Korean J Hematol.  2006 Mar;41(1):8-15. 10.5045/kjh.2006.41.1.8.

Selective Expansion of Natural Killer Cells from Peripheral Blood Mononuclear Cells by K562 Cell Line and IL-2

• Cho D
• Shin SW
• Park JS
• Kang HK
• Kim SK
• Pham TN
• Zhu XW
• Shin MG
• Suh SP
• Ryang DW
• Nam JH
• Kim YJ
• Lee JJ

• KMID: 2252322
• DOI: http://doi.org/10.5045/kjh.2006.41.1.8

Abstract

BACKGROUND: Several attempts have been made to expand human NK cells from peripheral blood mononuclear cells (PBMCs). This study examined the selective expansion of NK cells using interleukin 2 (IL-2) plus the K562 cell line, the expression of the NK cell receptors, and the cytotoxic activity.
METHODS
The PBMCs from seven healthy volunteers were cultured in a medium containing the IL-2 plus the K562 cell line for 14 days. The expression of the activating and inhibitory receptors on the resting NK cells and the 72 hr-expanded NK cells were analyzed. A flow cytometric cytotoxic assay was used to determined the killing activity of the non-expanded NK cells and the 7 day-expanded NK cells against the K562 target cells.
RESULTS
The NK cells from PBMCs expanded 4.5-fold after 7 days, and contained 56.5% CD3-CD56+ cells. The IL-2 or IL-2 plus K562 increased the expression levels of CD158b (MFI, mean florescence intensity), CD158e1/e2 (MFI), and NKp44 (MFI), while it decreased the expression levels of NKp30 (%), CD16 (MFI), and 2B4 (MFI). The non-expanded NK cells lysed 9.0% and 27.6% of the K562 target cells in the 1 : 1 and 5 : 1 effector and target ratio, respectively, and the 7-day expanded NK cells lysed 36.9% and 57.2% of the K562 target cells, respectively.
CONCLUSION
The selective expansion of CD3-CD56+ NK cells occurred only during 7 days of culture. IL-2 or IL-2 plus the K562 cells altered the expression of various activating and inhibitory receptors of NK cells, and the cytotoxicity of the expanded NK cells was higher than in the non-expanded cells.

Keyword

Natural killer cells; K562; IL-2; Expansion

MeSH Terms

Cell Line*
Healthy Volunteers
Homicide
Humans
Interleukin-2*
K562 Cells
Killer Cells, Natural*
Receptors, Natural Killer Cell
Interleukin-2
Receptors, Natural Killer Cell

Figure

• 

  Fig. 1 The change of CD158b expression on gated CD3- CD56+ NK cells in response to IL-2 alone and IL-2 plus K562 cell line. Representative data using PE-labeled CD 158b was shown. The increased expression was observed for CD 158b receptors, indicating that IL-2 alone (B) and IL2+K562 (C) induce up-regulation of CD158b compared to uncultured cells (A).

• 

  Fig. 2 Flow cytometric measurement of NK cytotoxicity using staining with FITC-congugated anti-CD45 and side scatter profiles to identify different cell populations and uptake of propidium iodide (PI) to detect cell death. Target cells and effectors (1：5) incubated for 4 hrs, are identified by region 1 (R1) and region 2 (R2) repectively. Percentages of dead cells in different regions are calculated from histograms showing PI uptake as measured by the intensity of fluorescence.

Reference

1). Kim JS., Lee WK., Suh JS, et al. T and B cell changes with aging. Korean J Lab Medicine. 2001. 21:135–40.

2). Lanier LL., Corliss B., Phillips JH. Arousal and inhibition of human NK cells. Immunol Rev. 1997. 155:145–54.

3). Domzig W., Stadler BM., Herberman RB. Interleukin 2 dependence of human natural killer (NK) cell activity. J Immunol. 1983. 130:1970–3.

4). Burns LJ., Weisdorf DJ., DeFor TE, et al. IL-2-based immunotherapy after autologous transplantation for lymphoma and breast cancer induces immune activation and cytokine release: a phase I/II trial. Bone Marrow Transplant. 2003. 32:177–86.

5). Harada H., Saijo K., Watanabe S, et al. Selective expansion of human natural killer cells from peripheral blood mononuclear cells by the cell line, HFWT. Jpn J Cancer Res. 2002. 93:313–9.

6). Perussia B., Ramoni C., Anegon I., Cuturi MC., Faust J., Trinchieri G. 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.

7). 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.

8). 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.

9). Godoy-Ramirez K., Franck K., Gaines H. A novel method for the simultaneous assessment of natural killer cell conjugate and formation and cytotoxicity at the single-cell level by multi-parameter flow cytometry. J Immunol Methods. 2000. 239:35–44.

10). Farag SS., Fehniger TA., Ruggeri L., Velardi A., Caligiuri MA. Natural killer cell receptors: new biology and insights into the graft-versus-leukemia effect. Blood. 2002. 100:1935–47.

11). Phillips JH., Lanier LL. A model for the differentiation of human natural killer cells. Studies on the in vitro activation of Leu-11+ granular lymphocytes with a natural killer-sensitive tumor cell, K562. J Exp Med. 1985. 161:1464–82.

12). Warren HS., Skipsey LJ. Phenotypic analysis of a resting subpopulation of human peripheral blood NK cells: the FcR gamma III (CD16) molecule and NK cell differentiation. Immunology. 1991. 72:150–7.

13). Ishikawa E., Tsuboi K., Saijo K, et al. Autologous natural killer cell therapy for human recurrent malignant glioma. Anticancer Res. 2004. 24:1861–71.

14). Ruggeri L., Capanni M., Urbani E, et al. Effectiveness of donor natural killer cell alloreactivity in mismatched hematopoietic transplants. Science. 2002. 295:2097–100.

15). Giebel S., Locatelli F., Lamparelli T, et al. Survival advantage with KIR ligand incompatibility in hematopoietic stem cell transplantation from unrelated donors. Blood. 2003. 102:814–9.

16). 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.

17). Kogure T., Mantani N., Sakai S., Shimada Y., Tamura J., Terasawa K. Natural killer cytolytic activity is associated with the expression of killer cell immunoglobulin-like receptors on peripheral lymphocytes in human. Mediators Inflamm. 2003. 12:117–21.

18). Shin EC., Choi KS., Kim SJ., Shin JS. Modulation of the surface expression of CD158 killer cell Ig-like receptor by interleukin-2 and transforming growth factor-beta. Yonsei Med J. 2004. 45:510–4.

19). Vitale M., Bottino C., Sivori S, et al. NKp44, a novel triggering surface molecule specifically expressed by activated natural killer cells, is involved in non-major histocompatibility complex-restricted tumor cell lysis. J Exp Med. 1998. 187:2065–72.

20). Castriconi R., Cantoni C., Della Chiesa M, et al. Transforming growth factor beta 1 inhibits expression of NKp30 and NKG2D receptors: consequences for the NK-mediated killing of dendritic cells. Proc Natl Acad Sci U S A. 2003. 100:4120–5.