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.

ILDR2 Is a Novel B7-like Protein That Negatively Regulates T Cell Responses.

The B7-like protein family members play critical immunomodulatory roles and constitute attractive targets for the development of novel therapies for human diseases. We identified Ig-like domain-containing receptor (ILDR)2 as a novel B7-like protein with robust T cell inhibitory activity, expressed in immune cells and in immune-privileged and inflamed tissues. A fusion protein, consisting of ILDR2 extracellular domain with an Fc fragment, that binds to a putative counterpart on activated T cells showed a beneficial effect in the collagen-induced arthritis model and abrogated the production of proinflammatory cytokines and chemokines in autologous synovial-like cocultures of macrophages and cytokine-stimulated T cells. Collectively, these findings point to ILDR2 as a novel negative regulator for T cells, with potential roles in the development of immune-related diseases, including autoimmunity and cancer.

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.

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.

The interaction of TIGIT with PVR and PVRL2 inhibits human NK cell cytotoxicity.

NK cell cytotoxicity is controlled by numerous NK inhibitory and activating receptors. Most of the inhibitory receptors bind MHC class I proteins and are expressed in a variegated fashion. It was recently shown that TIGIT, a new protein expressed by T and NK cells binds to PVR and PVR-like receptors and inhibits T cell activity indirectly through the manipulation of DC activity. Here, we show that TIGIT is expressed by all human NK cells, that it binds PVR and PVRL2 but not PVRL3 and that it inhibits NK cytotoxicity directly through its ITIM. Finally, we show that TIGIT counter inhibits the NK-mediated killing of tumor cells and protects normal cells from NK-mediated cytotoxicity thus providing an "alternative self" mechanism for MHC class I inhibition.

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.

A novel peptide agonist of formyl-peptide receptor-like 1 (ALX) displays anti-inflammatory and cardioprotective effects.

Activation of the formyl-peptide receptor-like (FPRL) 1 pathway has recently gained high recognition for its significance in therapy of inflammatory diseases. Agonism at FPRL1 affords a beneficial effect in animal models of acute inflammatory conditions, as well as in chronic inflammatory diseases. TIPMFVPESTSKLQKFTSWFM-amide (CGEN-855A) is a novel 21-amino acid peptide agonist for FPRL1 and also activates FPRL2. CGEN-855A was discovered using a computational platform designed to predict novel G protein-coupled receptor peptide agonists cleaved from secreted proteins by convertase proteolysis. In vivo, CGEN-855A displays anti-inflammatory activity manifested as 50% inhibition of polymorphonuclear neutrophil (PMN) recruitment to inflamed air pouch and provides protection against ischemia-reperfusion-mediated injury to the myocardium in both murine and rat models (36 and 25% reduction in infarct size, respectively). Both these activities are accompanied by inhibition of PMN recruitment to the injured organ. The secretion of inflammatory cytokines, including interleukin (IL)-6, IL-1beta, and tumor necrosis factor-alpha, was not affected upon incubation of human peripheral blood mononuclear cells with CGEN-855A, whereas IL-8 secretion was elevated up to 2-fold upon treatment with the highest CGEN-855A dose only. Collectively, these new data support a potential role for CGEN-855A in the treatment of reperfusion-mediated injury and in other acute and chronic inflammatory conditions.

A novel recombinant soluble splice variant of Met is a potent antagonist of the hepatocyte growth factor/scatter factor-Met pathway.

PURPOSE: The Met receptor tyrosine kinase and its ligand, hepatocyte growth factor/scatter factor (HGF/SF), are involved in a wide range of biological activities, including cell proliferation, motility, invasion, and angiogenesis. The HGF/SF-Met signaling pathway is frequently activated in a variety of cancers, and uncontrolled Met activation correlates with highly invasive tumors and poor prognosis. In this study, we investigated the inhibitory effect of a novel soluble splice variant of Met on the HGF/SF-Met pathway. EXPERIMENTAL DESIGN: Using our alternative splicing modeling platform LEADS, we have identified a novel splice variant of the Met receptor, which encodes a truncated soluble form of the receptor. This variant was produced as a recombinant Fc-fused protein named Cgen-241A and was tested in various cell-based assays representing different outcomes of the HGF/SF-Met pathway. RESULTS: Cgen-241A significantly inhibited HGF/SF-induced Met phosphorylation as well as cell proliferation and survival. In addition, Cgen-241A showed a profound inhibitory effect on cell scattering, invasion, and urokinase up-regulation. The inhibitory effects of Cgen-241A were shown in multiple human and nonhuman cell types, representing different modes of Met activation. Furthermore, Cgen-241A showed direct binding to HGF/SF. CONCLUSIONS: Taken together, our results indicate that Cgen-241A is a potent antagonist of the HGF/SF-Met pathway, underlining its potential as a therapeutic agent for the treatment of a wide variety of human malignancies that are dependent on this pathway.

Mouse PVRIG Has CD8+ T Cell-Specific Coinhibitory Functions and Dampens Antitumor Immunity.

