Use of Natural Killer Cells for Acute Leukemia Patients



Giovanni F. Torelli



Giovanni F. Torelli, Hematology, Department of Cellular Biotechnologies and Hematology, Sapienza University, Rome, Italy

Correspondence to: Giovanni F. Torelli, MD, PhD, Hematology, Department of Cellular Biotechnologies and Hematology, Sapienza University, Via Benevento 6, 00161 Rome, Italy.


Telephone: + 39-49974321         Fax: +390644241984

Received: March 28, 2015         Revised: April 17, 2015

Accepted: April 22, 2015

Published online: July 6, 2015



The management of acute leukemia has witnessed profound changes in recent years. The anti-leukemic potential of Natural Killer (NK) cells has raised considerable interest for the design of new therapeutic approaches based on the infusion of freshly-isolated or ex vivo manipulated effectors. Several points need to be addressed for the design of optimal protocols of NK infusion, including which disease can benefit most, the optimal timing during the course of the disease, the best preparative regimen and the origin of NK cells. This review aims at summarizing the biological and clinical data on the role played by NK cells in patients with acute leukemia; in addition, optimal good manufacturing practice compliant protocols for ex vivo expansion and activation of these effector cells are discussed.


© 2015 ACT. All rights reserved.


Key words: Anti-leukemic activity of NK cells; Ex vivo manipulation of NK cells; GMP protocols


Torelli GF. Use of Natural Killer Cells for Acute Leukemia Patients. International Journal of Hematology Research 2015; 1(2): 57-60 Available from: URL:


Definition of Natural Killer cells

Natural Killer (NK) cells are a subset of peripheral blood lymphocytes immunophenotypically characterized by the expression of the CD56 surface antigen and the lack of the CD3 and T cell receptor (TCR) proteins. These effectors play a central role in the defense against viral infections and tumor growth; they exert their cytotoxic capacity in a human leukocyte antigen (HLA)-unrestricted fashion, in addition to the production of various cytokines and chemokines, including tumor-necrosis-factor and interferon [1-3]. In humans, on the basis of the intensity of the CD56 antigen expression, it is possible to distinguish two main circulating NK cell subsets: CD56dim NK cells, which represent the major subset and mainly express a cytotoxic capacity, and the CD56bright subset which primarily produces cytokines[4].

    The cytolytic activity of these cells is finely regulated by the balance between inhibitory and activating signals derived from receptors expressed at the cell surface. In the late 80s, the “missing self hypothesis” was proposed by Ljunggren and Karre[5], based on the observation that NK cells could efficiently kill targets who had lost major histocompatibility complex (MHC)-class I. Specific inhibitory receptors expressed by NK cells and recognizing allelic forms of HLA-class I were then identified and collectively termed as killer immunoglobulin-like receptors (KIRs) (KIR2DL and KIR3DL)[6,7]. Activating KIRs (KIR2DS and KIR3DS) were also discovered, similar to the corresponding inhibitory KIRs in the extracellular domain but with substantial differences in the transmembrane and cytoplasmic portion[8]. Besides activating KIRs, other receptors involved in tumor recognition include the natural cytotoxicity receptors (NCRs) (NKp30, NKp44, NKp46), NKG2D and DNAM-1[9-13]. It is the selective engagement of these receptors, in the absence of efficient inhibitory signals, that render target cells susceptible to NK-cell mediated lysis. Interestingly, the known ligands for these receptors are typically over-expressed upon cell stress, such as after tumor transformation or viral infection, when the expression of HLA-I molecules is reduced rendering therefore the cell susceptible to NK lysis. While some of these receptors are still orphan of their ligands, MICA/B and ULBPs have been discovered to be the ligands for NKG2D[14], whereas the Poliovirus receptor (PVR, CD155) and Nectin-2 (Nec-2, CD112) interact with DNAM-1[15,16].


