Natural killer (NK) cells comprise 5–15% of human peripheral blood lymphocytes and play an important role in the clearance of virally infected cells, as well as the elimination of cancer cells (1, 2). The main population of NK cells differentiates from hematopoietic stem cells in the bone marrow, and fully matures as CD56⁺CD16⁺ NK cells in secondary lymphoid organs (3). While several recent studies have elegantly demonstrated the presence of a memory immune response in NK cells, previously only attributed to T and B cells of the adaptive immune system (4, 5), NK cells are generally recognized as innate immune cells by the fact that their receptors do not undergo DNA rearrangement processes. Instead NK cells express various germline-encoded activating and inhibitory NK receptors on their cell surface (6–8). Integrated signaling from both activating and inhibitory receptors is thought to enable NK cells to distinguish healthy “self” from infected or stressed unhealthy or “non-self” cells (9, 10). Therefore, activation of NK cells can be achieved via the ligation of a number of activating receptors with their corresponding ligand on the target cell (natural cytotoxicity). In addition, NK cells are also able to detect antibody-coated targets through the low affinity Fc receptor CD16 (FcγRIIIA) and thereby mediate antibody-dependent cellular cytotoxicity (ADCC) (11, 12). The main consequence of NK cell activation is the direct killing of bound unhealthy target cells through the release of perforin and granzymes from a preformed secretory lysosome known as the lytic granule (13, 14). In addition, NK cells can affect the overall immune response through the secretion of chemokines and cytokines (3, 15–18) as well as through direct interaction with other immune cells (2, 5).

Research conducted over the past quarter of a century has uncovered the process of NK cell development, identified and demonstrated the function of activating and inhibitory receptors, and provided a wealth of information regarding the signaling pathways that are linked to these receptors and the proteins that are critical to the delivery of the lethal cargo to the target cell. Significantly, the study of human primary immunodeficiency syndromes (PIDs) has provided invaluable knowledge and insight regarding the role of NK cells in the immune system, and in addition has also resulted in the identification of genes whose protein products regulate distinct steps that are critical to the development of cellular cytotoxicity by this population of innate immune cells. Among the PIDs, several NK cell deficiencies have been identified including rare isolated NK cell deficiencies, where there is total absence or very low number of NK cells in the peripheral blood, which is known as classical natural killer cell deficiency (CNKD) (19, 20). MCM4 and GATA2 are the only two genes identified thus far in which mutations are linked to CNKD, however there remain several cases of CNKD where the underlying genetic cause has not been identified (20). Clearly, sequencing of the genomes in patients with CNKD, which are not caused by mutation of either MCM4 or GATA2, will undoubtedly reveal other genes that play an important role in NK cell development or survival.

The term NK cell deficiency does not just mean an absence of NK cells in the immune system; it also includes the absence of specific NK cell effector functions, like cytotoxicity, despite the presence of normal NK cell numbers. This has been termed functional NK cell deficiency (FNKD) (19, 20). FNKDs have provided us with very valuable insights into the roles of specific NK cell functions in immunity as well as essential proteins required for NK cell effector activities, when the responsible gene has been identified. So far, the only known human genetic mutation leading to FNKD is in the FCGR3A gene, which encodes CD16 (20, 21). Similar to patients of CNKD, patients carrying homozygous missense mutations in CD16 (L66H) showed susceptibility to human papilloma virus (HPV) or herpesviridae members. In contrast to CKND, these patients had normal numbers of NK cells, but unexpectedly showed normal ADCC, whereas natural cytotoxicity was defective (20). The fact that the altered amino acid found in these patients is located outside of the immunoglobulin domain (Ig domain) responsible for IgG binding (22) suggests why ADCC of NK cells from the patients is normal. In addition, novel co-stimulatory roles of CD16 mediated by the distal Ig domain of CD16 (23) provided important insights that might explain why the patients’ NK cells showed defective natural cytotoxicity.

