A Natural Genetic Variant of Granzyme B Confers Lethality to a Common Viral Infection

Many immune response genes are highly polymorphic, consistent with the selective pressure imposed by pathogens over evolutionary time, and the need to balance infection control with the risk of auto-immunity. Epidemiological and genomic studies have identified many genetic variants that confer susceptibility or resistance to pathogenic micro-organisms. While extensive polymorphism has been reported for the granzyme B (GzmB) gene, its relevance to pathogen immunity is unexplored. Here, we describe the biochemical and cytotoxic functions of a common allele of GzmB (GzmBW) common in wild mouse. While retaining ‘Asp-ase ’ activity, GzmBW has substrate preferences that differ considerably from GzmBP, which is common to all inbred strains. In vitro, GzmBW preferentially cleaves recombinant Bid, whereas GzmBP activates pro-caspases directly. Recombinant GzmBW and GzmBP induced equivalent apoptosis of uninfected targets cells when delivered with perforin in vitro. Nonetheless, mice homozygous for GzmBW were unable to control murine cytomegalovirus (MCMV) infection, and succumbed as a result of excessive liver damage. Although similar numbers of anti-viral CD8 T cells were generated in both mouse strains, GzmBW-expressing CD8 T cells isolated from infected mice were unable to kill MCMV

The Murine Natural Cytotoxic Receptor NKp46/NCR1 Controls TRAIL Protein Expression in NK Cells and ILC1s

Summary: TRAIL is an apoptosis-inducing ligand constitutively expressed on liver-resident type 1 innate lymphoid cells (ILC1s) and a subset of natural killer (NK) cells, where it contributes to NK cell anti-tumor, anti-viral, and immunoregulatory functions. However, the intrinsic pathways involved in TRAIL expression in ILCs remain unclear. Here, we demonstrate that the murine natural cytotoxic receptor mNKp46/NCR1, expressed on ILC1s and NK cells, controls TRAIL protein expression. Using NKp46-deficient mice, we show that ILC1s lack constitutive expression of TRAIL protein and that NK cells activated in vitro and in vivo fail to upregulate cell surface TRAIL in the absence of NKp46. We show that NKp46 regulates TRAIL expression in a dose-dependent manner and that the reintroduction of NKp46 in mature NK cells deficient for NKp46 is sufficient to restore TRAIL surface expression. These studies uncover a link between NKp46 and TRAIL expression in ILCs with potential implications in pathologies involving NKp46-expressing cells. : Sheppard et al. find that mice deficient in the activating receptor NCR1/NKp46 (Ncr1−/−) fail to express the apoptosis-inducing ligand TRAIL at the surface of group 1 innate lymphoid cells (ILC1s). Keywords: NK cell, natural killer cell, NKp46, ILC1, TRAIL, IL-15, IL-

ILC1 Confer Early Host Protection at Initial Sites of Viral Infection

Infection is restrained by the concerted activation of tissue-resident and circulating immune cells. Whether tissue-resident lymphocytes confer early antiviral immunity at local sites of primary infection prior to the initiation of circulating responses is not well understood. Furthermore, the kinetics of initial antiviral responses at sites of infection remain unclear. Here, we show that tissue-resident type 1 innate lymphoid cells (ILC1) serve an essential early role in host immunity through rapid production of interferon (IFN)-γ following viral infection. Ablation of Zfp683-dependent liver ILC1 lead to increased viral load in the presence of intact adaptive and innate immune cells critical for mouse cytomegalovirus (MCMV) clearance. Swift production of interleukin (IL)-12 by tissue-resident XCR1+ conventional dendritic cells (cDC1) promoted ILC1 production of IFN-γ in a STAT4-dependent manner to limit early viral burden. Thus, ILC1 contribute an essential role in viral immunosurveillance at sites of initial infection in response to local cDC1-derived proinflammatory cytokines

Strain-specific antibody therapy prevents cytomegalovirus reactivation after transplantation

Cytomegalovirus infection is a frequent and life-threatening complication that significantly limits positive transplantation outcomes. We developed preclinical mouse models of cytomegalovirus reactivation after transplantation and found that humoral immunity is essential for preventing viral recrudescence. Preexisting antiviral antibodies decreased after transplant in the presence of graft-versus-host disease and were not replaced, owing to poor reconstitution of donor B cells and elimination of recipient plasma cells. Viral reactivation was prevented by the transfer of immune serum, without a need to identify and target specific antigenic determinants. Notably, serotherapy afforded complete protection, provided that the serum was matched to the infecting viral strain. Thus, we define the mechanisms for cytomegalovirus reactivation after transplantation and identify a readily translatable strategy of exceptional potency, which avoids the constraints of cellular therapies

Cytomegalovirus establishes a latent reservoir and triggers long-lasting inflammation in the eye

