Stipan Jonjić

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 cytoxicity thus providing an “alternative self” mechanism for MHC class I inhibition.

MCMV glycoprotein gp40 confers virus resistance to CD8+ T cells and NK cells in vivo.

The susceptibility of certain inbred mouse strains to murine cytomegalovirus (MCMV) is related to their inability to generate a strong natural killer (NK) cell response. We addressed here whether the MCMV susceptibility of the BALB/c strain is due to viral functions that control NK cell activation in a strain-specific manner. MCMV expresses two proteins, gp48 and gp40, that are encoded by the genes m06 and m152, respectively; they down-regulate major histocompatibility complex (MHC) class I expression at the plasma membrane. Using MCMV deletion mutants and revertants, we found that gp40 but not gp48 controls NK cell activation. Absence of gp40 improved antiviral NK cell control in BALB/c, but not C57BL/6, mice. Down-regulation of H-60, the high-affinity ligand for the NKG2D receptor, was the mechanism by which gp40 modulates NK cell activation. Thus, a single herpesvirus protein has a dual function in inhibiting both the adaptive as well as the innate immune response.

Gain of Virulence Caused by Loss of a Gene in Murine Cytomegalovirus

ABSTRACT Mouse strains are either resistant or susceptible to murine cytomegalovirus (MCMV). Resistance is determined by the Cmv1r (Ly49h) gene, which encodes the Ly49H NK cell activation receptor. The protein encoded by the m157 gene of MCMV has been defined as a ligand for Ly49H. To find out whether the m157 protein is the only Ly49H ligand encoded by MCMV, we constructed the m157 deletion mutant and a revertant virus. Viruses were tested for susceptibility to NK cell control in Ly49H+ and Ly49H− mouse strains. Deletion of the m157 gene abolished the viral activation of Ly49H+ NK cells, resulting in higher virus virulence in vivo. Thus, in the absence of m157, Ly49H+ mice react like susceptible strains. 129/SvJ mice lack the Ly49H activation NK cell receptor but express the inhibitory Ly49I NK cell receptor that binds to the m157 protein. The Δm157 inhibitory phenotype was weak because MCMV encodes a number of proteins that mediate NK inhibition, whose contribution could be shown by another mutant.

Binding of the Fap2 Protein of Fusobacterium nucleatum to Human Inhibitory Receptor TIGIT Protects Tumors from Immune Cell Attack

Bacteria, such as Fusobacterium nucleatum, are present in the tumor microenvironment. However, the immunological consequences of intra-tumoral bacteria remain unclear. Here, we have shown that natural killer (NK) cell killing of various tumors is inhibited in the presence of various F. nucleatum strains. Our data support that this F. nucleatum-mediated inhibition is mediated by human, but not by mouse TIGIT, an inhibitory receptor present on all human NK cells and on various T cells. Using a library of F. nucleatum mutants, we found that the Fap2 protein of F. nucleatum directly interacted with TIGIT, leading to the inhibition of NK cell cytotoxicity. We have further demonstrated that tumor-infiltrating lymphocytes expressed TIGIT and that T cell activities were also inhibited by F. nucleatum via Fap2. Our results identify a bacterium-dependent, tumor-immune evasion mechanism in which tumors exploit the Fap2 protein of F. nucleatum to inhibit immune cell activity via TIGIT.

Late phase inhibition of murine cytomegalovirus replication by synergistic action of interferon-gamma and tumour necrosis factor

We have shown previously that the antiviral function of CD4+ T lymphocytes against murine cytomegalovirus (MCMV) is associated with the release of interferon-gamma (IFN-gamma). We now demonstrate that IFN-gamma and tumour necrosis factor alpha (TNF-alpha) display synergism in their antiviral activity. As little as 2 ng/ml of IFN-gamma and TNF-alpha reduced the virus yield by about three orders of magnitude. There was no effect on immediate early (IE) and early (E) gene expression as far as the candidate genes IE1, E1 and those encoding the major DNA-binding protein and the DNA polymerase were concerned. Late gene transcription, assayed by the candidate genes encoding glycoprotein B and the MCMV homologue of ICP 18.5, was blocked and MCMV DNA replication was found to be reduced but not halted. The most prominent finding of the cytokine effect, seen by electron microscopy, was an alteration of nucleocapsid formation. Altogether, the synergism is multifaceted and acts at more than one stage during viral morphogenesis. Because the cytokines clearly do not act at an early stage of infection we conclude that the mode of cytokine activity differs between alpha- and betaherpesviruses.

NK cell activation through the NKG2D ligand MULT-1 is selectively prevented by the glycoprotein encoded by mouse cytomegalovirus gene m145

The NK cell–activating receptor NKG2D interacts with three different cellular ligands, all of which are regulated by mouse cytomegalovirus (MCMV). We set out to define the viral gene product regulating murine UL16-binding protein-like transcript (MULT)-1, a newly described NKG2D ligand. We show that MCMV infection strongly induces MULT-1 gene expression, but surface expression of this glycoprotein is nevertheless completely abolished by the virus. Screening a panel of MCMV deletion mutants defined the gene m145 as the viral regulator of MULT-1. The MCMV m145-encoded glycoprotein turned out to be necessary and sufficient to regulate MULT-1 by preventing plasma membrane residence of MULT-1. The importance of MULT-1 in NK cell regulation in vivo was confirmed by the attenuating effect of the m145 deletion that was lifted after NK cell depletion. Our findings underline the significance of escaping MULT-1/NKG2D signaling for viral survival and maintenance.

