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

Inflammatory monocytes and NK cells play a crucial role in DNAM-1-dependent control of cytomegalovirus infection

The poliovirus receptor (PVR) is a ubiquitously expressed glycoprotein involved in cellular adhesion and immune response. It engages the activating receptor DNAX accessory molecule (DNAM)-1, the inhibitory receptor TIG IT, and the CD96 receptor with both activating and inhibitory functions. Human cytomegalovirus (HCMV) down-regulates PVR expression, but the significance of this viral function in vivo remains unknown. Here, we demonstrate that mouse CMV (MCMV) also down-regulates the surface PVR. The m20.1 protein of MCMV retains PVR in the endoplasmic reticulum and promotes its degradation. A MCMV mutant lacking the PVR inhibitor was attenuated in normal mice but not in mice lacking DNAM-1. This attenuation was partially reversed by NK cell depletion, whereas the simultaneous depletion of mononuclear phagocytes abolished the virus control. This effect was associated with the increased expression of DNAM-1, whereas TIG IT and CD96 were absent on these cells. An increased level of proinflammatory cytokines in sera of mice infected with the virus lacking the m20.1 and an increased production of iNOS by inflammatory monocytes was observed. Blocking of CCL2 or the inhibition of iNOS significantly increased titer of the virus lacking m20.1. In this study, we have demonstrated that inflammatory monocytes, together with NK cells, are essential in the early control of CMV through the DNAM-1–PVR pathwa

Tumor Treating Fields (TTFields) Hinder Cancer Cell Motility through Regulation of Microtubule and Actin Dynamics

Tumor Treating Fields (TTFields) are noninvasive, alternating electric fields within the intermediate frequency range (100–300 kHz) that are utilized as an antimitotic cancer treatment. TTFields are loco-regionally delivered to the tumor region through 2 pairs of transducer arrays placed on the skin. This novel treatment modality has been FDA-approved for use in patients with glioblastoma and malignant pleural mesothelioma based on clinical trial data demonstrating efficacy and safety; and is currently under investigation in other types of solid tumors. TTFields were shown to induce an anti-mitotic effect by exerting bi-directional forces on highly polar intracellular elements, such as tubulin and septin molecules, eliciting abnormal microtubule polymerization during spindle formation as well as aberrant cleavage furrow formation. Previous studies have demonstrated that TTFields inhibit metastatic properties in cancer cells. However, the consequences of TTFields application on cytoskeleton dynamics remain undetermined. In this study, methods utilized in combination to study the effects of TTFields on cancer cell motility through regulation of microtubule and actin dynamics included confocal microscopy, computational tools, and biochemical analyses. Mechanisms by which TTFields treatment disrupted cellular polarity were (1) interference with microtubule assembly and directionality; (2) altered regulation of Guanine nucleotide exchange factor-H1 (GEF-H1), Ras homolog family member A (RhoA), and Rho-associated coiled-coil kinase (ROCK) activity; and (3) induced formation of radial protrusions of peripheral actin filaments and focal adhesions. Overall, these data identified discrete effects of TTFields that disrupt processes crucial for cancer cell motility

vMIP-II-Ig mainly recognizes the naïve CD56^Dim CD16^Pos NK cell population which express the CX3CR1 and CXCR1 chemokine receptors.

<p>(A) Freshly isolated naïve NK cells were double stained with anti-CD56 mAb together with control-Ig or with vMIP-II-Ig. The CD56^Dim CD16^Pos and CD56^Bright CD16^Neg NK cell populations are indicated by an arrow. The percentages of the various populations are indicated. (B) Expression of various chemokine receptors on freshly isolated naïve NK cells. Staining was performed with anti-CD56 mAb together with specific antibodies against CCR1, CCR2, CCR3, CCR5 and CXCR4. The percentages of the various populations are indicated. (C) CCR5 expression varies between different individuals. Staining of various donors (indicated on top of the dot plots) was performed with anti-CD56 mAb together with specific antibodies against CCR5. (D and E) Chemokine receptors expressed primarily by the CD56^Dim CD16^Pos population. Freshly isolated naïve NK cells were double stained with anti-CD56 mAb together with anti-CXCR1 (D) or anti-CX3CR1 (E). The percentages of the various populations are indicated. Figure shows one representative experiment out of three performed.</p

vMIP-II binds activated NK cells that express CCR5.

