The human cytomegalovirus protein UL147A downregulates the most prevalent MICA allele: MICA*008, to evade NK cell-mediated killing

Natural killer (NK) cells are innate immune lymphocytes capable of killing target cells without prior sensitization. One pivotal activating NK receptor is NKG2D, which binds a family of eight ligands, including the major histocompatibility complex (MHC) class I-related chain A (MICA). Human cytomegalovirus (HCMV) is a ubiquitous betaherpesvirus causing morbidity and mortality in immunosuppressed patients and congenitally infected infants. HCMV encodes multiple antagonists of NK cell activation, including many mechanisms targeting MICA. However, only one of these mechanisms, the HCMV protein US9, counters the most prevalent MICA allele, MICA*008. Here, we discover that a hitherto uncharacterized HCMV protein, UL147A, specifically downregulates MICA*008. UL147A primarily induces MICA*008 maturation arrest, and additionally targets it to proteasomal degradation, acting additively with US9 during HCMV infection. Thus, UL147A hinders NKG2D-mediated elimination of HCMV-infected cells by NK cells. Mechanistic analyses disclose that the non-canonical GPI anchoring pathway of immature MICA*008 constitutes the determinant of UL147A specificity for this MICA allele. These findings advance our understanding of the complex and rapidly evolving HCMV immune evasion mechanisms, which may facilitate the development of antiviral drugs and vaccines.     

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.     

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.     

Interaction of monocytes with NK cells upon Toll-like receptor-induced expression of the NKG2D ligand MICA

Reciprocal interactions between NK cells and dendritic cells have been shown to influence activation of NK cells, maturation, or lysis of dendritic cells and subsequent adaptive immune responses. However, little is known about the crosstalk between monocytes and NK cells and the receptors involved in this interaction. We report in this study that human monocytes, upon TLR triggering, up-regulate MHC class I-Related Chain (MIC) A, but not other ligands for the activating immunoreceptor NKG2D like MICB or UL-16 binding proteins 1-3. MICA expression was associated with CD80, MHC class I and MHC class II up-regulation, secretion of proinflammatory cytokines, and apoptosis inhibition, but was not accompanied by release of MIC molecules in soluble form. TLR-induced MICA on the monocyte cell surface was detected by autologous NK cells as revealed by NKG2D down-regulation. Although MICA expression did not render monocytes susceptible for NK cell cytotoxicity, LPS-treated monocytes stimulated IFN-gamma production of activated NK cells which was substantially dependent on MICA-NKG2D interaction. No enhanced NK cell proliferation or cytotoxicity against third-party target cells was observed after stimulation of NK cells with LPS-activated monocytes. Our data indicate that MICA-NKG2D interaction constitutes a mechanism by which monocytes and NK cells as an early source of IFN-gamma may communicate directly during an innate immune response to infections in humans.     

MicroRNA Editing Facilitates Immune Elimination of HCMV Infected Cells

The human cytomegalovirus (HCMV) is extremely prevalent in the human population. Infection by HCMV is life threatening in immune compromised individuals and in immune competent individuals it can cause severe birth defects, developmental retardation and is even associated with tumor development. While numerous mechanisms were developed by HCMV to interfere with immune cell activity, much less is known about cellular mechanisms that operate in response to HCMV infection. Here we demonstrate that in response to HCMV infection, the expression of the short form of the RNA editing enzyme ADAR1 (ADAR1-p110) is induced. We identified the specific promoter region responsible for this induction and we show that ADAR1-p110 can edit miR-376a. Accordingly, we demonstrate that the levels of the edited-miR-376a (miR-376a(e)) increase during HCMV infection. Importantly, we show that miR-376a(e) downregulates the immune modulating molecule HLA-E and that this consequently renders HCMV infected cells susceptible to elimination by NK cells.     

