**2. Activation of NK cells**

NK cell‐based immunotherapy has been explored for decades. Several animal experiments indi‐ cated the potential efficacy of NK cells in cancer treatment [1–3]. However, *ex vivo* NK cell expan‐ sion techniques have been insufficient regarding the numbers of cells, purity and antitumor activity to use in clinical settings. NK cells comprise only a minor population (i.e., 5–15% of peripheral lymphocytes), and only a small number of NK cells is isolated after a typical apheresis procedure. For example, approx. 77.8 ± 14.4 × 10<sup>9</sup> /L (range 62.7–95.9 × 10<sup>9</sup> /L) of leukapheretic products are the result of a single apheresis of peripheral blood in a normal adult human with a mean percentage of lymphocytes of 59.8% ± 6.1 (range 53.9–66.4%), and subsequently 5–10 × 10<sup>8</sup> NK cells can be obtained [4]. On the other hand, at least 2 × 10<sup>7</sup> /kg or 6 × 10<sup>6</sup> –6.5 × 10<sup>9</sup> /body NK cells are required for each effective injection with multiple administrations [5, 6]. In addition, the reported engraft‐ ment period of NK cells was 2–189 days (median 10 days), which showed no correlation with the number of NK cells administered [7]. The NK cell infusion should thus be repeated in order to maintain a sufficient number of NK cells meeting clinical requirements, but this would be a burden on patients.

Scientists have been working to develop various methods for proliferating NK cells *ex vivo* with high cytotoxicity and high purity. Several studies used an anti‐CD3 antibody (clone: OKT3) in the first few days of culture for the activation of autologous T cells to help the NK cell expansion, subsequently producing high numbers of undesirable T cells or NKT cells in the final product [8–10]. However, particularly in haploidentical NK cell transplantation, T cells must be excluded prior to the infusion in order to prevent graft versus host disease (GvHD). Other studies removed CD3+ cells by magnetic beads with or without CD56‐positive selection at the beginning of culture [11–13].

To acquire highly purified and expanded NK cells, an initial efficient depletion (<1%) of CD3<sup>+</sup> cells and a relatively long‐term culture (over 12 days) seem to be essential [12, 13]. Because only a minor fraction of circulating NK cells is reactive to target cells (tumor cells) *in vitro*, primary NK cells show insufficient cytotoxicity [14]. Various types of stimulation have thus been reported to enable NK cells to achieve their full effector potential, such as interleukin (IL)‐15 produced by dendritic cells (DCs) [15] or macrophages [16], IL‐2 [17], IL‐12 [18], IL‐18 [19] and IL‐21 [20]. Currently, the additional cytokines used in the cultivation of NK cells include IL‐2, IL‐15, and IL‐21. IL‐2, IL‐15, and IL‐21 share the receptor subunits IL‐2/15Rβ and common γ chain [21] on the NK cell surface and have a synergetic effect. Use of IL‐2 combined with IL‐15 for cultivation leads to good viability and good proliferation of NK cells [22]. IL‐2 is also important for NK cell infiltration and killing, and IL‐15 is important for both NK cell maturation and survival [21]. IL‐2 and IL‐15 induce the expression of KIRs and activating receptors (NKG2D and NKp44) on NK cell surface [23]. IL‐21 modifies the expression of killer cell immunoglobulin‐like receptors (KIRs) and NKp44 by reducing expression of DAP‐12, subsequently promote cell maturation, the ability of killing and survival [22, 23]. Several experiments used feeder cells to provide essential stimulation for NK cell cultivation through cytokine production or cell‐to‐cell contact [20, 23, 24]. The various cytokines and feeder cells used in some clinical trials are mentioned in a later section of this article.