A limitation to antitumor immunity is the dysfunction of T cells in the tumor microenvironment, in part due to upregulation of coinhibitory receptors such as PD-1. Here, we describe that poliovirus receptor-related immunoglobulin domain protein (PVRIG) acts as a coinhibitory receptor in mice. Murine PVRIG interacted weakly with poliovirus receptor (PVR) but bound poliovirus receptor-like 2 (PVRL2) strongly, making the latter its principal ligand. As in humans, murine NK and NKT cells constitutively expressed PVRIG. However, when compared with humans, less PVRIG transcript and surface protein was detected in murine CD8+ T cells ex vivo However, activated CD8+ T cells upregulated PVRIG expression. In the mouse tumor microenvironment, infiltrating CD8+ T cells expressed PVRIG whereas its ligand, PVRL2, was detected predominantly on myeloid cells and tumor cells, mirroring the expression pattern in human tumors. PVRIG-deficient mouse CD8+ T cells mounted a stronger antigen-specific effector response compared with wild-type CD8+ T cells during acute Listeria monocytogenes infection. Furthermore, enhanced CD8+ T-cell effector function inhibited tumor growth in PVRIG-/- mice compared with wild-type mice and PD-L1 blockade conferred a synergistic antitumor response in PVRIG-/- mice. Therapeutic intervention with antagonistic anti-PVRIG in combination with anti-PD-L1 reduced tumor growth. Taken together, our results suggest PVRIG is an inducible checkpoint receptor and that targeting PVRIG-PVRL2 interactions results in increased CD8+ T-cell function and reduced tumor growth.See related article on p. 257.

PVRIG and PVRL2 Are Induced in Cancer and Inhibit CD8+ T-cell Function.

Although checkpoint inhibitors that block CTLA-4 and PD-1 have improved cancer immunotherapies, targeting additional checkpoint receptors may be required to broaden patient response to immunotherapy. PVRIG is a coinhibitory receptor of the DNAM/TIGIT/CD96 nectin family that binds to PVRL2. We report that antagonism of PVRIG and TIGIT, but not CD96, increased CD8+ T-cell cytokine production and cytotoxic activity. The inhibitory effect of PVRL2 was mediated by PVRIG and not TIGIT, demonstrating that the PVRIG-PVRL2 pathway is a nonredundant signaling node. A combination of PVRIG blockade with TIGIT or PD-1 blockade further increased T-cell activation. In human tumors, PVRIG expression on T cells was increased relative to normal tissue and trended with TIGIT and PD-1 expression. Tumor cells coexpressing PVR and PVRL2 were observed in multiple tumor types, with highest coexpression in endometrial cancers. Tumor cells expressing either PVR or PVRL2 were also present in numbers that varied with the cancer type, with ovarian cancers having the highest percentage of PVR-PVRL2+ tumor cells and colorectal cancers having the highest percentage of PVR+PVRL2- cells. To demonstrate a role of PVRIG and TIGIT on tumor-derived T cells, we examined the effect of PVRIG and TIGIT blockade on human tumor-infiltrating lymphocytes. For some donors, blockade of PVRIG increased T-cell function, an effect enhanced by combination with TIGIT or PD-1 blockade. In summary, we demonstrate that PVRIG and PVRL2 are expressed in human cancers and the PVRIG-PVRL2 and TIGIT-PVR pathways are nonredundant inhibitory signaling pathways.See related article on p. 244.

Directed elimination of senescent cells by inhibition of BCL-W and BCL-XL.

Senescent cells, formed in response to physiological and oncogenic stresses, facilitate protection from tumourigenesis and aid in tissue repair. However, accumulation of such cells in tissues contributes to age-related pathologies. Resistance of senescent cells to apoptotic stimuli may contribute to their accumulation, yet the molecular mechanisms allowing their prolonged viability are poorly characterized. Here we show that senescent cells upregulate the anti-apoptotic proteins BCL-W and BCL-XL. Joint inhibition of BCL-W and BCL-XL by siRNAs or the small-molecule ABT-737 specifically induces apoptosis in senescent cells. Notably, treatment of mice with ABT-737 efficiently eliminates senescent cells induced by DNA damage in the lungs as well as senescent cells formed in the epidermis by activation of p53 through transgenic p14(ARF). Elimination of senescent cells from the epidermis leads to an increase in hair-follicle stem cell proliferation. The finding that senescent cells can be eliminated pharmacologically paves the way to new strategies for the treatment of age-related pathologies.

NK Cell Recognition of Candida glabrata through Binding of NKp46 and NCR1 to Fungal Ligands Epa1, Epa6, and Epa7.

Natural killer (NK) cells form an important arm of the innate immune system and function to combat a wide range of invading pathogens, ranging from viruses to bacteria. However, the means by which NK cells accomplish recognition of pathogens with a limited repertoire of receptors remain largely unknown. In the current study, we describe the recognition of an emerging fungal pathogen, Candida glabrata, by the human NK cytotoxic receptor NKp46 and its mouse ortholog, NCR1. Using NCR1 knockout mice, we observed that this receptor-mediated recognition was crucial for controlling C. glabrata infection in vitro and in vivo. Finally, we delineated the fungal ligands to be the C. glabrata adhesins Epa1, Epa6, and Epa7 and demonstrated that clearance of systemic C. glabrata infections in vivo depends on their recognition by NCR1. As NKp46 and NCR1 have been previously shown to bind viral adhesion receptors, we speculate that NKp46/NCR1 may be a novel type of pattern recognition receptor.