Beneficial effect of short-term exposure of human NK cells to IL15/IL12 and IL15/IL18 on cell apoptosis and function☆
Introduction
CD56+ cells display spontaneous MHC-unrestricted cytotoxicity and produce various cytokines with a pivotal role in the activation of both innate and adaptive immune responses, therefore representing a first line of defense against pathogens and transformed cells [1]. In addition, they express IgG receptors, which make them important antibody depended cytotoxicity (ADCC) mediating effector cells. All the above reasons have made CD56+ cells an attractive tool in cancer immunotherapy. Resting NK cells express a number of monocyte-derived cytokine (monokine) receptors, which may be activated for increased killer activity by numerous cytokines alone or in combinations [2], [3], [4], [5], [6].
IL15 was found to induce the proliferation, survival, and cytotoxicity in a dose-dependent fashion of resting CD56+ cells via components of IL-2R [6], [7]. IL12 is a proinflammatory and immunomodulatory cytokine inducing potent anti-tumor activity in a variety of murine tumor models [8], [9], [10]. IL18 is a proinflammatory monokine, which uses the IL1R-related protein to mediate intracellular signals [11] and to augment the killer activity of CD56+ cells in in vitro and in vivo models [12], [13]. IL15 has been demonstrated to act in concert with IL18 and the effect of IL15/IL18 has been reported to induce enhanced cytotoxicity and IFNγ production in total PBMC treated for 4 days [14], increased IFNγ production by isolated CD56+ cells treated for 24 h [15], [16] and high GM-CSF production by NK cells [16], [17]. The combination of IL15/IL12 has been more extensively investigated and in particular NK cells treated up to 3 days with IL15/IL12 exhibited high cytotoxicity and cytokine production [7], [17], [18]. In all cases mentioned above, the effect of both IL15/IL12 and IL15/IL18 was tested immediately after the end of the incubation period with the monokines. However, long-term incubation of CD56+ cells with IL15/IL12 was detrimental due to high apoptosis of the peripheral blood NK cells (detected within 48 h of exposure to the monokines) [19] and the umbilical cord blood NK cells [20].
The purpose of this study was to test the possible beneficial effect of short-term incubation (i.e., <48 h) with IL15/IL12 or IL15/IL18 on prevention of CD56+ cell apoptosis, while maintaining effective function in long-term cultures. Our work provides the first evidence that short-term exposure of CD56+ cells to the monokine combinations prevents high apoptosis while cells still exhibit high killer activity—that persists over the 18-day culture period tested after two additional 6 h cycles of exposure to the monokines applied every 8 days. Such treated CD56+ cells retain their ability for high cytokine production after each short-term stimulation. These repetitive short-term exposures to the monokine combinations result in long-lived CD56+ cells with lower rates of Fc receptor decline, therefore exhibiting increased ADCC responses when compared to the long-term presence of the monokine combinations in the culture medium.
Section snippets
Reagents and immunophenotyping
Human recombinant IL15 (rhIL15), rhIL12, and rhIL18 were purchased from R&D Systems (Europe). PC5-conjugated anti-human CD56 mAb and PC5-isotype control were purchased from Immunotech (Coulter Immunology, Hialeah, France). FITC- or PE-conjugated anti-human CD16 mAb and isotype control mAbs were purchased from Becton–Dickinson (Mountain View, CA, USA). Samples were analyzed using FACS-Calibur (Becton–Dickinson) and CellQuest analysis software.
Cell lines
The human Burkitt lymphoma cell line Daudi, the human
Short-term incubation of CD56+ cells with IL15/IL12 or IL15/IL18 prevents high apoptosis
IL15 in combination with IL12 has been demonstrated to induce cell death in NK cells within 48 h of incubation [19]. At the initiation of our studies, we determined the kinetic profile of such an effect mediated by IL15/IL12 as well as by IL15/IL18 on CD56+ cells (Fig. 1). In particular, cells were incubated either in the presence of IL12, IL15, and IL18 alone or in the presence of the above monokine combinations for the time period indicated (6, 18, 24, and 48 h), then washed, and further
Discussion
In the present study, we demonstrate for the first time that short-term incubation with IL15/IL12 or IL15/IL18 supports CD56+CD3− survival by conferring decreased levels of apoptosis. Furthermore, the combinations of both IL15/IL12 and IL15/IL18, as well as IL15 alone, are powerful enough to support, upon brief presence in the culture medium, NK cell-mediated cytotoxic responses, lasting as long as 8 days after monokine removal from the medium. Furthermore, NK cells showed sustained high levels
References (32)
- et al.
