Natural Killer (NK) cells were first characterized in the 1970s by their ability to detect and eliminate tumor cells without prior antigen sensitization (1). Following observations in the transplantation clinic almost 30 years later, NK cells were identified as one of the first populations to reconstitute following hematopoietic stem cell transplantation (HSCT) and were found to exhibit direct cytotoxicity against malignant cells (2). Indeed, this natural potency against tumor cells has sparked a great deal of interest in exploiting the NK cell platform to treat cancer. Although NK cell-based therapies have not yet achieved the same clinical success as adoptive T cell therapies, early successes in pre-clinical and clinical trials over the past decade have generated enthusiasm for maximizing their therapeutic potential. Several studies have sought to optimize the source from which therapeutic NK cells are derived and the ex vivo activation and expansion strategies by which their activity and persistence in vivo can be enhanced. However, although the NK cell repertoire is highly heterogeneous both between and within individuals, relatively little attention has been given to the initial selection of NK cells which harbor the greatest antitumor activity. Here, we review the current state of donor selection for peripheral blood NK (pb-NK) cell-based immunotherapies and discuss the factors which drive NK cell effector function along with the challenges associated with identifying highly potent NK cell populations for immunotherapy.

NK cells are a cytotoxic subset of innate lymphoid cells (ILCs) with marked potency against malignant cells. NK cells and other ILCs originate from the same bone marrow-derived common lymphoid progenitor cells (CLPs) as B and T lymphocytes (3). Although details of human NK cell development remain largely unknown, bone marrow (BM)-derived CD34⁺CD45RA⁺ CLPs are thought to migrate to various anatomical sites where they subsequently undergo interleukin-15 (IL-15) mediated differentiation along the NK cell lineage (4).

In humans, mature pb-NK cells can be divided into two major functional subsets traditionally characterized based on the relative cell-surface density of the CD56 molecule and expression of the low-affinity IgG Fc region receptor III (FcγRIII; CD16): CD56^brightCD16^- and CD56^dimCD16⁺. CD56^bright cells represent approximately 10% of the pb-NK cell population and primarily act as potent producers of pro-inflammatory cytokines such as interferon gamma (IFNγ) following cytokine stimulation (5). In contrast, CD56^dim cells comprise approximately 90% of the pb-NK cell population and produce IFNγ in response to direct interactions with target cells rather than via cytokine activation (6). In addition, CD56^dim cells are more strongly cytotoxic towards malignant cells than their CD56^bright counterparts and harbor high baseline levels of cytotoxic molecules such as perforin and granzyme B (7). The CD56^dim population can be further divided into CD57^- and CD57⁺ cells, the latter of which represent terminally differentiated NK cells typically considered to harbor the highest cytotoxic potential.

Unlike T and B lymphocytes, NK cell receptors do not undergo somatic rearrangement to generate antigen specificity. Rather, NK cells rely on the stochastic expression of germline-encoded activating and inhibitory receptors, with the complex integration and hierarchy of signals generated through these receptors tightly controlling NK cell function. NK cells express a suite of activating receptors which detect various molecules upregulated by malignant cells. Simultaneous engagement of multiple activating receptors is typically required to overcome an NK cell’s intrinsic activation threshold and trigger effector function (8). A notable exception is CD16, which is the only receptor that can activate NK cells in the absence of other activating signals (9). CD16 is the most potent activating receptor, with crosslinking of CD16 molecules by the Fc region of IgG antibody-opsonized target cells resulting in NK cell activation through a process known as antibody-dependent cell-mediated cytotoxicity (ADCC). Other activating receptors include: the natural cytotoxicity receptors (NCRs) NKp30, NKp44 and NKp46, which directly bind to a wide variety tumor-associated ligands (10); NKG2D, which recognizes the cell-stress induced major histocompatibility complex class I-related molecules MICA and MICB and the UL16-binding proteins (ULBP-1-6) (11); 2B4, which binds to CD48 (12, 13); DNAM-1, which recognizes two protein markers of cellular stress CD112 and CD155 (14); NKp80, which binds to activation-induced C-type lectin (AICL) (15); and the self-associating CD2-like receptor activating cytotoxic cells (CRACC/CD319/CS1/SLAMF7) (16).

NK cells also express a diverse repertoire of inhibitory receptors which recognize human leukocyte antigen (HLA) molecules and regulate self-tolerance to healthy tissues by dominantly inhibiting NK cell activation (17). Two major families of NK cell receptors recognize HLA molecules: the killer immunoglobulin-like receptor (KIR) family and the CD94/NKG2 family of C-type lectin receptors. Up to 15 genes are encoded within the KIR locus on chromosome 19 (18), resulting in 14 functional receptors comprising seven inhibitory KIR (-2DL1-2DL3, -2DL5, and -3DL1-3DL3), six activating KIR (-2DS1-2DS5 and -3DS1), and KIR-2DL4 which carries out both activating and inhibitory functions. These KIR genes are highly polymorphic and cluster into haplotypes that differ between individuals, creating at least 40-50 possible KIR genotypes and more than 20 haplotypes (18, 19). Haplotypes are divided into two groups characterized by their enrichment for inhibitory (Haplotype A) and activating (Haplotype B) KIRs. Although individual KIR recognize distinct allelic epitopes present in certain HLA-A, HLA-B or HLA-C allotypes, also referred to as KIR ligands, inhibitory KIR have higher avidity for their cognate ligands than activating KIR (20). Similarly, the non-classical HLA molecule, HLA-E, is recognized by both the inhibitory CD94/NKG2A and activating CD94/NKG2C receptors, though the CD94/NKG2A heterodimer binds with higher affinity (21).

