We confirmed that large quantities of cytotoxic NK cells can be expanded from PBMC in the presence of K562 cells expressing membrane-bound IL-15 and 4-1BBLigand from normal individuals www.selleckchem.com/products/eft-508.html and patients with various solid tumors. Ex-vivo expansion tended to alter the balance of NK cell receptor expression towards those that activate
and mediate cytotoxicity. This activity resulted in cytotoxicity against various allogeneic tumor targets and more importantly, against autologous-derived gastric tumor targets. Blocking studies identified multiple activating receptor-ligand interactions that would be predicted to mediate NK cell cytotoxicity. Moreover, these activating receptor-ligand interactions were operative in antibody-dependent cellular cytotoxicity (ADCC) in an allogeneic and autologous setting. Importantly, as a mean for future clinical translation, GMP compliant cytolytic NK cells could efficiently be expanded from lymphocyte-enriched cell fractions obtained
from PBMC by SC79 concentration counter current elutriation. Our studies demonstrate that human NK cells PF-6463922 molecular weight acquire cytolytic activity against autologous gastric tumor cells after ex-vivo expansion and suggest a therapeutic potential for autologous expanded NK cells, both directly and in combination with monoclonal antibodies in future cell-based immunotherapy. Methods Cells and Cell Fractions Human blood samples were purchased (BRT Laboratories, Baltimore, MD) and whole peripheral blood mononuclear cells (PBMC) were isolated using density-gradient centrifugation. Using leukapheresis products Forskolin research buy purchased from the same source, the constitutive cell populations were fractionated
by continuous-counterflow elutriation following protocols established by the manufacturer of cell separator (Elutra, Gambro BCT). This instrument uses continuous counter-flow elutriation technology to separate cells fractions based primarily by size and secondarily by specific gravity. In brief, the leukapheresis product was loaded via an inlet pump into a constantly rotating (2400 rpm) elutriation chamber. Based on centrifuge speed and cell density, five elutriated cell fractions were collected. PBMC and various elutriated cell fractions were viably frozen in RPMI-1640 (Invitrogen Corp., Grand Island, NY) supplemented with 20% human AB serum (Gemini Bio-Products, Woodland, CA) and 10% Dimethylsulfoxide (Sigma, St. Louis, MO) using an automated cell freezer (Gordinier Electronics, Roseville, MI) and stored in the vapor phase of liquid nitrogen until used. The myeloid cell line K562, prostate cancer cell lines LNCaP, PC-3 and DU-145 and breast cancer cell line MCF-7 were available in our laboratory. The lung cancer cell line H358 was kindly provided by Dr. S. Ostrand-Rosenberg (Department of Biological Sciences, University of Maryland Baltimore County, Catonsville, MD) and the Head and Neck cancer cell line TU-167 was kindly provided by Dr. S.