**2. Overview of natural killer cell biology**

NK cells represent a key component of innate lymphoid cells (ILC) and provide defense against microbial infection and malignant transformation. They belong to ILC1 group (according to their ability to produce type1 cytokine and transcription factors, they required for differentiation) and are so named for their capacity to mediate cytotoxicity toward cancer cells and virus-infected cells without prior sensitization [27–29]. NK cells constitute 10–15% of human peripheral blood lymphocytes and are defined by expression of CD56 without T cell marker CD3 [30]. Human NK cells can be further divided into two different subsets depending on the expression density of CD56 and CD16 [30]. The CD56dim CD16bright subsets account for majority (90%) of peripheral blood circulating NK cells and display cytotoxic function [31, 32], whereas CD56bright CD16bright subsets are predominantly found in lymph nodes and produce abundant amounts of inflammatory cytokines such as interferon-γ (IFN-γ), tumor necrosis factor alpha (TNF-α), tumor necrosis factor beta (TNF-β), IL-10, IL-13, granulocyte-macrophage colony stimulating factor (GM-CSF), etc., thereby promote adaptive immune responses [31, 33].

#### **2.1. Natural killer cell receptors**

graft-versus-host disease (GvHD), infections, and leukemia relapse [2, 3]. Natural killer (NK) cells represent a key component of innate lymphoid cells and provide defense against microbial infection and malignant transformation by direct cytotoxicity and cytokine production [4, 5]. The role of NK cells in allo-HSCT was first demonstrated in haploidentical transplants [6]. During T-cell depleted haploidentical HSCT, the rapid recovery of donorderived NK cells mediated potent graft-versus-leukemia (GvL) effect. Importantly, alloreactive NK cells mediated beneficial GvL effect without the occurrence of GvHD, which was

Although the rapid reconstitution of donor NK cells plays a critical role in their GvL effect of the graft, they still take about 6 months or more to acquire maturation phenotype and full functionality [9]. This immaturity and insufficient education status may result in impaired function of donor NK cells in the early stage post transplantation. In addition, since the alloreactivity of donor NK cells was proved to account for the clinical benefit, genotyping the polymorphisms of killer cell immunoglobulin-like receptor (KIR), human leukocyte antigen (HLA) and Fcγ receptor (FcγR) are important for donor selection to maximize the GvL effect [10]. Sufficient numbers of allogeneic NK cells with high purity can also be generated and expanded through several sources including peripheral blood mononuclear cells, umbilical

allo-HSCT [11, 12]. Many approaches involving the use of different feeder cells, engineered feeder cell, and cytokine stimulation were utilized to achieve sufficient numbers of donor NK cells with the most efficient GvL effect and clinical responses [13, 14]. Moreover, seven NK cell lines have been established to be used effectively during allo-HSCT, among which, NK-92 cell line has been shown to be safe and efficient in clinical trials [15–17]. Recently, genetic modification of NK cells has also been developed to enhance their function. Both gene transfer of chimeric antigen receptors (CARs) and expression of cytokine transgenes in NK cells are performed to improve the efficacy of NK cells therapy [18–20]. Furthermore, although traditionally considered as members of innate branch, increasing studies suggest that NK cells also "remember" prior certain stimulation like antigens, cytomegalovirus (CMV), or cytokines [21–26]. It draws particular interest to evaluate the role of adoptively transferred memory-like donor NK cells in allo-HSCT. Based on these research progresses of donor NK cell-mediated immunotherapy during allo-HSCT, in this chapter, we will describe the present state of donor NK cell therapy during allo-HSCT and its future direction aiming to improve therapeutic

NK cells represent a key component of innate lymphoid cells (ILC) and provide defense against microbial infection and malignant transformation. They belong to ILC1 group (according to their ability to produce type1 cytokine and transcription factors, they required for differentiation) and are so named for their capacity to mediate cytotoxicity toward cancer cells and virus-infected cells without prior sensitization [27–29]. NK cells constitute 10–15% of human

cells to be adoptively transferred after

consistently caused by donor T cells [7, 8].

128 Natural Killer Cells

cord blood (UCB), and bone marrow–derived CD34<sup>+</sup>

**2. Overview of natural killer cell biology**

benefit of donor NK cells.

NK cells immune function is mediated through an array of inhibitory and activating cell surface receptors. There are three main types of receptor families: KIR, C-type lectin receptors and natural cytotoxicity receptors (NCR) [34].

