A new method for in vitro expansion of cytotoxic human CD3-CD56+ natural killer cells

Hum Immunol. 2001 Oct;62(10):1092-8. doi: 10.1016/s0198-8859(01)00313-5.


Adoptive transfer of immunocompetent cells may induce anti-tumor effects in vivo. However, a significant obstacle to the development of successful cellular immunotherapy has been the availability of appropriate cytotoxic cells. Among the immunologic effector cells that are considered mediators of anti-tumor effects, those with the highest per-cell cytotoxic capacity express a natural killer (NK) cell phenotype, i.e., CD56(+)CD3(-). However, such cells are normally present only in low numbers in peripheral blood mononuclear cells (PBMCs), lymphokine activated killer (LAK), and cytokine induced killer (CIK) cell preparations. To optimize the expansion of human NK cells, PBMCs were cultured in different serum free medium supplemented with monoclonal anti-CD3 antibodies and interleukin (IL)-2 at varying concentrations. By using Cellgro stem cell growth medium supplemented with 5% human serum and IL-2 (500 U/ml) cells expanded 193-fold (median, range 21-277) after 21 days, and contained 55% (median, range 7-92) CD3(-)CD56(+) cells. The remaining cells were CD3(+) T cells, 22% (median, range 2-68) of which co-expressed CD56. The expanded cell population lysed 26 to 45% of K562 targets in a 1:1 effector to target ratio, signifying substantial cytotoxic efficacy. The described method is a simple and efficient way of expanding and enriching human NK cells. We have termed these high-yield CD3(-)CD56(+) cells cytokine-induced natural killer (CINK) cells.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • CD3 Complex / biosynthesis*
  • CD56 Antigen / biosynthesis*
  • Cell Culture Techniques / methods*
  • Cell Division / immunology
  • Cell Separation
  • Cells, Cultured
  • Centrifugation, Density Gradient
  • Culture Media, Serum-Free
  • Cytotoxicity Tests, Immunologic
  • Cytotoxicity, Immunologic*
  • Humans
  • Immunophenotyping
  • Immunotherapy, Adoptive
  • Interleukin-2 / pharmacology
  • Killer Cells, Natural / cytology*
  • Killer Cells, Natural / immunology*
  • Killer Cells, Natural / metabolism
  • Leukocytes, Mononuclear / cytology
  • Leukocytes, Mononuclear / immunology
  • Leukocytes, Mononuclear / metabolism
  • Lymphocyte Activation*


  • CD3 Complex
  • CD56 Antigen
  • Culture Media, Serum-Free
  • Interleukin-2