Down-regulation of the NKG2D ligand MICA by the human cytomegalovirus glycoprotein UL142

Human cytomegalovirus (HCMV) employs a variety of strategies to modify or evade the host immune response, and natural killer (NK) cells play a crucial role in controlling cytomegalovirus infections in mice and humans. Activation of NK cells through the receptor NKG2D/DAP10 leads to killing of NKG2D ligand-expressing cells. We have previously shown that HCMV is able to down-regulate the surface expression of some NKG2D ligands, ULBP1, ULBP2, and MICB via the viral glycoprotein UL16. Here, we show that the viral gene product UL142 is able to down-regulate another NKG2D ligand, MICA, leading to protection from NK cytotoxicity. UL142 is not able to affect surface expression of all MICA alleles, however, which may reflect selective pressure on the host to thwart viral immune evasion, further supporting an important role for the MICA-NKG2D interaction in immune surveillance.     

Effects of human cytomegalovirus infection on ligands for the activating NKG2D receptor of NK cells: up-regulation of UL16-binding protein (ULBP)1 and ULBP2 is counteracted by the viral UL16 protein

Human CMV (HCMV) interferes with NK cell functions at various levels. The HCMV glycoprotein UL16 binds some of the ligands recognized by the NK-activating receptor NKG2D, namely UL16-binding proteins (ULBP) 1 and 2 and MHC class I-related chain B, possibly representing another mechanism of viral immune escape. This study addressed the expression and function of these proteins in infected cells. HCMV induced the expression of all three ULBPs, which were predominantly localized in the endoplasmic reticulum of infected fibroblasts together with UL16. However, while at a lower viral dose ULBP1 and 2 surface expression was completely inhibited compared to ULBP3, at a higher viral dose cell surface expression of ULBP1 and ULBP2 was delayed. The induction of ULBPs correlated with an increased dependency on NKG2D for recognition; however, the overall NK sensitivity did not change (suggesting that additional viral mechanisms interfere with NKG2D-independent pathways for recognition). Infection with a UL16 deletion mutant virus resulted in a different pattern compared to the wild type: all three ULBP molecules were induced with similar kinetics at the cell surface, accompanied by a pronounced, entirely NKG2D-dependent increase in NK sensitivity. Together our findings show that upon infection with HCMV, the host cell responds by expression of ULBPs and increased susceptibility to the NKG2D-mediated component of NK cell recognition, but UL16 limits these effects by interfering with the surface expression of ULBP1 and ULBP2.     

UL16-binding proteins, novel MHC class I-related proteins, bind to NKG2D and activate multiple signaling pathways in primary NK cells.

The UL16-binding proteins (ULBPs) are a novel family of MHC class I-related molecules that were identified as targets of the human CMV glycoprotein, UL16. We have previously shown that ULBP expression renders a relatively resistant target cell sensitive to NK cytotoxicity, presumably by engaging NKG2D, an activating receptor expressed by NK and other immune effector cells. In this study we show that NKG2D is the ULBP counterstructure on primary NK cells and that its expression is up-regulated by IL-15 stimulation. Soluble forms of ULBPs induce marked protein tyrosine phosphorylation, and activation of the Janus kinase 2, STAT5, extracellular signal-regulated kinase, mitogen-activated protein kinase, and phosphatidylinositol 3-kinase (PI 3-kinase)/Akt signal transduction pathways. ULBP-induced activation of Akt and extracellular signal-regulated kinase and ULBP-induced IFN-gamma production are blocked by inhibitors of PI 3-kinase, consistent with the known binding of PI 3-kinase to DAP10, the membrane-bound signal-transducing subunit of the NKG2D receptor. While all three ULBPs activate the same signaling pathways, ULBP3 was found to bind weakly and to induce the weakest signal. In summary, we have shown that NKG2D is the ULBP counterstructure on primary NK cells and for the first time have identified signaling pathways that are activated by NKG2D ligands. These results increase our understanding of the mechanisms by which NKG2D activates immune effector cells and may have implications for immune surveillance against pathogens and tumors.     

ULBP4 is a novel ligand for human NKG2D

The ULBPs are a family of MHC class I-related molecules. We have previously shown that ULBPs 1, 2, and 3 are functional ligands of the NKG2D/DAP10 receptor complex on human natural killer (NK) cells. Here, we describe a new member of the ULBP family, ULBP4, which contains predicted transmembrane and cytoplasmic domains, unlike the other ULBPs, which are GPI-linked proteins. Transduction of ULBP4 into EL4 cells confers the ability to bind recombinant NKG2D and mediates increased cytotoxic activity by human NK cells, consistent with the role of ULBPs as ligands for the NKG2D/DAP10 activating receptors. Tissue expression of ULBP4 differs from other members of the family, in that it is expressed predominantly in the skin.     

