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 cytoxicity thus providing an ''alternative self'' mechanism for MHC class I inhibition.inhibitory receptors ͉ natural killers I n contrast to T cells, that possess a single dominant antigen receptor (1), NK cells rely on a vast combinatorial array of receptors to initiate effector functions (2). Both activating and inhibitory receptors expressed on NK cells regulate their activity when interacting with tumors, virus infected cells and bacteria, as well as normal self-cells (2). MHC class I-expressing cells are protected from NK-mediated lysis due to the recognition of various MHC class I proteins by the inhibitory receptors KIR, LIR and CD94-NKG2A (3). Other NK inhibitory receptors which do not interact with MHC class I also exist, such as CEACAM1 and IRp60 (4-8). The significance, however, of these non-MHC class I inhibitory receptors in normal conditions is still unclear. All of the inhibitory receptors share a common immune receptor tyrosinebased inhibitory motif (ITIM) in their cytoplasmic regions, which delivers the inhibitory signal (3).The NK cell-mediated killing is extracted by specific receptors, among which are the natural cytotoxicity receptors (NCRs), which include the NKp30 that interacts with pp65 of human cytomegalovirus (CMV), BAT3 and the recently identified B7-family member B7-H6 (9-11), and the NKp46/NKp44 receptors, which interact with various viral hemagglutinins (12, 13). The NKG2D receptor interacts with MICA, MICB and ULBP 1-5 (14) and NKp80 interacts with AICL (15). In addition, two other receptors, DNAM-1 and CD96, enhance NK cytotoxicity (16,17). Both DNAM-1 and CD96 recognize PVR (CD155), whereas DNAM-1 also recognizes PVRL2 (CD112) (16,17). It was recently shown that a new receptor, named TIGIT, for T cell Ig and ITIM domain, interacts with PVR and its related proteins and that TIGIT inhibits T cell activity indirectly through the manipulation of DC activity (18). Here, we show that TIGIT, through its ITIM, can directly inhibit NK cytotoxicity. ResultsTIGIT Inhibits YTS Killing Through Its ITIM Motif. While searching for new CD28 family-like receptors, based on bioinformatics analysis, we observed that a protein named VSIG9 or VSTM3 in the databases expresses an ITIM motif. We continued to work on this protein and found that it interacts with PVR (CD155) but not with any other NK ligands tested (supporting information (...

Members of the α- and β-subfamily of herpesviridae encode glycoproteins that specifically bind to the Fc part of immunoglobulin (Ig)G. Plasma membrane resident herpesviral Fc receptors seem to prevent virus-specific IgG from activating antibody-dependent effector functions. We show that the mouse cytomegalovirus (MCMV) molecule fcr-1 promotes a rapid down-regulation of NKG2D ligands murine UL16-binding protein like transcript (MULT)-1 and H60 from the cell surface. Deletion of the m138/fcr-1 gene from the MCMV genome attenuates viral replication to natural killer (NK) cell response in an NKG2D-dependent manner in vivo. A distinct N-terminal module within the fcr-1 ectodomain in conjunction with the fcr-1 transmembrane domain was required to dispose MULT-1 to degradation in lysosomes. In contrast, down-modulation of H60 required the complete fcr-1 ectodomain, implying independent modes of fcr-1 interaction with the NKG2D ligands. The results establish a novel viral strategy for down-modulating NK cell responses and highlight the impressive diversity of Fc receptor functions.

