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.

Transcription of the NKG2D ligand MICA is suppressed by the IRE1/XBP1 pathway of the unfolded protein response through the regulation of E2F1.

The unfolded protein response (UPR) is an adaptive signaling pathway activated in response to endoplasmic reticulum (ER) stress. The effectors of the UPR are potent transcription activators; however, some genes are suppressed by ER stress at the mRNA level. The mechanisms underlying UPR-mediated gene suppression are less known. Exploration of the effect of UPR on NK cells ligand expression found that the transcription of NK group 2 member D (NKG2D) ligand major histocompatibility complex class I polypeptide-related sequence A/B (MICA/B) is suppressed by the inositol-requiring enzyme 1 (IRE1)/X-box binding protein 1 (XBP1) pathway of the UPR. Deletion of IRE1 or XBP1 was sufficient to promote mRNA and surface levels of MICA. Accordingly, NKG2D played a greater role in the killing of IRE1/XBP1 knockout target cells. Analysis of effectors downstream to XBP1s identified E2F transcription factor 1 (E2F1) as linking UPR and MICA transcription. The inverse correlation between XBP1 and E2F1 or MICA expression was corroborated in RNA-Seq analysis of 470 primary melanoma tumors. While mechanisms that connect XBP1 to E2F1 are not fully understood, we implicate a few microRNA molecules that are modulated by ER stress and possess dual suppression of E2F1 and MICA. Because of the importance of E2F1 and MICA in cancer progression and recognition, these observations could be exploited for cancer therapy by manipulating the UPR in tumor cells.-Obiedat, A., Seidel, E., Mahameed, M., Berhani, O., Tsukerman, P., Voutetakis, K., Chatziioannou, A., McMahon, M., Avril, T., Chevet, E., Mandelboim, O., Tirosh, B. Transcription of the NKG2D ligand MICA is suppressed by the IRE1/XBP1 pathway of the unfolded protein response through the regulation of E2F1.

Decay of the Stress-Induced Ligand MICA Is Controlled by the Expression of an Alternative 3' Untranslated Region.

Recognition of the human stress-induced ligand MHC class I polypeptide-related sequence A (MICA) by the receptor NKG2D expressed on NK cells leads to NK cell-mediated killing of the target cells. Hence, the expression of MICA must be tightly regulated, and its cell surface expression needs to be quickly downregulated to avoid inappropriate activation of immune cells. In this article, we describe a transcript variant of human MICA that has not yet been studied, which contains a 3' untranslated region of 119 nt instead of 174. We identify its polyadenylation signal and demonstrate that, upon stresses, such as heat shock, butyrate treatment, and some oxidative and DNA-damaging treatments, the balance between the two MICA variants changes in favor of the less stable, longer variant. Mechanistically, we showed that this change is linked to microRNA activity and that poly-ADP ribose polymerase 1 is involved in the induction of the longer variant following stress. Thus, to our knowledge, we identify the first regulatory mechanism of a stress ligand's decay and also provide one of the first physiological examples for the biological function of a longer 3' untranslated region of a particular gene.

The Human Cytomegalovirus Protein UL148A Downregulates the NK Cell-Activating Ligand MICA To Avoid NK Cell Attack.

Natural killer (NK) cells are lymphocytes of the innate immune system capable of killing hazardous cells, including virally infected cells. NK cell-mediated killing is triggered by activating receptors. Prominent among these is the activating receptor NKG2D, which binds several stress-induced ligands, among them major histocompatibility complex (MHC) class I-related chain A (MICA). Most of the human population is persistently infected with human cytomegalovirus (HCMV), a virus which employs multiple immune evasion mechanisms, many of which target NK cell responses. HCMV infection is mostly asymptomatic, but in congenitally infected neonates and in immunosuppressed patients it can lead to serious complications and mortality. Here we discovered that an HCMV protein named UL148A whose role was hitherto unknown is required for evasion of NK cells. We demonstrate that UL148A-deficient HCMV strains are impaired in their ability to downregulate MICA expression. We further show that when expressed by itself, UL148A is not sufficient for MICA targeting, but rather acts in concert with an unknown viral factor. Using inhibitors of different cellular degradation pathways, we show that UL148A targets MICA for lysosomal degradation. Finally, we show that UL148A-mediated MICA downregulation hampers NK cell-mediated killing of HCMV-infected cells. Discovering the full repertoire of HCMV immune evasion mechanisms will lead to a better understanding of the ability of HCMV to persist in the host and may also promote the development of new vaccines and drugs against HCMV.IMPORTANCE Human cytomegalovirus (HCMV) is a ubiquitous pathogen which is usually asymptomatic but that can cause serious complications and mortality in congenital infections and in immunosuppressed patients. One of the difficulties in developing novel vaccines and treatments for HCMV is its remarkable ability to evade our immune system. In particular, HCMV directs significant efforts to thwarting cells of the innate immune system known as natural killer (NK) cells. These cells are crucial for successful control of HCMV infection, and yet our understanding of the mechanisms which HCMV utilizes to elude NK cells is partial at best. In the present study, we discovered that a protein encoded by HCMV which had no known function is important for preventing NK cells from killing HCMV-infected cells. This knowledge can be used in the future for designing more-efficient HCMV vaccines and for formulating novel therapies targeting this virus.