Interaction between these major inhibitory receptors and their specific HLA ligand is critical for NK cells to achieve functional maturation through a process known as “licensing” or NK cell education (22). Educated NK cells exhibit the highest reactive potential against target cells that have lost or downregulated HLA expression through a process known as “missing self” recognition, but are susceptible to inhibition by tumor cells that have retained HLA expression (23, 24). Interestingly, as any given inhibitory receptor is present on only a fraction of the NK cell repertoire, both uneducated and educated NK cell populations may coexist within an individual (23). Although the mechanisms underlying NK cell education remain poorly defined, several models have been proposed which debate the relative contributions of inhibitory and activating receptors towards this ongoing process of functional maturation [reviewed (25)].

Following activation, NK cells carry out a range of antitumor effector functions including the direct lysis of target cells and indirect modulation of both innate and adaptive antitumor immunity through the production of various immunomodulatory cytokines and chemokines (Figure 1). Given their potent antitumor activity, relatively low likelihood of severe adverse effects such as graft-versus-host disease, and potential for combination with other treatment strategies, NK cell-based therapies have emerged as promising candidates for the treatment of a variety of hematological malignancies and solid tumors (26).

The ongoing development of NK cell-based therapies has typically focused on optimizing two major factors: the source from which therapeutic NK cells are derived and the methods by which their activity can be enhanced. NK cells can be obtained from several allogeneic sources including the peripheral blood of related or unrelated donors, umbilical cord blood (UCB), induced pluripotent stem cells (iPSC), and immortalized NK cell lines, with each source harboring intrinsic advantages and disadvantages that must be considered when designing an optimal cellular therapy [reviewed (27, 28)]. Similarly, numerous ex vivo enhancements have been developed with the aim of increasing the in vivo activity, persistence, and tumor-targeting of the isolated NK cells following infusion. Common strategies include in vitro cytokine and feeder cell expansion, cytokine activation in the absence of NK cell expansion, and genetic modification to express a manufactured chimeric antigen receptor (CAR) directed against specific tumor-associated antigens [reviewed (26–28)]. Although several clinical trials are currently evaluating the efficacy of alternative NK cell sources [reviewed (28)], pb-NK cells have remained the most widely utilized source of therapeutic NK cells in clinical trials to date as they are relatively easy to source, have a mature phenotype, and harbor strong cytotoxic activity that can be further enhanced through cytokine stimulation prior to infusion (26). Indeed, pb-NK cells harbor stronger cytolytic activity and greater expression of activating receptors such as CD2 and CD16 than their UCB counterparts (29–31). Furthermore, whilst NK cell lines such as NK-92 represent a robust and renewable source of therapeutic NK cells with strong cytotoxic activity, the requirement for irradiation limits the in vivo persistence of the infused cells to a maximum of 48 hours and thus prevents the generation of a long-lasting clinical effect (32). More recently, studies have focused on the development of readily available “off-the-shelf” cellular therapies utilizing unrelated third-party donor-derived pb-NK cells or iPSC-derived NK cells. Indeed, several clinical trials are currently investigating the safety and efficacy of various “off-the-shelf” adoptive NK cell therapy strategies for the treatment of hematological malignancies (Table 1).

Despite the existence of a plethora of strategies to enhance the cytotoxicity and persistence of therapeutic NK cells in vivo, the initial selection of highly functional cells with strong innate potency is essential for the widespread success of NK cell therapies. Over the past decade it has become increasingly evident that not all NK cells have the same baseline capacity to eradicate leukaemic cells (33–37). In addition to the functional differences between traditional NK cell subsets (33–35), variability also exists in the functional capacity of NK cells derived from different individuals (36, 37). However, relatively little attention has been given to optimizing the particular subset or donor from which these cells are derived. We have recently reported that resting donor-derived pb-NK cells display marked variability in their capacity to mount an effector response against leukaemic target cells (36). Intriguingly, we identified a pool of donors with strong activity against multiple leukaemic cells, representing ideal candidates for the development of efficacious “off-the-shelf” NK cellular therapies. However, there is currently no standardized criteria by which NK cell donors are selected to improve clinical efficacy. We believe the intrinsic diversity in NK cell activity between individuals is an important consideration when developing and optimizing pb-NK cell-based therapies. Specifically, the selection of donors who harbor NK cells with high baseline anti-tumor activity may provide the opportunity to further improve the success of future NK cell therapies.

