**1. Introduction: cancer immunosurveillance by NK cells**

Natural killer (NK) cells were discovered more than four decades ago and were the focus of some of the earliest trials of cancer immunotherapy. With our more sophisticated understand‐ ing of their functional requirements, NK cells are once again attracting attention for their potential in cancer therapy [1]. Initially thought to be an artefact in cytotoxicity assays, NK cells are now known to play an important role in host immunity against tumourigenesis. The theory of cancer immunosurveillance was proposed by Burnet and Thomas in 1957, postu‐ lating that immune cells continuously monitor the body such that any threat to the immune system is detected and eliminated [2]. In 1975, NK cells were discovered in mice as a subpopu‐ lation of lymphocytes capable of killing tumour cells without prior sensitization [3–5]. This led

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to considerable enthusiasm over the possibility that they function as one of the main effector cells of immunosurveillance. Several studies in the 1980s reported a higher cancer incidence in individuals with genetic disorders such as Chediak‐Higashi syndrome and X‐linked lym‐ phoproliferative syndrome, which lead to defective NK cell function [6, 7]. Subsequent mouse studies showed increased tumour growth in mice with impaired NK cell activity or mice treated with an NK cell‐depleting agent [8, 9]. A long‐term epidemiological study following cancer patients reported that subjects with lower NK cell activity had a higher incidence of several types of cancer [10]. Collectively, data from both mouse and human studies support the theory of cancer immunosurveillance and the concept that NK cells play a critical role in tumour control and eradication [11, 12]. The two main effector functions observed by NK cells against tumour targets are target cell elimination and cytokine secretion [13]. Until recently, these two effector functions were thought to follow similar mechanisms of activation, but now it is recognized that cytokine secretion by NK cells is distinct from cytotoxicity [14].

#### **1.1. Target cell elimination**

NK cells kill tumour cells through granule exocytosis or death receptor ligation. Following NK cell activation, NK cells release the contents of their granules for target cell elimination. The membrane disrupting protein perforin, and a family of serine proteases termed gran‐ zymes, are the critical effector molecules contained in their granules [15]. Perforin results in the disruption of endosomal trafficking and binds in a calcium‐dependent manner to phos‐ pholipid components of the lipid bilayer to facilitate entry of granzymes into the target cell cytosol [16]. Once granzymes enter the target cell, they induce apoptosis. In addition to gran‐ ule exocytosis, NK cells can directly eliminate target cells through the engagement of cell sur‐ face death receptors. NK cells express Fas ligand (FasL) and TNF‐related apoptosis‐inducing ligand (TRAIL), which are both members of the TNF family and are shown to induce target cell apoptosis once bound to their respective receptors on target cells [16].

#### **1.2. Cytokine secretion**

Resting NK cells secrete a plethora of cytokines that help eliminate target cells and amplify acti‐ vation signals for a more efficient immune response. NK stimulation results in enhanced secre‐ tion of cytokines, which in turn influence the activity of other immune cells. Pro‐inflammatory cytokines secreted by NK cells, which include interleukin (IL)‐1, IL‐6, IL‐12, and the chemokine CXCL8 (also known as IL‐8), can enhance the activation and proliferation of T cells, dendritic cells (DCs) and macrophages [17]. By contrast, anti‐inflammatory cytokines such as IL‐4 and IL‐10 suppress T cell and macrophage function, but activate humoral responses. Chemokines, which are chemotactic cytokines, play an important role in directing various immune cells to target sites, such that more potent responses are achieved. Chemokines released by NK cells include, in addition to CXCL8, the macrophage inflammatory protein (MIP)‐1α and MIP‐1β; chemokine (C‐C motif) ligand 5 (CCL5), also known as "regulated on activation normal T cell expressed and secreted" (RANTES); monocytes chemoattractant protein (MCP)‐1; and eotaxin [18, 19]. The signalling pathways and mechanisms required for cytokine secretion also appear to be distinct from secretion of cytotoxic granules [14]. The localization and trafficking of IFN‐γ and TNF‐α were shown to take place in compartments and vesicles that do not overlap with perforin or other late endosome granule markers. Recycling endosomes (REs) are not needed for release of perforin, but are required for cytokine secretion in NK cells. Although perforin granules are released in a polarized fashion at lytic synapses, distinct carriers transport both IFN‐γ and TNF‐α to points all over the cell surface, including within the synapse, for non‐ polarized release.