Promiscuity and the single receptor: NKG2D

NKG2D (natural-killer group 2, member D) is a powerful activating receptor expressed by natural killer (NK) cells and T cells that regulates immune responses during infection, cancer and autoimmunity. NKG2D ligands comprise a diverse array of MHC-class-I-related proteins that are upregulated by cellular stress. Why is it beneficial for the host to have so many ligands for the same receptor? In this Opinion article, we propose that although competition with viruses is the most likely evolutionary drive for this diversity, there might be other explanations.     

Expression of the RAE-1 family of stimulatory NK-cell ligands requires activation of the PI3K pathway during viral infection and transformation

Natural killer (NK) cells are lymphocytes that play a major role in the elimination of virally-infected cells and tumor cells. NK cells recognize and target abnormal cells through activation of stimulatory receptors such as NKG2D. NKG2D ligands are self-proteins, which are absent or expressed at low levels on healthy cells but are induced upon cellular stress, transformation, or viral infection. The exact molecular mechanisms driving expression of these ligands remain poorly understood. Here we show that murine cytomegalovirus (MCMV) infection activates the phosphatidylinositol-3-kinase (PI3K) pathway and that this activation is required for the induction of the RAE-1 family of mouse NKG2D ligands. Among the multiple PI3K catalytic subunits, inhibition of the p110α catalytic subunit blocks this induction. Similarly, inhibition of p110α PI3K reduces cell surface expression of RAE-1 on transformed cells. Many viruses manipulate the PI3K pathway, and tumors frequently mutate the p110α oncogene. Thus, our findings suggest that dysregulation of the PI3K pathway is an important signal to induce expression of RAE-1, and this may represent a commonality among various types of cellular stresses that result in the induction of NKG2D ligands.     

Modulation of NKG2D-mediated cytotoxic functions of natural killer cells by viral protein R from HIV-1 primary isolates

HIV-1 viral protein R (Vpr) from laboratory-adapted virus strains activates the DNA damage/stress sensor ATR kinase and induces cell cycle arrest at the G(2)/M phase through a process that requires Vpr to engage the DDB1-CUL4A (VprBP/DCAF-1) E3 ligase complex. Activation of this DNA damage/stress checkpoint in G(2) by Vpr was shown to modulate NKG2D-dependent NK cell effector functions via enhancing expression of NKG2D ligands, notably ULBP2. However, it is unknown whether Vpr from HIV-1 primary isolates (groups M, N, O, and P) could modulate NKG2D-mediated cytotoxic functions of NK cells. Here, we report that Vpr from most HIV-1 primary isolates can upregulate ULBP2 expression and induce NKG2D-dependent NK cell killing. Importantly, these activities were always accompanied by an active G(2) cell cycle arrest function. Interestingly, Vpr variants from group P and a clade D isolate of group M were defective at enhancing NKG2D-mediated NK cell lysis owing to their inability to augment ULBP2 expression. However, distinct mechanisms were responsible for their failure to do so. While Vpr from group P was deficient in its ability to engage the DDB1-CUL4A (VprBP/DCAF-1) E3 ligase complex, the Vpr variant from group D was unable to properly localize to the nucleus, underlining the importance of these biological properties in Vpr function. In conclusion, the ability of Vpr from HIV-1 primary isolates to regulate NK cell effector function underscores the importance of this HIV-1 accessory protein in the modulation of the host's innate immune responses.     

Two Novel Human Cytomegalovirus NK Cell Evasion Functions Target MICA for Lysosomal Degradation

NKG2D plays a major role in controlling immune responses through the regulation of natural killer (NK) cells, αβ and γδ T-cell function. This activating receptor recognizes eight distinct ligands (the MHC Class I polypeptide-related sequences (MIC) A andB, and UL16-binding proteins (ULBP)1–6) induced by cellular stress to promote recognition cells perturbed by malignant transformation or microbial infection. Studies into human cytomegalovirus (HCMV) have aided both the identification and characterization of NKG2D ligands (NKG2DLs). HCMV immediate early (IE) gene up regulates NKGDLs, and we now describe the differential activation of ULBP2 and MICA/B by IE1 and IE2 respectively. Despite activation by IE functions, HCMV effectively suppressed cell surface expression of NKGDLs through both the early and late phases of infection. The immune evasion functions UL16, UL142, and microRNA(miR)-UL112 are known to target NKG2DLs. While infection with a UL16 deletion mutant caused the expected increase in MICB and ULBP2 cell surface expression, deletion of UL142 did not have a similar impact on its target, MICA. We therefore performed a systematic screen of the viral genome to search of addition functions that targeted MICA. US18 and US20 were identified as novel NK cell evasion functions capable of acting independently to promote MICA degradation by lysosomal degradation. The most dramatic effect on MICA expression was achieved when US18 and US20 acted in concert. US18 and US20 are the first members of the US12 gene family to have been assigned a function. The US12 family has 10 members encoded sequentially through US12–US21; a genetic arrangement, which is suggestive of an ‘accordion’ expansion of an ancestral gene in response to a selective pressure. This expansion must have be an ancient event as the whole family is conserved across simian cytomegaloviruses from old world monkeys. The evolutionary benefit bestowed by the combinatorial effect of US18 and US20 on MICA may have contributed to sustaining the US12 gene family.     

