**2. Repertoire of receptors orchestrating natural killer cell signals**

NK cells have a unique feature of discerning the infected or malignant cells from normal "self" cells via a complex balance between activating and inhibitory receptor-ligand interactions [27]. In addition, resting inactivated NK cell surface constitutively expresses a wide range of receptors that upon their activation by different ligands, initiation of several downstream signaling pathways take place, and finally resulting in boosting the NK cytotoxicity and cytokines production [28].

#### **2.1. Activating receptors and their ligands**

Upon instances of viral infection and malignant transformation, cellular stress often upregulate ligands for activating receptors and downregulate the self-marker major histocompatibility complex 1 (MHC class I) [29–31]. The interplay and cooperation of NK activating receptors creates a critical threshold of signaling that exceeds the counter balancing influence of the inhibitory receptors, in order for NK cells to mount a productive response [32]. Activating receptors embrace adaptor molecules such as DAP12 bearing immunoreceptor tyrosinebased activating motifs (ITAMs), while other receptors, such as NKG2D (natural killer group 2D), bind to non-ITAM-bearing proteins, providing NK cells with a strong stimulus [29–31]. Thoroughly, several activating receptors including NKG2D, the natural cytotoxicity receptors (NCRs), the nectin and nectin-like receptors, and NKp80 have now been characterized on NK cells [33–37]. These NCRs encompass NKp46, NKp44, and NKp30 collectively were found to play a central role in tumor cell recognition and killing [38]. Interaction of these receptors with their analogous ligands include the human leukocyte antigen-B-associated transcript 3 (BAT-3) and B7H6 for NKp30, a novel isoform of the mixed-lineage leukemia-5 protein (MLL5) for NKp44 and Nectin-2 (CD112) for DNAM-1, and MICA/B and ULBPs for NKG2D [39, 40] all depicted in **Figure 2**.

NKG2D is perhaps one of the best characterized among the activating NK cell receptors. It is a potent stimulatory, C-type lectin-like immunoreceptor that is expressed on NK cells, NKT cells, γδ+ T cells, and CD8+ T cells. It recognizes at least six ligands having MHC class I homology. MICA, MICB, and ULBP4 are transmembrane proteins [37, 41], while ULBP1, 2, and 3 are glycophosphatidylinositol (GPI)-anchored proteins [39]. Importantly, these NKG2D ligands are not expressed on normal tissues, but rather are induced during times of genotoxic or cellular stress as is seen with viral and malignant transformation [42]. Dissimilar to the other activating receptors, NKG2D does not utilize ITAM as a signaling peptide. However, upon receptor-ligand interaction, NKG2D phosphorylates an adaptor protein DAP-10 that recruits and activates phosphatidylinositol3 (PI-3) kinase, which in turn results in perforindependent cytotoxicity [43, 44]. Tumors exploit their common vulnerability and disarm NK cells and cytolytic T cell effector systems by targeting NKG2D. This draws an attention on the epithelial cancers that shed MICA/B ligands into the serum; these soluble ligands downmodulate NKG2D expression on these effector cells with consequent reduction in their cytolytic effectiveness [45–48].

Natural Killer Cells in the Near Future of Immuno-Oncological Therapeutic Approaches http://dx.doi.org/10.5772/intechopen.70567 35

**Figure 2.** The dynamic equilibrium regulating natural killer cell activation.

#### **2.2. Inhibitory receptors and their ligands**

**2. Repertoire of receptors orchestrating natural killer cell signals**

and cytokines production [28].

34 Natural Killer Cells

depicted in **Figure 2**.

effectiveness [45–48].

**2.1. Activating receptors and their ligands**

NK cells have a unique feature of discerning the infected or malignant cells from normal "self" cells via a complex balance between activating and inhibitory receptor-ligand interactions [27]. In addition, resting inactivated NK cell surface constitutively expresses a wide range of receptors that upon their activation by different ligands, initiation of several downstream signaling pathways take place, and finally resulting in boosting the NK cytotoxicity

Upon instances of viral infection and malignant transformation, cellular stress often upregulate ligands for activating receptors and downregulate the self-marker major histocompatibility complex 1 (MHC class I) [29–31]. The interplay and cooperation of NK activating receptors creates a critical threshold of signaling that exceeds the counter balancing influence of the inhibitory receptors, in order for NK cells to mount a productive response [32]. Activating receptors embrace adaptor molecules such as DAP12 bearing immunoreceptor tyrosinebased activating motifs (ITAMs), while other receptors, such as NKG2D (natural killer group 2D), bind to non-ITAM-bearing proteins, providing NK cells with a strong stimulus [29–31]. Thoroughly, several activating receptors including NKG2D, the natural cytotoxicity receptors (NCRs), the nectin and nectin-like receptors, and NKp80 have now been characterized on NK cells [33–37]. These NCRs encompass NKp46, NKp44, and NKp30 collectively were found to play a central role in tumor cell recognition and killing [38]. Interaction of these receptors with their analogous ligands include the human leukocyte antigen-B-associated transcript 3 (BAT-3) and B7H6 for NKp30, a novel isoform of the mixed-lineage leukemia-5 protein (MLL5) for NKp44 and Nectin-2 (CD112) for DNAM-1, and MICA/B and ULBPs for NKG2D [39, 40] all

