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Expansion of NK cells from cord blood with antileukemic activity using GMP-compliant substances without feeder cells

Leukemia volume 26, pages 1149–1152 (2012)Cite this article

Neonatal cord blood (CB) cells have been demonstrated to contain a high percentage of natural killer (NK) cells, but the NK cells are immature, with a low level of cytolytic activity. However, expression levels of perforin and granzyme have been reported to be high in CB NK cells, and it has been suggested that CB NK cells are phenotypically and functionally mature. It has been suggested that CB is a source of stem cells that is as safe and effective as bone marrow or mobilized peripheral blood.1, 2 Also, there are a number of progenitor cell populations in CB that can be differentiated to NK cells. Therefore, CB is a useful source to expand NK cells for adoptive immunotherapy, particularly against malignant cells that express a low level of human leukocyte antigen class I molecules. Resting CB NK cells rapidly respond to cytokine stimulation by increasing cytolytic activity. Adoptive transfer of allogeneic NK cells is a potential immunotherapy to induce a graft-versus-leukemia (GVL) effect, without causing a graft-versus-host disease (GVHD).3 In this study, we tried to expand NK cells from CB with antileukemic activity using good manufacturing practice (GMP)-compliant substances without feeder cells. We used tacrolimus (FK506) and low molecular weight heparin (dalteparin sodium) for expansion of NK cells. Kim et al.4 showed impaired interleukin (IL)-2 signaling and a reduction in activating receptors in NK cells by tacrolimus. However, we have reported that tacrolimus enhances the cytolytic activity of inhibitory NK cell receptor (CD94/NKG2A)-expressing CD8T cells.5 Also, Wang et al.6 showed that a related compound (cyclosporine A) has essentially no effect on cytolytic activity of NK cells. There is conflicting data on the effect of calcineurin inhibitors on NK cell. Anyways, tacrolimus can inhibit T-cell proliferation. Heparin was reported to bind to several types of cytokines and to activate them, and also to have an important role in the expansion of hematopoietic progenitor cells.7 Also, heparin sulfate and its related compounds were recognized by natural cytotoxicity receptors such as NKp30, NKp44 and NKp46, and soluble heparin enhanced the secretion of interferon-γ by NK cells.8 Spanholtz et al.9 reported efficient expansion of NK cells from CB CD34+ cells, using low molecular weight heparin-based media containing various cytokines. Therefore, we tried to use tacrolimus to inhibit T-cell proliferation and dalteparin to support NK-cell proliferation during NK-cell expansion using IL-2, IL-15 and OKT3 in vitro.

These expanded NK cells expressed stimulatory NK cell receptor NKG2D and intracellular cytotoxic molecule granzyme (Figure 1b, bars indicate means±s.d.s, n=5). Also, the expanded CD16+CD56+ NK cells expressed high levels of inhibitory NK receptors, but significantly higher levels of stimulatory NK cell receptors including NKG2C, NKG2D, NKp30, NKp46 and especially, NKp44, than the levels of these receptors on CD16+CD56+ NK cells in resting CB before culture were noted (Figure 1c, P<0.01, bars indicate means±s.d.s, n=10).

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References

  1. Rocha V, Labopin M, Sanz G, Arcese W, Schwerdtfeger R, Bosi A et al. Transplants of umbilical-cord blood or bone marrow from unrelated donors in adults with acute leukemia. N Engl J Med 2004; 351: 2276–2285.

    Article  CAS  Google Scholar 

  2. Eapen M, Rubinstein P, Zhang MJ, Stevens C, Kurtzberg J, Scaradavou A et al. Outcomes of transplantation of unrelated donor umbilical cord blood and bone marrow in children with acute leukaemia: a comparison study. Lancet 2007; 369: 1947–1954.

    Article  Google Scholar 

  3. Ruggeri L, Capanni M, Urbani E, Perruccio K, Shlomchik WD, Tosti A et al. Effectiveness of donor natural killer cell alloreactivity in mismatched hematopoietic transplants. Science 2002; 295: 2097–2100.

    Article  CAS  Google Scholar 

  4. Kim TJ, Kim N, Kang HJ, Kim EO, Kim ST, Ahn HS et al. FK506 causes cellular and functional defects in human natural killer cells. J Leukoc Biol 2010; 88: 1089–1097.

