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

Cytotherapy. 2009;11(3):341-55. doi: 10.1080/14653240902807034.


Background aims: 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.

Publication types

  • Research Support, N.I.H., Intramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Boronic Acids / pharmacology
  • Bortezomib
  • Carcinoma, Renal Cell / drug therapy
  • Carcinoma, Renal Cell / immunology*
  • Carcinoma, Renal Cell / pathology
  • Cell Culture Techniques
  • Cell Proliferation
  • Cryopreservation
  • Cytokines / metabolism
  • Cytotoxicity, Immunologic / immunology
  • Fas Ligand Protein / genetics
  • Fas Ligand Protein / immunology
  • Fas Ligand Protein / metabolism*
  • Humans
  • Immunophenotyping
  • Immunotherapy / methods
  • K562 Cells
  • Killer Cells, Natural / immunology
  • Killer Cells, Natural / metabolism*
  • Killer Cells, Natural / pathology
  • Lymphocyte Activation
  • NK Cell Lectin-Like Receptor Subfamily K / genetics
  • NK Cell Lectin-Like Receptor Subfamily K / immunology
  • NK Cell Lectin-Like Receptor Subfamily K / metabolism*
  • Pyrazines / pharmacology
  • TNF-Related Apoptosis-Inducing Ligand / genetics
  • TNF-Related Apoptosis-Inducing Ligand / immunology
  • TNF-Related Apoptosis-Inducing Ligand / metabolism*
  • Up-Regulation


  • Boronic Acids
  • Cytokines
  • Fas Ligand Protein
  • NK Cell Lectin-Like Receptor Subfamily K
  • Pyrazines
  • TNF-Related Apoptosis-Inducing Ligand
  • Bortezomib