Cytomegalovirus protein m154 perturbs the adaptor protein-1 compartment mediating broad-spectrum immune evasion

Cytomegaloviruses (CMVs) are ubiquitous pathogens known to employ numerous immunoevasive strategies that significantly impair the ability of the immune system to eliminate the infected cells. Here, we report that the single mouse CMV (MCMV) protein, m154, downregulates multiple surface molecules involved in the activation and costimulation of the immune cells. We demonstrate that m154 uses its cytoplasmic tail motif, DD, to interfere with the adaptor protein-1 (AP-1) complex, implicated in intracellular protein sorting and packaging. As a consequence of the perturbed AP-1 sorting, m154 promotes lysosomal degradation of several proteins involved in T cell costimulation, thus impairing virus-specific CD8+ T cell response and virus control in vivo. Additionally, we show that HCMV infection similarly interferes with the AP-1 complex. Altogether, we identify the robust mechanism employed by single viral immunomodulatory protein targeting a broad spectrum of cell surface molecules involved in the antiviral immune response

Reovirus infection of tumor cells reduces the expression of NKG2D ligands, leading to impaired NK-cell cytotoxicity and functionality

In recent years, reoviruses have been of major interest in immunotherapy because of their oncolytic properties. Preclinical and clinical trials, in which reovirus was used for the treatment of melanoma and glioblastoma, have paved the way for future clinical use of reovirus. However, little is known about how reovirus infection affects the tumor microenvironment and immune response towards infected tumor cells. Studies have shown that reovirus can directly stimulate natural killer (NK) cells, but how reovirus affects cellular ligands on tumor cells, which are ultimately key to tumor recognition and elimination by NK cells, has not been investigated. We tested how reovirus infection affects the binding of the NK Group-2 member D (NKG2D) receptor, which is a dominant mediator of NK cell anti-tumor activity. Using models of human-derived melanoma and glioblastoma tumors, we demonstrated that NKG2D ligands are downregulated in tumor cells post-reovirus-infection due to the impaired translation of these ligands in reovirus-infected cells. Moreover, we showed that downregulation of NKG2D ligands significantly impaired the binding of NKG2D to infected tumor cells. We further demonstrated that reduced recognition of NKG2D ligands significantly alters NK cell anti-tumor cytotoxicity in human primary NK cells and in the NK cell line NK-92. Thus, this study provides novel insights into reovirus-host interactions and could lead to the development of novel reovirus-based therapeutics that enhance the anti-tumor immune response

Presentation_1_Reovirus infection of tumor cells reduces the expression of NKG2D ligands, leading to impaired NK-cell cytotoxicity and functionality.pdf

In recent years, reoviruses have been of major interest in immunotherapy because of their oncolytic properties. Preclinical and clinical trials, in which reovirus was used for the treatment of melanoma and glioblastoma, have paved the way for future clinical use of reovirus. However, little is known about how reovirus infection affects the tumor microenvironment and immune response towards infected tumor cells. Studies have shown that reovirus can directly stimulate natural killer (NK) cells, but how reovirus affects cellular ligands on tumor cells, which are ultimately key to tumor recognition and elimination by NK cells, has not been investigated. We tested how reovirus infection affects the binding of the NK Group-2 member D (NKG2D) receptor, which is a dominant mediator of NK cell anti-tumor activity. Using models of human-derived melanoma and glioblastoma tumors, we demonstrated that NKG2D ligands are downregulated in tumor cells post-reovirus-infection due to the impaired translation of these ligands in reovirus-infected cells. Moreover, we showed that downregulation of NKG2D ligands significantly impaired the binding of NKG2D to infected tumor cells. We further demonstrated that reduced recognition of NKG2D ligands significantly alters NK cell anti-tumor cytotoxicity in human primary NK cells and in the NK cell line NK-92. Thus, this study provides novel insights into reovirus-host interactions and could lead to the development of novel reovirus-based therapeutics that enhance the anti-tumor immune response.</p

Cytomegalovirus restricts icosl expression on antigen-presenting cells disabling t cell co-stimulation and contributing to immune evasion