Lastly, there are several additional human PIDs that demonstrate defects in NK cell numbers and effector functions. Since many immune cells other than NK cells are also affected, there are additional complications and difficulties in understanding the complex immunological roles of NK cells in these diseases. However, the identification of specific gene mutations has illuminated molecular pathways that are important for NK cell development and effector functions, which are also shared in other immune cell types. In this review, we will specifically focus on PIDs where the mutated gene products impact the intracellular pathways that regulate the development of NK cell-mediated cytotoxicity (Table 1). For detailed discussions about human diseases involved in NK cell development and differentiation, NK cell signaling, or other NK cell effector functions, the reader is referred to other excellent reviews on these topics (19–21, 24).

Cell-mediated killing by NK cells and cytotoxic T lymphocytes (CTLs) is achieved by directed release of lytic granules toward bound target cells. Lytic granules are dual-function organelles that exhibit characteristics of a degradative lysosome, but can also secrete granule constituents for cell-mediated killing and are therefore often referred to as a secretory lysosome. This specialized organelle is mostly observed in hematopoietic lineage cells, but it is also found in melanocytes that produce pigment proteins called melanins (25, 26). Both secretory lysosomes and conventional lysosomes are acidic organelles (pH 5.1–5.4), morphologically similar, and contain proteins like acid hydrolases that are required for their degradative function as well as receptor proteins including members of the lysosomal-associated membrane protein (LAMP) family. On the other hand, the main distinction of secretory lysosomes from conventional lysosomes is that secretory lysosomes undergo a regulated secretion process and they contain additional contents that are cell-type-specific. In the case of NK cells and CTLs, these specific secreted contents include perforin and granzymes, which are essential for cytotoxic activity. On the other hand, the main secreted proteins of melanocytes are melanins, which are pigments responsible for skin color. Cells containing secretory lysosomes use common secretory machineries, but cell-type-specific secreted contents enable each cell type to perform different effector functions. This is why human genetic diseases such as Chediak–Higashi syndrome (CHS) and type 2 Hermansky–Pudlak syndrome (HPS), caused by impaired function of the secretory lysosome, are characterized not only by severe immune deficiencies including defective cytotoxicity by NK cells and CTLs but also by hypopigmentation due to defects in melanosome formation and excessive bleeding due to the absence of dense granules in platelets (26–28).

In contrast to CTLs, which need to be activated, the genes encoding perforin and granzymes are constitutively transcribed in NK cells thus allowing them to immediately kill an infected or stressed cell upon initial contact and without the need for additional gene expression (29, 30). Perforin is essential for NK cell-mediated cytotoxicity, since it is the only molecule that delivers apoptosis-inducing granzymes into the target cell. Perforin is initially synthesized as an inactive precursor in the ER and undergoes multiple posttranslational modifications including proteolysis and glycosylation during its transit to the Golgi and finally to the lytic granule (31). However, the exact pathway that sorts perforin from the trans-Golgi network (TGN) into lytic granules is unresolved. After arriving into lytic granules, perforin is further processed by cathepsin L to develop into a mature form. In the case of granzymes, five different granzymes, granzyme A, B, H, K, and M, are expressed in human NK cells and CTLs. They are synthesized as pro-enzymes and modified to have a mannose-6-phosphate moiety in the cis-Golgi, which facilitates their trafficking to the endosome by the mannose-6-phosphate receptor (M6PR) before finally arriving into the lytic granule (32, 33). In lytic granules, granzymes are further processed into an active form by various cathepsins (34–36). Granzymes are serine proteases and each granzyme has different substrate specificity and induces apoptosis in the target cell in both caspase-dependent and caspase-independent manners (37, 38).