Anti-leukemic activity of Natural Killer cells

It is from the setting of haploidentical stem cell transplantation (SCT) that the anti-leukemic capacity of NK cells became evident. In this context, alloreactive NK cells express inhibitory KIRs specific for HLA-class I alleles that are missing in the recipient, in addition to activating KIRs recognizing specific HLA ligands[17,18]; this further underlines the importance of both inhibitory and activating signals for tumor recognition.

    Alloreactive NK cells have been demonstrated to positively affect the outcome of both adult with acute myeloid leukemia (AML) and children with acute lymphoid leukemia (ALL) undergoing T-cell depleted haploidentical (SCT)[19,20]. The biological reasons responsible for the different susceptibility of adult and children ALL blasts to the lytic effect played by alloreactive NK cells are so far unknown. Nonetheless, we recently reported the significant increased expression in pediatric B-lineage ALL (B-ALL) of some of the ligands for NKG2D and DNAM-1, including Nec-2[21]. In addition, these data allowed to recognize a differential expression of ligands for NK cell activating receptors in molecularly-defined subgroups of ALL patients. This is the case of BCR-ABL+ ALL blasts, which display, together with the MLL-AF4+ subgroup, the highest intensity of ligand expression within the B-ALL adult context. Importantly, the high expression of ligands for NK cell activating receptors correlated with the degree of susceptibility to lysis by expanded allogeneic NK cells, further supporting the role played by these receptors during the process of recognition. These results provided for the first time an explanation to the differential susceptibility of age- or molecularly-defined subgroups of acute leukemia patients to the lytic action of cytotoxic NK cells.

    In the last years, the possibility of using either selected or ex vivo activated/expanded NK cells in the design of new immunotherapeutic strategies for the treatment of leukemic patients has gained much interest[22]. Isolated experiences are reported in the literature characterized by the infusion of NK cells derived from haploidentical donors[23-25] (see Table 1 for details) or from the hematopoietic stem cell donor when infused after a transplant[26-28] (see Table 2 for details), mainly for patient affected by AML, mielodysplastic syndrome or high risk lymphoma. Altogether these reports demonstrated that the infusion of these cells is safe and some encouraging results are described. Nonetheless several points need to be addressed for the design of optimal protocols of NK infusion, including which disease can benefit most, the optimal timing during the course of the disease, the best preparative regimen, the origin of NK cells whether autologous or allogeneic, and finally the ex vivo manipulation protocol.



Ex vivo manipulation of Natural Killer cells for adoptive immunotherapy

The need of large quantities of highly activated effector cells to produce an anti-cancer effect translates into the necessity of developing good manufacturing practice (GMP)-compliant methods for the efficient production of fully functional NK cells for clinical application. Several protocols for the ex vivo NK cell expansion and activation have been investigated, including the long-term culture with cytokines[29,30] and the use of different sources of allogeneic feeder cells[31-36]; very recently, a feeder-free particle-based technology which utilizes plasma membrane particles derived from transfected K562 cell line was also proposed[37]. Despite the excellent expansion fold, the majority of these protocols are not acceptable for clinical use in many countries, either because they do not utilize clinical grade materials or for the potential transmission of infectious diseases due to the use of allogeneic feeders. In addition, the proportion of contaminating CD3+ T lymphocytes remains high in the majority of these approaches, with an associated risk of graft-versus-host disease (GVHD) in the allogeneic context[38,39]; the probability of developing such a complication becomes even higher when the anti-CD3 monoclonal antibody (mAb) is added to the culture system[40,41], running the risk of expanding and activating residual CD3+ cells.

    We recently developed a new GMP compliant-method for ex-vivo NK cell expansion[42]. Peripheral blood mononuclear cells (PBMCs) are obtained from leukapheretic procedures and processed in the GMP facility. For NK-cell enrichment, a two-step immunomagnetic procedure consisting of a CD3+ T-cell depletion followed by a CD56+ cell positive selection is used. Isolated NK cells are suspended in serum-free medium containing autologous plasma, interleukin (IL)-2 and IL-15 in the presence of irradiated autologous feeder cells and cultured for 14 days at 37.

