**3. Natural killer cell development and education**

#### **3.1. Natural killer cell development**

NK cells develop from CD34<sup>+</sup> hematopoietic progenitor cells which can also give rise to T cells, B cells, and dendritic cells [54]. Particular transcription factors such as purine rich box-1 (PU.1), E26 transformation-specific (ETS-1), thymocyte selection-associated HMG box factor (TOX), the mammalian transcription factor E4 binding protein 4 (E4BP4), eomesodermin (Eomes), and T-box transcription factor (T-bet) are required for NK cell maturation stages [55–58]. Among these transcription factors, Eomes and T-bet are responsible for cytotoxicity and IFN-γ production in mature NK cells [59]. Furthermore, CD56dim NK cells express higher levels of T-bet and lower levels of Eomes than CD56bright NK cells, and T-bet/Eomes ratio increases with NK cells maturation [60]. A central cytokine required for NK cell development *in vivo* is IL-15, as mice lacking IL-15, CD122 (IL-15β-chain receptor), interleukin 15 receptor alpha (IL-15Rα), or signal transducer and activator of transcription 5 (STAT5) do not generate mature NK cells [61]. Stem cell factor (SCF), IL-7, and fms-like tyrosine kinase 3-ligand (Flt3-L) are also necessary for NK cell maturation since they induce pro-NK cell to express CD122 and CD132 (the common γ-chain receptor) so as to render cell to respond to IL-15 [62, 63]. Intriguingly, IL-15 is primarily accompanied by IL-15Rα and transpresented by accessory cells to support NK cell differentiation and proliferation *in vivo* rather than affecting NK cell in a soluble form [64].

After maturation in the bone marrow, NK cells migrate to peripheral circulation and take part in the defense against cancers and viral infections. Two major subsets in the peripheral blood are CD56dim and CD56bright NK cells [30]. These subsets differ in receptor expressions, functional capabilities, and tissue distribution. Although CD56dim NK cells have been considered to be cytotoxic population, it is now clearly proved that they also produce large amounts of cytokines upon receptor-induced NK cell triggering [65, 66]. These functional differences can be partly explained by different expression pattern of surface receptors. CD56dim NK cells express CD16, KIR and other activating or inhibitory receptors which are critical for their cytolytic functions, whereas CD56birght NK cells rarely express KIR and CD16 [67]. Interestingly, CD56bright NK cells could express KIR and CD16 after stimulation of IL-2 or IL-12 suggesting CD56bright NK cells are NK cell subsets in the stage of maturation [68, 69]. Thereafter, recent studies confirm that CD56dim NK cells indeed differentiate from CD56bright NK cells which display longer telomerase [70]. Due to different expression of chemokine receptors and adhesion molecules, CD56dim NK cells account for around 90% of peripheral blood NK cells, while CD56bright NK cells primarily exist in secondary lymphoid tissues or decidual tissues during early pregnancy [71].

#### **3.2. Generation of memory-like natural killer cells**

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 mono-

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.

**3. Natural killer cell development and education**

**3.1. Natural killer cell development**

NK cells develop from CD34<sup>+</sup>

Once NK cells are activated, they respond to target cells with function similar to CD8<sup>+</sup>

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.

B cells, and dendritic cells [54]. Particular transcription factors such as purine rich box-1 (PU.1), E26 transformation-specific (ETS-1), thymocyte selection-associated HMG box factor (TOX), the

hematopoietic progenitor cells which can also give rise to T cells,

T cells.

cytes [45]. However, the ligands of NCR have not been well identified yet.

**2.2. Natural killer cell function**

130 Natural Killer Cells

Immune memory is previously considered as an exclusive property of T cells and B cells and defined as quicker and more robust responses to recurrent antigens [72]. However, accumulating evidence proved NK cell also have memory-like feature after certain modes of challenge including antigen-specific stimulation and antigen-independent activation. The hepatic NK cell memory was first observed by the Von Andrian's group [73]. They found NK cells can mediate delayed-type hypersensitivity (DTH) reactions in *Rag2−/−* mice model. These hapten-specific NK cells are exclusively recruited to the liver and identified as CD49a<sup>+</sup> DX5− NK cell population, expressing chemokine receptor CXCR6 [21, 73, 74]. The mechanism that generate this antigen-specific NK cells whose receptors cannot be somatically rearranged remain unknown. Moreover, similar hepatic memory response has not been proved to occur in humans.

