Natural killer (NK) cells are lymphocytes that belong to the innate immune system known to eliminate transformed cells (malignant or virally infected cells) in a manner independent of the major histocompatibility complex class I (MHC-I). As it is known today, NK cell expansion and activation are highly regulated through a delicate balance between inhibitory and positive signals coming from the surrounding environment, which net-worth signal will at last determine the fate of the NK cells (Fig. 1) (Dunai et al., 2022). Therapeutic strategies that target this equilibrium are being exploited in NK cell-based therapies for the development of novel cancer treatment.

To prevent NK cells from eliminating healthy self-cells, NK cells stochastically express an array of inhibitory receptors that recognize MHC-I molecules. However, only those NK cells that express inhibitory receptors that bind to self-MHC-I molecules are set to have an important role in self-tolerance through a phenomenon first described in mice by Kim et al. (2005), Joncker et al. (2008), Joncker, Fernandez, Treiner, Vivier, and Raulet (2009) and later in humans by Anfossi et al. (2006) named licensing/arming or education respectively. According to this research, the licensed/educated NK cells are better equipped to eliminate transformed cells due to their hyperresponsiveness in the presence of activating signals when compared to unlicensed/uneducated NK cells (Alvarez et al., 2020, Alvarez et al., 2020, Alkayyal et al., 2017, Alvarez et al., 2014, Aguilar et al., 2021, Alvarez et al., 2020). However, upon activation, no differences are found between both subsets (Alvarez et al., 2019, Alvarez et al., 2016, Aguilar et al., 2021). Among the different inhibitory receptors displayed on human NK cells, the inhibitory killer immunoglobulin-like receptors (KIR) are part of a large family that recognize MHC-I molecules. The downstream signaling of KIRs is based on the presence of an immunoreceptor tyrosine-based inhibitory motif (ITIM) in their cytoplasmic domain which recruits Src homology 2-containing protein tyrosine phosphatase-1 (SHP-1) (Pende et al., 2019). In allogeneic hematopoietic stem cell transplantation (HSCT), the licensed/educated and unlicensed/uneducated NK cells have shown to modulate donor cell engraftment and allow tolerance (Alvarez et al., 2019, Ansell, 2017, Aste-Amezaga et al., 1994, Backhaus et al., 2019, Atkins et al., 1997). NKG2A is another inhibitory receptor that specifically recognize HLA-E and mediates NK cell inhibition and has also been linked to education and differentiation (Bailey et al., 2017, Barao et al., 2011). IgG4 humanized antagonist monoclonal antibodies (mAbs), IPH2101 (formerly 1–7F9) for KIR and monalizumab for NKG2A, have been developed to neutralize the suppression function of KIR and NKG2A on NK cells respectively and have been or are currently being evaluated in different clinical trials, mainly against hematological malignances. However, the results obtained from the therapeutic use of these compounds when given as monotherapy have been rather mild if any (Barrow et al., 2019, Barao et al., 2011). Surprisingly, one study found a contraction and hyporesponsiveness on the remaining NK cells upon IPH2101 infusion in multiple myeloma (MM) patients (Carlsten et al., 2016). A recent study showed that intratumoral administration of NK cells in combination with monalizumab reduced the tumor progression of head and neck squamous cell carcinoma (HNSCC) in a humanized xenograft mouse model (Benson et al., 2015, Berraondo et al., 2019). However, the reduction, although significant, was mild and unable to completely eradicate the tumor mass. Overall, these findings suggest the necessity to better explore the importance of the balance between activating and inhibitory molecules and how modulating one or the other might affect NK cell responses. Other inhibitory receptors known to suppress NK cell activation and function are TIGIT, CD96, LAG3, KLRG1, PD-1 and CTLA-4 (Fig. 1). A more in-depth analysis of the different inhibitory receptors presented on NK cells and strategies to neutralize them for cancer therapy have been explore elsewhere (Berrien-Elliott et al., 2023, Berraondo et al., 2019, Berrien-Elliott et al., 2022, Bhatt et al., 2017, Berrien-Elliott et al., 2022, Bessard et al., 2009).

Immunosuppression of NK cells can be also accomplished by the presence of immunosuppressive cells such as regulatory T cells (Tregs), myeloid derived suppressor cells (MDSC), M2-like tumor-associated macrophages and/or neutrophils. This inhibition can be due to the interaction with checkpoint inhibitor proteins and their ligands or to the release of immunosuppressive cytokines, mainly IL-10 and TGF-β. The secretion of reactive oxygen species (ROS), prostaglandin E2 (PGE2) and/or the generation of hypoxia within the tumor microenvironment (TME) by the tumor cells themselves or the immunosuppressive cells infiltrating that tumor are also contributing factors to the NK cell dysfunctionality (Tong et al., 2022).

As important as the inhibitory receptors are the activating receptors, which can help to directly recognize transformed cells by the expression of stress molecules on their surfaces (Fig. 1). MHC class I polypeptide-related sequence A/B (MICA/B) or Ul-16 binding proteins (UBLPs) are some examples of these stress signals present on the surface of virally infected or malignant cells that trigger a NK cell-mediated response through the activating receptor NKG2D. Poliovirus receptor (PVR) or Nectin-2, represent another example of stress molecules that drive NK cell cytotoxicity against transformed cells by their recognition through the activating receptor DNAM-1. Unlike the inhibitory KIRs, other activating receptor family involve the activating KIRs that also bind to MHC class I molecules but, signals through an immunoreceptor tyrosine-based activation motif (ITAM) and contains generally a shorter cytoplasmic tails (Blunt & Khakoo, 2020). CD16 (also known as FcγRIII) is an activating receptor that binds to the fragment common (Fc) portion of IgG antibodies and mediates antibody dependent cellular cytotoxicity (ADCC) with important implications in mAb-based therapies. Other set of activating receptors belongs to the natural cytotoxicity receptors (NCR) formed by NKp30, NKp44 and NKp46 that recognize non-MHC-I ligands on cancer or virally infected cells (Blunt and Khakoo, 2020, Bommareddy et al., 2024).

Lastly, activating and stimulatory cytokines also play an important role in the homeostasis, maturation, maintenance, activation and expansion of NK cells. In fact, different cytokines have been exploited in the clinic to drive more functional NK cells (Dunai et al., 2022). In this review, we analyze how these cytokines modulate NK cell activation and the therapeutic strategies that take advantage of them to maximize the cytotoxic potential of NK cells in cancer therapy.