## **2. NK tumour recognition**

to considerable enthusiasm over the possibility that they function as one of the main effector cells of immunosurveillance. Several studies in the 1980s reported a higher cancer incidence in individuals with genetic disorders such as Chediak‐Higashi syndrome and X‐linked lym‐ phoproliferative syndrome, which lead to defective NK cell function [6, 7]. Subsequent mouse studies showed increased tumour growth in mice with impaired NK cell activity or mice treated with an NK cell‐depleting agent [8, 9]. A long‐term epidemiological study following cancer patients reported that subjects with lower NK cell activity had a higher incidence of several types of cancer [10]. Collectively, data from both mouse and human studies support the theory of cancer immunosurveillance and the concept that NK cells play a critical role in tumour control and eradication [11, 12]. The two main effector functions observed by NK cells against tumour targets are target cell elimination and cytokine secretion [13]. Until recently, these two effector functions were thought to follow similar mechanisms of activation, but now

it is recognized that cytokine secretion by NK cells is distinct from cytotoxicity [14].

cell apoptosis once bound to their respective receptors on target cells [16].

NK cells kill tumour cells through granule exocytosis or death receptor ligation. Following NK cell activation, NK cells release the contents of their granules for target cell elimination. The membrane disrupting protein perforin, and a family of serine proteases termed gran‐ zymes, are the critical effector molecules contained in their granules [15]. Perforin results in the disruption of endosomal trafficking and binds in a calcium‐dependent manner to phos‐ pholipid components of the lipid bilayer to facilitate entry of granzymes into the target cell cytosol [16]. Once granzymes enter the target cell, they induce apoptosis. In addition to gran‐ ule exocytosis, NK cells can directly eliminate target cells through the engagement of cell sur‐ face death receptors. NK cells express Fas ligand (FasL) and TNF‐related apoptosis‐inducing ligand (TRAIL), which are both members of the TNF family and are shown to induce target

Resting NK cells secrete a plethora of cytokines that help eliminate target cells and amplify acti‐ vation signals for a more efficient immune response. NK stimulation results in enhanced secre‐ tion of cytokines, which in turn influence the activity of other immune cells. Pro‐inflammatory cytokines secreted by NK cells, which include interleukin (IL)‐1, IL‐6, IL‐12, and the chemokine CXCL8 (also known as IL‐8), can enhance the activation and proliferation of T cells, dendritic cells (DCs) and macrophages [17]. By contrast, anti‐inflammatory cytokines such as IL‐4 and IL‐10 suppress T cell and macrophage function, but activate humoral responses. Chemokines, which are chemotactic cytokines, play an important role in directing various immune cells to target sites, such that more potent responses are achieved. Chemokines released by NK cells include, in addition to CXCL8, the macrophage inflammatory protein (MIP)‐1α and MIP‐1β; chemokine (C‐C motif) ligand 5 (CCL5), also known as "regulated on activation normal T cell expressed and secreted" (RANTES); monocytes chemoattractant protein (MCP)‐1; and eotaxin [18, 19]. The signalling pathways and mechanisms required for cytokine secretion also appear to be distinct from secretion of cytotoxic granules [14]. The localization and trafficking of IFN‐γ and TNF‐α were shown to take place in compartments and vesicles that do not overlap with

**1.1. Target cell elimination**

16 Natural Killer Cells

**1.2. Cytokine secretion**

Prior to the discovery of NK cell receptors, it was unclear how NK cells could identify tumour targets for lysis. The 'missing‐self' recognition model was initially proposed based on the observation that NK cells kill targets with reduced or absent expression of major histocompat‐ ibility complex class I (MHC I) molecules [20, 21]. This model explains why tumour or virally‐ infected cells with deficient MHC class I expression are targeted by NK cells, whereas healthy autologous cells remain protected. It also explains the hybrid resistance phenomenon, in which F1 hybrid mice reject parental bone marrow cells donated by either parent, despite the fact that the transplant does not express foreign MHC molecules [22]. Early experiments supported the 'missing‐self' model by demonstrating selective rejection of an MHC class I‐deficient version of the tumour cell line RMA in mouse models, in which the results were reversed after treating mice with an NK‐depleting agent [20]. The characterization of NK cell inhibitory receptors fur‐ ther supported this recognition model by explaining the molecular mechanisms by which NK cells sensed the downregulation of MHC class I expression [23–29]. NK cell‐mediated killing of MHC class I‐deficient cells also provides a safeguard mechanism for MHC class I‐restricted elimination by cytotoxic T lymphocytes. However, the 'missing‐self' hypothesis alone failed to explain why NK cells spare autologous cells with absent MHC class I expression or kill tumour cells with sufficient MHC class I expression [30, 31]. The discovery of a wide array of activating NK cell receptors that detect stress‐induced ligands on damaged or stressed cells led to the proposition of the 'induced‐self' model, by which NK cells kill targets with upregu‐ lated expression of activating ligands. It is now understood that NK cell functions are tightly regulated by the integration of opposing signals from activating and inhibitory receptors [32]. Together these models suggest that NK cells detect changes in self‐ligands on the surface of autologous cells. NK cells can also be activated through antibody‐dependent cellular cytotoxic‐ ity (ADCC) whereby the NK cells are triggered directly through ligation of CD16 to kill tumour target cells to which the antibody has bound. The anti‐CD20 antibody, Rituximab mediated lysis of CD20+ve lymphoma cells through this mechanism. **Figure 1** summarizes tumour rec‐ ognition strategies by NK cells.