For several decades phenotypic analysis has played an integral role in inferring the identity, maturation state, and functional capacity of NK cell populations. For example, the classic model of NK cell maturation describes a gradual downregulation of CD56 expression and acquisition of CD16 and CD57 expression as pb-NK cells progress from an immunoregulatory CD56^brightCD16^- phenotype towards the cytotoxic CD56^dimCD16⁺ phenotype, before eventually transitioning into the terminally differentiated CD56^dimCD16⁺CD57⁺ population with the highest cytotoxic activity. Several other phenotypically and functionally distinct subsets have been described in both healthy and diseased states. For example, infection with cytomegalovirus (CMV) drives the expansion of the CD57⁺NKG2C⁺KIR⁺ adaptive NK cell population with an increased capacity for ADCC (35). In contrast, a population of CD56^- NK cells with impaired cytotoxicity and ADCC have been described at low frequencies within healthy individuals but are expanded following both acute (94) and chronic (95, 96) viral infections.

Although phenotyping remains an accessible means by which the heterogeneity of the NK cell repertoire can be explored, increasing evidence suggests these phenotypic classifications of maturity and functional state are inherently flawed. For example, the relative expression level of CD56 does not necessarily inform on maturation state, as CD56^dimCD16⁺ cells can up-regulate CD56 expression upon cytokine stimulation to become CD56^brightCD16⁺ (97). Similarly, a proportion of NKG2A^- clones have been reported to regain NKG2A expression and CD56^dimCD57⁺ clones lost CD57 expression following expansion with K562 feeder cells (98). Although typically considered a signature of NK cell functional maturation, KIR expression has also been observed on both CD56^dim and CD56^bright cells (99). Recent studies have raised further discrepancies in the classic functional roles assigned to the CD56^bright and CD56^dim populations. Although CD56^dimCD16⁺ cells are traditionally considered the cytotoxic subset, these cells have also been observed to carry out regulatory functions. Following culture with TGF-B, IL-15, and IL-18, CD56^dimCD16⁺ pb-NK cells demonstrated reduced cytotoxicity and pro-inflammatory cytokine production, but increased secretion of the immunoregulatory protein VEGF-A (100). Similarly, the traditionally “regulatory” CD56^bright NK cell subset is also capable of potent anti-tumor activity. Following priming with IL-15, Wagner and colleagues reported that CD56^brightCD16^- pb-NK cells displayed greater cytokine production, degranulation and killing of leukaemic targets than their CD56^dimCD16⁺ counterparts (101). A population of highly cytotoxic CD56^superbright NK cells have also recently been described following expansion of patient-derived NK cells with K562 feeder cells (102). In this study it was reported that NK cell degranulation, cytotoxicity, and IFNγ production increased alongside increasing expression of CD56. Furthermore, these expanded CD56^superbright NK cells were able to eliminate autologous ovarian tumors in vivo in patient-derived xenograft models (102). Consideration of other NK cell markers is similarly unable to address these discrepancies between NK cell phenotype and functional output. For example, both regulatory and cytotoxic NK cells can express high levels of activating receptors such as the NCRs and NKG2D, though stimulation through these receptors elicits distinct functional programs in each subset (100, 103, 104). Furthermore, both NK cell populations can express either high or low levels of inhibitory receptors such as NKG2A and KIRs (101, 102, 105, 106). Collectively, these studies suggest there is no specific combination of markers that can consistently distinguish regulatory and cytotoxic NK cell subsets. In a clinically relevant example, traditional phenotypic markers cannot be used to delineate the potency of a donor’s NK cell response against leukaemic cells (36). Indeed, methods to distinguish NK cells with highly potent antitumor activity remain elusive.