<div><p>Recent outbreaks of Ebola and Zika have highlighted the possibility that viruses may cause enduring infections in tissues like the eye, including the neural retina, which have been considered immune privileged. Whether this is a peculiarity of exotic viruses remains unclear, since the impact of more common viral infections on neural compartments has not been examined, especially in immunocompetent hosts. Cytomegalovirus is a common, universally distributed pathogen, generally innocuous in healthy individuals. Whether in immunocompetent hosts cytomegalovirus can access the eye, and reside there indefinitely, was unknown. Using the well-established murine cytomegalovirus infection model, we show that systemic infection of immunocompetent hosts results in broad ocular infection, chronic inflammation and establishment of a latent viral pool in the eye. Infection leads to infiltration and accumulation of anti-viral CD8⁺ T cells in the eye, and to the development of tissue resident memory T cells that localize to the eye, including the retina. These findings identify the eye as an unexpected reservoir for cytomegalovirus, and suggest that common viruses may target this organ more frequently than appreciated. Notably, they also highlight that infection triggers sustained inflammatory responses in the eye, including the neural retina.</p></div

Acute GVHD results in a severe DC defect that prevents T-cell priming and leads to fulminant cytomegalovirus disease in mice

Viral infection is a common, life-threatening complication after allogeneic bone marrow transplantation (BMT), particularly in the presence of graft-versus-host disease (GVHD). Using cytomegalovirus (CMV) as the prototypic pathogen, we have delineated the mechanisms responsible for the inability to mount protective antiviral responses in this setting. Although CMV infection was self-limiting after syngeneic BMT, in the presence of GVHD after allogeneic BMT, CMV induced a striking cytopathy resulting in universal mortality in conjunction with a fulminant necrotizing hepatitis. Critically, GVHD induced a profound dendritic cell (DC) defect that led to a failure in the generation of CMV-specific CD8(+) T-cell responses. This was accompanied by a defect in antiviral CD8(+) T cells. In combination, these defects dramatically limited antiviral T-cell responses. The transfer of virus-specific cells circumvented the DC defects and provided protective immunity, despite concurrent GVHD. These data demonstrate the importance of avoiding GVHD when reconstructing antiviral immunity after BMT, and highlight the mechanisms by which the adoptive transfer of virus-specific T cells overcome the endogenous defects in priming invoked by GVHD

MCMV infection causes inflammation in the iris and retina.

<p><b>(A)</b> Schematic diagram with the major compartments of the eye labelled. <b>(B)</b> Sections of the anterior chamber at the indicated times pi were stained with haematoxylin and eosin. Thickening of the iris was evident at day 5. Cellular infiltrates and keratic precipitates were present after infection (black arrows). Synechia (adherence of the iris to the cornea) was frequently observed (red arrows). <b>(C)</b> Haematoxylin and eosin stained sections of the retina show normal structure at day 5 pi. Enlarged vessels (two tailed arrow head), folds in the retina (asterisk) and infiltrating cells were present (black arrows) at day 10pi. At day 25 pi infiltrating cells within the retina (black arrow heads) and the vitreous (red arrow heads) were evident. Scale bar: 100 μm. Results are representative of those from 5 mice per time-point.</p

Defining the compartments of the eye infected by MCMV.

<p><b>(A)</b> Haematoxylin and eosin stained section of an uninfected mouse eye with the major compartments labelled. Scale bar: 500 μm. <b>(B)</b> Eyes were removed from MCMV-infected mice at day 5 pi, components of the eye dissected and whole-mounts prepared. The samples were stained with antibodies against MHC-II (red) and IE1 (green—for viral antigen) and microscopy performed. Scale bar: 200 μm. Results are representative of at least 5 independent experiments with n≥20. <b>(C)</b> Mice were infected with MCMV-LacZ and eyes removed at day 5 pi. Frozen tissue sections were prepared and stained with x-gal to identify virally infected cells (blue) and counterstained with neutral red to identify cell nuclei. Scale bar: 50 μm.</p

Pathological changes in the eye after systemic MCMV infection.

<p><b>(A)</b> SD-OCT image of the anterior segment of the eye from an uninfected mouse. <b>(B)</b> Cross-sections of the anterior chamber from mice infected with MCMV at the indicated times pi. Cellular deposits (white arrows), vessel dilation and iris thickening (yellow arrows). <b>(C)</b> A representative image demonstrating iris bombé, and the formation of synechia (yellow arrow). <b>(D)</b> The frequency of mice displaying the indicated pathological features following MCMV infection was quantified where n = 10 mice for each time point from at least 3 independent experiments. ND = not detected. <b>(E)</b> SD-OCT fundus images of the retina at the indicated times pi. Black arrows indicate retinal vessel enlargement (dilation and calibre variation). <b>(F)</b> Vessel diameter was measured at the indicated times pi with mean ± standard error of the mean (SEM) plotted, where n≥8 eyes (* P = 0.035; *** P = 0.0006).</p

Virus-specific CD8⁺ T cells infiltrate and accumulate in the iris and retina after systemic MCMV infection and form tissue resident memory (T_RM) populations.

<p>Mice were infected with MCMV, and at the indicated times pi single cell preparations were prepared and stained for T cell markers. (A) The number of CD8⁺ T cells, CD8⁺ IE1⁺ antiviral T cells and CD8⁺ IE1⁺ CD103^+/- CD69^+/- T cells are graphed as mean ± SEM for (<b>A</b>) the iris and (<b>B</b>) the retina. Samples represent n = 6 for uninfected mice, and n = 15 for MCMV infected mice for each time point from 3 independent experiments. Iris samples were pooled from 5 mice.</p