Immune cells, including natural killer (NK) cells, recognize transformed cells and eliminate them in a process termed immunosurveillance. It is thought that tumor cells evade immunosurveillance by shedding membrane ligands that bind to the NKG2D activating receptor on NK cells and/or T cells, and desensitize these cells. In contrast, we show that in mice, shedding of MULT1, a high affinity NKG2D ligand, causes NK cell activation and tumor rejection. Recombinant soluble MULT1 stimulated tumor rejection in mice. Soluble MULT1 functions, at least in part, by competitively reversing a global desensitization of NK cells imposed by engagement of membrane NKG2D ligands on tumor-associated cells, such as myeloid cells. The results overturn conventional wisdom that soluble ligands are inhibitory, and suggest a new approach for cancer immunotherapy.

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.

Poliovirus receptor (PVR, CD155) has recently been gaining scientific interest as a therapeutic target in the field of tumor immunology due to its prominent endogenous and immune functions. In contrast to healthy tissues, PVR is expressed at high levels in several human malignancies and seems to have protumorigenic and therapeutically attractive properties that are currently being investigated in the field of recombinant oncolytic virotherapy. More intriguingly, PVR participates in a considerable number of immunoregulatory functions through its interactions with activating and inhibitory immune cell receptors. These functions are often modified in the tumor microenvironment, contributing to tumor immunosuppression. Indeed, increasing evidence supports the rationale for developing strategies targeting these interactions, either in terms of checkpoint therapy (i.e., targeting inhibitory receptors) or in adoptive cell therapy, which targets PVR as a tumor marker.

Natural killer (NK) cells are innate cytotoxic lymphocytes that specialize in the defense against viral infection and oncogenic transformation. Their action is tightly regulated by signals derived from inhibitory and activating receptors; the later include proteins such as the Natural Cytotoxicity Receptors (NCRs: NKp46, NKp44 and NKp30). Among the NCRs, NKp46 is the only receptor that has a mouse orthologue named Ncr1. NKp46/Ncr1 is also a unique marker expressed on NK and on Lymphoid tissue inducer (LTI) cells and it was implicated in the control of various viral infections, cancer and diabetes. We have previously shown that human NKp46 recognizes viral hemagglutinin (HA) in a sialic acid-dependent manner and that the O-glycosylation is essential for the NKp46 binding to viral HA. Here we studied the molecular interactions between Ncr1 and influenza viruses. We show that Ncr1 recognizes influenza virus in a sialic acid dependent manner and that N-glycosylation is important for this binding. Surprisingly we demonstrate that none of the predicted N-glycosilated residues of Ncr1 are essential for its binding to influenza virus and we thus conclude that other, yet unidentified N-glycosilated residues are responsible for its recognition. We have demonstrated that N glycosylation play little role in the recognition of mouse tumor cell lines and also showed the in-vivo importance of Ncr1 in the control of influenza virus infection by infecting C57BL/6 and BALB/c mice knockout for Ncr1 with influenza.

Several essential viral proteins are proposed to participate in genome encapsidation of human cytomegalovirus (HCMV), among them pUL77 and pUL93, which remain largely uncharacterized. To gain insight into their properties, we generated an HCMV mutant expressing a pUL77-monomeric enhanced green fluorescent protein (mGFP) fusion protein and a pUL93-specific antibody. Immunoblotting demonstrated that both proteins are incorporated into capsids and virions. Conversely to data suggesting internal translation initiation sites within the UL93 open reading frame (ORF), we provide evidence that pUL93 synthesis commences at the first start codon. In infected cells, pUL77-mGFP was found in nuclear replication compartments and dot-like structures, colocalizing with capsid proteins. Immunogold labeling of nuclear capsids revealed that pUL77 is present on A, B, and C capsids. Pulldown of pUL77-mGFP revealed copurification of pUL93, indicating interaction between these proteins, which still occurred when capsid formation was prevented. Correct subnuclear distribution of pUL77-mGFP required pUL93 as well as the major capsid protein (and thus probably the presence of capsids), but not the tegument protein pp150 or the encapsidation protein pUL52, demonstrating that pUL77 nuclear targeting occurs independently of the formation of DNA-filled capsids. When pUL77 or pUL93 was missing, generation of unit-length genomes was not observed, and only empty B capsids were produced. Taken together, these results show that pUL77 and pUL93 are capsid constituents needed for HCMV genome encapsidation. Therefore, the task of pUL77 seems to differ from that of its alphaherpesvirus orthologue pUL25, which exerts its function subsequent to genome cleavage-packaging. IMPORTANCEThe essential HCMV proteins pUL77 and pUL93 were suggested to be involved in viral genome cleavage-packaging but are poorly characterized both biochemically and functionally. By producing a monoclonal antibody against pUL93 and generating an HCMV mutant in which pUL77 is fused to a fluorescent protein, we show that pUL77 and pUL93 are capsid constituents, with pUL77 being similarly abundant on all capsid types. Each protein is required for genome encapsidation, as the absence of either pUL77 or pUL93 results in a genome packaging defect with the formation of empty capsids only. This distinguishes pUL77 from its alphaherpesvirus orthologue pUL25, which is enriched on DNA-filled capsids and exerts its function after the viral DNA is packaged. Our data for the first time describe an HCMV mutant with a fluorescent capsid and provide insight into the roles of pUL77 and pUL93, thus contributing to a better understanding of the HCMV encapsidation network.T he life cycle of human cytomegalovirus (HCMV), the prototype member of the betaherpesviruses, comprises a nuclear phase that includes transcription of viral genes, replication of the double-stranded DNA genome, assembly of procapsids, packaging of the viral DNA into the preformed capsids, and maturation of the DNA-filled c...