The discovery of NK cell populations with a capacity for “memory-like” effector function has also stretched our understanding of how phenotype relates to functional potential. In a unique immunological phenomenon, CMV shapes the phenotypic and functional properties of the NK cell repertoire by driving the expansion of a subset of CD56^dimNKG2C⁺ adaptive NK cells with memory-like properties [reviewed (107)]. Although predominantly defined by expression of NKG2C, adaptive NK cells are also considered to have a mature phenotype as they typically lack NKG2A expression, express low levels of NKp30 and NKp46, and have high levels of KIR and CD57 expression (34, 108). Functionally, CMV-driven adaptive NK cells are specialized for enhanced ADCC, producing greater levels of IFNγ following activation through CD16 (109). Intriguingly, expansion of this NKG2C⁺ NK cell population is only observed in approximately one-third of CMV seropositive individuals (35). Whilst it has been revealed that the infecting strain of CMV impacts the degree of adaptive NK cell expansion through peptide-specific interactions between the UL40 peptide-HLA-E complex and the activating NKG2C receptor (110), recent findings have challenged the requirement for NKG2C in generating this memory-like functional fate. Indeed, a population of NK cells with a similar memory-like response to CMV has been described in NKG2C^-/- individuals (111). Moreover, adaptive NK cell responses have also been reported following CMV reactivation in patients that had received HSCT from NKG2C^-/- donors (112). Adaptive NK cells may actually be defined by an array of phenotypes including the loss of all, some, or none of following signaling proteins: FcϵRIγ, Eat-2, and Syk (35, 113, 114). Whilst it is quite possible that other viruses or environmental exposures may similarly impact upon the NK cell repertoire in vivo, most observations are confounded by donor CMV seropositivity (115). Furthermore, as many studies have focused solely on this pre-defined phenotypic classification of NKG2C⁺ adaptive NK cells, other markers that may delineate memory-like populations have not yet been identified. Indeed, not all memory-like NK cells display a classically mature phenotype. For example, a population of cytokine-induced memory-like (CIML) NK cells have been described following in vitro stimulation with IL-12/IL-15/IL-18 (116, 117). Although this CIML NK cell population is primarily composed of CD56^dim cells, the expression of NKG2A, CD25, CD69, CD94, and lack of KIR and CD57 expression suggests a more immature phenotype (116, 117). Strikingly, CIML NK cells display significantly enhanced IFNγ production following cytokine restimulation or target cell activation compared to conventional NK cells, and have since gone on to achieve preliminary success in a phase I clinical trial for the treatment of AML (118). A subset of tissue-resident NK cells with memory-like activity has also recently been described in the context of pregnancy (119). This population of pregnancy-trained decidual NK cells harbored a unique CD56^brightCD16^-NKG2C⁺ phenotype and displayed enhanced production of IFNγ and VEGF-A compared to conventional decidual NK cells (119). Taken together, these studies emphasize the vast heterogeneity of memory and memory-like NK cell responses. Moreover, the capacity for these stimuli to shape the functional fate of the NK cell repertoire, and drive a broad spectrum of different phenotypes, highlights the difficulty in assigning functional properties based on phenotypic analysis.

Further discrepancies between NK cell phenotype and function are encountered when attempting to describe dysfunctional NK cell populations. In cancer patients or the setting of chronic viral infection, dysfunctional NK cells are typically characterized by their reduced functional capacity including decreased cytotoxicity in response to target cell stimulation and reduced expression of IFNγ and granzyme B [reviewed (120, 121)]. However, as these are general markers of dysfunction, specific dysfunctional states such as exhaustion, anergy or senescence are difficult to define. Whilst several studies have reported that functional exhaustion of NK cells in tumors or chronic infections is accompanied by the downregulation of activating receptors such as NKG2D, CD16, NCRs, DNAM-1, and 2B4, or the upregulation of markers of T cell exhaustion such as PD-1, TIGIT, TIM-3 and LAG-3, it remains controversial whether NK cells even undergo exhaustion (120). As such, phenotypic markers of NK cell dysfunction remain ill-defined. Indeed, there is currently no established phenotype that can consistently distinguish dysfunctional from functional NK cell populations.

Collectively, these studies demonstrate that there remains no unifying phenotype by which NK cell maturation, functional state, or capacity for memory can be defined, which has important implications for the generation of NK cell products to achieve maximum therapeutic benefit. Indeed, if these classic phenotypes are truly unable to predict NK cell activity, then what other measures can be used to inform on an NK cell’s functional potential?

It is now apparent that cellular metabolism is not only a means by which cells generate energy and biochemical precursors required for homeostasis, but it is intrinsically tied to immune cell function. Recent studies have reported that immunometabolism plays a critical role in regulating NK cell development, education, activation, and memory response [reviewed (122, 123)]. As metabolism plays such an integral role in dictating NK cell biology, a new paradigm has emerged in which distinct “metabolic fingerprints” underpin NK cell functional fate (122). Specifically, differences in the capacity for and regulation of glucose-driven metabolic pathways may identify NK cells with enhanced cytotoxic potential (Figure 3).