    The application of this method resulted in the efficient production of effector cells presenting a significantly increased expression of activating receptors important in tumor cell recognition, including NKG2D, DNAM1, Nkp30 and Nkp44, when compared to freshly isolated NK cells. The two-step NK cell selection procedure ensures the highest purity of the final product, as demonstrated by a minimal contamination by CD3+ cells belonging to the T- and NK-T compartments; this is important in order to maximally reduce the risk of inducing a GVHD when the product is used in the allogeneic context. Furthermore, the high level of B-cell depletion reached with this system is also an important condition to prevent Epstein-Barr Virus (EBV) reactivation, which may potentially trigger lymphoproliferative diseases in immunocompromised patients.

    Several other advantages are also associated with the expansion strategy utilized in this protocol; these include (1) the lack of in vivo cytokine infusion which can cause systemic toxicities; (2) the use of clinical grade manufactured IL-2 and IL-15, which play essential roles in NK cell development, expansion, homeostasis and activation[43,44]; (3) the use of autologous feeder cells to prevent the unknown effects associated with the use of allogeneic feeders; and d) the lack of an anti-CD3 mAb in the culture system, thus reducing the risk of GVHD in the allogeneic setting.

    In our model, both cytokines and feeder cells are necessary to obtain optimal NK cell proliferation, since a lower expansion fold was observed in the presence of cytokines or feeder cells alone. Under these conditions, the degree of NK cell expansion was on average 15.7 fold. When tested in a cytotoxic assay against the K562 cell line, these expanded effectors proved to be highly active. This confirms recently published data from our group documenting the ability of expanded NK cells to recognize and kill primary acute leukemia blast cells[21,45]. Similar results were obtained when the expanded effectors were tested in a degranulation assay. Interestingly, these cells exerted a comparable cytotoxic activity when used prior to cryopreservation and after thawing; this is extremely important since ex vivo expanded cells must be cryopreserved before in vivo infusion in adoptive cell therapy protocols, in order to allow the necessary quality tests to be carried out before the batch of cells can be released for clinical use.



In conclusion, new adoptive immunotherapeutic approaches based on the in vivo infusion of ex vivo expanded and activated NK cells represent promising strategies for the treatment of patients affected by acute leukemia. Several points still need to be addressed for the design of optimal protocols of NK infusion. The development of GMP methods for efficient production of fully functional NK cells is mandatory for clinical application. Future clinical trials are warranted and may solve some of the still debated points.



The authors have no conflicts of interest to declare.



1         Trinchieri G. Biology of natural killer cells. Adv Immunol 1989; 47: 187-376.

2         Robertson MJ, Ritz J. Biology and clinical relevance of human natural killer cells. Blood 1990; 76: 2421-38.

3         Miller JS. The biology of natural killer cells in cancer, infection, and pregnancy. Exp Hematol 2001; 29: 1157-68.

4         Caligiuri MA. Human natural killer cells. Blood 2008; 112: 461-9.

5         Ljunggren HG, Karre K. In search of the “missing self”: MHC molecules and NK cell recognition. Immunol Today 1990; 11: 237-44.

6         Moretta A, Bottino C, Mingari MC et al. What is a natural killer cell. Nat Immunol 2002; 3: 6-8.

7         Moretta L, Pietra G, Montaldo E, et al. Human NK cells: from surface receptors to the therapy of leukemias and solid tumors. Frontiers in Immunology 2014; 5: 1-8.

8         Thielens A, Vivier E, Romagne F. NK cell MHC class I specific receptors (KIR): from biology to clinical intervention. Curr Opin Immunol 2012: 24: 239-45.

9         Moretta A, Bottino C, Vitale M, et al. Activating receptors and coreceptors involved in human natural killer cell-mediated cytolysis. Annu Rev Immunol 2001; 19: 197-223.

