Beneficial effect of short-term exposure of human NK cells to IL15/IL12 and IL15/IL18 on cell apoptosis and function

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Abstract

Monokines IL12, IL15, and IL18 have been shown to activate NK cell function, however with high apoptosis induced by their combination within 48 h. Here, we demonstrate for the first time that CD56+ cells incubated for only 18 h with the combination of IL15/IL12 or IL15/IL18, then washed, and further cultured in plain medium, exhibit low levels of apoptosis. These shortly activated CD56+ cells show high killer activity against NK- and LAK-sensitive tumor targets that persists over a culture period of 18 days after two additional 6 h cycles of exposure to the monokines applied every 8 days and also retain their ability for high cytokine production during each exposure. Moreover, these repetitive short-term exposures of CD56+ cells to the monokine combinations result in long-lived CD56+ cells with slower rates of FcγRIII receptor (CD16) decline, therefore exhibiting higher antibody depended cytotoxicity, as opposed to the continuous incubation with the monokine combinations. In conclusion, short-term exposure of CD56+ cells to IL15/IL12 or IL15/IL18 at 8-day intervals may hold a promise for improved clinical results in cellular adoptive cancer immunotherapy and for the in vivo injections of the monokines.

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)

  • J. Dunne et al.

    Selective expansion and partial activation of human NK cells and NK receptor-positive T cells by IL-2 and IL15

    J. Immunol.

    (2001)
  • W.E. Carson et al.

    Interleukin IL15 is a novel cytokine that activates human natural killer cells via components of the IL2 receptor

    J. Exp. Med.

    (1994)
  • M.J. Brunda et al.

    Antitumor and antimetastatic activity of interleukin 12 against murine tumors

    J. Exp. Med.

    (1993)
  • M.J. Smyth et al.

    The antitumor activity of IL12: mechanisms of innate immunity that are dose and model dependent

    J. Immunol.

    (2000)
  • C.N. Baxevanis et al.

    In vivo antitumor activity of NKT cells activated by the combination of IL12 and IL18

    J. Immunol.

    (2003)
  • C.A. Dinarello et al.

    Overview of interleukin 18: more than an interferon γinducing factor

    J. Leukoc. Biol.

    (1998)
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    Supported by a grant from the Regional Operational Program Attika No20, MIS code 59605GR to M.P.

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