Dynamic changes in the glucose-driven metabolic pathways glycolysis and oxidative phosphorylation (OxPhos) coincide with NK cell development and effector function. In mice, developing NK cells utilize both glycolysis and OxPhos to fuel the energy-intensive process of proliferation (124), whereas mature NK cells are considered metabolically quiescent at steady-state and preferentially use OxPhos to meet their homeostatic needs (125). Although less is known about the metabolic requirements of developing NK cells in humans, mature pb-NK cells also demonstrate low rates of glycolysis and OxPhos at resting state (126). Interestingly, metabolic differences have been reported between the CD56^bright and CD56^dim NK cell compartments. Resting CD56^dim NK cells have a greater mitochondrial mass and demonstrate higher rates of glycolysis and OxPhos compared to the CD56^bright subset (127). CD56^dimNKG2C⁺ adaptive NK cells isolated from CMV seropositive individuals also exhibit an increased capacity for glycolysis and OxPhos compared to donor-matched CD56^dimNKG2C^- canonical NK cells (128). However, basal levels of these pathways were comparable between adaptive and canonical NK cells, suggesting that adaptive NK cells may have a greater capacity to upregulate these metabolic pathways following activation (128). As NK cells are poised to respond rapidly following activation it is perhaps unsurprising that the rates of glycolysis and OxPhos remain unchanged during short-term cytokine stimulation (4 hours with IL-15 and/or IL-12 and/or IL-18) or receptor ligation (6 hours with anti-NK1.1 or anti-Ly49D) (125). However, inhibition of either pathway was shown to significantly impair IFNγ production, especially following receptor ligation. Taken together these findings suggest that the low basal metabolic rate of resting NK cells is sufficient to fuel acute NK cell effector responses. Interestingly, several studies have also reported that prolonged NK cell activation drives robust changes in cellular metabolism. Indeed, overnight stimulation with cytokines significantly increases the rates of glycolysis and OxPhos in both human and murine NK cells (124, 126, 129, 130). Accumulating evidence suggests that this increased level of glucose-driven metabolic fitness drives enhanced NK cell cytotoxicity. For example, NK cells stimulated with IL-15 for 3-5 days demonstrate higher rates and overall capacity for glycolysis and OxPhos and exhibit greater levels of IFNγ production in response to receptor-mediated activation compared to those that receive short-term stimulation of 4-24 hours (124, 125). Furthermore, this prolonged IL-15 stimulation eliminated the metabolic requirement for NK cell activation, with IFNγ production sustained following OxPhos inhibition (125). Metabolic reprogramming may also underpin the enhanced cytotoxic capacity of educated NK cells. Schafer and colleagues reported that following activation and expansion using IL-21-expressing K562 feeder cells, educated NK cells were metabolically reprogrammed towards glycolysis and mitochondrial-dependent glutaminolysis to support their increased cytolytic activity, whereas the uneducated subset relied solely upon OxPhos (131). In addition, metabolic fitness plays an important role in governing NK cell effector function within the tumor microenvironment (TME) [reviewed (132, 133)]. Limited nutrient availability within the TME may restrict NK cell metabolism, thus impairing effector function (134–136). Accumulation of tumor-derived metabolites within the TME has been reported to dampen NK cell activity through impairing key metabolic pathways. For example, adenosine has been shown to inhibit the metabolic activity of human NK cells by inhibiting OxPhos and reducing their glycolytic capacity (137) and uptake of lactic acid by NK cells leads to intracellular acidification and impaired energy production (138). More recently, Poznanski and colleagues reported that NK cell dysfunction within the TME is due to suppression of glucose-driven metabolic pathways via lipid peroxidation-associated oxidative stress (139). Strikingly, expanded NK cells reprogrammed towards complete metabolic substrate flexibility demonstrated greater metabolic fitness and enhanced antitumor activity against ovarian tumors in vivo (139). These findings suggest that an increased capacity for glucose-driven metabolism and high metabolic fitness may identify NK cells with enhanced cytotoxic activity and thus greater therapeutic potential.

Tight regulation of the mechanistic target of rapamycin (mTOR) is also critical for NK cell development and activation. mTOR is a highly evolutionarily conserved serine/threonine kinase comprised of two distinct complexes: mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). Together, these complexes act as master regulators of cellular metabolism and integrate signals for nutrient availability, growth, and activation to adjust the rates of glycolytic metabolism and biosynthesis accordingly (140). Several studies have reported that mTOR signaling plays a crucial role in the early stages of NK cell development (124, 141, 142). For example, IL-15-induced activation of the mTOR pathway is required for E4BP4 expression in developing BM-NK cells, which in turn promotes transcription of Eomes and drives commitment towards the NK cell lineage (141). More recently, crosstalk between mTORC1 and mTORC2 was found to promote NK cell maturation through controlling the expression of transcription factors Tbx21 and Eomes in a cooperative and non-redundant manner (143). Interestingly, mTORC1 and mTORC2 were also reported to regulate NK cell metabolism and anti-tumor activity in opposing ways. NK cells from mTORC2 deficient mice displayed greater cytolytic activity and increased metabolic rate compared to their wild-type counterparts, whilst cytotoxicity and cellular metabolism were significantly diminished in mTORC1 deficient NK cells (143). Other studies have also demonstrated that mTOR plays a critical role in controlling NK cell activation in both mice and humans, with IL-15 induced activation of the mTOR pathway required for priming of cytotoxicity in the periphery (124, 144). Marçais and colleagues reported that mTOR activity downstream of the IL-15 receptor increased granzyme B expression in both murine and human NK cells, whereas inhibition of mTOR by rapamycin abrogated NK cell cytotoxicity (124). Interestingly, IL-15 stimulation has been reported to activate the PI3K/Akt/mTOR pathway more robustly in CD56^bright NK cells compared to the CD56^dim population, corresponding with their potent increase in cytotoxicity following prolonged stimulation (101). Transforming growth factor-β (TGF-β), a major immuno-suppressive cytokine well-known for its role in inhibiting NK cell cytotoxicity, has also been reported to directly suppress NK cell activation through inhibition of mTORC1 and through mTORC1-independent inhibition of mitochondrial metabolism (130, 145). Indeed, in vitro treatment with TGF-β reduced the metabolic activity, cytotoxicity, and abundance of various NK cell receptors in both murine and human NK cells (130). Furthermore, deletion of the TGF-β receptor subunit TGF-βRII in murine NK cells enhanced mTOR activity and NK cell cytotoxicity in vivo (130). Together, these findings highlight the importance of mTOR activity in regulating NK cell antitumor function both in vitro and in the tumor microenvironment. Differences in mTOR activity, or in the relative activity of the mTORC1 and mTORC2 pathways, may therefore be useful in delineating NK cell cytotoxic potential. Moreover, boosting the metabolic activity of NK cells through targeting the mTOR pathway may be an effective strategy for enhancing the antitumor activity of NK cell-based therapies and thus warrants further investigation.