Interleukin 15: biology and relevance to human disease
Blood
(2001) - et al.
Defective NK cell activity and Th1 response in IL18-deficient mice
Immunity
(1998) - et al.
Augmentation by interleukin-18 of MHC-nonrestricted killer activity of human peripheral blood mononuclear cells in response to interleukin-12
Inter. J. Immunopharm.
(2000) - et al.
Inteleukin (IL)-15 antibody-dependent cellular cytotoxicity and natural killer activity in neonatal cells
Cell Immunol.
(1998) - et al.
Cytokine-induced apoptosis of human natural killer cells identifies a novel mechanism to regulate the innate immune response
Blood
(1997) - et al.
Inteleukin-2 and interleukin-15: immunotherapy for cancer
Cytokine Growth Factor Rev.
(2002) - et al.
Natural killer cells, viruses and cancer
Nat. Rev. Immunol.
(2001) - et al.
Functional consequences of interleukin-2 receptor expression on resting human lymphocytes. Identification of a novel natural killer cell subset with high affinity receptors
J. Exp. Med.
(1990) - et al.
Gene expression and secretion of cytokines and cytokine receptors from highly purified CD56+ natural killer cells stimulated with interleukin-2, interleukin-7 and interleukin-12
Eur. J. Immunol.
(1993) - et al.
Interleukin-21 and IL-21 receptor: novel effectors of NK and T cell responses
J. Leukoc. Biol.
(2002)
Selective expansion and partial activation of human NK cells and NK receptor-positive T cells by IL-2 and IL15
J. Immunol.
Interleukin IL15 is a novel cytokine that activates human natural killer cells via components of the IL2 receptor
J. Exp. Med.
Antitumor and antimetastatic activity of interleukin 12 against murine tumors
J. Exp. Med.
The antitumor activity of IL12: mechanisms of innate immunity that are dose and model dependent
J. Immunol.
In vivo antitumor activity of NKT cells activated by the combination of IL12 and IL18
J. Immunol.
Overview of interleukin 18: more than an interferon γinducing factor
J. Leukoc. Biol.
Cited by (11)
Early pregnancy decidual lymphocytes beside perforin use Fas ligand (FasL) mediated cytotoxicity
2007, Journal of Reproductive ImmunologyCitation Excerpt :NK cytolytic action is regulated by a dynamic maturation and activation dependent expression of NK cell inhibitory and activating receptors that bind to MHC class I or MHC class I like molecules (Ishitani et al., 2003). Th1 type cytokines can induce highly cytotoxic peripheral blood (PB) killer cells, particularly interleukin (IL)-2 (Verma et al., 2000b), IL-12, IL-15 and IL-18 (Sotiriadou et al., 2005). An increased expression of adhesion molecules (Liebau et al., 2002) and cytolytic mediators, perforin and TNFα in PB lymphocytes (Ozdemir et al., 2005) or perforin and Fas ligand (FasL) in interstitial intraepithelial lymphocytes (Ebert, 2004) on the mRNA and protein level is attributed to the increased cytolytic potential of LAK cells and can be induced by Th1 type cytokines.
Gamma-irradiation induces HER-2/neu overexpression in breast cancer cell lines and sensitivity to treatment with trastuzumab
2013, International Journal of Radiation BiologyCancer immunology and novel strategies for immunotherapy
2012, Molecular Oncology Principles and Recent Advances
- ☆
Supported by a grant from the Regional Operational Program Attika No20, MIS code 59605GR to M.P.