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.     

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.     

Immunohistochemical Validation and Expression Profiling of NKG2D Ligands in a Wide Spectrum of Human Epithelial Neoplasms

The MHC class I-chain-related proteins (MICs) and the UL16-binding proteins (ULBPs) are inducible stress response molecules that work as activators of a specific receptor, NKG2D, which is expressed on effector cells, such as NK cells and subsets of T cells. In this study, we sought to explore the biological significance of NKG2D ligands in human neoplasms by comprehensively examining the immunohistochemical expression profile of NKG2D ligands in a variety of human epithelial neoplasms. Following careful validation of the immunohistochemical specificity and availability of anti-human ULBP antibodies for formalin-fixed paraffin-embedded (FFPE) materials, the expression of NKG2D ligands was analyzed in FFPE tissue microarrays comprising 22 types of epithelial neoplastic tissue with their non-neoplastic counterpart from various organs. Hierarchical cluster analysis demonstrated a positive relationship among ULBP2/6, ULBP3, ULBP1, and ULBP5, whose expression patterns were similar across all of the neoplastic tissues examined. In contrast, MICA/B, as well as ULBP4, did not appear to be related to any other ligand. These expression profiles of NKG2D ligands in human neoplasms based on well-validated specific antibodies, followed by hierarchical cluster analysis, should help to clarify some functional aspects of these molecules in cancer biology, and also provide a path to the development of novel tumor-type-specific treatment strategies.     

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.     

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.     

The human cytomegalovirus glycoprotein UL16 traffics through the plasma membrane and the nuclear envelope

The human cytomegalovirus (HCMV) UL16 gene encodes a glycoprotein that interferes with the immune response to the virus-infected cell. In vitro, UL16 interacts with MICB and ULBPs that are ligands for the stimulatory receptor NKG2D, expressed on NK cells and CD8(+)T cells. UL16 expression has been shown to promote intracellular accumulation of MICB, ULBP1 and 2 and thus, interfere with the immune response to HCMV-infected cells. The mechanism that has been suggested for UL16-mediated MICB downmodulation is retention in the ER. Here, we studied the intracellular localization and maturation of UL16 and MICB in HCMV-infected cells and transfectant systems. UL16 trafficked through the ER, TGN and progressed to the plasma membrane, after which the protein was internalized. Strikingly, UL16 was also observed in the inner nuclear membrane. MICB was also localized in the TGN in HCMV-infected cells. These data suggest that MICB trafficking might be affected after its transit through the ER.     

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.     

Human cytomegalovirus-encoded UL16 discriminates MIC molecules by their alpha2 domains

Human CMV infection results in MHC class I down-regulation and induction of NKG2D ligand expression favoring NK recognition of infected cells. However, human CMV-encoded UL16 counteracts surface expression of several NKG2D ligands by intracellular retention. Interestingly, UL16 interacts with MICB, but not with the closely related MICA, and with UL16-binding proteins (ULBP) ULBP1 and ULBP2, which are only distantly related to MICB, but not with ULPB3 or ULBP4, although all constitute ligands for NKG2D. Here, we dissected the molecular basis of MICA-MICB discrimination by UL16 to elucidate its puzzling binding behavior. We report that the UL16-MICB interaction is independent of glycosylation and demonstrate that selective MICB recognition by UL16 is governed by helical structures of the MICB alpha2 domain. Transplantation of the MICB alpha2 domain confers UL16 binding capacity to MICA, and thus, diversification of the MICA alpha2 domain may have been driven by the selective pressure exerted by UL16.     

Structure of the HCMV UL16-MICB Complex Elucidates Select Binding of a Viral Immunoevasin to Diverse NKG2D Ligands

The activating immunoreceptor NKG2D promotes elimination of infected or malignant cells by cytotoxic lymphocytes through engagement of stress-induced MHC class I-related ligands. The human cytomegalovirus (HCMV)-encoded immunoevasin UL16 subverts NKG2D-mediated immune responses by retaining a select group of diverse NKG2D ligands inside the cell. We report here the crystal structure of UL16 in complex with the NKG2D ligand MICB at 1.8 Å resolution, revealing the molecular basis for the promiscuous, but highly selective, binding of UL16 to unrelated NKG2D ligands. The immunoglobulin-like UL16 protein utilizes a three-stranded β-sheet to engage the α-helical surface of the MHC class I-like MICB platform domain. Intriguingly, residues at the center of this β-sheet mimic a central binding motif employed by the structurally unrelated C-type lectin-like NKG2D to facilitate engagement of diverse NKG2D ligands. Using surface plasmon resonance, we find that UL16 binds MICB, ULBP1, and ULBP2 with similar affinities that lie in the nanomolar range (12–66 nM). The ability of UL16 to bind its ligands depends critically on the presence of a glutamine (MICB) or closely related glutamate (ULBP1 and ULBP2) at position 169. An arginine residue at this position however, as found for example in MICA or ULBP3, would cause steric clashes with UL16 residues. The inability of UL16 to bind MICA and ULBP3 can therefore be attributed to single substitutions at key NKG2D ligand locations. This indicates that selective pressure exerted by viral immunoevasins such as UL16 contributed to the diversification 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.     