It is now apparent that metabolism plays an essential role in dictating NK cell functional fate. As metabolism is intrinsically tied to NK cell survival and antitumor activity, the potential to manipulate NK cells ex vivo for enhanced metabolic fitness holds promise for enhancing the efficacy of NK cell-based therapies. Indeed, several strategies have been proposed to genetically or pharmacologically “rewire” NK cell metabolism to promote in vivo longevity, improve tumor recognition, sustain antitumor function, increase trafficking to the tumor site, and protect the adoptively transferred NK cells from the tumor microenvironment itself (146). As more tools than ever before are now available to interrogate NK cell metabolism [reviewed (147)], future investigations should also seek to define specific “metabolic fingerprints” which can be used to identify NK cells with the highest therapeutic potential. Although our understanding of NK cell metabolism is currently in its infancy, a deeper appreciation of the interplay between metabolism and molecular regulators of NK cell functional fate, as discussed below, holds promise for unlocking the full potential of NK cell therapies.

The fields of transcriptomics and epigenomics are rapidly advancing. Recent breakthroughs in the development of high-resolution and high-throughput sequencing technologies have enabled researchers to explore the transcriptional and epigenetic landscape of NK cells in more depth than ever before. However, our understanding of the molecular regulation of NK cell functional fate is still in its infancy. Relatively little is known about the molecular pathways and regulatory programs that underly NK cell development, effector function, and memory response. As a central goal of genetic and epigenetic studies involves understanding the factors that drive individual variation, a deeper understanding of NK cell biology at the molecular level may also aid in identifying optimal donors for NK cell immunotherapy.

The development of high-resolution transcriptomic analyses such as RNA sequencing (RNA-seq) and single cell RNA-seq (scRNA-seq) has provided researchers with unprecedented insight into the developmental and functional plasticity within the NK cell compartment. A particular interest has arisen in unravelling the developmental trajectory of human NK cells. Based on phenotypic analyses, the current model of NK cell differentiation describes a linear relationship between the immature CD56^bright and terminally differentiated CD57⁺ NK cell populations. However, the well-established loss of CD56^bright but not CD56^dim NK cells in GATA2-deficient individuals challenges this current dogma of NK cell development (148). Using scRNA-seq to analyze NK-lineage cells derived from a donor with the GATA2^T354M mutation, Yang et al. confirmed the loss of CD56^bright cells in this donor due in part to a higher rate of apoptosis compared to GATA2-sufficient cells (149). Furthermore, whilst the heterogeneity of this donor’s NK cell repertoire was mostly intact compared to healthy controls, defects in steady-state activation were also observed (149). Although the developmental trajectory of these GATA2-deficient NK cells remains unclear, transcriptomic analyses of healthy donor-derived NK cells supports the linear model of differentiation, suggesting that the CD56^bright subset is a precursor to the CD56^dim population with CD57⁺ cells representing the terminal stage of NK cell differentiation (149, 150). Interestingly, analysis of CD56^brightCD16^- and CD56^dimCD16⁺ NK cell populations derived from various tissues suggests that NK cell developmental and functional fate is shaped by the tissue site from which they are derived (151). Indeed, tissue-specific transcriptional patterns of maturation, distribution, and function were largely maintained across donor age, sex and CMV serostatus (151). Yang et al. have also described the shared presence of five distinct NK cell clusters across the BM and peripheral blood derived from healthy donors (149). Interestingly, two of these five clusters (“Mature NK” and “Terminal NK”) were predicted to form the CD56^dimCD57⁺ population together, suggesting that this classically terminally differentiated NK cell subset is not as homogenous as previously thought (149). An additional three novel subsets of pb-NK cells have been described using scRNA-seq, including type I IFN-responding CD56^neg NK cells, CIML NK cells, and a small population of NK cells with reduced ribosomal expression, decreased OxPhos and markers of cellular activation (152). Heterogeneity within the CD56^dimCD16⁺CD57^- subset was also observed, with two distinct subpopulations distinguished based on the relative abundance of chemokine mRNA and frequency of KIR-like receptor expression (152). In addition to this high level of variation within an individual’s NK cell repertoire, several studies have also reported the presence of strong donor phenotypes in scRNA-seq datasets (149, 153, 154), likely reflecting the unique genetic profile and immunological history of each donor. As these donor phenotypes are present under physiological conditions, exploration of the transcriptomic differences between donors may uncover an even deeper level of NK cell functional heterogeneity than previously described by phenotypic analysis alone. Indeed, several studies have reported the presence of adaptive NK cell clusters in CMV seropositive individuals (149, 153), and an “inflamed” NK cell cluster specific to one BM donor (149). However, the development of new annotation tools and more robust sequencing technologies may be required to interrogate the full heterogeneity of the NK cell repertoire between healthy donors. More recently, Crinier and colleagues have reported strong donor phenotypes in BM-NK cells derived from AML patients at diagnosis (153). Remarkably, the extent of transcriptomic heterogeneity between AML patients was so high that traditional annotation tools were unable to identify conserved NK cell subsets, even when considering patients at the same classification of AML (153). Interestingly, the overall transcriptomic profile of BM-NK cells from AML patients was enriched for genes involved in cytokine and type I IFN signaling pathways, whereas healthy BM-NK cells displayed a transcriptomic profile enriched for genes involved in NK cell cytotoxicity (153). These findings highlight the strong donor-specific effects AML carries out on the endogenous NK cell repertoire. However, the relationship between these distinct transcriptomic profiles and patient outcomes has not yet been elucidated. Similarly, the extent to which other hematological malignancies impact on the endogenous NK cell compartment, or on adoptively transferred NK cells, remains unexplored. Indeed, further investigation is needed to fully appreciate the role transcriptomic regulation plays in controlling NK cell activity in health and disease.

