Natural killer (NK) cells represent up to 15% of human peripheral blood mononuclear cells (PBMC), and range from 5-20% of peripheral blood lymphocytes. NK cells generally fall into three subtypes: CD56dim CD16+, CD56brightCD16+/- and CD56– CD16+ NK cells, the prevalence and functions of which I have previously discussed. NK cells are considered to be a promising avenue in cell-based anti-tumor immunotherapeutics. However, the relatively low numbers of these cell types in PBMC have constituted a technical challenge in these efforts and in other studies needing large numbers of NK cells. In the April 2013 issue of Clinical & Experimental Immunology, Wang et. al, describe an in vitro method for the preferential expansion of human NK cells from PBMC.
NK cell expansion in vitro systems requires multiple signals for survival, proliferation, and activation. In a previous study, Fujisaki et. al (2009) demonstrated that highly cytotoxic CD56+ NK cells could be highly preferentially expanded when cultured with a version of the chronic myeloid leukemia K562 cell line, which was genetically altered to express a membrane-bound form of IL-15 and the 41BB ligand (CD137L). Under this protocol, NK cells expanded an average of 21.6-fold after 7 days and 277-fold after 21 days in culture, and at 21 days reached a purity of 98.6%. CD3+ T cells on the other hand fell to an average of 3.1% of the cells remaining after 21 days. Importantly however, is not only the expansion of NK cells, but the functionality of the expanded cell product. The NK cells generated by this method had enhanced killing potential in vitro. In xenograft models of acute myeloid leukemia (AML) in immune deficient NOD/scid-IL2RGnull mice, these NK cells were able to elicit potent anti-leukemic activity. Thus, this method generates large numbers of highly functional human NK cells.
In the current study by Wang et. al, a similar method was utilized in which the K562 cell line was engineered to express a membrane-bound form of IL-21 along with CD137L. On average under these conditions, NK cells expanded from less than 30% of PBMC to over 85% after 7 days and 95% after 3 weeks, while CD3+ T cells went from 60% initially to 6% at seven days and 1% at three weeks. Proliferation of NK cells was continual over eight weeks in culture, and by 3 weeks reached over 100-fold, although the exact numbers and ranges were not explicitly stated in the paper. Thus, NK cells are highly selectively expanded using this method, similarly to the method used by Fujisaki et. al.
In answer to the functionality of NK cells generated under these conditions, Wang et. al demonstrated enhanced expression of activating and inhibitory NK receptors. Significantly enhanced cytotoxic killing potential after culture was shown, being maximal after one and three weeks in culture whereafter it decreased but still remained higher than resting NK cells. Thus, these expanded NK cells are also highly functional.
It would be interesting to see a direct comparison of the extent and quality of NK cell expansion from human PBMC by CD137L combined with the membrane-bound form of IL-15 as was done by Fujisaki et. al versus the membrane-bound form of IL-21 developed by Wang et. al. IL-21 is a strong and preferential activator of STAT3. Wang et al did establish a role for STAT3 in the induction of these cells. IL-15 is a strong activator of STAT5 and activates STAT3 to a lesser extent. However, IL-15 has been shown to strongly induce expression of the STAT3-activating cytokine IL-10. Thus, for optimal clinical applications of expanded NK cells, it is important to determine how the different cytokine-STAT signals contribute to NK cell proliferation, survival, and activation.
Membrane-bound interleukin-21 and CD137 ligand induce functional human natural killer cells from peripheral blood mononuclear cells through STAT-3 activation. Wang X, Lee DA, Wang Y, Wang L, Yao Y, Lin Z, Cheng J, Zhu S. Clin Exp Immunol. 2013 Apr;172(1):104-12. doi: 10.1111/cei.12034.
Expansion of highly cytotoxic human natural killer cells for cancer cell therapy. Fujisaki H, Kakuda H, Shimasaki N, Imai C, Ma J, Lockey T, Eldridge P, Leung WH, Campana D. Cancer Res. 2009 May 1;69(9):4010-7. doi: 10.1158/0008-5472.CAN-08-3712. Epub 2009 Apr 21.
Prospects for the use of NK cells in immunotherapy of human cancer. Ljunggren HG, Malmberg KJ. Nat Rev Immunol. 2007 May;7(5):329-39.
Properties of the K562 cell line, derived from a patient with chronic myeloid leukemia. Klein E, Ben-Bassat H, Neumann H, Ralph P, Zeuthen J, Polliack A, Vánky F. Int J Cancer. 1976 Oct 15;18(4):421-31.
Characterization of cytokine differential induction of STAT complexes in primary human T and NK cells. Yu CR, Young HA, Ortaldo JR. J Leukoc Biol. 1998 Aug;64(2):245-58.
IL-15-induced IL-10 increases the cytolytic activity of human natural killer cells. Park JY, Lee SH, Yoon SR, Park YJ, Jung H, Kim TD, Choi I. Mol Cells. 2011 Sep;32(3):265-72. doi: 10.1007/s10059-011-1057-8. Epub 2011 Jul 29.