Influenza virus uses its neuraminidase protein to evade the recognition of two activating NK cell receptors.

Natural Killer (NK) cells play a central role in the defense against viral infections and in the elimination of transformed cells. The recognition of pathogen-infected and tumor cells is controlled by inhibitory and activating receptors. We have previously shown that among the activating (killer) NK cell receptors the natural cytotoxicity receptors, NKp44 and NKp46, interact with the viral hemagglutinin (HA) protein expressed on the cell surface of influenza-virus-infected cells. We further showed that the interaction between NKp44/NKp46 and viral HA is sialic-acid dependent and that the recognition of HA by NKp44 and NKp46 leads to the elimination of the infected cells. Here we demonstrate that the influenza virus developed a counter-attack mechanism and that the virus uses its neuraminidase (NA) protein to prevent the recognition of HA by both the NKp44 and NKp46 receptors, resulting in reduced elimination of the infected cells by NK cells.

Mouse TIGIT inhibits NK-cell cytotoxicity upon interaction with PVR.

The activity of natural killer (NK) cells is controlled by a balance of signals derived from inhibitory and activating receptors. TIGIT is a novel inhibitory receptor, recently shown in humans to interact with two ligands: PVR and Nectin2 and to inhibit human NK-cell cytotoxicity. Whether mouse TIGIT (mTIGIT) inhibits mouse NK-cell cytotoxicity is unknown. Here we show that mTIGIT is expressed by mouse NK cells and interacts with mouse PVR. Using mouse and human Ig fusion proteins we show that while the human TIGIT (hTIGIT) cross-reacts with mouse PVR (mPVR), the binding of mTIGIT is restricted to mPVR. We further demonstrate using surface plasmon resonance (SPR) and staining with Ig fusion proteins that mTIGIT binds to mPVR with higher affinity than the co-stimulatory PVR-binding receptor mouse DNAM1 (mDNAM1). Functionally, we show that triggering of mTIGIT leads to the inhibition of NK-cell cytotoxicity, that IFN-γ secretion is enhanced when mTIGIT is blocked and that the TIGIT-mediated inhibition is dominant over the signals delivered by the PVR-binding co-stimulatory receptors. Additionally, we identify the inhibitory motif responsible for mTIGIT inhibition. In conclusion, we show that TIGIT is a powerful inhibitory receptor for mouse NK cells.

Virus-mediated inhibition of natural cytotoxicity receptor recognition.

Natural killer (NK) cells are a part of the innate immune system that functions mainly to kill transformed and infected cells. Their activity is controlled by signals derived from a panel of activating and inhibitory receptors. The natural cytotoxicity receptors (NCRs): NKp30, NKp44, and NKp46 (NCR1 in mice) are prominent among the activating NK cell receptors and they are, notably, the only NK-activating receptors that are able to recognize pathogen-derived ligands. In addition, the NCRs also recognize cellular ligands, the identity of which remains largely unknown. In this review, we summarize the current knowledge regarding viruses that are recognized by the NCRs, focusing on the diverse immune-evasion mechanisms employed by viruses to escape this detection. We also discuss the unique role the NCRs have in regulating NK cell activity with particular emphasis on the in vivo function of NKp46/NCR1.

CLPTM1L is a GPI-anchoring pathway component targeted by HCMV.

The GPI-anchoring pathway plays important roles in normal development and immune modulation. MHC Class I Polypeptide-related Sequence A (MICA) is a stress-induced ligand, downregulated by human cytomegalovirus (HCMV) to escape immune recognition. Its most prevalent allele, MICA*008, is GPI-anchored via an uncharacterized pathway. Here, we identify cleft lip and palate transmembrane protein 1-like protein (CLPTM1L) as a GPI-anchoring pathway component and show that during infection, the HCMV protein US9 downregulates MICA*008 via CLPTM1L. We show that the expression of some GPI-anchored proteins (CD109, CD59, and MELTF)-but not others (ULBP2, ULBP3)-is CLPTM1L-dependent, and further show that like MICA*008, MELTF is downregulated by US9 via CLPTM1L during infection. Mechanistically, we suggest that CLPTM1L's function depends on its interaction with a free form of PIG-T, normally a part of the GPI transamidase complex. We suggest that US9 inhibits this interaction and thereby downregulates the expression of CLPTM1L-dependent proteins. Altogether, we report on a new GPI-anchoring pathway component that is targeted by HCMV.