Epigenetic alterations are reversible and heritable changes to the genome that do not alter the DNA sequence itself, but have profound impacts on gene expression, cell phenotype, and functional fate. Broadly, NK cell development and effector function is regulated through various epigenetic alterations including DNA methylation, histone modification, transcription factor (TF) changes, and microRNA (miRNA) expression [reviewed (155)]. NK cells undergo profound epigenetic remodeling throughout their development. For example, gradual demethylation of gene promoters at the KIR and IFNG loci during NK cell differentiation corresponds with acquisition of KIR expression (156) and the ability to produce IFNγ (157), respectively. The dynamic interplay between chromatin accessibility and gene expression levels throughout NK cell development has recently been described (158). Using the Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) to assess changes in genome-wide chromatin accessibility levels of different developmental stages of in vitro-derived NK cells, Li and colleagues revealed the presence of two distinct TF clusters that regulate NK cell differentiation (158). Additionally, two novel TFs were identified (FOSL2 and EGR2) and found to be essential for controlling NK cell maturation and function (158). However, as this study utilized an in vitro model of NK cell differentiation it is unclear how these findings will translate to the natural in vivo process of NK cell development. Indeed, Li and colleagues have reported differences in chromatin accessibility between these in vitro-derived NK cells and their naturally occurring counterparts (158). Nevertheless, the identification and characterization of TFs and transcriptional regulatory networks involved in NK cell differentiation presents an opportunity to artificially drive NK cell functional fate along a desired pathway in vitro. Indeed, pharmacological inhibition of GSK3 kinase during ex vivo NK cell expansion with IL-15 is currently used to drive late-stage maturation and enhanced effector function of FATE-NK100 cells through upregulating the expression of TFs such as T-BET, ZEB2, and BLIMP-1 (91). Chromatin dynamics also play an important role in regulating the function of the mature NK cell repertoire. For example, target cell recognition drives alterations in the NK cell histone methylation state that correspond with changes in gene expression levels (159). Interestingly, small-molecule compounds targeting H3K4 and H3K27 methyltransferases and demethylases were able to mimic these activation-induced histone modification states in the NK92MI cell line, inducing significantly greater levels of degranulation (UNC1999) and expression of IFNγ and TNFα (OG-L002 and MM102) compared to untreated controls (159). Entinostat, a histone deacetylase inhibitor, also modulates NK cell effector function through modifying chromatin accessibility (160). Mechanistically, treatment with entinostat was reported to increase chromatin accessibility of the IFIT1 gene promoter region, driving the epigenetic upregulation of the IFIT1-mediated IRF1, STAT4 and STING pathways, and resulting in increased NK cell cytotoxicity against tumor targets (160). Although entinostat was found to enhance NK cell cytotoxicity, it remains unclear how other epigenetic modifying drugs (several of which are currently in clinical trials for the treatment of various cancers) may directly or indirectly impact upon NK cell function (155). Furthermore, the breadth of other exposures which can imprint upon the NK cell epigenetic landscape and thus modulate NK cell activity is currently unknown.

