【FAQ】An important target point for NK cell culture that has been neglected

As an important part of the classic immune cell therapy in the field of immuno-oncology, NK cells have been widely used in the treatment of various malignant tumor diseases and the improvement of immunity. When faced with cancer cells, natural killer cells (NK cells) can directly recognize and kill cancer cells without the activation and sensitization of other cells. Therefore, NK cells are also known as important players in the human body's first line of defense against cancer, and have long-term applications and good development prospects in immune cell therapy of tumors.

?How do NK cells play a role in anti-tumor?

?NK cells have been identified as core cells of innate immunity because of their important dual functions: cytotoxicity and immunomodulatory effects. First, NK cells are not restricted by MHC, and can clear tumor cells through death receptors or directly release perforin and granzymes without being sensitized by antigens or involving antibodies; Cytokines and chemokines to activate the occurrence of adaptive immune activity.

?These two characteristics of NK cells also make them occupy a very important position in the treatment of cancer, making NK immune cell therapy a possibility to treat tumors. The activation of NK cells depends on the balance between inhibitory receptors and activating receptors expressed on the surface of NK cells, which interact with ligands on tumor cells [1].

?Analysis of the way of NK cell expansion

Generally, there are four main sources of NK cells, namely: peripheral blood, induced pluripotent stem cells, NK cell line (NK-92) and mesenchymal stem cells. However, the number of NK cells in the human body is very small, accounting for only 5% to 10% of the peripheral blood; in addition, a small amount of NK cells can also be seen in the liver, lung, thymus, lymph nodes and other parts; in the cells in the spleen, NK cells also maintain. 3% to 4%.

Because scarce NK cells are so important, in experiments, researchers have developed a variety of NK cell expansion methods to study the various functions of NK cells and the impact of external stimuli on NK cell-mediated immune activity. At present, the methods of NK cell expansion mainly include cytokine expansion method, feeder layer cell expansion method and superantigen stimulation method. Among them, IL-2, IL-7, IL-12, IL-15, IL-18, IL-21 and IFN-γ are important regulators of NK cell proliferation, promoting their differentiation and maturation, and exerting cytotoxicity. It can stimulate the proliferation and activity of NK cells, especially IL-2 and IL-15. The common additions of feeder cell expansion are chronic myeloid leukemia K562 cells and stimulated autologous PBMC cells (ie, peripheral blood mononuclear cells) [2]. The expansion of a large number of high-purity, highly active NK cells is the secret of their use in therapy.

Especially when PBMC (or NK cells separated by magnetic beads) are used as raw materials for expansion, NK cells can be specifically expanded in vitro through the stimulation of specific cytokines. This method is currently considered to be a promising method. One of [3, 4]. Relatively large-scale NK cell preparation can be carried out through in vitro stimulation culture, thereby ensuring the development of clinical treatment.

Comparison of NK cell expansion methods

How to make NK cells expand more efficiently?

01. IL-12

In August 2022, researchers from the University of California published an article entitled "Differential IL-12 signaling induces human natural killer cell activating receptor-mediated ligand-specific expansion" in the journal J. Exp. Med.[ 5].

Low concentrations of IL-12 promote NK receptor ligand-specific expansion in long-term cultured IL-2 primary NK cells

The results of this study show that low concentrations of IL-12 can effectively activate NK receptors to mediate NK cell expansion and promote the persistence of NK cell expression, thereby establishing ligand specificity for IL-12 signaling in human NK cells. The role of IL-12 and NK cells in the sexual memory signature may contribute to the realization of the combination of IL-12 and NK cells in the immunotherapy of cancer.

02. IL-15/IL-21

In 2009, in the journal "Cancer Research", Wing et al. published an article titled "EXPANSION OF HIGHLY CYTOTOXIC HUMAN NATURAL KILLER CELLS FOR CANCER CELL THERAPY" [6]. A practical method for preferential expansion of NK cells from PBMCs.

Wing et al. demonstrated that the expression of NK cell receptors activated and inhibited on the surface of these cells was enhanced, and their cell killing ability was also greatly enhanced (after 1-3 weeks, the killing ability of NK cells reached a peak, and then decreased, but still higher than resting NK cells). At the same time, the role of the STAT3 signaling pathway in the induction of these NK cells was also confirmed, and it was found that IL-21 was a strong activator of the STAT3 signaling pathway; IL-15 was a strong activator of STAT5, but had a weaker ability to activate STAT3 . However, IL-15 has been shown to induce high expression of the STAT3 activator IL-10. Therefore, these findings are helpful for clinical expansion of NK cells.

Summary: At present, both foreign and domestic, NK cell immunotherapy has made major breakthroughs in the field of tumor treatment, but most of the candidate drugs are still in the relatively early stage of clinical trials. According to the existing relevant clinical results, we are well aware of the efficacy and safety of NK cell therapy, and can also predict the "great future" of NK cell immunotherapy. We are convinced that in the near future, the expansion and anti-tumor activity of NK cells will also be popularized, and the prospect of NK cell therapy will be widely expected in the future!

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references:

[1] Adapted from Cancer Immunol Immunother 2020 May, 69(5):879-899

[2]Gong, Y., et al., Chimeric antigen receptor natural killer (CAR-NK) cell design and engineering for cancer therapy. Journal of Hematology & Oncology, 2021. 14(1): p. 73.

[3]Klingemann HG and Martinson J.Ex vivo expansion of nature killer cells for clinical applications [J].C ytoth erapy,2004, 6(1):15-22.

[4] Li Xiaohong, Ma Jian, Wang Feifei, et al. Study on in vitro expansion of high-purity human peripheral blood-derived NK cells [J]. Chinese Journal of Experimental Hematology, 2007, 15(2): 373-377.

[5]?Avishai Shemesh et al. Differential IL-12 signaling induces human natural killer cell activating receptor-mediated ligand-specific expansion. J Exp Med. 2022 Aug 1;219(8):e20212434. doi: 10.1084/jem.20212434.