Background Natural Killer (NK) cells are potent in attacking cancers that escape from the T cell immune surveillance. Recent discoveries have reported that memory NK cells can be effective in cancer immunotherapy. However, there is no research available on the in vivo induction, identification, and potential of memory-like NK cells in in vivo models. In this study we report on the identification and induction of memory-like NK cells by a combination of a lipid nanoparticle (LNP) loaded with a stimulator of interferon genes (STING) agonist (STING-LNP) and CpG-ODN, and the potential of memory-like NK cells against melanoma lung metastasis model.

Methods The antitumor effects after monotherapy with either the STING-LNP or CpG-ODN or their combination were evaluated using a B16-F10 lung metastasis model. The combined treatment effect was also evaluated by measuring cytokine production. Memory-like NK cells was demonstrated by flow cytometry and confirmed through its preventative effect.

Results The best dosage of STING-LNP and CpG-ODN for induction of the memory-like NK cells were optimized through screening the cytokine levels required for memory-like NK cell induction. The combined therapy enhanced the production levels of interleukin 12 (IL-12), IL-18, and exerted an antitumor effect. The combination therapy induced the production of CD11b^high CD27^low NK cells, the most mature phenotype of NK cells. Two cycle treatments were required for the induction of this phenotype. Moreover, although monotherapies failed to show preventative effects, the combination therapy induced surprisingly drastic preventative effect. This preventative effect confirmed that CD11b^high CD27^low is the phenotype of memory-like NK cells, a phenotype first to be confirmed to characterize the memory-like NK cells.

Conclusions The findings reported herein reveal for the first time the in vivo induction of memory-like NK cells and its preventative effect against B16-F10 lung metastasis using a combination of STING-LNP and CpG-ODN. This represents the first report to identify and confirm the phenotype of memory-like NK cells through a prophylactic effect. Our study is the first to provide novel insights regarding the in vivo induction and the phenotypical characterization of the memory-like NK cells using a combination of immunotherapeutic drugs paving the way for the development of efficient immunotherapies.

Acknowledgements This work was supported by JSPS KAKENHI (Grant Number 20H03373) (T.N.), the Special Education and Research Expenses from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) (H.H.).

Ethics Approval All animal experiments were approved by the Ethics of Pharmaceutical Science Animal Committee of Hokkaido University (approval number: 20–0126). All experiments were carried out in accordance with the National University Corporation Hokkaido University Regulations on Animal Experimentation.