Perhaps the most notable example of an environmental exposure driving epigenetic remodeling of the NK cell repertoire is the CMV-driven expansion of adaptive NK cells. The unique phenotypic and functional characteristics of this “memory-like” adaptive NK cell population corresponds with epigenetic imprinting at the regulatory regions of genes encoding IFNγ, FcϵRIγ, EAT-2, and PLZF (35, 113, 161). For example, demethylation of the IFNG locus increases the accessibility of the CNS1 region and drives the characteristic increase in IFNγ expression displayed by adaptive NK cells (161). Conversely, hypermethylation of the FCER1G and SH2D1B (encoding EAT-2) loci corresponds with the reduced expression of these signaling proteins (35). Hypermethylation of an intronic region of the ZBTB16 locus (encoding PLZF) was also observed, corresponding with a striking 77% downregulation of this transcript in adaptive NK cells compared to conventional NK cell populations (35). Demethylation of the IFNG locus at CNS1 has also been reported in NKG2C^-/- adaptive NK cells, further highlighting the crucial role epigenetic remodeling plays in driving this unique functional state. Although described only in the context of CMV to date, it remains possible that a broader range of exposures to other viral, bacterial, or even eukaryotic pathogens may also contribute towards a memory-like functional fate (162). However, as many studies have focused solely on known adaptive NK cell phenotypes, distinct functional or epigenetic profiles driven by other environmental stimuli have not been identified. Indeed, it remains unclear how a lifetime of exposures may imprint upon the epigenetic landscape of an individual’s NK cell repertoire. It is also unclear whether ex vivo expansion methods or the tumor microenvironment itself may drive epigenetic changes in adoptively transferred NK cells, potentially affecting their persistence and antitumor activity in vivo. Nevertheless, as epigenetic alterations are both reversible and druggable, identification of the specific epigenetic signatures underlying enhanced antitumor immunity will yield novel targets that can be exploited to further improve NK cell-based therapies. More broadly, a deeper appreciation of the epigenetic determinants of NK cell functional potential may also aid in identifying and selecting optimal NK cell populations or donors for immunotherapy.

Transcriptomic and epigenomic analyses have uncovered a greater level of heterogeneity, both within and between the NK cell repertoires of different individuals, than previously observed by phenotype alone. As emerging evidence continues to highlight the high level of complexity and plasticity within the NK cell compartment, new studies into the molecular regulation of NK cell functional fate hold great promise for revolutionizing the field of NK cell immunotherapy. Indeed, a comprehensive understanding of the transcriptional and epigenetic programs underlying enhanced NK cell activity or longevity in vivo may reveal a plethora of molecular phenotypes and targets that can be exploited to improve future NK cell-based therapies.

NK cells have tremendous potential to revolutionize the field of cancer immunotherapy. Coupled with their innate potency against cancer and ability to be transferred between donors and patients without mediating severe adverse effects, NK cells have emerged as ideal candidates for the development of readily available “off-the-shelf” therapies. Whilst a range of NK cell sources and ex vivo manipulation strategies have been extensively investigated over the past two decades, there remains no standard criteria by which NK cells with enhanced therapeutic potential can be identified and selected for immunotherapy. Indeed, it is now apparent that NK cells displaying similar functions can express a wide variety of phenotypic markers, and individual NK cells within a defined phenotypic population can fulfil a range of distinct functional roles. As accumulating evidence continues to expose discrepancies between the NK cell phenotype and functional output, there is a need to develop new strategies by which NK cell donors or populations with enhanced antitumor potency can be identified.

Advancements in the fields immunometabolism, transcriptomics, and epigenomics have led to an exciting new era for NK cell research, highlighting a deeper level of complexity and plasticity within the NK cell compartment than previously described. Recent studies have leveraged these multi-omics technologies to describe novel determinants of enhanced NK cell activity, including increased rates of glycolytic metabolism, greater metabolic fitness, and epigenetic remodeling towards a “poised” effector state. However, it remains unclear how these metabolic and molecular “fingerprints” can be used to select NK cell donors or populations for greater antitumor activity in vivo. Whilst several measures of metabolic fitness and mTOR activity can be assessed simultaneously using flow cytometric analyses [reviewed (147)], efficient profiling of NK cells based on epigenomic or transcriptomic signatures remains unachievable. However, these unbiased approaches are indispensable for gaining a greater appreciation for the heterogeneity in the NK cell repertoire both within and between donors. A comprehensive understanding of how these molecular regulatory programs interact with cellular metabolism and drive NK cell functional fate will aid in developing new strategies for profiling NK cells based on functional potential. Furthermore, a deeper knowledge of these important regulatory pathways will uncover new targets that can be exploited to enhance the efficacy of future NK cell-based therapies.

SB, IT, EJ, and BF designed, wrote, and edited the manuscript. All authors contributed to the article and approved the submitted version.

The preparation of this review article received no external funding. This work was supported by the Australian Government Research Training Program Scholarship at The University of Western Australia (scholarship to SB), and the Cancer Council Western Australia and the Western Australia Department of Health (research funding to BF).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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