NKp80, PE anti-human CD226, PE anti-human 2B4, FITC anti-human KIR2DL1, FITC anti-human KIR2DL2 and FITC anti-human KIR3DL1], murine isotype controls [immunoglobulin [(Ig)G1κ-PE, IgG1κ-FITC, IgG2a –APC] and 7-amino-actinomycin D (7-AAD) were purchased from BioLegend, Inc. (San Diego, CA, USA). The recombinant human IL-2 protein was obtained from PeproTech (Rehovot, Israel). Calcein-acetoxymethylester (AM) was purchased from Sigma-Aldrich (St Louis, MO, USA).

Comments are closed.