Viral infections are controlled, and very often cleared, by activated T lymphocytes. The inducible co-stimulator (ICOS) mediates its functions by binding to its ligand ICOSL, enhancing T-cell activation and optimal germinal center (GC) formation. Here, we show that ICOSL is heavily downmodulated during infection of antigen-presenting cells by different herpesviruses. We found that, in murine cytomegalovirus (MCMV), the immunoevasin m138/fcr-1 physically interacts with ICOSL, impeding its maturation and promoting its lysosomal degradation. This viral protein counteracts T-cell responses, in an ICOS-dependent manner, and limits virus control during the acute MCMV infection. Additionally, we report that blockade of ICOSL in MCMV-infected mice critically regulates the production of MCMV-specific antibodies due to a reduction of T follicular helper and GC B cells. Altogether, these findings reveal a novel mechanism evolved by MCMV to counteract adaptive immune surveillance, and demonstrates a role of the ICOS:ICOSL axis in the host defense against herpesviruses.ISSN:2050-084

Cytomegalovirus restricts ICOSL expression on antigen-presenting cells disabling T cell co-stimulation and contributing to immune evasion

Viral infections are controlled, and very often cleared, by activated T lymphocytes. The inducible co-stimulator (ICOS) mediates its functions by binding to its ligand ICOSL, enhancing T-cell activation and optimal germinal center (GC) formation. Here, we show that ICOSL is heavily downmodulated during infection of antigen-presenting cells by different herpesviruses. We found that, in murine cytomegalovirus (MCMV), the immunoevasin m138/fcr-1 physically interacts with ICOSL, impeding its maturation and promoting its lysosomal degradation. This viral protein counteracts T-cell responses, in an ICOS-dependent manner, and limits virus control during the acute MCMV infection. Additionally, we report that blockade of ICOSL in MCMV-infected mice critically regulates the production of MCMV-specific antibodies due to a reduction of T follicular helper and GC B cells. Altogether, these findings reveal a novel mechanism evolved by MCMV to counteract adaptive immune surveillance, and demonstrates a role of the ICOS:ICOSL axis in the host defense against herpesviruses

Treg cells show an activated phenotype after MCMV infection.

<p>BALB/c mice were i.v. injected with 2x10⁵ PFU of WT MCMV (clone MW97.01) or left uninfected. (<b>A</b>) Absolute number of Treg cells in spleen and liver is shown. (<b>B</b>) Representative FACS plots and (<b>C</b>) graphs showing percentages and (<b>D</b>) median fluorescence intensity (MFI) of Ki-67 expression by naive Treg cells. (<b>F</b>) Bcl-2 expression by naive Treg cells. (<b>E</b>) Mice were treated with BrdU in drinking water for 6 days starting at the day of infection. Percentage of BrdU positive Treg cells on day 7 was determined. (<b>G</b>) Histograms show a representative expression of different markers by Treg cells from uninfected and 7 days infected mice. (<b>H</b>) Representative FACS plots and (<b>I</b>) graphs showing percentages and (<b>J</b>) median fluorescence intensity (MFI) of ST2 expression by Treg cells isolated from the spleen and liver of naive BALB/c and ST2^-/- mice. Data are shown as mean ± SEM of n = 3–5 mice from one representative experiment out of three. *p<0.05; ** p<0.01; ***p<0.001 from two tailed, unpaired Student’s t-test.</p

ST2^-/- mice show increased liver damage and mortality rate after MCMV infection.

<p>(<b>A</b>) BALB/c and ST2^-/- mice were i.v. injected with 2x10⁵ PFU of WT MCMV (MW97.01) and lymphocytes from spleen and liver were analyzed on day 7 p.i. Absolute number of Treg cells is shown. (<b>B</b>-<b>E</b>) BALB/c and ST2^-/- mice were i.v. injected with 10⁶ PFU of WT MCMV (pSM3fr-MCK-2fl clone 3.3) and analyzed on day 5 p.i. (<b>B</b>) AST and ALT serum levels were determined. (<b>C</b>) Scores of cumulative liver pathology for apoptosis, intranuclear inclusion bodies (INIBs), inflammation, and necrosis. Bars correspond to the mean score for each parameter. The height of each bar represents the mean of the total histological score (out of 12). (<b>D</b>) Representative H&E and (<b>E</b>) Caspase-3 staining of paraffin embedded liver sections. (<b>F</b>) BALB/c and ST2^-/- mice were i.p. injected with indicated doses of SGV MCMV. Survival rates were monitored daily. Data are shown as mean ± SEM of n = 3–5 mice from one representative experiment out of three. For survival monitoring n = 7. *p <0.05 and **p<0.01 from two tailed, unpaired Student’s t-test.</p

Intrinsic requirement for ST2 expression in Treg cells.

