**3. The importance of NK cell-activating receptors in NK cell cytotoxicity**

Although both NK and CD8<sup>+</sup> T cells have the ability to injure target cells, these cells also possess many different characteristics, one of which is the machinery required for the recognition of targets followed by signal transduction linked to cell injury. The diversity of antigen specificity in T cells is dependent on the T cell receptor (TCR) complex on the cell surface, and accounts for the ability of CD8<sup>+</sup> T cells to recognize and kill any kind of target cell by the strong interaction between the TCR and MHC antigen peptide complex. However, as naïve T cells are not ready to exert target injury and antigen specificity differs among cells, with only a small amount being present within each clone, CD8<sup>+</sup> T cells need to be activated before injuring targets. In contrast, NK cells are capable of killing targets with no activation required and are equipped with various kinds of receptors to recognize targets, referred to as NK cell-activating receptors (KARs) [13–16]. NKG2D is the most-studied of these receptors and belongs to the NKG2 family of proteins which are characterized by the presence of a lectin-like domain. NKG2D binds to MHC class I polypeptide-related sequence A and B (MICA/B) and UL16-binding protein (ULBP) [17–22], which are often expressed in tumor cells [23, 24]. Natural cytotoxicity receptors (NCRs) also play a role in killing various kinds of tumors, and NKp30, NKp44, NKp46 and NKp80 are members of the NCR family of proteins [14]. Moreover, the signaling lymphocyte activating molecule (SLAM) family are another group of players involved in the recognition of targets by NK cells, and 2B4 (CD244), a representative member of the SLAM family, recognizes CD48 and leads to cytotoxicity [25–29]. It is thought that the variety of activating receptors on a single cell impart NK cells with the ability to exert cytotoxicity against various target cells without clonal selection and expansion as with T lymphocytes. Those activating receptors share the same mechanism of signal transduction, by which a microtubule organizing center (MTOC) is induced to polarize cytotoxic granules, including perforin and granzymes, near the plasma membrane, and those intragranular molecules are subsequently released against targets *via* degranulation (**Figure 2**). The ligation of those activating receptors allow Src family kinases (SFK) to trigger the pathways from phosphoinositide -3 kinase (PI3K) to extracellular signal-regulated kinase (ERK) and from phospholipase C (PLC) γ to c-jun N-terminal kinase (JNK) to facilitate polarization [30–32]. Those findings led us to surmise that some alteration in cell surface expression levels of activating receptors on NK cells might influence lytic activity against targets. We previously investigated the toxicological effects of asbestos on NK cell function, and demonstrated that asbestos exposure caused impaired cytotoxicity of NK cells with altered expression of several activating receptors [1, 2, 4, 5]. Continuous exposure of the human NK cell line YT-A1 to asbestos resulted in decreased levels of cell surface NKG2D and 2B4, as well as impaired cytotoxicity against K562 cells. Furthermore, it was confirmed that the degranulation induced by stimulation with antibodies to NKG2D or

densities prepared separately by two individuals. Following pre-culturing, all targets were stained with DiO and incubated with PBMCs in a 96-well U-bottom culture plate for 4 h. Following incubation, cells were stained with PI before being analyzed by flow cytometry,

cells in the total number of DiO<sup>+</sup>

NK cell activity (%) = ([percentage of lysed cells] − [percentage of spontaneously dead cells])/ 

In this formula, the percentage of spontaneously dead cells represented the percentage of dead cells in target cells harvested from wells without effector cells. In that experiment, the values of NK cell activity clearly differed among the four kinds of pre-cultured K562 cells and showed a maximum twofold difference at an E/T ratio of 10. These results indicate that the values of NK cell activity vary between assays, a phenomenon which is unavoidable when using target cells, that is, bioassay. If researchers were not bound to use target cells for the determination of NK cell activity without targets, the results obtained from the assays might

would be unnecessary. Therefore, we attempted to develop a new method to determine NK

**3. The importance of NK cell-activating receptors in NK cell cytotoxicity**

possess many different characteristics, one of which is the machinery required for the recognition of targets followed by signal transduction linked to cell injury. The diversity of antigen specificity in T cells is dependent on the T cell receptor (TCR) complex on the

target cell by the strong interaction between the TCR and MHC antigen peptide complex. However, as naïve T cells are not ready to exert target injury and antigen specificity dif-

need to be activated before injuring targets. In contrast, NK cells are capable of killing targets with no activation required and are equipped with various kinds of receptors to recognize targets, referred to as NK cell-activating receptors (KARs) [13–16]. NKG2D is the most-studied of these receptors and belongs to the NKG2 family of proteins which are characterized by the presence of a lectin-like domain. NKG2D binds to MHC class I polypeptide-related sequence A and B (MICA/B) and UL16-binding protein (ULBP) [17–22], which are often expressed in tumor cells [23, 24]. Natural cytotoxicity receptors (NCRs) also play a role in killing various kinds of tumors, and NKp30, NKp44, NKp46 and NKp80 are members of the NCR family of proteins [14]. Moreover, the signaling lymphocyte activating molecule (SLAM) family are another group of players involved in the recognition of targets by NK cells, and 2B4 (CD244), a representative member of the SLAM family, recognizes CD48 and leads to cytotoxicity [25–29]. It is thought that the

fers among cells, with only a small amount being present within each clone, CD8<sup>+</sup>

(100 <sup>−</sup> [percentage of spontaneously dead cells]) × 100 (1)

target cells. Finally, the percentage of natural cytotoxicity was calculated as follows:

cells was measured as lysed

incubator and a clean bench for cell culture

T cells to recognize and kill any kind of

T cells

T cells have the ability to injure target cells, these cells also

where the percentage of DiO<sup>+</sup>

184 Natural Killer Cells

PI+

be more stable, and requirements such as a CO2

cell surface, and accounts for the ability of CD8<sup>+</sup>

cell activity without the use of cell culture.

Although both NK and CD8<sup>+</sup>

**Figure 2.** The summarized machinery of target cell injury caused by target recognition with activating receptors. Recognition of target cells with various activating receptors induces Src family kinases (SFK) to trigger the two pathways from phosphoinositide-3 kinase (PI3K) to extracellular signal-regulated kinase (ERK) and from phospholipase C (PLC) γ to c-jun N-terminal kinase (JNK). Both of these pathways induce polarization of cytotoxic granules *via* a microtubule organizing center (MTOC), whereby granules move to a region near the plasma membrane. Finally, the fusion between plasma membrane and granular membrane occurs to induce degranulation, and perforin and granzymes are released from those granules to induce death of the target cells by apoptosis.

2B4 was low in those asbestos-exposed cells. Moreover, we examined the characteristics of human primary NK cells in PBMCs cultured with asbestos and found a decrease in cell surface NKp46 in patients with malignant mesothelioma, a tumor disease caused by inhalation of asbestos, and also showed impaired natural cytotoxicity. Less information is known about the natural ligands of NKp46. However, a previous investigation demonstrated that cell surface expression levels of NKp46 were correlated with the natural cytotoxicity of K562 and that reverse antibody-dependent cell-mediated cytotoxicity (ADCC) of P815 was correlated with antibodies to NKp46 [33]. Additionally, our previous study demonstrated that NK cells in healthy individuals with high natural cytotoxicity showed high expression of NKG2D, NKp46 and phosphorylation of ERK following stimulation *via* those receptors, whereas NK cells in individuals with low natural cytotoxicity showed the converse [2]. These results led us to surmise that determination of the gene expression level of activating receptors might be one important parameter in estimating the natural cytotoxicity of effector cells such as PBMCs *in lieu* of employing methods involving incubation with NK-sensitive target cells.