10     Coudert JD, Held W. The role of the NKG2D receptor for tumor immunity. Semin Cancer Biol 2006; 16: 333-43.

11     Eagle RA, Trowsdale J. Promiscuity and the single receptor: NKG2D. Nat Rev Immunol 2007; 7: 737-44.

12     Shibuya A, Campbell D, Hannum C, et al. DNAM-1, a novel adhesion molecule involved in the cytolytic function of T lymphocytes. Immunity 1996; 4: 573-81.

13     Lanier LL. NK cell recognition. Annu Rev Immunol 2005; 23: 225-274.

14     Pende D, Rivera P, Marcenaro S, et al. Major histocompatibility complex class I-related chain A and UL 16-binding protein expression on tumor cell lines of different histotypes: analysis of tumor susceptibility to NKG2D-dependent natural killer cell cytotoxicity. Cancer Res 2002; 62: 6178-86.

15     Bottino C, Castriconi R, Pende D, et al. Identification of PVR (CD155) and Nectin-2 (CD112) as cell surface ligands for the human DNAM-1 (CD226) activating molecule. J Exp Med 2003; 198: 557-67.

16     Fuchs A, Colonna M. The role of NK cell recognition of nectin and nectin-like proteins in tumor immunosurveillance. Semin Cancer Biol 2006; 16: 359-66.

17     Freud AG, Caligiuri MA. Human natural killer cell development. Immunol Rev 2006; 214: 56-72.

18     Moretta L, Locatelli F, Pende D, et al. Killer Ig-like receptor-mediated control of natural killer cell alloreactivity in haploidentical hematopoietic stem cell transplantation. Blood 2011, 117: 764-71.

19     Ruggeri L, Mancusi A, Capanni M, et al. Donor natural killer cell allorecognition of missing self in haploidentical hematopoietic transplantation for acute myeloid leukemia: challenging its predictive value. Blood 2007; 110: 433-40.

20     Pende D, Marcenaro S, Falco M, et al. Anti-leukemia activity of alloreactive NK cells in KIR ligand-mismatched haploidentical HSCT for pediatric patients: evaluation of the functional role of activating KIR and redefinition of inhibitory KIR specificity. Blood 2009; 113: 3119-29.

21     Torelli GF, Peragine N, Raponi S, et al. Recognition of adult and pediatric acute lymphoblastic leukemia blasts by natural killer cells. Haematologica 2014; 99: 1248-54.

22     Locatelli F, Moretta F, Brescia L, et al. Natural killer cells in the treatment of high-risk acute leukemia. Seminars in Immunology 2014; 26: 173-9.

23     Miller JS, Soignier Y, Panoskaltsis-Mortari A, et al. Successful adoptive transfer and in vivo expansion of human haploidentical NK cells in patients with cancer. Blood 2005; 105: 3051-7.

24     Rubnitz JE, Inaba H, Ribeiro RC, et al. NKAML: a pilot study to determine the safety and feasibility of haploidentical natural killer cell transplantation in childhood acute myeloid leukemia. J Clin Oncol 2010; 28: 955-9.

25     Curti A, Ruggeri L, D’Addio A, et al. Successful transfer of alloreactive haploidentical KIR ligand-mismatched natural killer cells after infusion in elderly high risk acute myeloid leukemia patients. Blood 2011; 118: 3273-9.

26     Yoon SR, Lee YS, Yang SH, et al. Generation of donor natural killer cells from CD34+ progenitor cells and subsequent infusion after HLA-mismatched allogeneic hematopoietic cell transplantation: a feasibility study. Bone Marrow Transplant 2010; 45: 1038-46.

27     Stern M, Passweg JR, Meyer-Monard S, et al. Pre-emptive immunotherapy with purified natural killer cells after haploidentical SCT: a prospective phase II study in two centers. Bone Marrow Transplant 2013; 48: 433-8.