An important question to be asked is: How do cytotoxic lymphocytes protect themselves from these dangerous molecules? Significantly, the activity of perforin is achieved by the membrane attack complex/perforin (MACPF) domain, which has cytolytic activity, and a C2 domain that enables binding to membrane in a calcium-dependent manner (37, 39, 40). Importantly, the low level of Ca²⁺ in lytic granules maintains perforin in an inactive conformation. In addition, calreticulin, another component of the lytic granule, inhibits perforin activity (41), and the acidic environment of lytic granules provides protection to NK cells since the activity of both perforin and granzymes are inhibited at this low pH. Furthermore, the low pH environment favors association of serglycin, a proteoglycan matrix found within lytic granules, with both perforin and granzymes. This interaction keeps both perforin and granzymes in an inactive state until they are secreted into a neutral pH environment (42). Interestingly, the mechanism by which perforin facilitates the transport of granzymes into the target cell is still unclear (37, 40). According to the prevailing model, perforin is released into the cytotoxic synaptic cleft where it binds calcium, oligomerizes on the target membrane, and forms pores. The high concentration of calcium as well as the neutral pH at the interface between NK cells and target cells relieves perforin from its inhibited conformation and triggers its activity (40). It is assumed that the established pore is large enough for granzymes to get into target cells by simple diffusion (21, 37, 40). Last but not least, how do NK cells prevent themselves from potential self-destruction from released cytotoxic components? Cell surface expression of cathepsin B has been suggested to provide protection of cytotoxic lymphocytes by cleaving perforin (43). In addition, it was recently shown that the surface expression of LAMP-1 (CD107a) also protects NK cells from degranulation-associated damage by inhibiting binding of perforin to NK cells (44). Therefore, it seems there exist multiple layers of protection at the final degranulation step that ensure unidirectional cytotoxicity. In the following paragraphs, we will focus our discussion on PIDs with impaired NK cell cytotoxicity caused by: (1) defects in the contents of lytic granules and (2) impaired biogenesis and maturation of lytic granules.

Natural killer cells rapidly accumulate at the site of inflammation through the stimulation of inflammatory chemokine receptors and adhesion molecules expressed on their cell surface (1). Although the roles of weak adhesion molecules like selectin family members have not been directly examined in NK cell – target cell binding, it is generally appreciated that the first step toward the development of NK cell cytotoxicity is weak integrin-mediated adhesion to target cells (99). Signals from this weak adhesion, along with the initial signaling resulting from the engagement of activating/inhibitory receptors on the NK cell with corresponding ligands on the target cell, determines whether the NK cell should detach (strong inhibitory signal) or establish a firm adhesion (strong activation signals) leading to the formation of the cytotoxic synapse (CS). Along with high affinity interactions toward their ligands, integrins were also observed to cluster at the CS of NK cells, strengthening adhesion (avidity) (100–102) and promoting the scanning of target cells (Figure 1) (11, 12, 99). In addition to being an important mediator of cell–cell adhesion, integrins can serve as co-stimulatory molecules enhancing cytotoxicity signals from other activating NK receptors (103–105), as well as promoting lytic granule polarization following ligand engagement (104, 106–108).

Essential signaling molecules like NK activating/inhibitory receptors are accumulated at the center of the interface known as the central supramolecular activation cluster (cSMAC), whereas F-actin and integrins are localized at the periphery of the interface forming a ring-shaped peripheral supramolecular activation cluster (pSMAC). Among these distinct molecular patterns at the CS, F-actin accumulation at the pSMAC is thought to be one of the early and critical events in NK cell-medicated cytotoxicity, since clustering of NK receptors as well as adhesion molecules are observed to be dependent on F-actin polymerization (13, 99, 101). Two primary immunodeficiencies causing defective F-actin accumulation at the CS of NK cells have provided some clues on how F-actin accumulation might be regulated at the CS.

Another important step required for NK cell-mediated cytotoxicity is the polarization of lytic granules toward the CS. This event occurs after the rearrangement of F-actin at the CS and requires both F-actin polymerization and microtubule function, since lytic granules fail to polarize toward the CS when either F-actin accumulation or the microtubule network are disrupted (101). As NK cells start to form conjugates with target cells, lytic granules are rapidly clustered around the MTOC (Figure 2) (107). This retrograde, minus-end-directed transport of lytic granules on microtubules is Src kinase-dependent, and mediated by the dynein/dynactin motor complex that constitutively associates with lytic granules and tethers them to the microtubules (107, 114, 168). In conjunction with the clustering of the lytic granules, the MTOC polarizes toward the CS, thereby delivering the lethal cargo to the site of target cell contact (101, 107, 168). Importantly, this process occurs along with maturation of the CS, as it requires F-actin accumulation, rearrangement of NK receptors, and clustering of adhesion molecules.