A short isoform of human cytomegalovirus US3 functions as a dominant negative inhibitor of the full-length form

Human cytomegalovirus encodes four unique short (US) region proteins, each of which is independently sufficient for causing the down-regulation of major histocompatibility complex (MHC) class I molecules on the cell surface. This down-regulation enables infected cells to evade recognition by cytotoxic T lymphocytes (CTLs) but makes them vulnerable to lysis by natural killer (NK) cells, which lyse those cells that lack MHC class I molecules. The 22-kDa US3 glycoprotein is able to down-regulate the surface expression of MHC class I molecules by dual mechanisms: direct endoplasmic reticulum retention by physical association and/or tapasin inhibition. The alternative splicing of the US3 gene generates two additional products, including 17-kDa and 3.5-kDa truncated isoforms; however, the functional significance of these isoforms during viral infection is unknown. Here, we describe a novel mode of self-regulation of US3 function that uses the endogenously produced truncated isoform. The truncated isoform itself neither binds to MHC class I molecules nor prevents the full-length US3 from interacting with MHC class I molecules. Instead, the truncated isoform associates with tapasin and competes with full-length US3 for binding to tapasin; thus, it suppresses the action of US3 that causes the disruption of the function of tapasin. Our results indicate that the truncated isoform of the US3 locus acts as a dominant negative regulator of full-length US3 activity. These data reflect the manner in which the virus has developed temporal survival strategies during viral infection against immune surveillance involving both CTLs and NK cells.     

Human NK cells inhibit cytomegalovirus replication through a noncytolytic mechanism involving lymphotoxin-dependent induction of IFN-beta

NK cells play a key role in host defense against the beta-herpesvirus CMV through perforin-dependent cytolysis. In this study, we show that human NK cells can also control human CMV (HCMV) infection by a noncytolytic mechanism involving induction of IFN-beta in the virus-infected cell. Both IL-2-activated primary NK cells and an IL-2-dependent NK cell line (NK-92) exhibited potent, noncytolytic anti-HCMV activity at very low E:T cell ratios (<0.1:1). Activated NK cells expressed lymphotoxin (LT)alphabeta on their cell surface, and secreted LTalpha and TNF, all of which contributed to the NF-kappaB-dependent release of IFN-beta from infected fibroblasts. IFN-beta produced by fibroblasts and NK cell-produced IFN-gamma combined to inhibit HCMV replication after immediate early gene expression. These results highlight an efficient mechanism used by NK cells to activate IFN-beta expression in the infected target cell that contributes to the arrest of virion production and virus spread without cellular elimination.     

Cutting edge: down-regulation of MICA on human tumors by proteolytic shedding

The immunoreceptor NKG2D stimulates tumor immunity through activation of CD8 T cells and NK cells. Its ligand MICA has been shown to be broadly expressed on human tumors of epithelial origin. MICA expression correlates with an enrichment of Vdelta1 T cells in tumor tissue. We report that human tumor cells spontaneously release a soluble form of MICA encompassing the three extracellular domains, which is present at high levels in sera of patients with gastrointestinal malignancies, but not in healthy donors. Release of MICA from tumor cells is blocked by inhibition of metalloproteinases, concomitantly causing accumulation of MICA on the cell surface. Shedding of MICA by tumor cells may modulate NKG2D-mediated tumor immune surveillance. In addition, determination of soluble MICA levels may be implemented as an immunological diagnostic marker in patients with epithelial malignancies.     

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.     

The activating NKG2D ligand MHC class I-related chain A transfers from target cells to NK cells in a manner that allows functional consequences

Recently, it has become apparent that surface proteins commonly transfer between immune cells in contact. Inhibitory receptors and ligands exchange between cells during NK cell surveillance and we report here that NK cells also acquire activating ligands from target cells. Specifically, the stress-inducible activating ligand for NKG2D, MHC class I-related chain A (MICA), transferred to NK cells upon conjugation with MICA-expressing target cells. Acquisition of MICA from target cells was dependent on cell contact and occurred after accumulation of MICA at the immunological synapse. Moreover, transfer of MICA was facilitated by specific molecular recognition via NKG2D and augmented by Src kinase signaling. Importantly, MICA associated with its new host NK cell membrane in an orientation that allowed engagement with NKG2D in trans and indeed could down-regulate NKG2D in subsequent homotypic interactions with other NK cells. MICA captured from target cells could subsequently transfer between NK cells and, more importantly, NK cell degranulation was triggered in such NK cell-NK cell interactions. Thus, NK cells can influence other NK cells with proteins acquired from target cells and our data specifically suggest that NK cells could lyse other NK cells upon recognition of activating ligands acquired from target cells. This mechanism could constitute an important function for immunoregulation of NK cell activity.     

