Functional characterization of interleukin-15 gene transduction into the human natural killer cell line NKL
Introduction
Natural killer (NK) cells are a key component of the innate immune system that is involved in the early defense against virus-infected or transformed cells by non-major histocompatibility complex (MHC)-, non-T-cell receptor (TCR)-restricted mechanisms [1, 2]. NK cell activation is thought to be regulated by the signaling balance between inhibitory and activating receptors, and is immediately followed by cytolysis against target cells and secretion of immunoregulatory cytokines if activated, such as interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), granulocyte–macrophage colony-stimulating factor (GM-CSF), G-CSF, M-CSF and others [3., 4., 5., 6., 7., 8., 9.]. NK cells not only mediate spontaneous anti-tumor effector functions but also regulate innate and adaptive immune responses. Adoptive transfer of activated or allogeneic NK cells is effective in the treatment of certain types of leukemia and solid tumors [10, 11]. Given the importance of NK cells to immunity, a variety of approaches have been taken to try and selectively augment NK cell responses to tumors, including genetic modification.The cytokine interleukin-15 (IL-15) was originally identified in media conditioned by a monkey kidney epithelial cell line (CVI/EBNA) [12] and is present in a wide variety of tissues and cell types [13., 14., 15., 16.]. IL-15 shares many biologic activities with IL-2 and can directly induce CD34+ cells to differentiate into NK cells in the absence of IL-2 [17, 18]. This cytokine is also a potent regulator of NK cell proliferation, survival and cytolytic activity [19., 20., 21.]. IL-15 signals through the β- and γ-subunits of the IL-2R and the unique private α-subunit [22]. Thus, based upon complex regulation, it is likely that the functions of IL-15 and IL-15Rα differ from those of IL-2 and IL-2Rα. In previous work, we have reported that IL-15 gene-modified NK-92 cells exerted more efficiency against cancer [23] but extensive utilization of this gene-modified human NK-92 cell line is limited by its tumor spectrum.
Compared with primary NK-92 cells, NKL cells appear to have a different anti-tumor spectrum. In recent work, some human cancer cells were more sensitive to NKL cell cytotoxicity than NK-92 cells, such as the human gastric carcinoma cell line SGC7901 [24]. NKL is one of six malignant NK cell lines that have currently been established and are sufficiently well characterized. The NKL cell line was established from the peripheral blood of a patient with CD3− CD16+ CD56+ large granular lymphocyte (LGL) leukemia and grows in the presence of IL-2 at a concentration of 100 U/mL. The morphology of NKL cells resembles that of normal activated NK cells, with a CD16+ CD56dim phenotype and very similar natural killing, antibody-dependent cellular cytotoxicity (ADCC) and proliferative responses [25]. Among all the NK cell lines, NKL is probably the one that has retained the most original features of NK cells and could therefore potentially be used as effector cells in adoptive immunotherapy [26., 27., 28.].
In this report, the human (h)IL-15 gene was stably transfected into NKL cells. We analyzed the characterization of hIL-15 gene-modified NKL cells and investigated the molecular mechanisms for IL-15 gene modification in improving the proliferation, anti-apoptosis activity of NKL cells and enhancing the natural cytotoxicity against hepatocarcinoma cells.
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Cell culture and cell lines
Human hepatocellular carcinoma cell lines HepG2, H7402 and PLC/PRF-5, conserved in our laboratory, were cultured in RPMI-1640 medium (GIBCO/BRL) supplemented with 100 U/mL penicillin, 100 µg/mL treptomycin and 10% fetal bovine serum (FBS). NKL was gifted from Professor Jin BQ (Department of Immunology, Fourth Military Medical University, Xi'an, PR China), conserved in our laboratory and cultured in complete IL-2-containing (100 U/mL) RPMI-1640 medium. All these cells were incubated at of 37°C, 5%
Establishment of the hIL-15 gene-modified NKL cell line
Numerous studies have convincingly shown that IL-15 is critical for NK cell development [3, 31]. It is the major physiologic growth factor responsible for NK cell ontogeny. In this study, the IL-15 gene was transfected into NKL cells by an electroporation method and the cells were selected by G418. Levels of hIL-15 gene expression in transfectant NK cells were evaluated by an RT-PCR method. As shown in Figure 1A, hIL-15 gene-modified NKL (NKL-IL15) cells expressed significantly higher IL-15
Discussion
NK cells play a crucial role as a first line of defense against virus infection and tumor cells, and have been used as a strategic weapon in cancer cell therapy and hematopoietic stem cell transplantation, through activation of endogenous NK cells and adoptive transfer of in vitro-activated autologous NK cells or permanent NK cell lines among which the NK-92 cell line has already finished clinical trials [32, 33]. Numerous studies have been conducted to improve the anti-tumor effect of NK
Acknowledgements
This work was supported by the Natural Science Foundation of China (30671901) and Ministry of Science and Technology of China (2007AA021000; 2006CB504300; 2004CB518807).
