**4. The roles executed by NK cells with molecules in cytotoxic granules, the cell surface ligand and secreted proteins**

As mentioned above, the cell surface expression of activating receptors on NK cells is a key event which defines the performance of those cells. What precisely occurs in NK cells following stimulation with KARs? NK cells execute two different events following recognition of target cells with activating receptors (**Figure 3**). The first event exerts natural cytotoxicity against the targets by releasing perforin and granzymes in cytotoxic granules into the space of the immune synapse between NK and target cells. Perforin is thought to function in generating a pore in the plasma membrane as complement proteins, and then granzymes enter through the pore and mediate apoptosis by deploying their serine protease activity [34]. Alternatively, NK cells also express cell surface FasL, which can also induce apoptosis through Fas receptors on the target cells [35]. Moreover, TNF-related apoptosis-inducing ligand (TRAIL) is also produced by NK cells and induces apoptosis of target cells like FasL [36–38]. This natural cytotoxicity itself highlights the importance of early removal of abnormal cells, which transiently appear in the body, and directly contributes to preventing the development of tumor diseases. However, it also has another role linked to antigen-specific cytotoxicity by cytotoxic T lymphocytes (CTLs) in acquired immunity. CTLs have to be primed by dendritic cells (DCs) with a complex comprising antigen peptide and MHC class I molecule before they can become effective cytotoxic cells from naïve cells. In this priming, extracellular antigen is exceptionally presented on MHC class I for CTLs by a particular subset of DCs, in a process referred to as "cross-presentation" [39–41]. These type of DCs endocytose dead target cells, digest their antigens and express a complex of MHC class I and antigen peptide on the cell surface [42], and the dead cells can be provided from early injury of target cells by NK cells to DCs [43]. That is why NK cells are linked to acquired

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 incu-

**4. The roles executed by NK cells with molecules in cytotoxic granules,** 

As mentioned above, the cell surface expression of activating receptors on NK cells is a key event which defines the performance of those cells. What precisely occurs in NK cells following stimulation with KARs? NK cells execute two different events following recognition of target cells with activating receptors (**Figure 3**). The first event exerts natural cytotoxicity against the targets by releasing perforin and granzymes in cytotoxic granules into the space of the immune synapse between NK and target cells. Perforin is thought to function in generating a pore in the plasma membrane as complement proteins, and then granzymes enter through the pore and mediate apoptosis by deploying their serine protease activity [34]. Alternatively, NK cells also express cell surface FasL, which can also induce apoptosis through Fas receptors on the target cells [35]. Moreover, TNF-related apoptosis-inducing ligand (TRAIL) is also produced by NK cells and induces apoptosis of target cells like FasL [36–38]. This natural cytotoxicity itself highlights the importance of early removal of abnormal cells, which transiently appear in the body, and directly contributes to preventing the development of tumor diseases. However, it also has another role linked to antigen-specific cytotoxicity by cytotoxic T lymphocytes (CTLs) in acquired immunity. CTLs have to be primed by dendritic cells (DCs) with a complex comprising antigen peptide and MHC class I molecule before they can become effective cytotoxic cells from naïve cells. In this priming, extracellular antigen is exceptionally presented on MHC class I for CTLs by a particular subset of DCs, in a process referred to as "cross-presentation" [39–41]. These type of DCs endocytose dead target cells, digest their antigens and express a complex of MHC class I and antigen peptide on the cell surface [42], and the dead cells can be provided from early injury of target cells by NK cells to DCs [43]. That is why NK cells are linked to acquired

bation with NK-sensitive target cells.

186 Natural Killer Cells

**the cell surface ligand and secreted proteins**

**Figure 3.** Summarized illustration of the roles executed by NK cells following the recognition of targets with activating receptors and subsequent responses in acquired immunity. (1) NK cells recognize target cells with activating receptors including NKG2D and NKp46, which trigger the machinery for target cell injury. (2) NK cells exert the action of killing targets by perforin/granzymes, FasL or TRAIL. (3) Dead target cells are endocytosed as antigen (Ag) by DCs. (4) The particular subset of DCs which have endocytosed lysed target cells play a role in "cross-presentation" to stimulate naïve CD8<sup>+</sup> T lymphocytes to develop into mature CTLs. (5) The stimulated NK cells also produce cytokines including TNF-α and IFN-γ, (6)–(8) which stimulate DCs to produce IL-12 and other proinflammatory cytokines, thereby promoting Th1 cell polarization and CTL development.

immunity as well as function in innate immunity by themselves. Secondly, the production of cytokines including TNF-α and IFN-γ by NK cells after recognition of targets also plays an important role in DC maturation [43, 44]. Those cytokines stimulate DCs to produce IL-12 and other proinflammatory cytokines, which promote Th1 cell polarization and CTL development with specificity against target cells. Additionally, IFN-γ produced by NK cells is able to effect Th1 polarization directly. Those findings relating to the production of cytokines by NK cells, in particular, as IFN-γ is a key cytokine which is produced by NK cells and supports tumor immunity, leads us to hypothesize that the production of IFN-γ by NK cells in an individual might be utilized alone to estimate the performance of natural cytotoxicity in those cells. However, it is known that NK cells can be divided into two populations comprising CD56bright and CD56dim cells, which show different natural cytotoxicity and production of IFN-γ. CD56bright NK cells have high production of IFN-γ and low natural cytotoxicity, whereas CD56dim NK cells have low production of IFN-γ and high natural cytotoxicity [45, 46]. Those findings demonstrate that measurement of IFN-γ production by NK cells is insufficient to estimate the natural cytotoxicity of those cells and that determination of multiple parameters related to NK cells is necessary in order to effectively evaluate the performance of natural cytotoxicity in an indirect manner. All of this information indicates that the performance of NK cells is reflected by the strength of stimulation through cell surface activating receptors as well as the subsequent production of functional molecules as described above.
