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. 2018 Mar 9;14(3):331-340.
doi: 10.7150/ijbs.22809. eCollection 2018.

V体育ios版 - Effect of IL-18 on the Expansion and Phenotype of Human Natural Killer Cells: Application to Cancer Immunotherapy

Hiroaki Senju  1   2   3 , Asuka Kumagai  2 , Yoichi Nakamura  1   3 , Hiroyuki Yamaguchi  1   3 , Katsumi Nakatomi  1   3 , Shota Fukami  2 , Kengo Shiraishi  2 , Yuka Harada  2 , Mitsuhiro Nakamura  2 , Haruki Okamura  4 , Yoshimasa Tanaka  2   4 , Hiroshi Mukae  1   3
Affiliations

Effect of IL-18 on the Expansion and Phenotype of Human Natural Killer Cells: Application to Cancer Immunotherapy

Hiroaki Senju et al. Int J Biol Sci. .

Abstract

When pathogenic stresses are recognized by innate immune cells, inflammasomes are assembled and caspase-1 is activated, resulting in the conversion of pro-IL-18 into mature IL-18. Because natural killer (NK) cells express IL-18 receptors, IL-18 may play roles in immune functions of NK cells. In the present study, we examined the effect of IL-18 on NK cells derived from lung cancer patients and healthy adult volunteers. When peripheral blood NK cells were stimulated with IL-2, the cells formed clusters beginning on day 5-6 and proliferated thereafter, in which the number of NK cells increased by 10-fold in 10 days. When IL-18 was added, cell clusters were observed as early as on day 4 and NK cells proliferated vigorously. On day 10, the expansion rate was 56-fold on average, showing that IL-18 promoted the expansion of NK cells VSports手机版. It was also notable that IL-18 enhanced the expression of CD80, CD86, HLA-DR and HLA-DQ on NK cells, suggesting that IL-18 conferred NK cells an APC-like phenotype. When cellular cytotoxicity was determined, APC-like NK cells efficiently killed tumor cells and anti-tumor activity was augmented by the addition of tumor antigen-specific mAbs. In addition, IFN-γ was produced by APC-like NK cells in response to tumor cells, and the cytokine production was further enhanced by mAbs. Taken together, IL-18 not only promoted the expansion of NK cells, but also changed the phenotype of NK cells. IL-2/IL-18-induced NK cells might, therefore, serve as a bridge between innate immunity and adaptive immunity and be useful for cancer immunotherapy. .

Keywords: IL-18; NK cells; PD-1; antigen-presenting cells; immune checkpoint. V体育安卓版.

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Conflict of interest statement

Competing Interests: YT is a co-inventor of Japanese Patent 2014-73475 on the development of a non-radioactive cellular cytotoxicity assay using a precursor of a novel Eu chelate-forming compound. The other authors have no conflicts of interest V体育ios版.

Figures

Figure 1
Figure 1
Effect of IL-18 on the IL-2-mediated expansion of human NK cells. (A) Microscopic analysis of the effect of IL-18 on the expansion of NK cells stimulated with IL-2. Heparinized PBMCs were obtained from a healthy adult volunteer and a lung cancer patient and CD3- PBMCs were purified by negative selection using anti-CD3 mAb-coated beads. The cell suspensions were stimulated with either IL-2 or IL-2/IL-18 at 37oC with 5% CO2 and cell clustering was observed under a microscope on days 3, 4, 5 and 6. (B) Flow cytometric analysis of CD3- PBMCs stimulated with IL-2 or IL-2/IL-18. CD3- PBMCs derived from lung cancer patients (LC01 and LC02) and healthy donors (HD01 and HD02) were incubated with IL-2 or IL-2/IL-18 for 10 days and the expanded cells were analyzed through flow cytometry. (C) Effect of IL-18 on the number of NK cells after stimulation with IL-2. Before and after expansion of CD3- PBMCs derived from 13 lung cancer patients with IL-2 or IL-2/IL-18 for 10 days, the number of NK cells was counted by trypan blue dye exclusion. A p value between the numbers of IL-2- and IL-2/IL-18-induced NK cells is shown.
Figure 2
Figure 2
Effect of IL-18 on the expression of surface markers on NK cells. CD3- PBMCs derived from a healthy adult volunteer were incubated with IL-2 or IL-2/IL-18 and the expression of cell surface markers on CD56+ cells were detected through flow cytometry using FITC-conjugated anti-CD56 mAb and PE-conjugated anti-CD80, CD86, HLA-DR, HLA-DQ, ICOS and CD25 mAbs on days 0, 5, 7 and 10. The flow cytometric profiles of CD56+ cells incubated with IL-2 are displayed as open histograms with dotted contours and that with IL-2/IL-18 as filled histograms with solid contours. The proportions of cell surface marker-positive cells in CD56+ cells after expansion are indicated.
Figure 3
Figure 3
Cellular cytotoxicity and ADCC against tumor cell lines exhibited by IL-2/IL-18-stimulated NK cells. CD3- PBMCs derived from a lung cancer patient (A) and a healthy adult volunteer (B) were stimulated with IL-2/IL-18 for 10 days and cellular cytotoxicity and ADCC against K562, PC-9, ACHN, VMRC-RCW, Raji and RAMOS-RAI exhibited by NK cells were determined using a non-radioactive cellular cytotoxicity assay kit. PC-9, ACHN and VMRC-RCW were pretreated with anti-EGFR mAb at concentrations of 0 (♦), 0.05 (■) or 0.5 μg/mL (●) and Raji and RAMOS-RAI with anti-CD20 mAb at concentrations of 0 (♦), 0.01 (■) or 0.1 μg/mL (●) at 37oC with 5% CO2 for 15 min. The spontaneous release (%) was less than 20% in all the experiments. All experiments were done in triplicate.
Figure 4
Figure 4
Effector functions exhibited by IL-2/IL-18-induced NK cells. (A) CD107a degranulation assay in IL-2/IL-18-induced NK cells in response to tumor cell lines. PC-9 cells were preincubated with 0 or 0.5 μg/mL of anti-EGFR mAbs for 20 min on ice. IL-2/IL-18-stimulated NK cells from an adult donor were incubated with PC-9 or K562 cells at an effector-to-target ratio of 1 : 3 and the degree of degranulation was analyzed by flow cytometry using PE-conjugated anti-CD107a mAbs. The experiments were done in triplicate and a representative dot plot is shown. (B) IFN-γ production from IL-2/IL-18-induced NK cells in response to tumor cells. PC-9 cells and VMRC-RCW cells were preincubated with 0 or 1 μg/mL of anti-EGFR mAbs for 15 min on ice. IL-2/IL-18-stimulated NK cells were incubated with PC-9 or VMRC-RCW cells at an effector-to-tumor ratio of 1 : 1 and IFN-γ production was measured through a flow cytometer using PE-conjugated anti-IFN-γ mAb. The experiments were done in triplicate and p values between IFN-γ productions in the absence and presence of mAb are shown.
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