28     Choi I, Yoon SR, Park SY, et al. Donor-derived natural killer cells infused after human leukocyte antigen-haploidentical hematopoietic cell transplantation: a dose-escalation study. Biol Blood Marrow Transplant 2014; 20: 696-704.

29     Carlens S, Gilljam M, Chambers BJ, et al. A new method for in vitro expansion of cytotoxic human CD3-CD56+ natural killer cells. Hum Immunol 2001; 62: 1092-8.

30     McKenna DH, Jr, Sumstad D, Bostrom N, et al. Good manufacturing practices production of natural killer cells for immunotherapy: a six-year single-institution experience. Transfusion 2007; 47: 520-8.

31     Torelli GF, Guarini A, Maggio R, et al. Expansion of natural killer cells with lytic activity against autologous blasts from adult and pediatric acute lymphoid leukemia patients in complete hematologic remission. Haematologica 2005; 90: 785-92.

32     Luhm J, Brand JM, Koritke P, et al. Large-scale generation of natural killer lymphocytes for clinical application. J Hematother Stem Cell Res 2002; 11: 651-7.

33     Fujisaki H, Kakuda H, Shimasaki N, et al. Expansion of highly cytotoxic human natural killer cells for cancer cell therapy. Cancer Res 2009; 69: 4010–17.

34     Perussia B, Ramoni C, Anegon I, et al. Preferential proliferation of natural killer cells among peripheral blood mononuclear cells cocultured with B lymphoblastoid cell lines. Nat Immun Cell Growth Regul 1987; 6: 171-88.

35     Miller JS, Oelkers S, Verfaillie C, McGlave P. Role of monocytes in the expansion of human activated natural killer cells. Blood 1992; 80: 2221-9.

36     Boissel L, Tuncer HH, Betancur M, et al. Umbilical cord mesenchymal stem cells increase expansion of cord blood natural killer cells. Biol Blood Marrow Transplant 2008; 14: 1031-8.

37     Oyer JL, Igarashi RY, Kulikowski AR, et al. Generation of highly cytotoxic natural killer cells for treatment of acute myelogenous leukemia using a feeder-free, particle-based approach. Biol Blood Marrow Transplant 2015; Apr; 21: 632-9.

38     Suck G, Koh MB. Emerging natural killer cell immunotherapies: large-scale ex vivo production of highly potent anticancer effectors. Hematol Oncol Stem Cell Ther 2010; 3: 135-42.

39     Miller JS. Should natural killer cells be expanded in vivo or ex vivo to maximize their therapeutic potential? Cytotherapy 2009; 11: 259-60.

40     Ahn YO, Kim S, Kim TM, et al. Irradiated and activated autologous PBMCs induce expansion of highly cytotoxic human NK cells in vitro. J Immunother 2013; 36: 373-81.

41     Siegler U, Meyer-Monard S, Jörger S, et al. Good manufacturing practice-compliant cell sorting and large-scale expansion of single KIR-positive alloreactive human natural killer cells for multiple infusions to leukemia patients. Cytotherapy 2010; 12: 750-63.

42     Torelli GF, Rozera C, Santodonato L, et al. A good manufacturing practice method to ex vivo expand natural killer cells for clinical use. Blood Transfus 2015; in press: doi 10.2450/2015.0231-14.

43     Liao W, Lin J-X, Leonard WJ. Interleukin-2 at the Crossroads of Effector Responses, Tolerance, and Immunotherapy. Immunity 2013; 38: 13-25.

44     Croce M, Orengo AM, Azzarone B, et al. Immunotherapeutic applications of IL-15. Immunotherapy 2012; 4: 957-69.

45     Peragine N, Torelli GF, 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 2015; 64: 201-11.


Peer reviewer: Georgia D Kaiafa, Aristotle University, AHEPA University Hospital, 1st Propedeutic Dept of Internal Medicine, Heamatology Unit, Stilponos Kyriakidi 1, PC 546 36, Thessaloniki, Greece.




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