IFN-gamma production dominates the early human natural killer cell response to Coxsackievirus infection

Coxsackieviruses (CV) are important human pathogens that have been implicated in the pathogenesis of several diseases, including myocarditis and pancreatitis. How the human immune system recognizes and controls CV infections is not well understood. Studies in mice suggest that natural killer (NK) cells play a critical role in viral clearance and host survival, but the mechanism(s) by which human NK cells may contribute to the host anti-CV defence has not been investigated. Here we show that CVB3 infection markedly reduces HLA class I cell surface expression but does not increase the expression of the activating NK cell receptor ligands MICA/B and ULBP1-3 on human cells. We also demonstrate that the lowered target cell HLA class I surface expression does not correlate with an increased susceptibility to NK cell-mediated killing. However, NK cells responded with a robust production of interferon γ (IFN-γ) when peripheral blood mononuclear cells were cocultured with infected cells. In summary, this study shows that CVB3 interferes with the expression of NK cell receptor ligands on infected cells and indicates that IFN-γ production, rather than cytotoxicity, marks the early human NK cell response to CVB3 infection.     

Modulation of MICA on the surface of Chlamydia trachomatis-infected endocervical epithelial cells promotes NK cell-mediated killing

Chlamydia trachomatis serovars D-K are obligate intracellular bacteria that have tropism for the columnar epithelial cells of the genital tract. Chlamydia trachomatis infection has been reported to induce modifications in immune cell ligand expression on epithelial host cells. In this study, we used an in vitro infection model that resulted in a partial infection of C. trachomatis-exposed primary-like immortalized endocervical epithelial cells (A2EN). Using this model, we demonstrated that expression of the natural killer (NK) cell activating ligand, MHC class I-related protein A (MICA), was upregulated on C. trachomatis-infected, but not on noninfected bystander cells. MICA upregulation was concomitant with MHC class I downregulation and impacted the susceptibility of C. trachomatis-infected cells to NK cell activity. The specificity of MICA upregulation was reflected by a higher cytolytic activity of an NK cell line (NK92MI) against C. trachomatis-infected cells compared with uninfected control cells. Significantly, data also indicated that NK cells exerted a partial, but incomplete sterilizing effect on C. trachomatis as shown by the reduction in recoverable inclusion forming units (IFU) when cocultured with C. trachomatis-infected cells. Taken together, our data suggest that NK cells may play a significant role in the ability of the host to counter C. trachomatis infection.     

Intracellular retention of the NKG2D ligand MHC class I chain-related gene A in human melanomas confers immune privilege and prevents NK cell-mediated cytotoxicity

Most tumors grow in immunocompetent hosts despite expressing NKG2D ligands (NKG2DLs) such as the MHC class I chain-related genes A and B (MICA/B). However, their participation in tumor cell evasion is still not completely understood. Here we demonstrate that several human melanomas (cell lines and freshly isolated metastases) do not express MICA on the cell surface but have intracellular deposits of this NKG2DL. Susceptibility to NK cell-mediated cytotoxicity correlated with the ratio of NKG2DLs to HLA class I molecules but not with the amounts of MICA on the cell surface of tumor cells. Transfection-mediated overexpression of MICA restored cell surface expression and resulted in an increased in vitro cytotoxicity and IFN-gamma secretion by human NK cells. In xenografted nude mice, these melanomas exhibited a delayed growth and extensive in vivo apoptosis. Retardation of tumor growth was due to NK cell-mediated antitumor activity against MICA-transfected tumors, given that this effect was not observed in NK cell-depleted mice. Also, mouse NK cells killed MICA-overexpressing melanomas in vitro. A mechanistic analysis revealed the retention of MICA in the endoplasmic reticulum, an effect that was associated with accumulation of endoH-sensitive (immature) forms of MICA, retrograde transport to the cytoplasm, and degradation by the proteasome. Our study identifies a novel strategy developed by melanoma cells to evade NK cell-mediated immune surveillance based on the intracellular sequestration of immature forms of MICA in the endoplasmic reticulum. Furthermore, this tumor immune escape strategy can be overcome by gene therapy approaches aimed at overexpressing MICA on tumor cells.     

