**3. NK cell cytotoxicity**

Natural killer cells have diverse biological functions including killing pathogen-infected cells and cancer cells as well as an immunoregulatory role [2]. Natural killer cells can discriminate between normal cells and cells that do not express adequate amounts of major histocompat-

NK cell cytotoxicity is regulated by a balance between activating and inhibitory signals delivered by receptors expressed at the cell surface. These cells are known to directly recognize and kill malignant cells or induce apoptosis. However, tumor cells have the ability to evade immunosurveillance by using multiple mechanisms. Furthermore, tumors harbor a population of cancer stem cells (CSC), which is responsible of tumor progression and therapeutical

Therapeutic applications of NK cells in cancer and NK cells targeting cancer stem cells (CSCs)

NK cells originate from common lymphoid progenitor cells and further differentiate into immature/mature NK cells in bone marrow. They are then distributed in peripheral lymphoid and nonlymphoid organs and tissues [3–5], including bone marrow, spleen, peripheral blood, placenta, lung, liver, uterus [6], and peritoneal cavity while limited numbers are localized in

NK cells were originally described as large granular lymphocytes with natural cytotoxicity against tumor cells. NK cells were later recognized as a separate lymphocyte lineage, with both cytotoxicity and immunoregulatory role, as they are involved in the production of cytokines [9]. More recently, data revealed that activated NK cells may also influence the outcome of helminth infections. CD4-NK cells increasing early following nematode infection with *Brugia pahangi* are able to produce IL-4 and then could polarize the immune response toward a Th2 profile [10]. In fact, protection against helminthic infections are usually mediated by Th2 immune response characterized by secretion of IL-4, IL-5, and IL-13, secretion of IgE antibodies, and activation of mast cells [11, 12]. Studies revealed that the clearance of these parasites is more efficient and complete in the presence of NK cells. In the case of Th2 immunity disruption, NK cells may become an important source of IL-13 during murine gastrointestinal nematode infections [13, 14]. Human NK cells can be classified into two major subsets CD56dim and CD56bright depending on their immunophenotype and functions and more recently in terms of their homing properties [15, 16]. CD56dim NK cells are fully mature, make up about 90% of the NK cells in peripheral blood and inflammatory sites, and they express perforin and exhibit a high cytotoxic activity after encountering target cells [17, 18]. These CD56dim NK cells are cytotoxic and produce interferon γ (IFN-γ) upon interaction with tumor cells in vitro [19]. In contrast, CD56bright cells are more immature, make up about 5–15% of total NK cells, and have been considered primarily as cytokine producers, while playing a limited role in cytolytic responses. Approximately, 90% of NK cells in lymph nodes belong

lymph nodes [7]. Human NK cell turnover in blood is around 2 weeks [8].

ibility complex (MHC) class I molecules.

**2. NK cells' biology and function**

represent a promising strategy for cancer immunotherapy.

resistance.

52 Natural Killer Cells

NK cell cytotoxicity is tightly regulated by a balance between activating and inhibitory signals [35] delivered by a multitude of receptors expressed at the cell surface [36] (**Figure 1**). The inhibitory NK cell receptors interact with MHC class I molecules expressed on almost all nucleated cells, preventing NK cell activation against healthy cells (**Figure 2a**). NK cell activation is blocked through engagement of their KIR receptors [37]. This explains selftolerance and prevention of host cell killing. NK cells can discriminate between normal host cells and infected or abnormal cells by recognition of MHC class I molecules. It was earlier discovered that NK cells are activated when they encounter cells that lack self-MHC class I molecule. For example, under stress conditions, such as cellular transformation, cells downregulate MHC-I expression causing NK cells to lose inhibitory signaling and be activated in

**Figure 1.** Examples of activating and inhibitory NK cell receptors and their respective ligands. AICL: activation-induced C-type lectin; B7-H6: Member of the B7 family of immunoreceptors; DNAM-1: DNAX accessory molecule 1; HLA: human leucocyte antigen; KIR2DL: killer-cell immunoglobulin-like receptor 2DL; KIR3DL: Killer-cell immunoglobulinlike receptor 3DL; KIR2D5: killer-cell immunoglobulin-like receptor 2D5; KIR3D5: killer-cell immunoglobulin-like receptor 3D5; LIR-1: leukocyte inhibitory receptor 1; MICA: MHC class I polypeptide-related sequence A; MICB: MHC class I polypeptide-related sequence B; NKG2A: natural killer group protein 2 family member A; NKG2C: natural killer group protein 2 family member C; NKp30: natural killer Cell P30-related Protein; NKp46: natural killer Cell P46-related Protein; NKp80: Natural killer Cell P80-related Protein; PD1: programmed cell death 1; PD-L1: programmed deathligand 1; PD-L2: programmed death-ligand 2; PVR: polio virus receptor; TIGIT: T cell immunoreceptor with Ig and ITIM domains.

a process called "missing-self recognition" [38]. This model is based on the fact that NK cell activity is normally controlled by self-MHC molecules that interact with a large repertoire of inhibitory NK receptors. In this condition, activation receptors are no longer suppressed and they induce potent stimulatory signals, resulting in NK cell activation including cytokine production and granule release leading to cytotoxicity [39, 40]. Abnormal cells can also upregulate the expression of ligands to activate receptors on the NK cells that can overcome the inhibitory signals.