NKG2D is perhaps one of the best characterized among the activating NK cell receptors. It is a potent stimulatory, C-type lectin-like immunoreceptor that is expressed on NK cells, NKT cells, γδ+ T cells, and CD8+ T cells. It recognizes at least six ligands having MHC class I homology. MICA, MICB, and ULBP4 are transmembrane proteins [37, 41], while ULBP1, 2, and 3 are glycophosphatidylinositol (GPI)-anchored proteins [39]. Importantly, these NKG2D ligands are not expressed on normal tissues, but rather are induced during times of genotoxic or cellular stress as is seen with viral and malignant transformation [42]. Dissimilar to the other activating receptors, NKG2D does not utilize ITAM as a signaling peptide. However, upon receptor-ligand interaction, NKG2D phosphorylates an adaptor protein DAP-10 that recruits and activates phosphatidylinositol3 (PI-3) kinase, which in turn results in perforindependent cytotoxicity [43, 44]. Tumors exploit their common vulnerability and disarm NK cells and cytolytic T cell effector systems by targeting NKG2D. This draws an attention on the epithelial cancers that shed MICA/B ligands into the serum; these soluble ligands downmodulate NKG2D expression on these effector cells with consequent reduction in their cytolytic

Self-immune-surveillance of NK cells activity is inhibited through the recognition of the MHC class I, which is expressed by all healthy cells; by NK cell inhibitory receptors under normal physiological conditions [49, 50]. These receptors comprise the inhibitory killer immunoglobulin-like receptors (KIRs), monomeric type 1 glycoproteins of the immunoglobulin super family that bind to classical MHC class Ia ligands (HLA-A, B, and C), and the inhibitory CD94-NKG2A heterodimeric, C-type lectin-like receptor that bind the non-classical MHC class Ib (HLA-E) as shown in **Figure 2** [29, 51, 52]. Recognition of HLA-E by NKG2A/CD94 receptors enables NK cells to monitor the expression of other HLA class I proteins on cells. CD94 dominates the interaction with HLA-E, whereas NKG2A is more peripheral to the interface [53]. Since, HLA-E expression on the cell surface bound to peptides, depends on classical and non-classical MHC-I proteins production; from which these peptides are derived [53–55]. This double-check mechanism ensures that cells are producing MHC-I molecules in a normal manner [56].

Even in the absence of KIRs-MHC class Ia mediated self-tolerance, other inhibitory receptorligand systems help the NK cells to determine whether tolerance of the host tissue is appropriate. Backing up the other inhibitory NK receptors, is the C-type lectin-like receptor NKR-P1A (CD161) that interacts with a host encoded non-MHC ligand "lectin-like transcript-1 (LLT-1)" illustrated in **Figure 2** [57, 58]. Interestingly, engagement of NK inhibitory receptors including NKG2A with their ligands activates the same signaling mechanisms. Upon phosphorylation of the inhibitory receptors' cytoplasmic immunoreceptor tyrosine-based inhibition motifs (ITIMs), the downstream targets: Src homology 2 domain-containing phosphatases (SHP-1 and SHP-2) are recruited directly to antagonize the signaling pathways of the activating receptors [59–63]. In an elaboration, these tyrosine phosphatases suppress NK cell responses by dephosphorylating the protein substrates of the tyrosine kinases "ITAMs" linked to the activating receptors. Consequently, they terminate Ca2+ influx, degranulation, cytokine production, and proliferation of NK cells. Notably, these events are transient, spatially localized, and do not interfere with the ability of the same NK cells to get activated upon encounter with subsequent viral or tumor target cells [32].

NK cells rely on a vast combinatorial array of receptors, rather than possessing one dominant receptor to initiate its effector functions. Upon instances of viral infection and malignant transformation, cellular stress often upregulate ligands for activating receptors and downregulate MHC class I expression. These receptors comprise the inhibitory killer immunoglobulin-like receptors (KIRs) that bind classical HLA-A, B, and C, and the inhibitory CD94-NKG2A that bind the non-classical MHC class Ib (HLA-E). Backup inhibitory receptors like NKR-P1A (CD161) that interacts with a non-MHC ligand "lectin-like transcript-1 (LLT-1)." Balancing off, the activating receptors comprise, NCRs (NKp30, NKp44, and NKp46) that interact with their ligands including the human leukocyte antigen-B-associated transcript 3 (BAT-3) and B7H6 for NKp30 and the mixed-lineage leukemia-5 protein (MLL5) for NKp44, and also, nectin-2 for DNAM-1, and MICA/B and ULBPs for NKG2D activating receptors [56–58].