<p>(A) vMIP-II-Ig binding to activated NK cells (Act-NK) (black open histogram). The filled grey histogram is the secondary antibody staining. (B) Activated NK cells were double stained with anti-CD56 together with anti-CX3CR1 (left dot plot) or with anti CXCR1 (right dot plot). Numbers represent percentages. (C) Activated NK cells were double stained with anti-CD56 mAb and antibodies against CCR1, CCR2, CCR3, CCR5 and CXCR4 (indicated in the X axis). Numbers represent percentages. (D and E) rhRANTES (D) or rvMIP-II (E) were placed in the bottom chamber of transwell plates and the migration of activated NK was quantified by FACS following a 3 hours incubation period at 37°C. The migration of the NK cells without the appropriate chemokine was set as 1 and the results are presented as fold increase (FI). ***P<0.001. NS - not significant. Figure shows one representative experiment out of four performed.</p

vMIP-II blocks the migration of freshly isolated naïve NK cells to Fractalkine.

<p>(A) Freshly isolated naïve NK cells were double stained with Fck-Ig and with anti-CD56 mAb. The percentages of the various populations are indicated in the figure. (B) CX3CR1 expression on the transfectant 293T-CX3CR1 cells (black open histogram) or on 293T parental cells (filled grey histogram). (C) Binding of Fck-Ig (left histogram) or vMIP-II-Ig (right histogram) to 293T-CX3CR1 transfectant (black open histogram) or to the 293T parental cells (filled grey histogram). (D) 293T-CX3CR1 cells were incubated with (black empty histogram) and without (dark gray empty histogram) rvMIP-II for 1 hour in 4°C and then stained with Fck-Ig. The light gray filled histogram is the staining of Fck-Ig on the 293T parental cells. (E) Freshly isolated naïve NK cells were incubated at 4°C for 1 hour with and without the proteins indicated in the x axis. RhFck was placed in the bottom chamber and the numbers of migrated cells was determined by FACS following 3 hours incubation at 37°C. The migration of the NK cells without the appropriate chemokine was set as 1 and the results are presented as fold increase (FI). **P<0.01. NS - not significant. Figure shows one representative experiment out of four performed.</p

vMIP-II-Ig binds PBLs and naïve NK cells.

<p>Freshly isolated PBLs (A), freshly isolated naïve NK cells (B), Monocytes (C) and Neutrophils (D), (the various cell types are indicated in the right of the figure), were stained with different KSHV chemokines fused to human IgG1: vIL6-Ig, vMIP-I-Ig, vMIP-II-Ig or vMIP-III-Ig (X axis, indicated in the top of the figure). PBLs were double stained with the indicated chemokines fused to IgG and with anti-CD3. The percentages of various populations are indicated inside the dot plot. The Median Fluorescence Intensity (MFI) of the vMIP-II-Ig staining of CD3+ cells (A) and of NK cells (B) is indicated and is marked by an arrow. Figure shows one representative staining out of more than 3 performed.</p

vMIP-II in the supernatant of KSHV infected cells inhibits the migration of naïve and activated NK cells.

<p>(A) RvMIP-II was used as standard protein in ELISA assay to determine the concentration of vMIP-II in the supernatant of KSHV infected cells (black square). (B) Naïve NK cells were incubated at 4°C for 1 hour with rvMIP-II or with concentrated supernatant from KSHV infected cells (KSHV supernatant). RhFck was placed in the bottom chamber and the numbers of migrated cells was determined by FACS following 3 hours incubation at 37°C. The migration of the NK cells towards Fck was set as 100%. (C) Activated NK cells were incubated at 4°C for 1 hour with rvMIP-II or with concentrated supernatant from KSHV infected cells (KSHV supernatant). RhRANTES was placed in the bottom chamber and the numbers of migrated cells was determined by FACS following 3 hours incubation at 37°C. The migration of the NK cells towards RANTES was set as 100%. **P<0.01. ***P<0.001. Figure shows one representative experiment out of two performed.</p

vMIP-II blocks the migration of activated NK cells towards RANTES.

<p>(A) Activated NK cells were stained with RANTES-Ig (black open histogram). The filled grey histogram is the secondary antibody staining. (B) U87-CCR5 (black open histogram) or U87 parental cells (filled grey histogram) were stained with anti-CCR5 mAb. (C) FACS staining of RANTES-Ig (left histogram) or vMIP-II-Ig (right histogram) binding to U87-CCR5 cells (staining in both histograms is represented by the black open histogram). The gray filled histograms are the staining of the U87 parental cells with the indicated fusion proteins. (D) U87-CCR5 cells were incubated with (black empty histogram) and without (dark gray empty histogram) rvMIP-II for 1 hour in 4°C and then stained with RANTES-Ig. The light gray filled histogram is the staining of RANTES-Ig on the U87 parental cells. (E) Activated NK cells were incubated at 4°C for 1 hour with and without the proteins indicated in the x axis. RhRANTES was placed in the bottom chamber and the numbers of migrated cells was determined by FACS following 3 hours incubation at 37°C. The migration of the NK cells without the appropriate chemokine was set as 1 and the results are presented as fold increase. ***P<0.001. The figure shows one representative experiment out of four performed.</p