Current knowledge suggests that activation of extracellular-signal-regulated kinase (Erk), Vav1, and protein tyrosine kinase 2 (PYK2) are required for MTOC polarization in NK cells (169–171). In addition to these molecules, several other proteins have been identified that impact the polarization of lytic granules to the CS (Figure 2). For example, interrupting the cellular function of the CDC42-interacting protein-4 (CIP4) was shown to impair MTOC polarization as well as NK cell-mediated cytotoxicity (172). CIP4 was suggested to link the microtubule and F-actin network at the CS through its ability to interact with microtubules and WASP. In this regard, the scaffold protein IQGAP1 also has the potential to interact with F-actin and microtubules, and silencing of IQGAP1 was also shown to impair NK cell cytotoxicity due to defective MTOC polarization toward the CS (173). However, MTOC polarization and cell-mediated killing were found to be normal in mouse CTLs lacking IQGAP1 (174), highlighting again that distinct mechanisms regulating cell-mediated killing are engaged in these two cell types. The Ral GTPase, RalA, also regulates MTOC polarization toward the CS (175). Very recently, the small GTP-binding protein, Arl8b, was found to interact with KIF5B, the heavy chain of the microtubule plus-end-directed motor kinesin-1, and mediate MTOC polarization in NK cells (176). How and where the kinesin-1 motor is recruited to mediate movement of both the MTOC and lytic granules toward the CS remains unclear. In CTLs, a Rab27a/Slp3/kinesin-1 complex was reported to be critical for final transport of polarized lytic granules along with microtubules toward the CS (Figure 2). Interestingly, polarization of the MTOC toward the CS was normal when kinesin-1 was interrupted in CTLs (177). It will be interesting to analyze these seemingly different roles of kinesin-1 in both NK cells and CTLs. Lastly, although not examined in NK cells, activity of CDC42 might also be essential for MTOC polarization considering similar roles in other immune and non-immune cells (163–167).

Following the clustering of the lytic granules at the MTOC, the MTOC docks near the plasma membrane (PM) facilitating fusion of lytic granules into the secretory cleft formed between the NK cell and target cell (168, 178). Previous studies using conventional confocal microscopy suggested that the lytic granules pass through an actin-cleared region at the center of the CS (101, 102); however, recent approaches using super-resolution microscopy suggest that the secretory region at the center of the CS is not totally absent of F-actin. Instead, a hypodense branched F-actin meshwork exists in the central region of the CS that is rearranged to produce small pores through which the lytic granules travel (179, 180). The previously suggested role of myosin IIA, an actin motor protein, in the final transit of lytic granules through the actin-rich region of the CS are consistent with this new finding and will be discussed in the next section (Figure 2). It still remains to be determined how the F-actin network at the secretory region is regulated to allow lytic granule movement by the myosin motor protein.

Exocytosis of lytic granules requires membrane fusion between lytic granules and the PM. An increasing number of studies suggest that small GTPases, including Rab–GTPases, known to regulate membrane trafficking and fusion are important regulators of lytic granule exocytosis in NK cells (191, 192). Following GDP to GTP exchange, these small GTPases are able to interact with their effector proteins involved in protein sorting, motor activity, and tethering, thereby impacting membrane trafficking. Therefore, they are suggested to play important roles, at least in determining specificity of the initial tethering step (Figure 2) (193, 194). In fact, mutation in Rab27a is known to be responsible for a PID known as Griscelli syndrome type 2 (GS2), which will be discussed in detail below (195, 196).

