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IL-12 regulates the expansion, phenotype, and function of murine NK cells activated by IL-15 and IL-18

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Cancer Immunology, Immunotherapy Aims and scope Submit manuscript

Abstract

NK cells, which are composed of phenotypically and functionally heterogeneous subpopulations, play critical roles in immunity against cancer. The mechanism of generation of distinct subsets such as the effector and regulatory subtypes is unclear. Here, we show that this process comprises several steps, including generation of proliferating, highly cytotoxic cells activated by IL-15/IL-18 and differentiation into distinct cell populations induced with IL-12. Freshly prepared murine splenic NK cells expressed IL-15Rs and IL-18Rs and rapidly began to proliferate following stimulation with IL-15/IL-18. The proliferating NK cells highly expressed various activation markers such as B220, CD49b (DX5), lysosome-associated membrane glycoprotein 1 (LAMP-1), DNAX accessory molecule 1, perforin, and granzyme B and showed reduced expression of natural killer cell p46-related protein (NKp46) and IL-18Rα. These cells exerted strong cytotoxicity against YAC-1 cells, but did not secrete cytokines. IL-12 rapidly activated STAT4 in these cells, induced IFN-γ production, and then upregulated p21 and p27, leading to withdrawal from the cell cycle. In parallel, IL-12-stimulated cells gradually reduced cytotoxicity, decreased expression of activation markers, and instead increased expression of Sca-1, CD25, CD49a, and NKp46. Some IL-15/IL-18-induced cells strongly expressed PD-1, whereas NK cells induced with IL-15/IL-18 and IL-12 expressed high levels of T cell immunoglobulin mucin-3, LAG-3, and natural killer group 2 A. Furthermore, these cells spontaneously secreted IL-10 and TGF-β following prolonged incubation. Thus, IL-12 regulates expansion of NK cells activated with IL-15/IL-18, influences the population size of highly cytotoxic cells, and induces differentiation to unique cells sharing some phenotypes of ILCs.

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Abbreviations

CFSE:

5-(And-6)-carboxyfluorescein diacetate succinimidyl ester

CTLA-4:

Cytotoxic T-lymphocyte antigen 4

DNAM-1:

DNAX accessory molecule 1

DX5:

Anti-integrin alpha 2/CD49b

Eomes:

Eomesodermin

FSC:

Forward scattered light

Id2:

DNA-binding protein inhibitor

ILC:

Innate lymphoid cell

KO:

Knock out

LAMP-1:

Lysosome-associated membrane glycoprotein 1

mNK:

Mature NK

NKG2:

Natural killer group 2

NKp46:

Natural killer cell p46-related protein

Nlrp3:

Nucleotide-binding domain-like receptor protein 3

PI:

Propidium iodide

Sca-1:

Stem cell antigen-1

SSC:

Side-scattered light

T-bet:

T-box expressed in T cells

TIM-3:

T cell immunoglobulin mucin-3

WT:

Wild type

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Acknowledgments

We gratefully acknowledge the excellent technical help of Masaki Hata, Fumiko Yoshida and Atsuko Tamamoto.

Funding

This work was partly supported by Grants-in-Aid for Scientific Research (Japan Society for the Promotion of Science, KAKENHI: numbers JP25462671 and JP16K11220) and the Practical Research Project for Rare/Intractable Diseases from the Japan Agency for Medical Research and Development (AMED: number JP18nk0101355h0102).

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Authors and Affiliations

  1. Department of Otorhinolaryngology-Head and Neck Surgery, Hyogo College of Medicine, Nishinomiya, Hyogo, Japan

    Naoto Oka, Kenzo Tsuzuki & Masafumi Sakagami

  2. Department of Pharmacology, Medical University, Sofia, Bulgaria

    Tzvetanka Markova

  3. Laboratory of Tumor Immunology and Cell Therapy, Hyogo College of Medicine, Nishinomiya, Hyogo, Japan

    Wen Li & Haruki Okamura

  4. Department of Immunology, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Tokyo, Japan

    Yosif El-Darawish

  5. Department of Biology, Medical University, Sofia, Bulgaria

    Magdalena Pencheva-Demireva

  6. Department of Neuropsychiatry, Hyogo College of Medicine, Nishinomiya, Hyogo, Japan

    Kyousuke Yamanishi

  7. Hirakata General Hospital for Developmental Disorders, Hirakata, Osaka, Japan

    Hiromichi Yamanishi

  8. Center for Bioinformatics and Molecular Medicine, Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki, Japan

    Yoshimasa Tanaka

  9. Laboratory of Tumor Immunology and Immunotherapy, Hyogo College of Medicine, Nishinomiya, Hyogo, 663-8501, Japan

    Haruki Okamura

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  1. Naoto Oka
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Contributions

Conception and design of the study were carried out by HO, WL, and MP-D; TM, YE-D, MP-D, and WL acquired data; analysis and interpretation of data were done by KY, HY, YE-D, MP-D, and HO; KT, MS, and HO drafted the manuscript.

Corresponding author

Correspondence to Haruki Okamura.

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Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval and ethical standards

All mouse experiments were conducted in line with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health and guidelines provided by the European Parliament for the protection of animals. All mice were bred and maintained at our animal facility in accordance with the guidelines for the care and use of experimental animals at the Hyogo College of Medicine, Japan. All experiments were conducted with approval (#28041, #15-029, #16-021, #16-030, #16-034, #17-018 and #19-017) from the Animal Care Committee of the Hyogo College of Medicine.

Animal source

WT mice were purchased from SLC Japan Co, Ltd. (Shizuoka, Japan). C57BL/6 J IFN-γKO male mice (9 weeks old) were purchased from Jackson Laboratory (Bar Harbor, ME, USA). C57BL/6JN IL-18KO male mice were backcrossed for more than eight generations at the National Institute for Agrobiological Sciences (Ibaraki, Japan). Homozygous mutant mice were used for breeding and experiments at the animal facilities of the Hyogo College of Medicine.

Cell line authentication

Murine YAC-1 was obtained from the American Type Culture Collection (Manassas, VA, USA). Authentication of the cell line was not required as cells were purchased from professional cell line vendor and our master cell banks were generated soon after placing the original vials of cells into culture.

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Oka, N., Markova, T., Tsuzuki, K. et al. IL-12 regulates the expansion, phenotype, and function of murine NK cells activated by IL-15 and IL-18. Cancer Immunol Immunother 69, 1699–1712 (2020). https://doi.org/10.1007/s00262-020-02553-4

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