Mica Micb

Granule Movement

family are the key effectors in executing NK-mediated cytotoxicity. Upon receptor engagement of its ligand, adaptor proteins that associate with the receptor through charged interactions activate a signal cascade either via PI3K or Syk.Zap70, which ultimately results in the phosphorylation of ERK. ERK activation leads to granule exocytosis and lysis of the target cell.

to induce cytoxicity through the DAP10 protein.

NKG2D is a highly conserved and important receptor on NK cells for lysis of altered self. Its ligands are present in embryonal tissues and are lost in mature organs. On the other hand, these ligands are upregu-lated in cellular distress situations, as occurs during viral infection and transformation (Cosman et al, 2001; Moretta et al, 2006). Heat shock proteins produced during transformation can promote MICA and MICB gene expression through the heat shock elements present on the promoter regions of these genes (Bauer et al, 1999). Indeed, a variety of tumors, including leukemia, hep-atoma, melanoma, and prostate cancer, have been demonstrated to express MICA and

MICB, thereby providing ligands for NK recognition (Groh et al, 2002; Salih et al, 2003; Holdenrieder et al, 2006). Other equally important ligands of NKG2D were discovered through studies of human cyto-megalovirus (HCMV). HCMV expresses the protein UL16 that functions in immune evasion by blocking the expression of ULBPs, such as RAET1 that bind to UL16 (Cosman et al., 2001). Notably, these proteins were found to be expressed on tumor cells (Radosavljevic et al., 2002). All NKG2D ligands, although diverse in nature, can associate with NKG2D because they express the same amino acid residues in key binding regions as well as have a similar alpha domain structure (McFarland et al, 2003). Thus, NK cell promiscuity in killing unre-

Kills Target Protects Target

HLA-Class I

HLA-Class I Ligand

___Target Cell Membrane

FIGURE 5.2 Model of activating and inhibitory KIRs. Activating KIRs are structurally distinct from inhibiting receptors in that activating receptors have a short cytoplasmic tail. As a result of the shorter cytoplasmic tail, activating KIRs do not contain an ITIM. The ITAM-containing adaptor protein DAP12 associates with the activating KIR. Upon ligand engagement, the tyrosines in the ITAM (YxxL x 6-8 YxxL) of DAP12 are phosphorylated and become a binding site for the two SH2 domains of SYK/ZAP70 kinase. Activation of SYK/ ZAP70 ultimately leads to ERK activation and granule movement. In the case of inhibitory KIRs, binding to an HLA-class I ligand leads to phosphorylation of the tyrosines in the ITIM (YxxL x 28-32 YxxL) in the cytoplasmic tail. These phosphotyrosines specifically bind the SH2 domains of the tyrosine phosphatase SHP-1. Recruitment of SHP-1 leads to its activation and results in inhibition of NK-mediated cytotoxicity by dephosphorylating essential signal molecules, such as Syk/ZAP70 and ERK. For an inhibiting KIR to prevent NK stimulation, it must be in close proximity to the activating KIR. This occurs during a process known as KIR clustering, and the initial kinases that are triggered by an activating receptor are responsible for phosphorylation of the nearby ITIM in the inhibitory receptor. Thus, inhibitory receptors work to shut down the function of an activating receptor.

FIGURE 5.2 Model of activating and inhibitory KIRs. Activating KIRs are structurally distinct from inhibiting receptors in that activating receptors have a short cytoplasmic tail. As a result of the shorter cytoplasmic tail, activating KIRs do not contain an ITIM. The ITAM-containing adaptor protein DAP12 associates with the activating KIR. Upon ligand engagement, the tyrosines in the ITAM (YxxL x 6-8 YxxL) of DAP12 are phosphorylated and become a binding site for the two SH2 domains of SYK/ZAP70 kinase. Activation of SYK/ ZAP70 ultimately leads to ERK activation and granule movement. In the case of inhibitory KIRs, binding to an HLA-class I ligand leads to phosphorylation of the tyrosines in the ITIM (YxxL x 28-32 YxxL) in the cytoplasmic tail. These phosphotyrosines specifically bind the SH2 domains of the tyrosine phosphatase SHP-1. Recruitment of SHP-1 leads to its activation and results in inhibition of NK-mediated cytotoxicity by dephosphorylating essential signal molecules, such as Syk/ZAP70 and ERK. For an inhibiting KIR to prevent NK stimulation, it must be in close proximity to the activating KIR. This occurs during a process known as KIR clustering, and the initial kinases that are triggered by an activating receptor are responsible for phosphorylation of the nearby ITIM in the inhibitory receptor. Thus, inhibitory receptors work to shut down the function of an activating receptor.

lated target cells can be explained by various target cells expressing different NKG2D ligands that trigger NK cells via a common receptor.