KIRs are proteins belonging to immunoglobulin superfamily that are encoded by a gene complex located on chromosome 19q13.4. Individuals may have up to 15 different KIR genes and two pseudogenes encoding for relevant receptors [35]. KIRs express stochastically on mature NK cells and recognize HLA molecules in a manner independent of antigen presentation. When bound to their ligands, these receptors transmit either inhibitory or activating signals. KIR genes are inherited as haplotypes due to the different number of KIR gene, and their high degree of diversity induced by various KIR gene content and allelic polymorphism [36]. Two major groups of KIR haplotypes have been distinguished based on gene content. Group A haplotypes contain a fix number of genes that are mainly inhibitory KIR, including KIR2DL1, KIR2DL3, KIR3DL1, KIR2DS4, and the pseudogene KIR2DP1. On the other hand, group B haplotypes are alterable both in numbers and content of KIR gene and have at least one of the following genes: KIR2DL2, KIR2DL5A, KIR2DL5B, KIR2DS2, KIR2DS5, and KIR3DS1 [37]. Group B haplotypes contain more activating KIR (up to 5) compared with group A haplotypes (only one).

C-type lectin receptors are heterodimers and comprise of two subunits including CD94 protein and a C-type lectin natural killer group 2 (NKG2) molecule [38]. The gene of this receptor family located on chromosome 12 encoding six different NKG2 proteins: NKG2A, NKG2C, NKG2E, NKG2F, NKG2B, NKG2H, as well as CD94 protein. Among these receptors, CD94/NKG2A and CD94/NKG2B are inhibitory receptors and bind HLA-E, whereas other receptors are activating. Activating receptor NKG2D also belongs to NKG2 family and is expressed on the surface of NK cells as a homodimer. NKG2D can recognize two different families of cognate ligands including nonclassical major histocompatibility complex (MHC) molecules (MIC-A and MIC-B) and UL16 binding protein family [39, 40]. The expression of NKG2D ligands is upregulated under the influence of cellular stress such as viral infection, inflammation, and tumor formation.

Natural cytotoxicity receptors including NKp30, NKp44, and NKp46 are activating receptors expressed on the surface of NK cells [41]. NKp30 can recognize ligands expressed on tumor cells such as the nuclear factor HLA-B-associated transcript-3 (BAT-3) and B7-H6 [42]. NKp30 also binds to viral ligands (CMV pp65 protein) [42]. Both NKp46 and NKp44 bind to viral-derived proteins such as influenza hemagglutinin (HA) [43, 44]. Moreover, NKp44 has been shown to recognize the envelope glycorpoteins from West Nile and dengue viruses and NKp46 has been shown to recognize vimentin expressed on *Mycobacterium tuberculosis*–infected human monocytes [45]. However, the ligands of NCR have not been well identified yet.

#### **2.2. Natural killer cell function**

Upon education, the "licensed" NK cells acquire cytotoxic activity toward target cells lacking self MHC class-I molecules, meanwhile they are tolerant to normal cells expressing self MHC class-I molecules, namely "missing self" hypothesis. However, this hypothesis seems to oversimplify NK function regulation. Activating receptors have also been shown to play an important role to make the decision "to kill or not." For example, during murine allo-HSCT model, alloreactive donor NK cells do not respond to host's epithelial cells although donor NK cells lacking inhibitory receptors specific for host MHC class-I, mainly due to their low expression level of ligands specific for activating receptors of donor NK cells [6]. In some cases, the extremely high strength of activating signals may even overcome weaker inhibitory signals resulting in activation of NK cells [46, 47]. Therefore, triggering of NK cell activation is finally depended on the balance of activating and inhibitory receptors of NK cells.

Once NK cells are activated, they respond to target cells with function similar to CD8<sup>+</sup> T cells. Immune synapse forms and perforin and granzyme are released to induce apoptosis of target cells through activation of Caspase 3 [48]. NK cell activation also upregulates the expression of factor associated suicide (FAS) and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and kill the target cells through Caspase 8 activation induced apoptosis [49, 50]. Furthermore, NK cells can also crosstalk with adaptive immune system through cytokine release and antibodies. Virus infected cells and tumor cells are recognized by B cells. These cells are then marked with antibodies covering their cell surface. NK cells recognize and bind to these cells through the interaction between activating receptor CD16 and the Fc end of antibodies [51]. This recognition mediates a potent activating signal in NK cells and results in target cells lysis. Moreover, NK cells are able to secret cytokines and chemokines upon activation, such as TNF-α, IFN-γ, regulated upon activation, norma, T-cell expressed and secreted (RANTES), GM-CSF, macrophage inflammatory protein-1-alpha and beta (MIP1-α and-β). NK cells can promote dentritic cells maturation through TNF-α and IFN-γ secretion [52]. IFN-γ production also affects helper T cell subset 1 (Th1) migration and effector function. Additionally, Th1 polarization in secondary lymphogenous organs is enhanced by IFN-γ secretion [53]. Thus, IFN-γ secretion of NK cells is critical for forming a bridge between the innate and adaptive immune responses.