<p>Mixed bone marrow chimeras were generated by irradiation of C57BL/6 CD45.1⁺CD45.2⁺ mice followed by i.v. injection of 1:1 mixture of wild-type (WT; CD45.1⁺) and knockout (ST2^-/-; CD45.2⁺) bone marrow cells. After reconstitution, mixed chimeras were infected with 2x10⁵ PFU of Δm157 MCMV and analyzed on day 7 p.i. (<b>A</b>) Percentage of donor WT and KO Treg cells is shown. (<b>B</b>) The ratio between WT and KO Treg cells is shown. Dotted line represents initial ratio of transferred bone marrow cells. (<b>C</b>) Percentage of Ki-67 expression by donor Treg cells is shown. Data are shown as mean ± SEM of n = 4–5 mice per group from one representative experiment out of two. *p <0.05 and **p<0.01 from two tailed, unpaired Student’s t-test.</p

ST2 deficiency does not affect viral control.

<p>(<b>A</b>) WT and ST2^-/- mice were injected i.v. with 2x10⁵ PFU of WT MCMV (MW97.01) and lymphocytes from the spleen and liver were isolated on day 4 p.i. The percentages of IE1-specific and m164-specific CD8⁺ T are shown. Data are shown as mean ± SEM. (<b>B</b>) WT and ST2^-/- mice were injected i.v. with 10⁶ PFU of WT MCMV (pSM3fr-MCK-2fl clone 3.3). Viral titers in indicated organs 5 days post infection were determined by the plaque assay. A circle depicts the titer for each individual mouse; a small horizontal line indicates the median. n = 4–5 mice from one representative experiment out of three. **p<0.01 from two tailed, unpaired Student’s t-test.</p

Identification of the cell type that expresses IL-33 in inflammatory foci in infected liver tissue as F4/80⁺ macrophages.

<p>BALB/c mice were injected i.v. with 5x10⁵ PFU of WT MCMV (MW97.01) and liver tissue was harvested on day 5 p.i. (<b>A</b>) Consecutive serial 1-μm sections of liver tissue focusing on an infected hepatocyte (Hc) that is delimited from uninfected tissue by a sheath made up by a mononuclear cell infiltrate. The expression of the indicated marker molecules was tested in a two-color IHC (2C-IHC) staining. (<b>a</b>-<b>d</b>) Identification of the infected Hc by red staining of the intranuclear viral IE1 protein. (<b>a</b>) Focus-forming mononuclear cells are not CD31^<b>+</b> black-stained endothelial cells (EC). (<b>b</b>) Focus-forming mononuclear cells are not CD3ε^<b>+</b> black-stained cells, thus excluding α/ß and γ/δ T cells as well as NKT cells. (<b>c</b>) Identification of focus-forming mononuclear cells as black-stained F4/80^<b>+</b> macrophages (Mø). (<b>d</b>) IL33-expressing cells stained in turquoise-green color colocalize with focus-forming F4/80 macrophages in the neighboring section of image <b>c</b>. Counterstaining with hematoxylin. Arrows point to the indicated cell types exemplarily. The bar markers represent 50 μm throughout. (<b>B</b>) 2C-IHC verifying colocalization of F4/80 and IL33 on the cellular level. (<b>a</b>) Higher resolution image of an advanced, aged focus consisting of a cluster of dual-stained F4/80⁺ (red) IL33⁺ (turquoise-green) macrophages (Mø) surrounding an infected hepatocyte (Hc) that is identified by an intranuclear inclusion body. Note that dually-expressing macrophages localize also to liver tissue outside of a focus. (<b>b</b>) A young focus in which dual-stained F4/80^<b>+</b> (red) IL33⁺ (turquoise-green) macrophages cling to an infected hepatocyte (Hc) that shows the pathocytomorphology of an owl’s eye cell with an intranuclear inclusion body that indicates the late phase (L phase) in the viral gene expression program. Counterstaining with hematoxylin. Arrows point to sites of interest. The bar markers represent 50 μm.</p