#### **2.1. Killer cell immunoglobulin‐like receptors (KIRs)**

NK cells express KIRs, most of which are inhibitory (partially activating) receptors that rec‐ ognized MHC class I molecules. In the 1980s, KIRs were first described as explaining the NK cell‐mediated rejection of allogeneic bone marrow transplants from a homozygous donor to a hemizygous host in lymphoma and in F1‐hybrid anti‐parental resistance in a rodent model [25]. In these situations, the graft fails to express at least one MHC class I allele of the host, and NK cells are highly capable of identifying the difference, thus causing rejection. This can be explained by the concept that NK cells have inhibitory receptors and begin to attack target cells if they do not express ligands that interact with the specific inhibitory receptors. This phenomenon was termed the "missing‐self hypothesis", which is now accepted as one of the complex target recognition mechanisms of NK cells.

#### *2.1.1. Regulation of NK cell activity*

NK cells, which are thought to have emerged much later than B cells and T cells based on the evolutionary convergence of variable receptors, rarely cause autoimmune diseases. We will

NK cell‐based immunotherapy has been explored for decades. Several animal experiments indi‐ cated the potential efficacy of NK cells in cancer treatment [1–3]. However, *ex vivo* NK cell expan‐ sion techniques have been insufficient regarding the numbers of cells, purity and antitumor activity to use in clinical settings. NK cells comprise only a minor population (i.e., 5–15% of peripheral lymphocytes), and only a small number of NK cells is isolated after a typical apheresis procedure.

/L (range 62.7–95.9 × 10<sup>9</sup>

result of a single apheresis of peripheral blood in a normal adult human with a mean percentage

for each effective injection with multiple administrations [5, 6]. In addition, the reported engraft‐ ment period of NK cells was 2–189 days (median 10 days), which showed no correlation with the number of NK cells administered [7]. The NK cell infusion should thus be repeated in order to maintain a sufficient number of NK cells meeting clinical requirements, but this would be a burden

Scientists have been working to develop various methods for proliferating NK cells *ex vivo* with high cytotoxicity and high purity. Several studies used an anti‐CD3 antibody (clone: OKT3) in the first few days of culture for the activation of autologous T cells to help the NK cell expansion, subsequently producing high numbers of undesirable T cells or NKT cells in the final product [8–10]. However, particularly in haploidentical NK cell transplantation, T cells must be excluded prior to the infusion in order to prevent graft versus host disease

To acquire highly purified and expanded NK cells, an initial efficient depletion (<1%) of CD3<sup>+</sup> cells and a relatively long‐term culture (over 12 days) seem to be essential [12, 13]. Because only a minor fraction of circulating NK cells is reactive to target cells (tumor cells) *in vitro*, primary NK cells show insufficient cytotoxicity [14]. Various types of stimulation have thus been reported to enable NK cells to achieve their full effector potential, such as interleukin (IL)‐15 produced by dendritic cells (DCs) [15] or macrophages [16], IL‐2 [17], IL‐12 [18], IL‐18 [19] and IL‐21 [20]. Currently, the additional cytokines used in the cultivation of NK cells include IL‐2, IL‐15, and IL‐21. IL‐2, IL‐15, and IL‐21 share the receptor subunits IL‐2/15Rβ and common γ chain [21] on the NK cell surface and have a synergetic effect. Use of IL‐2 combined with IL‐15 for cultivation leads to good viability and good proliferation of NK cells [22]. IL‐2 is also important for NK cell infiltration and killing, and IL‐15 is important for both NK cell maturation and survival [21]. IL‐2 and IL‐15 induce the expression of KIRs and activating receptors (NKG2D and NKp44) on NK cell surface [23]. IL‐21 modifies the expression of killer

/kg or 6 × 10<sup>6</sup>

–6.5 × 10<sup>9</sup>

cells by magnetic beads with or without CD56‐positive

of lymphocytes of 59.8% ± 6.1 (range 53.9–66.4%), and subsequently 5–10 × 10<sup>8</sup>

/L) of leukapheretic products are the

/body NK cells are required

NK cells can be

introduce the applicability of the NK cell to cancer treatment.