Role of NK cell-activating receptors and their ligands in the lysis of mononuclear phagocytes infected with an intracellular bacterium

We studied the role of NK cell-activating receptors and their ligands in the lysis of mononuclear phagocytes infected with the intracellular pathogen Mycobacterium tuberculosis. Expression of the activating receptors NKp30, NKp46, and NKG2D were enhanced on NK cells by exposure to M. tuberculosis-infected monocytes, whereas expression of DNAX accessory molecule-1 and 2B4 was not. Anti-NKG2D and anti-NKp46 inhibited NK cell lysis of M. tuberculosis-infected monocytes, but Abs to NKp30, DNAX accessory molecule-1, and 2B4 had no effect. Infection of monocytes up-regulated expression of the NKG2D ligand, UL-16 binding protein (ULBP)1, but not expression of ULBP2, ULBP3, or MHC class I-related chain A or chain B. Up-regulation of ULBP1 on infected monocytes was dependent on TLR2, and anti-ULBP1 abrogated NK cell lysis of infected monocytes. The dominant roles of NKp46, NKG2D, and ULBP1 were confirmed for NK cell lysis of M. tuberculosis-infected alveolar macrophages. We conclude that NKp46 and NKG2D are the principal receptors involved in lysis of M. tuberculosis-infected mononuclear phagocytes, and that ULBP1 on infected cells is the major ligand for NKG2D. Furthermore, TLR2 contributes to up-regulation of ULBP1 expression.     

Expression, purification and crystallization of the human UL16-binding protein ULBP1

UL16-binding proteins (ULBPs) are markers of cellular stress which are upregulated on the surface of virus-infected and tumor cells. Recognition of ULBP1 by the activating receptor NKG2D on the surface of cytotoxic natural killer (NK) and T cells promotes lysis of cells expressing ULBP1 and is an important mechanism of immune surveillance. We report a robust method for the generation of large quantities of crystal-grade recombinant ULBP1 protein. The extracellular portion of human ULBP1 was cloned into a T7 expression vector for expression in Escherichia coli. Unpaired cysteines in the sequence which are predicted not to be involved in the intramolecular disulfide bond formation were mutated to serine. ULBP1 was expressed in E. coli BL21 (DE3) pLysS cells as inclusion bodies. Purified inclusion bodies were solubilized by denaturation in guanidine, and refolded by slow dilution. The refolded protein was purified by size exclusion gel filtration and anion exchange chromatography. Furthermore, we have identified conditions optimal for the crystallization of this protein and have obtained initial diffraction data to 4.6? from these crystals.     

Ras activation induces expression of raet1 family NK receptor ligands

NK cells play a crucial role in innate immunity against tumors. In many human tumors, Ras is chronically active, and tumor cells frequently express ligands for the activating NK cell receptor NKG2D. In this study, we report that Ras activation upregulates the expression of Raet1 protein family members Rae1α and Rae1β in mouse and ULBP1-3 in human cells. In addition, Ras also induced MHC class I chain-related protein expression in some human cell lines. Overexpression of the constitutively active H-RasV12 mutant was sufficient to induce NKG2D ligand expression. H-RasV12-induced NKG2D ligand upregulation depended on Raf, MAPK/MEK, and PI3K, but not ATM or ATR, two PI3K-like kinases previously shown to induce NKG2D ligand expression. Analysis of the 5' untranslated regions of Raet1 family members suggested the presence of features known to impair translation initiation. Overexpression of the rate-limiting translation initiation factor eIF4E induced Rae1 and ULBP1 expression in a Ras- and PI3K-dependent manner. Upregulation of NKG2D ligands by H-RasV12 increased sensitivity of cells to NK cell-mediated cytotoxicity. In summary, our data suggest that chronic Ras activation is linked to innate immune responses, which may contribute to immune surveillance of H-Ras transformed cells.     