A cytomegaloviral protein reveals a dual role for STAT2 in IFN-γ signaling and antiviral responses

A mouse cytomegalovirus (MCMV) gene conferring interferon (IFN) resistance was identified. This gene, M27, encodes a 79-kD protein that selectively binds and down-regulates for signal transducer and activator of transcription (STAT)-2, but it has no effect on STAT1 activation and signaling. The absence of pM27 conferred MCMV susceptibility to type I IFNs (α/β), but it had a much more dramatic effect on type II IFNs (γ) in vitro and in vivo. A comparative analysis of M27+ and M27 − MCMV revealed that the antiviral efficiency of IFN-γ was partially dependent on the synergistic action of type I IFNs that required STAT2. Moreover, STAT2 was directly activated by IFN-γ. This effect required IFN receptor expression and was independent of type I IFNs. IFN-γ induced increasing levels of tyrosine-phosphorylated STAT2 in M27− MCMV-infected cells that were essential for the antiviral potency of IFN-γ. pM27 represents a new strategy for simultaneous evasions from types I and II IFNs, and it documents an unknown biological significance for STAT2 in antiviral IFN-γ responses.

Cytomegaloviral control of MHC class I function in the mouse.

Summary: Cytomegaloviruses (CMVs) represent prototypic viruses of the (i‐subgroup of herpesviruses, Murine cytomegalovirus (MCMV) infects mice as its natural host. Among viruses, CMVs have evolved the most extensive genetic repertoire to subvert MHC class I functions. To date three MCMV proteins have been identified which affect MHC I complexes. They are encoded by members of large virus‐specific gene families located at either flanking region of the 235 kb MCMV genome. The MHC I subversive genes belong to the early class of genes and code for type I transmembrane glycoproteins. The ml52‐encoded 37/40 kDa glycoprotein interacts with MHC I transiently and retains class I complexes in the endoplasmic reticulum (ER) Golgi intermediate compartment on its journey to the endolysosome. In contrast, the m06‐encoded glycoprotein of 48 kDa complexes tightly with ternary MHC class I molecules in the ER, Due to sorting signals in its cytoplasmic tail, gp48 redirects MHC I to endolysosomal compartments for proteolytic destruction. Likewise, the 34 kDa glycoprotein encoded by mO4 binds tightly to MHC class I complexes in (he ER but the gp34/MHC I complex reaches the plasma membrane. The CD8+ T‐cell‐dependent attenuation of a m152 deletion mutant virus proves for the first time that inhibition of antigen presentation is indeed essential for the biological fitness of CMVs in vivo.

Degradation of cellular mir-27 by a novel, highly abundant viral transcript is important for efficient virus replication in vivo.

Cytomegaloviruses express large amounts of viral miRNAs during lytic infection, yet, they only modestly alter the cellular miRNA profile. The most prominent alteration upon lytic murine cytomegalovirus (MCMV) infection is the rapid degradation of the cellular miR-27a and miR-27b. Here, we report that this regulation is mediated by the ∼1.7 kb spliced and highly abundant MCMV m169 transcript. Specificity to miR-27a/b is mediated by a single, apparently optimized, miRNA binding site located in its 3′-UTR. This site is easily and efficiently retargeted to other cellular and viral miRNAs by target site replacement. Expression of the 3′-UTR of m169 by an adenoviral vector was sufficient to mediate its function, indicating that no other viral factors are essential in this process. Degradation of miR-27a/b was found to be accompanied by 3′-tailing and -trimming. Despite its dramatic effect on miRNA stability, we found this interaction to be mutual, indicating potential regulation of m169 by miR-27a/b. Most interestingly, three mutant viruses no longer able to target miR-27a/b, either due to miRNA target site disruption or target site replacement, showed significant attenuation in multiple organs as early as 4 days post infection, indicating that degradation of miR-27a/b is important for efficient MCMV replication in vivo.

Participation of endogenous tumour necrosis factor α in host resistance to cytomegalovirus infection

Interferon gamma (IFN gamma) represents an essential cytokine involved in murine cytomegalovirus (MCMV) clearance from the salivary gland and the control of horizontal transmission. Because IFN gamma cannot be responsible for all cytokine effects during recovery from MCMV infection we have now tested the potential participation of tumour necrosis factor alpha (TNF alpha) in the antiviral defence. Neutralization of endogenous TNF alpha abolished the antiviral activity of CD4 T cells in immunocompetent as well as in CD8 subset-deficient mice. These data suggest that the antiviral effect of the CD4 subset requires the presence of at least two cytokines, namely IFN gamma and TNF alpha. Depletion of endogenous TNF alpha in adoptive cell transfer recipients diminished the antiviral function of CD8 T lymphocytes suggesting that TNF alpha also participates in CD8 T cell effector functions. Furthermore, endogenous cytokines were found to be required for survival after infection with lethal doses of MCMV, whereas immunotherapy with recombinant TNF alpha and IFN gamma could not limit virus replication in vivo. The results suggest that, similar to IFN gamma, TNF alpha is an integral part of the protective mechanisms involved in cytomegalovirus clearance.