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

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Abstract

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 CD3−CD56+ 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.

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Abbreviations

ALL:

Acute lymphoblastic leukemia

AML:

Acute myeloid leukemia

51Cr:

51Chromium

CR:

Complete remission

EBV:

Epstein–Barr virus

FBS:

Fetal bovine serum

GMP:

Good manufactory practice

GVHD:

Graft-versus-host disease

HLA:

Human leukocyte antigen

IL-2:

Interleukin-2

KIRs:

Killer cell immunoglobulin-like receptors

LAK:

Cells lymphokine-activated killer cells

mAbs:

Monoclonal antibodies

MFI:

Mean fluorescence intensity

NCRs:

Natural cytotoxicity receptors

NK:

Cells natural killer cells

PBMCs:

Peripheral blood mononuclear cells

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Acknowledgments

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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Authors and Affiliations

  1. Hematology, Department of Cellular Biotechnologies and Hematology, “Sapienza” University, Via Benevento 6, 00161, Rome, Italy

    Nadia Peragine, Giovanni F. Torelli, Paola Mariglia, Simona Pauselli, Antonella Vitale, Anna Guarini & Robin Foà

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  1. Nadia Peragine
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  2. Giovanni F. Torelli
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  3. Paola Mariglia
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  5. Antonella Vitale
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  6. Anna Guarini
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  7. Robin Foà
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Correspondence to Robin Foà.

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Peragine, N., Torelli, G.F., Mariglia, P. et al. Immunophenotypic and functional characterization of ex vivo expanded natural killer cells for clinical use in acute lymphoblastic leukemia patients. Cancer Immunol Immunother 64, 201–211 (2015). https://doi.org/10.1007/s00262-014-1614-z

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  • DOI: https://doi.org/10.1007/s00262-014-1614-z

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