A slowly cleaved viral signal peptide acts as a protein-integral immune evasion domain.

Stress can induce cell surface expression of MHC-like ligands, including MICA, that activate NK cells. Human cytomegalovirus (HCMV) glycoprotein US9 downregulates the activating immune ligand MICA*008 to avoid NK cell activation, but the underlying mechanism remains unclear. Here, we show that the N-terminal signal peptide is the major US9 functional domain targeting MICA*008 to proteasomal degradation. The US9 signal peptide is cleaved with unusually slow kinetics and this transiently retained signal peptide arrests MICA*008 maturation in the endoplasmic reticulum (ER), and indirectly induces its degradation via the ER quality control system and the SEL1L-HRD1 complex. We further identify an accessory, signal peptide-independent US9 mechanism that directly binds MICA*008 and SEL1L. Collectively, we describe a dual-targeting immunoevasin, demonstrating that signal peptides can function as protein-integral effector domains.

Human Metapneumovirus Escapes NK Cell Recognition through the Downregulation of Stress-Induced Ligands for NKG2D.

The Pneumoviridae family includes human metapneumovirus (HMPV) and human orthopneumovirus, which is also known as a respiratory syncytial virus (HRSV). These are large enveloped, negative single-strand RNA viruses. HMPV and HRSV are the human members, which commonly infect children. HMPV, which was discovered in 2001, infects most children until the age of five, which causes an influenza-like illness. The interaction of this virus with immune cells is poorly understood. In this study, we show that HMPV evades natural killer (NK) cell attack by downregulating stress-induced ligands for the activating receptor NKG2D including: Major histocompatibility complex (MHC) class I polypeptide-related sequences A and B (MICA, MICB), UL16 binding proteins ULBP2, and ULBP3, but not ULBP1. Mechanistically, we show that the viral protein G is involved in the downregulation of ULBP2 and that the viral protein M2.2 is required for MICA and MICB downregulation. These findings emphasize the importance of NK cells, in general, and NKG2D, in particular, in controlling HMPV infection, which opens new avenues for treating HMPV.

Keeping PPE barriers in COVID-19 wards while doing proper auscultation.

The emerging COVID-19 pandemic poses many difficulties to medical professionals. One of them is the need to use personal protective equipment (PPE) in order to protect themselves and their families, while not compromising their care. Physical examination is one of the cornerstones of medical assessment but parts of it are nearly impossible to do while wearing protective equipment. In this brief report we demonstrate a novel wireless stethoscope and its use for treating suspected and proven COVID-19 patients, as a representative to other infectious diseases.

The integrated stress response promotes B7H6 expression.

The B7 family member, B7H6, is a ligand for the natural killer cell receptor NKp30. B7H6 is hardly expressed on normal tissues, but undergoes upregulation on different types of tumors, implicating it as an attractive target for cancer immunotherapy. The molecular mechanisms that control B7H6 expression are poorly understood. We report that in contrast to other NK cell ligands, endoplasmic reticulum (ER) stress upregulates B7H6 mRNA levels and surface expression. B7H6 induction by ER stress requires protein kinase R-like ER kinase (PERK), one of the three canonical sensors of the unfolded protein response. PERK phosphorylates eIF2α, which regulates protein synthesis and gene expression. Because eIF2α is phosphorylated by several kinases following different stress conditions, the program downstream to eIF2α phosphorylation is called the integrated stress response (ISR). Several drugs were reported to promote the ISR. Nelfinavir and lopinavir, two clinically approved HIV protease inhibitors, promote eIF2α phosphorylation by different mechanisms. We show that nelfinavir and lopinavir sustainably instigate B7H6 expression at their pharmacologically relevant concentrations. As such, ER stress and ISR conditions sensitize melanoma targets to CAR-T cells directed against B7H6. Our study highlights a novel mechanism to induce B7H6 expression and suggests a pharmacological approach to improve B7H6-directed immunotherapy. KEY MESSAGES: B7H6 is induced by ER stress in a PERK-dependent mechanism. Induction of B7H6 is obtained pharmacologically by HIV protease inhibitors. Exposure of tumor cells to the HIV protease inhibitor nelfinavir improves the recognition by B7H6-directed CAR-T.

IFNG-AS1 Enhances Interferon Gamma Production in Human Natural Killer Cells.