In addition to viral infection and tumor transformation, other pathological conditions can significantly increase the expression of NKG2D ligands. DNA damage in cells has been shown to have a key role in this expression. DNA damaging agents and DNA replication inhibitors induced the expression of NKG2D ligands in cells, while inhibition of ATM or ATR, which are involved in detecting double-stranded DNA breaks or stalled DNA replication, respectively, blocked induction of NKG2D ligands (Gasser et al, 2005). This DNA damage response may also operate in precancerous lesions, resulting in cells that display NKG2D ligands that tag themselves for NK recognition. Genotoxic stress, microbial infection, and cell transformation all constitute types of cellular alterations in which induction of NKG2D ligands acts as an indicator of cell stress. Therefore, elevating the expression of NKG2D ligands is a common natural means to alert the immune system to the presence of disease. It is also important to note that NKG2D can trigger NK cytotoxicity despite MHC class I expression on the target cell, suggesting that this receptor, when activated, can override the protective signal of self MHC class I as long as the danger signal provided by the NKG2D ligand is available (Diefenbach et al, 2001).

In the mouse system, NKG2D is able to bind three subfamilies of RAET1 genes, Rael, histocompatibility 60 (H60), and mouse UL-16 binding protein 1 (Multl) (Dienfenbach et al, 2001). Although these genes are structurally related and localized on chromosome 10, they share little amino acid sequence homology. Rae1 is a lipid-linked membrane protein, and H60 is a transmembrane protein. No MIC analog has been found in mice. As in humans, tumor formation in mice is associated with upregulation of these NKG2D ligands. Interestingly, IFN-y has been well-known to induce NK resistance in tumor cells. Indeed, it was found that IFN-y downregulates the expression of the NKG2D ligand H60 on methylcholanthrene (MCA)-induced sarcomas concurrently with enhancing the expression of MHC class I molecules (Bui et al., 2006). These cooperative effects resulted in a dramatic decrease in the sensitivity of MCA-induced sarcomas to NK-mediated cytotoxicity. On the other hand, IFN-y treatment has the opposite effect of activating CTLs, an event that requires the specific interaction of MHC class I with the target antigen. Therefore, it has been proposed that IFN-y may serve to inhibit innate immunity while activating adaptive immunity, thereby facilitating progression toward a long-term T cell memory response (Bui et al, 2006). In contrast to this mouse model, IFN-y has been found to have a different effect on human acute myeloid lymphoma (AML). AML cells are typically very resistant to lysis by NK cells. In vivo, treatment of these cells with IFN-y and growth factors that promote myeloid differentiation resulted in a robust upregulation of ULBP1 and other NK receptor ligands, whereas treatment with the growth factors alone had only a minimal effect (Nowbakht et al., 2005). The combination treatment resulted in gain of sensitivity of AML cells to lysis by NK cells. However, increased sensitivity to NK-mediated cell lysis was accomplished only by blocking MHC class I expression. Therefore the response to IFN-y may be complex depending on the type of tumor and the factors that are present in the environment.

Like all other NK activation receptors, NKG2D has a positively charged amino acid in its transmembrane region that is vitally important for its association with the adapter protein, DAP 10, which contains a negative charge in its transmembrane domain. NKG2D is the only NK receptor that associates with DAP10 (Wu et al, 1999). Interestingly, DAP10 does not contain an ITAM like the other adapter proteins but rather an YxxM motif (x donates any amino acid) that serves as a consensus PI3K binding site. Upon ligand interaction with NKG2D, the tyrosine in the YxxM motif of DAP10 becomes phosphorylated to bind the SH2 domain of PI3K. Binding to DAP triggers PI3K activation, leading to downstream signal events that result in lytic granule movement (Jiang et al, 2000). A sequential phosphorylation of Rac, p21-activated kinase (PAK), extracellular signal-regulated kinase (ERK), and mitogen-activated protein kinase/ERK (MEK) occurs after PI3K activation, and it is ERK that appears to mobilize the lytic granules, as shown in Figure 5.2 (Djeu et al, 2002).

Besides directly inducing target cell lysis, NKG2D engagement also results in the pro duction of multiple cytokines and chemo-kines. Specifically, binding of NKG2D to MICA/B or ULBP binding results in the production of IFN-y TNF-a, lymphotoxin, and granulocyte-macrophage colony-stimulating factor (GM-CSF) (Bauer et al, 1999; Cosman et al, 2001). In addition, the chemokines CCL4 and CCL1 are secreted upon NKG2D-ULBP binding. These cytokines and chemokines augment the immune processes to eliminate microbial pathogens and transformed cells.

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