**2. Activation of NK cells**

For example, approx. 77.8 ± 14.4 × 10<sup>9</sup>

(GvHD). Other studies removed CD3+

selection at the beginning of culture [11–13].

on patients.

88 Natural Killer Cells

obtained [4]. On the other hand, at least 2 × 10<sup>7</sup>

The activation of NK cells is regulated by various receptors including KIRs, CD94–NKG2 family, leukocyte immunoglobulin‐like receptors (LILRs), natural cytotoxicity receptors (NCRs), and FcγRIIIa (CD16) [26–28]. Among these receptors, KIRs, CD94/NKG2A heterodimers, and LILRs belong to the large family of inhibitory receptors of MHC class I, mediating NK cell function by signaling through intracytoplasmic immunoreceptor tyrosine‐based inhibition motifs (ITIMs) [29]. Each NK cell has a threshold of activation through a balance of total stimulation between inhibitory and activating signals [30] (**Figure 1**). In other words, NK cells selectively kill tar‐ get cells that down‐regulate MHC class I molecules and/or up‐regulate other activating ligands [28] such as MHC class I chain (MIC)‐related antigens MICA, MICB and UL‐16 binding protein (ULBP). MICA, MICB, ULBPs are ligands of NKG2D homodimer, which belong to C‐type lectin receptor NKG2 family expressed on the surface of NK cells and CD8+ T‐lymphocytes. NKG2A/ CD94 and NKG2B/CD94 heterodimers transmit inhibitory signals, while NKG2C/CD94, NKG2E/CD94, NKG2H/CD94 heterodimers and NKG2D homodimer are activating receptors.

#### *2.1.2. Genetics of KIRs*

The KIR gene family includes 14 loci (KIR2DL1, KIR2DL2/3, KIR2DL4, KIR2DL5A/B, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, KIR2DS5, KIR3DL1/S1, KIR3DL2, KIR3DL3 and two pseudo‐ genes, KIR2DP1 and KIR3DP1) [31]as shown in **Table 1**. These loci are located on chromosome 19q13.4, which is known as the leukocyte receptor cluster (LRC); each haplotype has 9–15 KIR genes in a row [32]. Different NK cells within individuals each express a subset of the available KIR repertoire, leading to an allelic polymorphism of KIRs. Based on studies of KIR genotype variation, two major KIR haplotype groups termed the groups 'A' and 'B' are defined [33].

**Figure 1.** The mechanisms of cytotoxicity by NK cells.


**Table 1.** Human KIRs.

Each haplotype is separated into two regions: the centromeric half (Cen) and the telomeric half (Tel). Cen and Tel motifs can be divided into Cen‐A, Cen‐B and Tel‐A, Tel‐B by the KIR genes they contain. Haplotype A is a combination of Cen‐A and Tel‐A, which consists of mainly inhibitory KIRs (KIR3DL3, 2DL3, 2DL1, 3DL1, 2DS4, 3DL2 and two pseudogenes). Other combinations are termed Haplotype B (such as Cen‐A and Tel‐B or Cen‐B and Tel‐B), composed of a large variation of genes characterized by the presence of more activating KIRs. All individuals can be categorized according to their haplotype: A/A, which is homozygous for group A haplotypes, or B/x, which contains either one (A/B) or two (B/B homozygotes) group B haplotypes[34]. As a consequence of this genetic variation, several studies report that donor‐derived NK cells can mediate the graft‐versus‐leukemia (GVL) effect or even the graft‐ versus‐tumor (GVT) effect after allogeneic hematopoietic cell transplantation (HCT) [34–36]. These are the results of a KIR‐ligand mismatch, the details of which are described later in this article.