Long, non-coding RNAs (lncRNAs) are involved in the regulation of many cellular processes. The lncRNA IFNG-AS1 was found to strongly influence the responses to several pathogens in mice by increasing interferon gamma (IFNγ) secretion. Studies have looked at IFNG-AS1 in T cells, yet IFNG-AS1 function in natural killer cells (NKs), an important source of IFNγ, remains unknown. Here, we show a previously undescribed sequence of IFNG-AS1 and report that it may be more abundant in cells than previously thought. Using primary human NKs and an NK line with IFNG-AS1 overexpression, we show that IFNG-AS1 is quickly induced upon NK cell activation, and that IFNG-AS1 overexpression leads to increased IFNγ secretion. Taken together, our work expands IFNG-AS1's activity to the innate arm of the type I immune response, helping to explain its notable effect in animal models of disease.

Human cytomegalovirus escapes immune recognition by NK cells through the downregulation of B7-H6 by the viral genes US18 and US20.

Human cytomegalovirus (HCMV) is a major human pathogen, causing serious diseases in immunocompromised populations and congenially infected neonates. One of the main immune cells acting against the virus are Natural Killer (NK) cells. Killing by NK cells is mediated by a small family of activating receptors such as NKp30 that interact with the cellular ligand B7-H6. The outcome of B7-H6-NKp30 interaction was, so far, mainly studied with regard to NK recognition and killing of tumors. Here, we demonstrated that the expression of B7-H6 is upregulated following HCMV infection and that HCMV uses two of its genes: US18 and US20, to interfere with B7-H6 surface expression, in a mechanism involving endosomal degradation, in order to evade NK cell recognition.

Cytokine secretion and NK cell activity in human ADAM17 deficiency.

Genetic deficiencies provide insights into gene function in humans. Here we describe a patient with a very rare genetic deficiency of ADAM17. We show that the patient's PBMCs had impaired cytokine secretion in response to LPS stimulation, correlating with the clinical picture of severe bacteremia from which the patient suffered. ADAM17 was shown to cleave CD16, a major NK killer receptor. Functional analysis of patient's NK cells demonstrated that his NK cells express normal levels of activating receptors and maintain high surface levels of CD16 following mAb stimulation. Activation of individual NK cell receptors showed that the patient's NK cells are more potent when activated directly by CD16, albeit no difference was observed in Antibody Depedent Cytotoxicity (ADCC) assays. Our data suggest that ADAM17 inhibitors currently considered for clinical use to boost CD16 activity should be cautiously applied, as they might have severe side effects resulting from impaired cytokine secretion.

Dynamic Co-evolution of Host and Pathogen: HCMV Downregulates the Prevalent Allele MICA∗008 to Escape Elimination by NK Cells.

Natural killer (NK) cells mediate innate immune responses against hazardous cells and are particularly important for the control of human cytomegalovirus (HCMV). NKG2D is a key NK activating receptor that recognizes a family of stress-induced ligands, including MICA, MICB, and ULBP1-6. Notably, most of these ligands are targeted by HCMV proteins and a miRNA to prevent the killing of infected cells by NK cells. A particular highly prevalent MICA allele, MICA∗008, is considered to be an HCMV-resistant "escape variant" that confers advantage to human NK cells in recognizing infected cells. However, here we show that HCMV uses its viral glycoprotein US9 to specifically target MICA∗008 and thus escapes NKG2D attack. The finding that HCMV evolved a protein dedicated to countering a single host allele illustrates the dynamic co-evolution of host and pathogen.

MiR-520d-5p directly targets TWIST1 and downregulates the metastamiR miR-10b.

MicroRNAs are key players in most biological processes. Some microRNAs are involved in the genesis of tumors and are therefore termed oncomiRs, while others, termed metastamiRs, play a significant role in the formation of cancer metastases. Previously, we identified ten different cellular microRNAs that downregulate the expression of MICB, a ligand of the activating NK receptor NKG2D. Interestingly, several of the ten MICB-targeting microRNAs, such as miR-10b, are involved in tumor formation and metastasis. In this work, we identify a complex interplay between these different microRNAs. Specifically, we demonstrate that three of the MICB-targeting microRNAs: miR-20a, miR-17-5p and miR-93, also target the same site in the 3'UTR of TWIST1, a transcription factor implicated in cancer metastasis. Additionally, we show that miR-520d-5p targets a different site in the 3'UTR of TWIST1. We next show that the miR-520d-5p-mediated decrease of TWIST1 expression results in reduced expression of one of its targets, miR-10b, and in the restoration of E-Cadherin expression, which in turn results in reduced cellular motility and invasiveness. Finally, we show that miR-520d-5p leads to reduced proliferation of tumor cells, and that high levels of miR-520d-5p correlate with higher survival rates of cancer patients.