**4. Natural killer cell signaling pathways**

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

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, nec-

tin-2 for DNAM-1, and MICA/B and ULBPs for NKG2D activating receptors [56–58].

NK cells effector functions is a matter of convoluted balance between activating and inhibitory receptors previously explained. The unique feature of those cells to recognize their targets without prior activation has perplexed researchers along the way since early 1990s. NK cells checks for the presence of MHC class I expression that are only present on normal "self" cells just like checking for an ID, and to consider everything lacking this "self" as foreign, which is the case in transformed or virally infected cells; this was first explained by Kärre and colleagues postulating the "missing self" hypothesis [64]. Recognition of the MHC class I molecules is achieved via the inhibitory receptors of NK cells that include members of the killer cell Ig-like receptors (i.e., KIR) [65], in addition to a CD94/NKG2A heterodimer [66].

subsequent viral or tumor target cells [32].

36 Natural Killer Cells

**3. Dictation of natural killer cell function**

The integration of signal transduction pathways is modulated *in vivo* by several soluble immune cytokines, including IL-2, IL-12, IL-15, and IFN-α/β [4, 13, 70] as well as cell-to-cell interactions involving different cell types primarily dendritic cells (DC) [13, 71], macrophages, and mesenchymal stromal cells [72, 73] determine NK cell effector responses [74, 75].

For instance, interleukin-2 (IL-2) binds to its corresponding receptor; IL-2 receptor (IL-2R) is composed of three subunits: IL-2Rα (CD25), IL-2Rβ (CD122), and the common γ chain (γc, CD132). Subsequently, activating downstream intracellular signaling pathways JAK-STAT pathway, MAPK-ERK pathway, and PI3K-AKT-mTOR pathway promote NK cell proliferation, cytotoxicity, and survival [28, 76, 77]. Intricately, IL-15R is a heterotrimeric receptor consisting of a unique α chain, a shared β subunit with IL-2, and a common ɣ subunit with several cytokines [78]. Engagement of IL-15R on NK cells causes auto-phosphorylation and activation of Janus kinases (JAK1 and JAK3), which induces at least three parallel signaling cascades: Ras-Raf-MEK, PI3K-AKT-mTOR, and STAT5 pathways [28, 78]. In addition, it was recently demonstrated that the PI3K-AKT-mTOR pathway is critically activated by the most potent interleukin-15 (IL-15) enabling NK cell homeostasis, maturation, and activation. Furthermore, this pathway is also implicated in a broad range of IL-15-induced NK cell effector functions such as proliferation, cytokine production, and cytotoxicity [79]. Co-activation of NK cells by IL-12 and IL-18 or IL-15, results in the production of IFN-γ and deactivation of the TGF-β; the immunosuppressive signaling cascade [80]. IL-21 binds to γc receptor shared with IL-2 and IL-15 and the IL-21Rβ receptor which is similar to IL-2Rβ, and then activates a series of intracellular signaling pathways. Thus, it stimulates cell proliferation, survival, secretion of IFN-γ, and cytotoxicity of NK cells [81, 82].

#### **4.1. Functions of natural killer cells**

In the immune system, the NK cells are of crucial importance due to its important functions that can be classified into three categories: (A) cytotoxicity, (B) cytokine and chemokine release, and (C) co-stimulation of other immune cells.

#### **4.2. Cytotoxicity**

Through their cytotoxic activity, NK cells are capable of killing viral infected and malignant cells [83]. Different cytotoxic pathways in NK cell have been characterized. First, the Prf- and Gzmmediated cytotoxic pathway is the most common killing pathway of NK cell. In such pathway, the well-known effector molecules (perforin-1 and granzyme B) are exocytosed from their cytoplasmic granules in NK cells to the vicinity of the target cells (immunological synapse) [70]. Prf1, which is a pore performing protein, polymerizes forming pores in the phospholipid bilayer of the target cell facilitating the delivery of granzymes into the cytosol of the target cell; whereas, the serine protease GzmB cleaves several procaspases and other intracellular substrates to initiate the classical apoptotic pathways [84]. Second, the killing process may also be mediated in a perforin-independent manner through the CD95 (Fas)-CD178 (Fas ligand) pathway. Upon binding of the NK cell to the Fas expressing stressed target cells, death inducing signaling complex is formed and subsequent activation of caspases promoting the apoptotic process of the target cell [85]. Third, the antibody-dependent cellular cytotoxicity (ADCC) which is used by leukocytes that express CD16 (Fc receptors), including NK cells to kill antibody-coated target cells [86].

#### **4.3. Cytokine and chemokine release**

It is thought that NK cells participate in a complex interaction network with other lymphocytes, dendritic cells, and macrophages to effectively control infection and malignancy. NK cells mediate their modulatory function on the immune cells through the production of cytokines and chemokines following either cytokine- or activating-receptor stimulation on the NK cell surface. The prototype effector cytokine produced by NK cells is IFN-γ, which has pleotropic effector actions on other immune cells, antigen-presenting cells, and virally infected or malignant target cells [7]. In viral models of infection, IFN-γ production by NK cells has been shown to be a key event in successful resolution of infection [87]. In liver, the IFN-γ contributes to the anti-viral [88], anti-fibrotic [89], anti-regenerative [90], and anti-tumor [91] activities of resident NK cells there. NK cells release several other cytokines including interleukin-3 (IL-3) and granulocyte-macrophage colony-stimulating factor (GM-CSF), tissue necrosis factor-α (TNF-α) and transforming growth factor-β (TGF-β) and chemokines (MIP-1α, MIP-1β, and RANTES), which depends on the NK cell stimulation type and the time course after activation [92].

#### **4.4. Co-stimulation**

NK cells can also interact with other immune cells through contact-dependent cell co-stimulation, this takes place upon the expression of several co-stimulatory ligands on the NK cell including CD40L (CD154) and OX40L, thus allowing NK cells to provide a co-stimulatory signal to T cells or B cells [93, 94]. So from that, NK cells serve as a bridge in an interactive loop between innate and adaptive immunity. Dendritic cells (DC) stimulate NK cells, which then deliver a co-stimulatory signal to T- or B cells allowing for an optimal immune response. In addition to signals received from soluble mediators, activation of NK cells are also done through cell contact by their receptors that recognize ligands on other cells [95].