NK cells lyse T regulatory cells that expand in response to an intracellular pathogen

We evaluated the capacity of NK cells to influence expansion of CD4(+)CD25(+)FoxP3(+) regulatory T cells (Tregs) in response to microbial Ags, using Mycobacterium tuberculosis as a model. We previously found that Tregs expand when CD4(+) cells and monocytes are exposed to M. tuberculosis. Addition of NK cells that were activated by monokines (IL-12, IL-15, and IL-18) or by exposure to M. tuberculosis-stimulated monocytes reduced Treg expansion in response to M. tuberculosis. NK cell inhibition of Treg expansion was not mediated through IFN-gamma. Activated NK cells lysed expanded, but not freshly isolated Tregs. Although monokines increased NK cell expression of the activating receptors NKp46, NKG2D, 2B4, CD16, and DNAM-1, only anti-NKG2D and anti-NKp46 inhibited NK cell lysis of expanded Tregs. Of five NKG2D ligands, only UL16-binding protein 1 (ULBP1) was up-regulated on M. tuberculosis-expanded Tregs, and anti-ULBP1 inhibited NK cell lysis of expanded Tregs. M. tuberculosis-stimulated monocytes activated NK cells to lyse expanded Tregs, and this was also inhibited by anti-NKG2D and anti-ULBP1, confirming the physiological relevance of this effect. Our study identifies a potential new role for NK cells in maintaining the delicate balance between the regulatory and effector arms of the immune response.     

An identical miRNA of the human JC and BK polyoma viruses targets the stress-induced ligand ULBP3 to escape immune elimination

The human polyoma viruses JCV and BKV establish asymptomatic persistent infection in 65%-90% of humans but can cause severe illness under immunosuppressive conditions. The mechanisms by which these viruses evade immune recognition are unknown. Here we show that a viral miRNA identical in sequence between JCV and BKV targets the stress-induced ligand ULBP3, which is a protein recognized by the killer receptor NKG2D. Consequently, viral miRNA-mediated ULBP3 downregulation results in reduced NKG2D-mediated killing of virus-infected cells by natural killer (NK) cells. Importantly, when the activity of the viral miRNA was inhibited during infection, NK cells killed the infected cells more efficiently. Because NKG2D is also expressed by various T cell subsets, we propose that JCV and BKV use an identical miRNA that targets ULBP3 to escape detection by both the innate and adaptive immune systems, explaining how these viruses remain latent without being eliminated by the immune system.     

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.     

Syk regulation of phosphoinositide 3-kinase-dependent NK cell function

Emerging evidence suggests that NK-activatory receptors use KARAP/DAP12, CD3zeta, and FcepsilonRIgamma adaptors that contain immunoreceptor tyrosine-based activatory motifs to mediate NK direct lysis of tumor cells via Syk tyrosine kinase. NK cells may also use DAP10 to drive natural cytotoxicity through phosphoinositide 3-kinase (PI3K). In contrast to our recently identified PI3K pathway controlling NK cytotoxicity, the signaling mechanism by which Syk associates with downstream effectors to drive NK lytic function has not been clearly defined. In NK92 cells, which express DAP12 but little DAP10/NKG2D, we now show that Syk acts upstream of PI3K, subsequently leading to the specific signaling of the PI3K-->Rac1-->PAK1-->mitogen-activated protein/extracellular signal-regulated kinase (ERK) kinase-->ERK cascade that we earlier described. Tumor cell ligation stimulated DAP12 tyrosine phosphorylation and its association with Syk in NK92 cells; Syk tyrosine phosphorylation and activation were also observed. Inhibition of Syk function by kinase-deficient Syk or piceatannol blocked target cell-induced PI3K, Rac1, PAK1, mitogen-activated protein/ERK kinase, and ERK activation, perforin movement, as well as NK cytotoxicity, indicating that Syk is upstream of all these signaling events. Confirming that Syk does not act downstream of PI3K, constitutively active PI3K reactivated all the downstream effectors as well as NK cytotoxicity suppressed in Syk-impaired NK cells. Our results are the first report documenting the instrumental role of Syk in control of PI3K-dependent natural cytotoxicity.     

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.     

The protein-tyrosine phosphatase TCPTP regulates epidermal growth factor receptor-mediated and phosphatidylinositol 3-kinase-dependent signaling.

In this study we have investigated the down-regulation of epidermal growth factor (EGF) receptor signaling by protein-tyrosine phosphatases (PTPs) in COS1 cells. The 45-kDa variant of the PTP TCPTP (TC45) exits the nucleus upon EGF receptor activation and recognizes the EGF receptor as a cellular substrate. We report that TC45 inhibits the EGF-dependent activation of the c-Jun N-terminal kinase, but does not alter the activation of extracellular signal-regulated kinase 2. These data demonstrate that TC45 can regulate selectively mitogen-activated protein kinase signaling pathways emanating from the EGF receptor. In EGF receptor-mediated signaling, the protein kinase PKB/Akt and the mitogen-activated protein kinase c-Jun N-terminal kinase, but not extracellular signal-regulated kinase 2, function downstream of phosphatidylinositol 3-kinase (PI 3-kinase). We have found that TC45 and the TC45-D182A mutant, which is capable of forming stable complexes with TC45 substrates, inhibit almost completely the EGF-dependent activation of PI 3-kinase and PKB/Akt. TC45 and TC45-D182A act upstream of PI 3-kinase, most likely by inhibiting the recruitment of the p85 regulatory subunit of PI 3-kinase by the EGF receptor. Recent studies have indicated that the EGF receptor can be activated in the absence of EGF following integrin ligation. We find that the integrin-mediated activation of PKB/Akt in COS1 cells is abrogated by the specific EGF receptor protein-tyrosine kinase inhibitor tyrphostin AG1478, and that TC45 and TC45-D182A can inhibit activation of PKB/Akt following the attachment of COS1 cells to fibronectin. Thus, TC45 may serve as a negative regulator of growth factor or integrin-induced, EGF receptor-mediated PI 3-kinase signaling.