Another differentiation pathway for antigen-specific NK cell memory response is CMV infection. CMV-triggered NK cell memory was first observed by the Lanier laboratory demonstrating that Ly49H<sup>+</sup> NK cells can rapidly expand and response robustly after murine CMV (MCMV) re-infection [75]. NK cell co-activating receptor DNAX adhesion molecule-1 (DNAM-1) is required for the initial expansion of MCMV-induced memory NK cells [76]. Furthermore, transcription factor Zbtb32 and pro-inflammatory cytokine pathway also play important role in MCMV-induced expansion [77, 78]. In human, natural killer Group 2, member C positive (NKG2C<sup>+</sup> ) NK cells also expand during acute human CMV (HCMV) infection and display a CD56dimCD57<sup>+</sup> NKG2A− phenotype [79, 80]. These HCMV-induced memory NK cells have increased expression of IFN-γ, which may be induced by epigenetic remodeling of *IFNG* locus [81, 82]. The role of HCMV-induced NK memory during HSCT will be described in section 6 of this chapter.

Antigen-independent NK cell memory response was first observed by the Yokoyama laboratory [24]. They showed mouse NK cells which were pre-activated with the combination of IL-12/15/18 overnight produced increased level of IFN-γ upon re-stimulation. Additionally, these cytokine-induced memory-like NK cells displayed enhanced proliferation and upregulation of CD25 expression in response to re-stimulation with cytokines [24, 25]. Intriguingly, human NK cells also can give rise to cytokine-induced memory-like NK cells with similar key properties [26]. Series of studies explored the antitumor responses of cytokine-induced memory-like NK cells, and we will discuss their role during HSCT in section 7 and 9 of this chapter.

#### **3.3. Natural killer cell education**

#### *3.3.1. Inhibitory receptors are critical for natural killer cell education*

The genes encoding for KIR are located on chromosome 19, while HLA genes are on chromosome 6 [83]. Therefore, KIR genes and HLA genes are inherited independently, and this randomness may result in the expression of KIR on NK cells in the absence of their corresponding HLA ligands, thereby NK cells may have chance to attack normal cells. In fact, NK cells are tolerant to our body most of the time. It is interesting to understand how NK cells are educated to become tolerant? Inhibitory receptors have been shown to play an important role in this education process. During NK cell maturation, when the inhibitory receptors recognize self-MHC class I, the stochastic expression of KIR genes is switched off, and signals are transmitted to promote NK cell maturation and generate effector function. Oppositely, when the inhibitory receptors of NK cells fail to recognize self-MHC class I, they remain in a state of hyporesponsiveness and are not able to attack normal autologous cells [35, 84]. Collectively, only NK cells expressing "at least one" self-recognizing inhibitory receptor are "licensed" and acquire effector function, whereas "unlicensed" NK cells that recognize non-self-MHC are hyporesponsive and tolerant to normal cells. This process of "licensing" was proved in "peptide transporter-associated antigen processing" (TAP)-deficient patients [85]. Cells from these patients hardly express HLA class I molecules on their surface, thereby potentially render self-cells to be targets of NK cells. However, most of these patients do not have autoimmune disorders because of the hyporesponsiveness of NK cells. Additionally, NK cells isolated from TAP-deficient patients are also unresponsive to autologous HLA class I-negative B lymphoblastoid cell lines [86].

#### *3.3.2. The state of "unlicensed" natural killer cells is reversible*

that Ly49H<sup>+</sup>

132 Natural Killer Cells

chapter.

positive (NKG2C<sup>+</sup>

display a CD56dimCD57<sup>+</sup>

in section 6 of this chapter.