Autocrine/paracrine IL-15 that is required for type I IFN-mediated dendritic cell expression of MHC class I-related chain A and B is impaired in hepatitis C virus infection

We previously reported that monocyte-derived dendritic cells activate resting NK cells by expressing MHC class I-related chain A and B (MICA/B), ligands for NKG2D, in response to IFN-alpha, but the MICA/B expression was severely impaired in patients with chronic hepatitis C virus (HCV) infection. In the present study, we examined induction of MICA/B on DCs by various innate cytokines and found that DCs from either healthy donors or HCV-infected individuals, upon IL-15 stimulation, express MICA/B and can activate NK cells, which is solely dependent on MICA/B-NKG2D interaction. Of interest is the finding that IL-15- and type I IFN-mediated induction of MICA/B in healthy donors is completely inhibited when DCs are incubated in the presence of anti-IFN-alpha/betaR or anti-IL-15Ralpha, respectively, suggesting interdependent roles of these cytokines in MICA/B expression. Indeed, DCs produced IL-15 in response to type I IFN, whereas they directly produced IFN-beta, in response to IL-15, which was followed by the production of IFN-alpha. In HCV-infected individuals, type I IFN-mediated production of IL-15 was virtually absent, but IL-15-mediated production of type I IFN was not compromised, which is consistent with the distinct ability of these cytokines to induce MICA/B in these patients. The present study demonstrates that IL-15 and type I IFN lead to DC expression of MICA/B and subsequent DC activation of NK cells, which is critically dependent on each other's autocrine/paracrine effect, and suggests that impaired IL-15 production is one of the mechanisms of the aberrant response of DC to type I IFN in HCV-infected patients.     

A hypoxia-induced decrease of either MICA/B or Hsp70 on the membrane of tumor cells mediates immune escape from NK cells

Recent findings suggest that hypoxia of the tumor microenvironment contributes to immune escape from natural killer (NK) cell-mediated cytotoxicity. Heat shock protein 70 (Hsp70) and the stress-regulated major histocompatibility class I chain-related protein A and B (MICA/B) both serve as ligands for activated NK cells when expressed on the cell surface of tumor cells. Herein, we studied the effects of hypoxia and hypoxia-inducible factor-1α (HIF-1α) on the membrane expression of these NK cell ligands in H1339 with high and MDA-MB-231 tumor cells with low basal HIF-1α levels and its consequences on NK cell-mediated cytotoxicity. We could show that a hypoxia-induced decrease in the membrane expression of MICA/B and Hsp70 on H1339 and MDA-MB-231 cells, respectively, is associated with a reduced sensitivity to NK cell-mediated lysis. A knockdown of HIF-1α revealed that the decreased surface expression of MICA/B under hypoxia is dependent on HIF-1α in H1339 cells with high basal HIF-1α levels. Hypoxia and HIF-1α did not affect the MICA/B expression in MDA-MB-231 cells but reduced the Hsp70 membrane expression which in turn also impaired NK cell recognition. Furthermore, we could show that the hypoxia-induced decrease in membrane Hsp70 is independent of HIF-1α in MDA-MB-231. Our data indicate that hypoxia-induced downregulation of both NK cell ligands MICA/B and Hsp70 impairs NK cell-mediated cytotoxicity, whereby only MICA/B appears to be regulated by HIF-1α.     

Critical role of MHC class I-related chain A and B expression on IFN-alpha-stimulated dendritic cells in NK cell activation: impairment in chronic hepatitis C virus infection

Dendritic cells (DCs) augment effector functions of NK cells, but the underlying mechanisms are not fully understood. Here we show in an in vitro coculture system that human monocyte-derived DCs enhance IFN-gamma production, CD69 expression, and K562 cytolytic ability of NK cells when DCs are prestimulated with various maturation stimuli such as IFN-alpha or LPS. Of interest is the finding that NK cell activation mediated by LPS-stimulated DCs was dependent on IL-12 produced in DC/NK coculture, but that IFN-alpha-stimulated DC-mediated activation was not. Alternatively, MHC class I-related chain A and B (MICA/B), ligands for NKG2D activating receptor, were found to be induced on DCs upon IFN-alpha stimulation and to be responsible for the NK activation because mAb-mediated masking of MICA/B as well as inhibition of direct cell-to-cell contact using transwell insert completely abolished DC-dependent NK cell activation by IFN-alpha. Finally, DCs recovered from chronic hepatitis C virus-infected patients showed defects in the induction of MICA/B and impaired ability to activate NK cells in response to IFN-alpha stimulation. These findings suggested that MICA/B induction on DCs may be one of the mechanisms by which IFN-alpha activates NK cells; this impairment might affect IFN-alpha responsiveness in hepatitis C virus infection.