Interactions of human NKG2D with its ligands MICA, MICB, and homologs of the mouse RAE-1 protein family

NKG2D is an activating receptor that is expressed on most natural killer (NK) cells, CD8 alphabeta T cells, and gammadelta T cells. Among its ligands is the distant major histocompatibility complex class I homolog MICA, which has no function in antigen presentation but is induced by cellular stress. To extend previous functional evidence, the NKG2D-MICA interaction was studied in isolation. NKG2D homodimers formed stable complexes with monomeric MICA in solution, demonstrating that no other components were required to facilitate this interaction. MICA glycosylation was not essential but enhanced complex formation. Soluble NKG2D also bound to cell surface MICB, which has structural and functional properties similar to those of MICA. Moreover, NKG2D stably interacted with surface molecules encoded by three newly identified cDNA sequences (N2DL-1, -2, and -3), which are identical to the human ULBP proteins and may represent homologs of the mouse retinoic acid-early inducible family of NKG2D ligands. Because of the substantial sequence divergence among these molecules, these results indicated promiscuous modes of receptor binding. Comparison of allelic variants of MICA revealed large differences in NKG2D binding that were associated with a single amino acid substitution at position 129 in the alpha2 domain. Varying affinities of MICA alleles for NKG2D may affect thresholds of NK-cell triggering and T-cell modulation.     

Differential Mechanisms of Shedding of the Glycosylphosphatidylinositol (GPI)-anchored NKG2D Ligands

Tumor cells release NKG2D ligands to evade NKG2D-mediated immune surveillance. The purpose of our investigation was to explore the cellular mechanisms of release used by various members of the ULBP family. Using biochemical and cellular approaches in both transfectant systems and tumor cell lines, this paper shows that ULBP1, ULBP2, and ULBP3 are released from cells with different kinetics and by distinct mechanisms. Whereas ULBP2 is mainly shed by metalloproteases, ULBP3 is abundantly released as part of membrane vesicles known as exosomes. Interestingly, exosomal ULBP3 protein is much more potent for down-modulation of the NKG2D receptor than soluble ULBP2 protein. This is the first report showing functionally relevant differences in the biochemistry of the three members of the ULBP family and confirms that in depth study of the biochemical features of individual NKG2D ligands will be necessary to understand and manipulate the biology of these proteins for therapy.     

The NKG2D receptor: sensing stressed cells

The activating killer cell lectin-like receptor NKG2D plays a key role in the natural killer (NK) cell-mediated lysis of tumours and infected cells. Unlike other receptors, the ligands recognised by NKG2D are 'induced-self' ligands on stressed cells. This system requires precise regulation because inappropriate expression of NKG2D ligands might compromise NK cell activation. For therapeutic purposes it is essential to understand the mechanisms that regulate the expression and function of the NKG2D system. This review focuses on the importance of the signalling pathways involved in the regulation of the NKG2D receptor and its ligand expression in arming the immune response against infected or tumour cells and for the identification of new molecular targets and therapeutic strategies.     

Generation, affinity maturation, and characterization of a human anti-human NKG2D monoclonal antibody with dual antagonistic and agonistic activity

In humans, NKG2D is an activating receptor on natural killer (NK) cells and a costimulatory receptor on certain T cells and plays a central role in mediating immune responses in autoimmune diseases, infectious diseases, and cancer. Monoclonal antibodies that antagonize or agonize immune responses mediated by human NKG2D are considered to be of broad and potent therapeutic utility. Nonetheless, monoclonal antibodies to NKG2D that are suitable for clinical investigations have not been published yet. Here, we describe the generation, affinity maturation, and characterization of a fully human monoclonal antibody to human NKG2D. Using phage display technology based on a newly generated naïve human Fab library in phage display vector pC3C followed by a tandem chain shuffling process designed for minimal deviation from natural human antibody sequences, we selected a human Fab, designated KYK-2.0, with high specificity and affinity to human NKG2D. KYK-2.0 Fab blocked the binding of the natural human NKG2D ligands MICA, MICB, and ULBP2 as potently as a commercially available mouse anti-human NKG2D monoclonal antibody in immunoglobulin G (IgG) format. Conversion of KYK-2.0 Fab to IgG1 resulted in subnanomolar avidity for human NKG2D. KYK-2.0 IgG1 was found to selectively recognize defined subpopulations of human lymphocytes known to express NKG2D, that is, the majority of human CD8+, CD16+, and CD56+ cells as well as a small fraction of human CD4+ cells. In solution, KYK-2.0 IgG1 interfered with the cytolytic activity of ex vivo expanded human NK cells. By contrast, immobilized KYK-2.0 IgG1 was found to strongly induce human NK cell activation. The dual antagonistic and agonistic activity promises a wide range of therapeutic applications for KYK-2.0 IgG1 and its derivatives.     