**3.3. Natural killer cell education**

blastoid cell lines [86].

NKG2A−

*3.3.1. Inhibitory receptors are critical for natural killer cell education*

NK cells can rapidly expand and response robustly after murine CMV (MCMV)

) NK cells also expand during acute human CMV (HCMV) infection and

phenotype [79, 80]. These HCMV-induced memory NK

re-infection [75]. NK cell co-activating receptor DNAX adhesion molecule-1 (DNAM-1) is required for the initial expansion of MCMV-induced memory NK cells [76]. Furthermore, transcription factor Zbtb32 and pro-inflammatory cytokine pathway also play important role in MCMV-induced expansion [77, 78]. In human, natural killer Group 2, member C

cells have increased expression of IFN-γ, which may be induced by epigenetic remodeling of *IFNG* locus [81, 82]. The role of HCMV-induced NK memory during HSCT will be described

Antigen-independent NK cell memory response was first observed by the Yokoyama laboratory [24]. They showed mouse NK cells which were pre-activated with the combination of IL-12/15/18 overnight produced increased level of IFN-γ upon re-stimulation. Additionally, these cytokine-induced memory-like NK cells displayed enhanced proliferation and upregulation of CD25 expression in response to re-stimulation with cytokines [24, 25]. Intriguingly, human NK cells also can give rise to cytokine-induced memory-like NK cells with similar key properties [26]. Series of studies explored the antitumor responses of cytokine-induced memory-like NK cells, and we will discuss their role during HSCT in section 7 and 9 of this

The genes encoding for KIR are located on chromosome 19, while HLA genes are on chromosome 6 [83]. Therefore, KIR genes and HLA genes are inherited independently, and this randomness may result in the expression of KIR on NK cells in the absence of their corresponding HLA ligands, thereby NK cells may have chance to attack normal cells. In fact, NK cells are tolerant to our body most of the time. It is interesting to understand how NK cells are educated to become tolerant? Inhibitory receptors have been shown to play an important role in this education process. During NK cell maturation, when the inhibitory receptors recognize self-MHC class I, the stochastic expression of KIR genes is switched off, and signals are transmitted to promote NK cell maturation and generate effector function. Oppositely, when the inhibitory receptors of NK cells fail to recognize self-MHC class I, they remain in a state of hyporesponsiveness and are not able to attack normal autologous cells [35, 84]. Collectively, only NK cells expressing "at least one" self-recognizing inhibitory receptor are "licensed" and acquire effector function, whereas "unlicensed" NK cells that recognize non-self-MHC are hyporesponsive and tolerant to normal cells. This process of "licensing" was proved in "peptide transporter-associated antigen processing" (TAP)-deficient patients [85]. Cells from these patients hardly express HLA class I molecules on their surface, thereby potentially render self-cells to be targets of NK cells. However, most of these patients do not have autoimmune disorders because of the hyporesponsiveness of NK cells. Additionally, NK cells isolated from TAP-deficient patients are also unresponsive to autologous HLA class I-negative B lymphoIt is important to note that the hyporesponsive state of "unlicensed" NK cells is reversible. During viral infection, "unlicensed" NK cells regain cytotoxic activity and are even more effectively in controlling murine cytomegalovirus (MCMV) infection compared with "licensed" NK cells owing to the lack of inhibitory effect of self MHC class-I expression on target cells [87]. The activity of "unlicensed" NK cells can also be restored after *in vitro* culture in the presence of IL-2 or IL-12+IL-18 or through activating receptors mediated strong signals [88].

The education process is more complex during allo-HSCT as donor-derived progenitors of NK cells posses both possibilities to undergo either donor type or host type education. Besides this, adoptively transferred mature NK cells can undergo "re-education" following MHC mismatched HSCT. It is now believed that donor HSC-derived NK cells generate a KIR repertoire of donor type, thanks to the high number of infused donor cells during haplo-HSCT and both nonhematopoietic as well as hematopoietic cells may present self MHC class-I molecules to precursors of NK cells thereby are involved in NK cell education [34, 89].