#### *2.1.3. KIRs subtypes*

**Inhibitory Ligands Ligand missing Notes**

B73 negative HLA‐B46, B73

Activating Ligands Ligand missing Notes

Others Ligands Ligand missing Notes KIR2DP1 − − Pseudogene KIR3DP1 ‐ ‐ Pseudogene

KIR2DS4 HLA‐A\*1102,(A\*1101, C\*0304, C\*0501) HLA‐A\*1102 negative

KIR2DS1 HLA‐C2 HLA‐C1/C1

HLA‐C1/C1

HLA‐C2/C2(except for C\*02, \*05) and HLA‐B46,

HLA‐C2/C2 and HLA‐ B46, B73 negative

HLA‐Bw6/Bw6 and HLA‐A23/24/32 negative

free of EBV

HLA‐A03, A11 negative,

Expression level: \*01502, \*020

\*001 is homo‐dimer A03/A11 could not promote NK cell

> \*001, \*007 >\*004

Licensing

KIR2DL1 HLA‐C2 group (Cw2, C\*0307,Cw4,

**Figure 1.** The mechanisms of cytotoxicity by NK cells.

90 Natural Killer Cells

KIR2DL2 HLA‐C1 group (Cw1, Cw3, Cw7, Cw8,

KIR2DL3 HLA‐C1 group (Cw1, Cw3, Cw7, Cw8,

KIR3DL1 HLA‐Bw4 epitope(including HLA‐A23,

KIR3DL2 HLA‐A03, A11(+ EBNA peptide),HLA‐

HLA‐B46, B73

A24, A32)

B27 dimer

KIR2DS2 Unknown(HLA‐C1?) KIR3DS1 Unknown(HLA‐B\*2705?)

KIR2DL5B Unknown KIR2DL5T Unknown

KIR2DS3/5 Unknown KIR2DL4 HLA‐G

**Table 1.** Human KIRs.

Cw5, Cw6, C\*0707, C\*0709, C\*1204, C\*1205, Cw15, C\*1602, Cw17, Cw18)

Cw12, Cw13, Cw14, C\*1507, C\*1601/4)

HLA‐C2(C\*02, C\*05)(weak interaction)

Cw12, Cw13, Cw14, C\*1507, C\*1601/4)

KIRs are type I transmembrane glycoproteins expressed on the surface of NK cells, composed of two (2D) or three (3D) extracellular Ig‐like domains and a cytoplasmic short (activating) or long (inhibitory) tail [31]. The length of the intracytoplasmic part determines the function; for example, receptors with long cytoplasmic tails with one or two ITIMs that bind to phospha‐ tase SHP‐1, 2 allow the transduction of inhibitory signals through its dephosphorylation. In contrast, receptors with short cytoplasmic tails possess a positively charged residue (lysine) in the transmembrane domain that enables it to associate with adaptor proteins including DAP12 and process the immunoreceptor tyrosine‐based activation motifs (ITAMs) [29]. There is one exception: KIR2DL4, with a long cytoplasmic tail, binds to the activation motif of FcεRIγ and thus seems to transmit the activating signal [31].

MHC class I molecules are well‐known ligands for KIRs, but HLA‐C molecules, in particu‐ lar, are the main ligands contributing greatly to NK cell activity. Polymorphisms in amino acids at positions 77 and 80 of HLA‐C show specificity for its target KIRs. Group 1 HLA‐C ligands (C1) include allele‐encoded molecules with serine and asparagine (Ser77 and Asn80), whereas group 2 ligands (C2) are characterized by allele‐encoded molecules with asparagine and lysine (Asn77 and Lys80). C1 epitopes bind specifically to KIR2DL2/3, and C2 epitopes are ligands for KIR2DL1 [37]. However, it was shown that KIR2DL2/3 might also bind to certain HLA‐C2 epitopes (C\*0501, C\*0202, C\*0401) and some HLA‐B epitopes (HLA‐B\*4601, B\*7301) with very low affinity [38].