The NKG2D receptor is one of the most potent activating natural killer cell receptors involved in antiviral responses. The mouse NKG2D ligands MULT-1, RAE-1, and H60 are regulated by murine cytomegalovirus (MCMV) proteins m145, m152, and m155, respectively. In addition, the m138 protein interferes with the expression of both MULT-1 and H60. We show here that one of five RAE-1 isoforms, RAE-1␦, is resistant to downregulation by MCMV and that this escape has functional importance in vivo. Although m152 retained newly synthesized RAE-1␦ and RAE-1␥ in the endoplasmic reticulum, no viral regulator was able to affect the mature RAE-1␦ form which remains expressed on the surfaces of infected cells. This differential susceptibility to downregulation by MCMV is not a consequence of faster maturation of RAE-1␦ compared to RAE-1␥ but rather an intrinsic property of the mature surface-resident protein. This difference can be attributed to the absence of a PLWY motif from RAE-1␦. Altogether, these findings provide evidence for a novel mechanism of host escape from viral immunoevasion of NKG2D-dependent control.Cytomegaloviruses (CMVs) are ubiquitous pathogens causing morbidity in immune suppressed and immunodeficient hosts (34). Since CMVs are strictly species-specific viruses, the infection of mice with murine CMV (MCMV) represents a widely used model for studying CMV infection and disease (22,40).Natural killer (NK) cells play a crucial role in the control of many viruses and are among the first cells to sense proinflammatory cytokines, as well as the perturbations in the expression of major histocompatibility complex (MHC) class I molecules and other surface molecules induced by viral infection (13). Both human CMV (HCMV) and MCMV have evolved strategies to compromise innate immunity-mediated by NK cells (20,49).Although proinflammatory cytokines released during the early stage of MCMV infection induce NK cell activation, this is usually not sufficient for virus control (11). Namely, most mouse strains fail to mount an effector phase of NK cell response against infected cells (42), in spite of the fact that MCMV infection causes the downmodulation of MHC I molecules (17), which should activate NK cells via a "missing-self" mechanism (28). The lack of NK cell activation by MCMV is even more puzzling considering that NK cells possess activating receptors that recognize cellular ligands induced by infection. Among these is the activating receptor NKG2D, a C-type lectinlike receptor encoded by a single gene in humans and rodents (39). Engagement of NKG2D transduces a strong activating signal to promote NK cell stimulation. NKG2D also serves as a costimulatory receptor on CD8 ϩ T cells (2). Several NKG2D ligands have been described in mice: MULT-1, H60a, H60b, H60c, and RAE-1␣, -1␤, -1␥, -1␦, and -1ε isoforms (4-6, 10, 14, 32, 35, 44). What prevents the activation of NK cells via the NKG2D receptor during MCMV infection? We and others have characterized four MCMV proteins involved in the downmodulation of NKG2D ligands (15,23...

Human cytomegalovirus (HCMV) is a human pathogen that causes severe disease primarily in the immunocompromised or immunologically immature individual. To date, no vaccine is available. We describe use of a spread-deficient murine CMV (MCMV) as a novel approach for betaherpesvirus vaccination. To generate a spread-deficient MCMV, the conserved, essential gene M94 was deleted. Immunization with MCMV-⌬M94 is apathogenic and protective against wild-type challenge even in highly susceptible IFN␣␤R ؊/؊ mice. MCMV-⌬M94 was able to induce a robust CD4؉ and CD8 ؉ T-cell response as well as a neutralizing antibody response comparable to that induced by wild-type infection. Endothelial cells were identified as activators of CD8 ؉ T cells in vivo. Thus, the vaccination with a spread-deficient betaherpesvirus is a safe and protective strategy and allows the linkage between cell tropism and immunogenicity. Furthermore, genomes of MCMV-⌬M94 were present in lungs 12 months after infection, revealing first-target cells as sites of genome maintenance.

Congenital HCMV infection is a leading infectious cause of long-term neurodevelopmental sequelae. Infection of newborn mice with mouse cytomegalovirus (MCMV) intraperitoneally is a well-established model of congenital human cytomegalovirus infection, which best recapitulates the hematogenous route of virus spread to brain and subsequent pathology. Here, we used this model to investigate the role, dynamics, and phenotype of CD8 T cells in the brain following infection of newborn mice. We show that CD8 T cells infiltrate the brain and form a pool of tissue-resident memory T cells (T cells) that persist for lifetime. Adoptively transferred virus-specific CD8 T cells provide protection against primary MCMV infection in newborn mice, reduce brain pathology, and remain in the brain as T cells. Brain CD8 T cells were long-lived, slowly proliferating cells able to respond to local challenge infection. Importantly, brain CD8 T cells controlled latent MCMV and their depletion resulted in virus reactivation and enhanced inflammation in brain.