#### **2.1. NK cell inhibitory receptors**

Human NK cell inhibitory receptors fall into two groups: the killer immunoglobulin‐like receptors (KIRs), and the lectin‐like receptor NKG2A, which forms a heterodimeric complex with CD94. KIRs bind to human leukocyte antigen (HLA)‐A, ‐B, or ‐C, whereas the NKG2/ CD94 complexes ligate HLA‐E. Human KIRs contain either two (KIR2D) or three (KIR3D) immunoglobulin (Ig)‐like domains in their extracellular domain. KIR2D receptors recognize

**Figure 1.** Tumour recognition strategies by NK cells. A) Balanced signals delivered by activating and inhibitory NK cells receptors are recognized as healthy and spared from NK cell‐mediated lysis. B) Tumour cells downregulate MHC class I molecules, and are recognized by NK cells through 'missing self' for lysis. C) The upregulation of stress‐ or damage‐ related ligands is recognized by activating NK cell receptors and can overcome inhibitory signals to result in tumour lysis through the 'induced‐self'. D) Antigen‐specific antibodies can bind CD16 on NK cells to result in ADCC. ADCC: antibody‐dependent cell‐mediated cytotoxicity; MHC: major histocompatibility complex; NK: natural killer cell.

HLA‐C alleles, whereas KIR3D receptors recognize HLA‐A or HLA‐B alleles. The common pathway generated by ligation of inhibitory receptors is characterized by tyrosine phosphory‐ lation of immune tyrosine‐based inhibitory motifs (ITIM) that recruit tyrosine phosphatases such as the Src homology 2 domain‐containing phosphatase (SHP)‐1 and SHP‐2, which are responsible for the inhibition of various NK cell effector functions [33].

#### **2.2. NK cell education**

NK cell education refers to the mechanisms through which inhibitory input by MHC class I during development translates into functional responsiveness in mature NK cells [34]. Unlike the educational processes in T‐ or B‐cell development, NK cell education remains a topic of intense debate, with several models proposed to explain how NK cell respon‐ siveness relates to inhibitory signalling. NK cells that lack ITIM‐bearing inhibitory recep‐ tors for self‐MHC‐I and NK cells from hosts that lack MHC‐I ligands for ITIM‐bearing inhibitory receptors have a reduced responsiveness to activation signals, such as stimula‐ tion by sensitive target cells or cross‐linking of NK cell activating receptors [34–37]. These results have led to the two main models in NK cell education. The first 'disarming' model proposes that in the absence of inhibition, continuous stimulation of NK cells leads to a state of hyporesponsiveness [38]. The second model proposes that inhibitory receptors pro‐ vide an ITIM‐dependent signal to the NK cells that renders them responsive [39]. This model is referred to as 'arming' or 'licensing', although the latter term is now understood to include any process by which NK cells that receive signals through inhibitory receptors for self‐MHC‐I gain responsiveness [40]. Studies reporting that NK cell responsiveness is calibrated according to the strength of inhibitory signals received [36, 41, 42], have led to a third 'rheostat' model that aimed to reconcile the two opposing models, and account for the quantitative tuning of NK cell responsiveness [42–44]. The rheostat model postulates that NK cell responsiveness is dynamically calibrated based on the strength of inhibitory signals received. More recent data demonstrating that NK cell 'tuning' or 'licensing' may be set by transient signals and can be reversible have led to an updated model known as the 'revo‐ cable license' [45]. The revocable license model argues that NK cells can keep their license as long as they are tightly regulated by inhibitory signals, but once this inhibitory input is lost, their license is revoked. Many questions regarding the molecular basis of licens‐ ing and the effect of subsequent activation signals on licensed vs. unlicensed cells remain unanswered. In many cases, the original concept of 'missing‐self' and the self‐tolerance of NK cells in an MHC‐I‐devoid environment cannot be explained without the involvement of NK cell activating receptors.