Among the inhibitory KIRs, KIR2DL1 results in a stronger inhibition compared to the KIR 2DL2/3 [38]. The third inhibitory KIR is KIR3DL1, which binds to HLA‐Bw4 epitopes and a subset of HLA‐A epitopes (A\*23, A\*24, and A\*32). All HLA‐B have either the Bw4 or Bw6 epitope, but only the Bw4 epitope is a ligand for KIRs [39]. KIR3DL2 is a framework gene, and it recognizes HLA‐A\*03 and HLA‐A\*11 with a low level of inhibition [40]. Although haplotype A includes only a single activating KIR (2DL4), at least two to five activating KIR genes (2DS1, 2DS2, 2DS3, 2DS5 and 3DS1) are subject to haplotype B. However, KIR2DS1 alone is now confirmed to have matched ligand HLA‐C2 with lower avidity compared to KIR2DL1 [41].

#### **2.2. Death Ligands**

NK cells express members of the tumor necrosis factor (TNF) superfamily, the so‐called death ligands. The cognate of the receptor on target cells by these ligands on NK cells which include TNF‐related apoptosis‐inducing ligand (TRAIL), Fas ligand (FasL) and TNF‐ like weak inducer of apoptosis (TWEAK) — results in classical caspase‐dependent apoptosis [42, 43]. In the TRAIL/TRAIL receptor system, at least five receptors have been identified and two of them, TRAIL‐R1 (DR4) and TRAIL‐R2 (DR5), contain cytoplasmic death domains and are able to transduce an apoptotic signal [44]. Other TRAIL receptors — TRAIL‐R3 (DcR1) and TRAIL‐R4 (DcR2) and a soluble receptor called osteoprotegerin (OPG: TRAIL‐R5) lack a death domain, but they are dedicated as decoy receptors to regulate TRAIL‐mediated cell death [45]. In target cell expressing TRAIL receptors, the ligation of TRAIL can lead to the activation of caspase‐8 and subsequently caspase‐3 to induce apoptosis [46, 47]. TRAIL is up‐regulated after the stimulation of interferon‐gamma (IFN‐γ) *in vitro* [48, 49] and also *in vivo*, which demonstrates that TRAIL is required for the IFN‐γ‐mediated prevention of tumors [50].

FasL is expressed on activated NK cells and cytotoxic T lymphocytes (CTLs) [42]. Although it is known that FasL is expressed on NK cells at a low level, significant amounts are stored intracellularly [51]. An activating signal of rodent NK1.1 up‐regulated the expression of FasL [52]. After FasL has bound to Fas, the Fas associated with two specific proteins, Fas‐ associated death domain (FADD) and caspase‐8, to form the death‐inducing signal complex (DISC). Fas is expressed on various tissues, but the molecule is downregulated in cancers during its progression [53]. NK cells are capable of directly inducing Fas expression on tumor cells via IFN‐γ secretion, and NK cells show cytotoxicity to tumor cells expressing Fas [45].

#### **2.3. Perforin and granzyme**

Cytolytic killing using perforin and granzyme is a major mechanism in the elimination of infected cells and tumor cells by NK cells. NK cells contain cytoplasmic granules including perforin (a membrane‐disrupting protein) and granzyme (a family of serine proteases). Once NK cells recognize target cells, they form an immunological synapse, and the secretory gran‐ ules fuse with the presynaptic membrane and release perforin and granzyme into the syn‐ aptic cleft. Released perforin provides transmembrane pores on the target cell and enables granzyme to diffuse into the cell. Granzyme then initiates the apoptosis of the target cells, and the NK cells detach from the dying cells and can interact with other target cells to accom‐ plish serial killing [54]. The release of such granules stored in the NK cells is dependent on the polarization of both microtubules and actin filaments in the cytoskeleton. The increase of intracellular calcium concentration triggered by a positive balance of activating and inhibitory signals initiate the rapid move of microtubule‐organizing center (MTOC) in the cytoplasm towards the target cell, and then cytotoxic granules migrate along the MTOC [54]. Granules fuse with the presynaptic membrane, subsequently, the lytic granules can be released at the NK cell‐target cell interface[55] (**Figure 2**).

**Figure 2.** Interaction of an NK cell with a target cell.