自然杀伤细胞受体及其配体表达的转录和表观遗传调控

自然杀伤(natural killer cell,NK)细胞受体及其配体在NK细胞发挥抗病毒、抗肿瘤和免疫调节作用中起重要作用.NK细胞功能的发挥取决于NK细胞受体及其配体的表达水平和其所传递信号的综合.病毒、肿瘤和热休克等刺激可以通过激活相应的转录调节因子,提高启动子活性而上调NKG2家族受体及其配体的表达,而启动子区DNA的甲基化状态、组蛋白的乙酰化和甲基化等表观遗传调控,在NK细胞受体及其配体的表达方面亦起重要作用,并决定NK细胞受体的克隆性分布.深入探讨NK细胞受体及其配体的表达调控机制,将为提高NK.细胞抗肿瘤和抗感染疗效提供新的策略.     

Importance of NKG2D-NKG2D ligands interaction for cytolytic activity of natural killer cell

In this study, we investigate the relationship between natural killer (NK) cell susceptibility and the surface markers of cancer cells. Through phenotypic analysis, we found evidence that more susceptible cancer cell lines (K562 and Jurkat) express more NKG2D ligands. Major histocompatibility complex (MHC) class I chain-related A/B (MIC-A/B) and UL16 binding protein (ULBP) 1-5 molecules are typical ligands of NKG2D. The high killing activity of NK cells against K562 was abolished through the addition of a NKG2D blocking antibody. Upon in vitro stimulation with quercetin, low susceptible cancer cells increased NKG2D ligand expression, leading to enhancement of NK cell cytolytic activity. These results suggested that the anti-cancer activity of NK cells is not dependent on the origin and growth style of the target cells, but is dependent on the surface markers of the target cells.     

NKG2D is required for NK cell activation and function in response to E1-deleted adenovirus

Despite high transduction efficiency in vivo, the application of recombinant E1-deleted adenoviral vectors for in vivo gene therapy has been limited by the attendant innate and adaptive immune responses to adenoviral vectors. NK cells have been shown to play an important role in innate immune elimination of adenoviral vectors in vivo. However, the mechanisms underlying NK cell activation and function in response to adenoviral vectors remain largely undefined. In this study, we showed that NK cell activation upon adenoviral infection was dependent on accessory cells such as dendritic cells and macrophages and that cell contact-dependent signals from the accessory cells are necessary for NK cell activation. We further demonstrated that ligands of the NK activating receptor NKG2D were upregulated in accessory cells upon adenoviral infection and that blockade of NKG2D inhibited NK cell activation upon adenoviral infection, leading to a delay in adenoviral clearance in vivo. In addition, NKG2D was required for NK cell-mediated cytolysis on adenovirus-infected targets. Taken together, these results suggest that efficient NK cell activation and function in response to adenoviral infection is critically dependent on the NKG2D pathway, which understanding may assist in the design of effective strategies to improve the outcome of adenovirus-mediated gene therapy.     

Direct TLR2 Signaling Is Critical for NK Cell Activation and Function in Response to Vaccinia Viral Infection

Natural killer (NK) cells play an essential role in innate immune control of poxviral infections in vivo. However, the mechanism(s) underlying NK cell activation and function in response to poxviruses remains poorly understood. In a mouse model of infection with vaccinia virus (VV), the most studied member of the poxvirus family, we identified that the Toll-like receptor (TLR) 2-myeloid differentiating factor 88 (MyD88) pathway was critical for the activation of NK cells and the control of VV infection in vivo. We further showed that TLR2 signaling on NK cells, but not on accessory cells such as dendritic cells (DCs), was necessary for NK cell activation and that this intrinsic TLR2-MyD88 signaling pathway was required for NK cell activation and played a critical role in the control of VV infection in vivo. In addition, we showed that the activating receptor NKG2D was also important for efficient NK activation and function, as well as recognition of VV-infected targets. We further demonstrated that VV could directly activate NK cells via TLR2 in the presence of cytokines in vitro and TLR2-MyD88-dependent activation of NK cells by VV was mediated through the phosphatidylinositol 3-kinase (PI3K)-extracellular signal-regulated kinase (ERK) pathway. Taken together, these results represent the first evidence that intrinsic TLR signaling is critical for NK cell activation and function in the control of a viral infection in vivo, indicate that multiple pathways are required for efficient NK cell activation and function in response to VV infection, and may provide important insights into the design of effective strategies to combat poxviral infections.