#### **2.3. NK cell activation receptors**

NK cell activation receptors can be grouped into three categories: those that associate with immunoreceptor tyrosine‐based activation motif (ITAM)‐containing subunits, the DAP10‐ associated NK group 2 member D (NKG2D) receptor and a number of other receptors including DNAX accessory molecule‐1 (DNAM‐1), CD2 and 2B4. Receptors that associate with the ITAM‐containing adapter proteins transmit signals through the recruitment of tyro‐ sine kinases Syk or ZAP70, and include CD16, which mediates antibody‐dependent cellu‐ lar cytotoxicity, and the natural cytotoxicity receptors (NCRs) NKp30, NKp44, NKp46 and NKp80, which are known to play an important role in NK‐mediated cytotoxicity against tumour cells [46]. NKp30 and NKp46 are constitutively expressed on all peripheral blood NK cells, whereas NKp44 is expressed only on activated NK cells. NKp30 binds the nuclear factor HLA‐B‐associated transcript (BAT)‐3, NKp46 binds to influenza haemagglutinin and the cellular ligand for NKp80 is the activation‐induced C‐type lectin (AICL) [47]. NKG2D associates with the DAP10 adaptor protein and signals through a phosphoinositide 3‐kinase (PI3K)‐binding motif. It binds several ligands associated with stress, infection or transforma‐ tion including MHC I chain‐related protein A and B (MICA/B) and the UL16‐binding proteins 1–4 (UBLP1‐4) [48].

#### **2.4. NK cell activation**

HLA‐C alleles, whereas KIR3D receptors recognize HLA‐A or HLA‐B alleles. The common pathway generated by ligation of inhibitory receptors is characterized by tyrosine phosphory‐ lation of immune tyrosine‐based inhibitory motifs (ITIM) that recruit tyrosine phosphatases such as the Src homology 2 domain‐containing phosphatase (SHP)‐1 and SHP‐2, which are

**Figure 1.** Tumour recognition strategies by NK cells. A) Balanced signals delivered by activating and inhibitory NK cells receptors are recognized as healthy and spared from NK cell‐mediated lysis. B) Tumour cells downregulate MHC class I molecules, and are recognized by NK cells through 'missing self' for lysis. C) The upregulation of stress‐ or damage‐ related ligands is recognized by activating NK cell receptors and can overcome inhibitory signals to result in tumour lysis through the 'induced‐self'. D) Antigen‐specific antibodies can bind CD16 on NK cells to result in ADCC. ADCC: antibody‐dependent cell‐mediated cytotoxicity; MHC: major histocompatibility complex; NK: natural killer cell.

NK cell education refers to the mechanisms through which inhibitory input by MHC class I during development translates into functional responsiveness in mature NK cells [34]. Unlike the educational processes in T‐ or B‐cell development, NK cell education remains a topic of intense debate, with several models proposed to explain how NK cell respon‐ siveness relates to inhibitory signalling. NK cells that lack ITIM‐bearing inhibitory recep‐ tors for self‐MHC‐I and NK cells from hosts that lack MHC‐I ligands for ITIM‐bearing inhibitory receptors have a reduced responsiveness to activation signals, such as stimula‐ tion by sensitive target cells or cross‐linking of NK cell activating receptors [34–37]. These results have led to the two main models in NK cell education. The first 'disarming' model

responsible for the inhibition of various NK cell effector functions [33].

**2.2. NK cell education**

18 Natural Killer Cells

NK cells require the co‐engagement of multiple activating receptors in order to exhibit natu‐ ral cytotoxicity against tumour target cells [49]. Upon encounter with potential target cells, an immunological synapse forms at the point of contact between the NK cell and the target cell, where NK cell receptors can interact with their respective ligands. Given sufficient activation signals, NK cell cytoskeletal rearrangements are initiated, which result in the polarization of NK cell lytic granules toward the immunological synapse, where they eventually fuse and release their cytotoxic contents on to the target cell [50]. In contrast to CTLs, NK cells have their cytotoxic granules preformed before target cell recognition, and so their release is initially constrained until sufficient signalling is achieved. NK cells have also been shown to establish cytoskeletal polarity more slowly than CTLs, and to have a unique sensitivity to minor interference with cytoskeletal dynamics [51]. This stepwise progression in activa‐ tion events with specific requirements for synergistic signalling may provide a mechanistic explanation of how the spontaneous cytotoxic capacity of NK cells is regulated [52]. **Figure 2** outlines NK cell activation events at the immunological synapse with a tumour target cell.

**Figure 2.** Activating immunological synapse between NK cell and tumour target. NK cell encounter with a tumour cell target generates an immunological synapse at the point of contact. If the ligand combination on the tumour target engages NK cell activating receptors sufficiently, cytoskeletal rearrangements take place resulting in granule polarization and the eventual release of cytotoxic granules on to the target cell. NK: natural killer cell.