#### **3.1. Activating NK cell receptors**

NK cells require external signals to begin the process of cell activation, which usually occurs via triggering receptors. A number of receptors have been identified that allow NK cells to become activated. The major activating receptors expressed on human NK cells include the natural cytotoxicity receptors (NCRs: NKp30, NKp44, NKp46), the immunoglobulin gamma Fc-region receptor III (FcγRIII/CD16), activating forms of killer cell Ig-like receptors (KIR:

**Figure 2.** NK cell functions. (a) Inhibitory NK cell receptors interact with MHC class I molecules expressed on nucleated cells, preventing NK cell activation and lysis against normal cells. (b) NK cells can eliminate tumors cells that downregulate major histocompatibility complex (MHC) class I molecules causing NK cells to lose inhibitory signaling and be activated in a process called "missing-self recognition." (c) NK cells can kill tumor cells that retain full expression of MHC class I but overexpress induced stress ligands recognized by activating NK cell receptors, which override the inhibitory signals and elicit target cell lysis.

KIR2DS and KIR3DS), NKG2D, C-type lectin receptors (CD94/NKG2C, NKG2E/H, and NKG2F), NKp80, and 2B4 [41]. NKG2D and NCRs are particularly important receptors for triggering NK cell responses toward tumor cells [42].

a process called "missing-self recognition" [38]. This model is based on the fact that NK cell activity is normally controlled by self-MHC molecules that interact with a large repertoire of inhibitory NK receptors. In this condition, activation receptors are no longer suppressed and they induce potent stimulatory signals, resulting in NK cell activation including cytokine production and granule release leading to cytotoxicity [39, 40]. Abnormal cells can also upregulate the expression of ligands to activate receptors on the NK cells that can overcome

**Figure 1.** Examples of activating and inhibitory NK cell receptors and their respective ligands. AICL: activation-induced C-type lectin; B7-H6: Member of the B7 family of immunoreceptors; DNAM-1: DNAX accessory molecule 1; HLA: human leucocyte antigen; KIR2DL: killer-cell immunoglobulin-like receptor 2DL; KIR3DL: Killer-cell immunoglobulinlike receptor 3DL; KIR2D5: killer-cell immunoglobulin-like receptor 2D5; KIR3D5: killer-cell immunoglobulin-like receptor 3D5; LIR-1: leukocyte inhibitory receptor 1; MICA: MHC class I polypeptide-related sequence A; MICB: MHC class I polypeptide-related sequence B; NKG2A: natural killer group protein 2 family member A; NKG2C: natural killer group protein 2 family member C; NKp30: natural killer Cell P30-related Protein; NKp46: natural killer Cell P46-related Protein; NKp80: Natural killer Cell P80-related Protein; PD1: programmed cell death 1; PD-L1: programmed deathligand 1; PD-L2: programmed death-ligand 2; PVR: polio virus receptor; TIGIT: T cell immunoreceptor with Ig and ITIM

NK cells require external signals to begin the process of cell activation, which usually occurs via triggering receptors. A number of receptors have been identified that allow NK cells to become activated. The major activating receptors expressed on human NK cells include the natural cytotoxicity receptors (NCRs: NKp30, NKp44, NKp46), the immunoglobulin gamma Fc-region receptor III (FcγRIII/CD16), activating forms of killer cell Ig-like receptors (KIR:

the inhibitory signals.

domains.

54 Natural Killer Cells

**3.1. Activating NK cell receptors**

A new family of receptors that recognize nectin and nectin-like molecules has recently emerged as a critical regulator of NK cell functions — DNAX accessory molecule 1 (DNAM-1, CD226) is an adhesion molecule that controls NK cell cytotoxicity and interferon-γ production against a wide range of cancer and infected cells [43].

The B7 family member B7-H6 is a tumor cell ligand for the activating natural killer cell receptor NKp30 in humans [44]. Activating KIR receptor recognizes classical MHC-I molecules [45], whereas NKG2D recognizes the nonclassical MHC-I molecules, MICA/MICB, retinoic acid early transcript 1E protein (RAET1E), RAET1G, RAET1H, RAET1I, RAET1L, and RAET1N (also known as ULBP1–ULBP6) [46, 47]. These ligands are not present on the cell surface of most normal cells, but are upregulated at the cell surface after cellular stress, on rapidly proliferating cells, infected cells, transformed cells, and tumor cells [48], further increasing the NK cell activity [49]. CD16 binds the Fc portion of IgG antibodies to initiate antibodydependent cellular cytotoxicity (ADCC) and provides NK cells with the ability to recognize and kill target cells coated with antibodies [50]. DNAM-1 ligands CD112 and CD155 have been described in different pathological conditions, and recent evidence indicates that their expression is regulated by cellular stress.

All of these activating receptors promote cytotoxicity and cytokine production responses through stimulating intracellular protein tyrosine kinase cascades.

#### **3.2. Inhibitory NK cell receptors**

Inhibitory receptors are able to prevent the activation of NK cells and have been thought of as fail-safe mechanisms to prevent attack on normal cells and tissues. In general, these receptors express one or more immunoreceptor tyrosine-based inhibition motifs (ITIM), and they recruit SH2-containing phosphatase-1 (SHP1), SH2-containing phosphatase-2 (SHP2), and/ or SH2-containing inositol phosphatase (SHIP) proteins upon binding to their ligands [51]. These phosphatases prevent the activation of cellular signaling cascades by inhibiting phosphorylation of proteins.

The inhibitory receptors encompass two distinct classes: the monomeric type I glycoprotein of the immunoglobulin superfamilies KIR2DL and KIR3DL [51], leukocyte immunoglobulinlike receptors (ILT2), and the hetero-dimeric C-type lectin-like receptor (CTLR) called CD94/ NKG2A (natural killer group protein 2 family member A) [52, 53].
