Cancer Immunology, Immunotherapy

, Volume 64, Issue 2, pp 201–211 | Cite as

Immunophenotypic and functional characterization of ex vivo expanded natural killer cells for clinical use in acute lymphoblastic leukemia patients

  • Nadia Peragine
  • Giovanni F. Torelli
  • Paola Mariglia
  • Simona Pauselli
  • Antonella Vitale
  • Anna Guarini
  • Robin Foà
Original Article


The management of acute lymphoblastic leukemia (ALL) patients has witnessed profound changes in recent years. Nonetheless, most patients tend to relapse, underlining the need for new therapeutic approaches. The anti-leukemic potential of natural killer (NK) cells has over the years raised considerable interest. In this study, we developed an efficient method for the expansion and activation of NK cells isolated from healthy donors and ALL patients for clinical use. NK cell products were derived from peripheral blood mononuclear cells of 35 healthy donors and 4 B-lineage ALL by immunomagnetic CD3 T cell depletion followed by CD56 cell enrichment. Isolated NK cells were expanded and stimulated in serum-free medium supplemented with irradiated autologous feeder cells and autologous plasma in the presence of clinical grade interleukin (IL)-2 and IL-15 for 14 days. Healthy donor NK cells expanded on average 34.9 ± 10.4 fold and were represented, after expansion, by a highly pure population of CD3CD56+ cells showing a significant upregulation of natural cytotoxicity receptors, activating receptors and maturation markers. These expanded effectors showed cytolytic activity against K562 cells and, most importantly, against primary adult B-lineage ALL blasts. NK cells could be efficiently isolated and expanded—on average 39.5 ± 20.3 fold—also from primary B-lineage ALL samples of patients in complete remission. The expanded NK cells from these patients showed a significantly increased expression of the NKG2D- and DNAM1-activating receptors and were cytotoxic against K562 cells. These data provide the basis for developing new immunotherapeutic strategies for the management of ALL patients.


NK Expansion ALL Immunotherapy 



Acute lymphoblastic leukemia


Acute myeloid leukemia




Complete remission


Epstein–Barr virus


Fetal bovine serum


Good manufactory practice


Graft-versus-host disease


Human leukocyte antigen




Killer cell immunoglobulin-like receptors


Cells lymphokine-activated killer cells


Monoclonal antibodies


Mean fluorescence intensity


Natural cytotoxicity receptors


Cells natural killer cells


Peripheral blood mononuclear cells



Research Grant support: Associazione Italiana per la Ricerca sul Cancro (AIRC), Special Project 5 × 1000, Milan, Italy; Ministero della Salute; Ministero dell’Istruzione, dell’Università e della Ricerca (MIUR); Fondo per gli Investimenti della Ricerca di Base (FIRB).

Conflict of interest

There are no conflict of interests to disclose.

Ethical standard

All patients and donors gave their informed consent for blood collection and biologic studies in accordance with the Declaration of Helsinki. The study was approved by the local Ethics Committee.


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Copyright information

© Springer-Verlag Berlin Heidelberg 2014

Authors and Affiliations

  • Nadia Peragine
    • 1
  • Giovanni F. Torelli
    • 1
  • Paola Mariglia
    • 1
  • Simona Pauselli
    • 1
  • Antonella Vitale
    • 1
  • Anna Guarini
    • 1
  • Robin Foà
    • 1
  1. 1.Hematology, Department of Cellular Biotechnologies and Hematology“Sapienza” UniversityRomeItaly

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