References (38)
- et al.
Biology and clinical relevance of human natural killer cells
Blood
(1990) Recognition of tumor cells by the innate immune system
Curr Opin Immunol
(2001)- et al.
Exploitation of alloreactive NK cells in adoptive immunotherapy of cancer
Curr Opin Immunol
(2005) - et al.
On the road to a tumor cell vaccine: 20 years of cellular immunotherapy
Vaccine
(2004) - et al.
Functional characterization of human interleukin-15 receptor α chain and close linkage of IL-15RA and IL-2RA genes
J Biol Chem
(1995) - et al.
Role of interleukin-15 in the development of human CD56+ natural killer cells from CD34+ hematopoietic progenitor cells
Blood
(1996) - et al.
Interleukin 15: biology and relevance to human disease
Blood
(2001) - et al.
Expression of functional interleukin-15 receptor and autocrine production of interleukin-15 as mechanisms of tumor propagation in multiple myeloma
Blood
(2000) - et al.
Interleukin-15 improves cytotoxicity of natural killer cells via up-regulating NKG2D and cytotoxic effector molecule expression as well as STAT1 and ERK1/2 phosphorylation
Cytokine
(2008) - et al.
Efficient gene transfer into the human natural killer cell line, NKL, using the Amaxa nucleofection system™
J Immunol Methods
(2004)
Cost efficient and effective gene transfer into the human natural killer cell line, NK92
J Immunol Methods
Cloning and expression of rabbit interleukin-15
Vet Immunol Immunopathol
Novel approaches using natural killer cells in cancer therapy
Semin Cancer Biol
In vivo evidence for a dependence on interleukin 15 for survival of natural killer cells
Blood
IL-15 is an essential mediator of peripheral NK-cell homeostasis
Blood
IL-15: targeting CD8+ T cells for immunotherapy
Cytotherapy
The biology of the human natural killer cell
J Clin Immunol
Natural killer cells, viruses and cancer
Nat Rev Immunol
Predominance of group A KIR haplotypes in Japanese associated with diverse NK cell repertoires of KIR expression
Immunogenetics
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2017, Seminars in ImmunologyCitation Excerpt :The introduction of CD16a into the NK cell line NK-92 can increase the ADCC effect [147]. Genetic modifications of NK cells to produce cytokines, such as IL-15 and IL-2, can improve their survival, persistence, proliferation and function in vivo [148–150]. Recent studies have focused on the introduction of tumor-associated antigens into NK cells to direct these antigens to specific target cells.
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2015, Cancer LettersCitation Excerpt :This also resulted in increased cytotoxic activity of NK-92 cells in vitro and in vivo. Similarly, IL-15 gene modified human NK cell showed increased proliferative rate and enhanced cytotoxic activity [111,112]. From the aforementioned studies it is clear that cytokine gene transfer approach induces NK cell proliferation and increases survival capacity, further enhancing their activation.
HIL-15 gene-modified human natural killer cells (NKL-IL15) augments the anti-human hepatocellular carcinoma effect in vivo
2014, ImmunobiologyCitation Excerpt :Indeed, IL-15 is the major physiologic growth factor responsible for NK cell ontogeny (Fehniger and Caligiuri, 2001) and is also a potent regulator of NK cell proliferation, survival, and cytolytic activity (Grund et al., 2004; He et al., 2004). In a previous study, we successfully established an hIL-15 gene-modified NKL cell line (NKL-IL15) and confirmed that the addition of hIL-15 augmented the NKL cell-mediated anti-hepatocellular carcinoma (HCC) effect in vitro (Jiang et al., 2008). However, whether this anti-HCC effect could also be improved in an in vivo setting was unknown.