In addition to Rab proteins, the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins, which are known to mediate most membrane fusion events in eukaryotic cells, also participate in exocytosis (197–199). There are approximately 40 SNARE family proteins in humans with different combinations of SNARE proteins expressed in different cell types. In addition, each SNARE protein exhibits a distinct localization pattern suggesting that selective pairings of SNARE members in specific organelles (like lytic granules) constrain their target binding (197–199). All SNARE proteins contain a coiled-coil SNARE motif at the center, and are structurally categorized as either an R-SNARE or Q-SNARE (further subclassified as Qa-, Qb-, Qc-, or Qb,c-SNARE). Membrane fusion is induced when an R-SNARE protein, such as those found on the lytic granule membrane binds to its cognate Q-SNARE proteins expressed on the PM (Figure 2). Bundling of four SNARE motifs (one from the R-SNARE, and three motifs from two or three Q-SNAREs) makes a stable SNARE complex, and the exerted mechanical force pulls the two membranes closer to promote membrane fusion. Interestingly, familial hemophagocytic lymphohistiocytosis type 4 (FHL4) is the result of a mutation in the gene encoding a Qa-SNARE protein called syntaxin-11 (STX11) (200, 201). In addition to the selectivity of SNARE proteins, accessory proteins including SM (Sec1/Munc18-like), Munc13-like, and synaptotagmin associate with SNARE proteins to regulate membrane fusion both temporally and spatially (197–199, 202). Mutations in Munc13-4 and Munc18-2/syntaxin-binding protein 2 (STXBP2) are responsible for familial hemophagocytic lymphohistiocytosis type 3 (FHL3) and familial hemophagocytic lymphohistiocytosis type 5 (FHL5), respectively (Figure 2) (203–205). Lastly, the entry of extracellular Ca²⁺ is essential for lytic granule exocytosis (206). Although PIDs regarding this category are not discussed in this review (please refer to reviews (21, 207) for more details), NK cells from patients deficient in either the ER calcium sensor STIM1 or the PM calcium channel ORAI1 showed defective cytotoxicity due to impaired exocytosis of lytic granules, although polarization of lytic granules toward the CS was normal (206). Significantly, Munc13 proteins and synaptotagmin proteins contain two calcium-dependent phospholipid-binding C2 domains and are known to regulate activity of the SNARE complex in a Ca²⁺-regulated manner (197, 199, 202). Likewise, members of the synaptotagmin-like protein (Slp) family also share C-terminal tandem C2 domains. Slps were also shown to interact with activated Rab27 (Rab27–GTP) via the Slp homology domain (SHD) and participate in docking granules to the membrane (208). Unfortunately, we still do not have a clear picture as to which of these proteins are regulating the membrane fusion steps in NK cell-mediated cytotoxicity. However, as discussed below, clues to understanding this seemingly complicated and tightly regulated mechanism of lytic granule docking and fusion have been uncovered by functionally antagonizing these molecular pathways and examining NK cells from PID patients deficient in these key molecules.

Advances in clinical diagnostics has substantially increased the identification of patients with PIDs that affect NK cell numbers or effector functions. In addition to furthering our understanding of NK cells in the immune system (CKND and FKND), this group of diseases has substantiated the important roles that NK cells play in immune surveillance. Furthermore, the quantitative and/or qualitative defects in NK cells derived from these patients have highlighted essential molecular machineries shared among immune cells and their importance in NK cell-mediated cytotoxicity. In fact, these loss-of-function mutations have provided invaluable insight into the molecular processes regulating the development of cell-mediated killing by NK cells. However, there are many issues that still remain unanswered. First, how can IL-2-mediated signaling pathways restore NK cell functions in multiple PIDs? Secondly, what are the NK cell-specific mechanisms that differentiate the cytotoxic processes engaged in NK cells from those of CTLs (from biogenesis to exocytosis of lytic granules)? Lastly, how do the signaling molecules that regulate the detailed secretory pathway of lytic granules in NK cells cooperate with the multiple tethering and fusion molecules to promote lytic granule exocytosis? It is clear that PIDs have substantially impacted our understanding of NK cell biology and the cellular mechanisms that control the tightly regulated process of lytic granule release. Further mechanistic insight into the process of NK cell-mediated killing will hopefully reveal novel therapeutic approaches to treat PID patients.

The authors declare that the research was conducted without any commercial or financial relationships that could be construed as a potential conflict of interest.