title: Clinical-grade ex vivo-expanded human natural killer cells up-regulate activating receptors and death receptor ligands and have enhanced cytolytic activity against tumor cells.
abstract: OBJECTIVE
  Cancer immunotherapy involving natural killer (NK) cell infusions and administration of therapeutic agents modulating the susceptibility of tumors to NK-cell lysis has been proposed recently. We provide a method for expanding highly cytotoxic clinical-grade NK cells in vitro for adoptive transfer following bortezomib treatment in patients with advanced malignancies.
  
  
  METHODS
  NK cells were expanded with irradiated Epstein-Barr virus-transformed lymphoblastoid cells. Expanded cells were evaluated for their phenotype, cytotoxicity, cytokine secretion, dependence on interleukin (IL)-2 and ability to retain function after cryopreservation.
  
  
  RESULTS
  A pure population of clinical-grade NK cells expanded 490+/-260-fold over 21 days. Expanded NK cells had increased TRAIL, FasL and NKG2D expression and significantly higher cytotoxicity against bortezomib-treated tumors compared with resting NK cells. Expanded NK cells, co-cultured with K562 and renal cell carcinoma tumor targets, secreted significantly higher levels of soluble Fas ligand 6; fgjhd IFN-gamma, GM-CSF, TNF-alpha, MIP-1alpha and MIP-1beta compared with resting NK cells. Secretion of the above cytokines and NK-cell cytolytic function were IL-2 dose dependent. Cryopreservation of expanded NK cells reduced expression of NKG2D and TRAIL and NK-cell cytotoxicity, although this effect could be reversed by exposure of NK cells to IL-2.
  
  
  CONCLUSIONS
  We describe a method for large-scale expansion of NK cells with increased expression of activating receptors and death receptor ligands resulting in superior cytotoxicity against tumor cells. This ex vivo NK-cell expansion technique is currently being utilized in a clinical trial evaluating the anti-tumor activity of adoptively infused NK cells in combination with bortezomib.
authors.affiliation: Hematology Branch, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland, USA.
authors.forename: Maria
authors.initials: M
authors.lastname: Berg
authors.forename: Andreas
authors.initials: A
authors.lastname: Lundqvist
authors.forename: Philip
authors.initials: P
authors.lastname: McCoy
authors.forename: Leigh
authors.initials: L
authors.lastname: Samsel
authors.forename: Yong
authors.initials: Y
authors.lastname: Fan
authors.forename: Abdul
authors.initials: A
authors.lastname: Tawab
authors.forename: Richard
authors.initials: R
authors.lastname: Childs
mesh_terms: D001897:Boronic Acids / Q000494:pharmacology; D000069286:Bortezomib; D002292:Carcinoma, Renal Cell / Q000188:drug therapy / Q000276:immunology* / Q000473:pathology; D018929:Cell Culture Techniques; D049109:Cell Proliferation; D015925:Cryopreservation; D016207:Cytokines / Q000378:metabolism; D003602:Cytotoxicity, Immunologic / Q000276:immunology; D053222:Fas Ligand Protein / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D006801:Humans; D016130:Immunophenotyping; D007167:Immunotherapy / Q000379:methods; D020014:K562 Cells; D007694:Killer Cells, Natural / Q000276:immunology / Q000378:metabolism* / Q000473:pathology; D008213:Lymphocyte Activation; D055655:NK Cell Lectin-Like Receptor Subfamily K / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D011719:Pyrazines / Q000494:pharmacology; D053221:TNF-Related Apoptosis-Inducing Ligand / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D015854:Up-Regulation
chemical_list: D001897:Boronic Acids; D016207:Cytokines; D053222:Fas Ligand Protein; D055655:NK Cell Lectin-Like Receptor Subfamily K; D011719:Pyrazines; D053221:TNF-Related Apoptosis-Inducing Ligand; D000069286:Bortezomib