    Article  CAS  Google Scholar 

  5. Tanaka J, Toubai T, Iwao N, Tsutsumi Y, Kato N, Miura Y et al. The immunosuppressive agent FK506 enhances the cytolytic activity of inhibitory natural killer cell receptor (CD94/NKG2A)-expressing CD8 T cells. Transplantation 2005; 80: 1813–1815.

    Article  CAS  Google Scholar 

  6. Wang H, Grzywacz B, Sukovich D, McCullar V, Cao Q, Lee AB et al. The unexpected effect of cyclosporin A on CD56+CD16- and CD56+CD16+ natural killer cell subpopulations. Blood 2007; 110: 1530–1539.

    Article  CAS  Google Scholar 

  7. Gupta P, Oegema Jr TR, Brazil JJ, Dudek AZ, Slungaard A, Verfaillie CM . Human LTC-IC can be maintained for at least 5 weeks in vitro when interleukin-3 and a single chemokine are combined with O-sulfated heparan sulfates: requirement for optimal binding interactions of heparan sulfate with early-acting cytokines and matrix proteins. Blood 2000; 95: 147–155.

    CAS  PubMed  Google Scholar 

  8. Hecht ML, Rosental B, Horlacher T, Hershkovitz O, De Paz JL, Noti C et al. Natural cytotoxicity receptors NKp30, NKp44 and NKp46 bind to different heparan sulfate/heparin sequences. J Proteome Res 2009; 8: 712–720.

    Article  CAS  Google Scholar 

  9. Spanholtz J, Tordoir M, Eissens D, Preijers F, van der Meer A, Joosten I et al. High log-scale expansion of functional human natural killer cells from umbilical cord blood CD34-positive cells for adoptive cancer immunotherapy. PLoS One 2010; 5: e9221.

    Article  Google Scholar 

  10. Alici E, Sutlu T, Björkstrand B, Gilljam M, Stellan B, Nahi H et al. Autologous antitumor activity by NK cells expanded from myeloma patients using GMP-compliant components. Blood 2008; 111: 3155–3162.

    Article  CAS  Google Scholar 

  11. Ayello J, van de Ven C, Cairo E, Hochberg J, Baxi L, Satwani P et al. Characterization of natural killer and natural killer-like T cells derived from ex vivo expanded and activated cord blood mononuclear cells: implications for adoptive cellular immunotherapy. Exp Hematol 2009; 37: 1216–1229.

    Article  CAS  Google Scholar 

  12. Passweg JR, Tichelli A, Meyer-Monard S, Heim D, Stern M, Kühne T et al. Purified donor NK-lymphocyte infusion to consolidate engraftment after haploidentical stem cell transplantation. Leukemia 2004; 18: 1835–1838.

    Article  CAS  Google Scholar 

  13. Miller JS, Soignier Y, Panoskaltsis-Mortari A, McNearney SA, Yun GH, Fautsch SK et al. Successful adoptive transfer and in vivo expansion of human haploidentical NK cells in patients with cancer. Blood 2005; 105: 3051–3057.

    Article  CAS  Google Scholar 

  14. Nguyen S, Béziat V, Norol F, Uzunov M, Trebeden-Negre H, Azar N et al. Infusion of allogeneic natural killer cells in a patient with acute myeloid leukemia in relapse after haploidentical hematopoietic stem cell transplantation. Transfusion 2011; 51: 1769–1778.

    Article  Google Scholar 

  15. Yoon SR, Lee YS, Yang SH, Ahn KH, Lee JH, Lee JH 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: 1038–1046.

    Article  CAS  Google Scholar 

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Acknowledgements

We thank Ms M Yamane, Ms M Mayanagi and Ms Y Ishimaru for their technical assistance.

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  1. Department of Hematology and Oncology, Hokkaido University Graduate School of Medicine, Sapporo, Japan

    J Tanaka, J Sugita, S Shiratori, A Shigematu, S Asanuma, K Fujimoto, M Nishio, T Kondo & M Imamura

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Correspondence to J Tanaka.

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A patent application for the composition for expanding NK cells and the use of them has been filed with Junji Tanaka as a sole inventor.

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Tanaka, J., Sugita, J., Shiratori, S. et al. Expansion of NK cells from cord blood with antileukemic activity using GMP-compliant substances without feeder cells. Leukemia 26, 1149–1152 (2012). https://doi.org/10.1038/leu.2011.345

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