Late-rising CD4 T cells resolve mouse cytomegalovirus persistent replication in the salivary gland.

Conventional antiviral memory CD4 T cells typically arise during the first two weeks of acute infection. Unlike most viruses, cytomegalovirus (CMV) exhibits an extended persistent replication phase followed by lifelong latency accompanied with some gene expression. We show that during mouse CMV (MCMV) infection, CD4 T cells recognizing an epitope derived from the viral M09 protein only develop after conventional memory T cells have already peaked and contracted. Ablating these CD4 T cells by mutating the M09 genomic epitope in the MCMV Smith strain, or inducing them by introducing the epitope into the K181 strain, resulted in delayed or enhanced control of viral persistence, respectively. These cells were shown to be unique compared to their conventional memory counterparts; producing higher IFNγ and IL-2 and lower IL-10 levels. RNAseq analyses revealed them to express distinct subsets of effector genes as compared to classical CD4 T cells. Additionally, when M09 cells were induced by epitope vaccination they significantly enhanced protection when compared to conventional CD4 T cells alone. These data show that late-rising CD4 T cells are a unique memory subset with excellent protective capacities that display a development program strongly differing from the majority of memory T cells.

Histamine-releasing factor in severe asthma and rhinovirus-associated asthma exacerbation.

BACKGROUND: Histamine-releasing factor (HRF) is implicated in allergic diseases. We previously showed its pathogenic role in murine models of asthma. OBJECTIVE: We aim to present data analysis from 3 separate human samples (sera samples from asthmatic patients, nasal washings from rhinovirus [RV]-infected individuals, and sera samples from patients with RV-induced asthma exacerbation) and 1 mouse sample to investigate correlates of HRF function in asthma and virus-induced asthma exacerbations. METHODS: Total IgE and HRF-reactive IgE/IgG as well as HRF in sera from patients with mild/moderate asthma or severe asthma (SA) and healthy controls (HCs) were quantified by ELISA. HRF secretion in culture media from RV-infected adenovirus-12 SV40 hybrid virus transformed human bronchial epithelial cells and in nasal washings from experimentally RV-infected subjects was analyzed by Western blotting. HRF-reactive IgE/IgG levels in longitudinal serum samples from patients with asthma exacerbations were also quantified. RESULTS: HRF-reactive IgE and total IgE levels were higher in patients with SA than in HCs, whereas HRF-reactive IgG (and IgG1) level was lower in asthmatic patients versus HCs. In comparison with HRF-reactive IgElow asthmatic patients, HRF-reactive IgEhigh asthmatic patients had a tendency to release more tryptase and prostaglandin D2 on anti-IgE stimulation of bronchoalveolar lavage cells. RV infection induced HRF secretion from adenovirus-12 SV40 hybrid virus transformed bronchial epithelial cells, and intranasal RV infection of human subjects induced increased HRF secretion in nasal washes. Asthmatic patients had higher levels of HRF-reactive IgE at the time of asthma exacerbations associated with RV infection, compared with those after the resolution. This phenomenon was not seen in asthma exacerbations without viral infections. CONCLUSIONS: HRF-reactive IgE is higher in patients with SA. RV infection induces HRF secretion from respiratory epithelial cells both in vitro and in vivo. These results suggest the role of HRF in asthma severity and RV-induced asthma exacerbation.

Profiling Human Cytomegalovirus-Specific T Cell Responses Reveals Novel Immunogenic Open Reading Frames.

Despite the prevalence and medical significance of human cytomegalovirus (HCMV) infections, a systematic analysis of the targets of T cell recognition in humans that spans the entire genome and includes recently described potential novel open reading frames (ORFs) is not available. Here, we screened a library of epitopes predicted to bind HLA class II that spans over 350 different HCMV ORFs and includes ∼150 previously described and ∼200 recently described potential novel ORFs by using an ex vivo gamma interferon (IFN-γ) FluoroSpot assay. We identified 235 unique HCMV-specific epitopes derived from 100 ORFs, some previously described as immunodominant and others that were not previously described to be immunogenic. Of those, 41 belong to the set of recently reported novel ORFs, thus providing evidence that at least some of these are actually expressed in vivo in humans. These data reveal that the breadth of the human T cell response to HCMV is much greater than previously thought. The ORFs and epitopes identified will help elucidate how T cell immunity relates to HCMV pathogenesis and instruct ongoing HCMV vaccine research. IMPORTANCE To understand the crucial role of adaptive immunity in controlling cytomegalovirus infection and disease, we systematically analyzed the CMV "ORFeome" to identify new CMV epitopes targeted primarily by CD4 T cells in humans. Our study identified >200 new T cell epitopes derived from both canonical and novel ORFs, highlighting the substantial breadth of the anti-CMV T cell response and providing new targets for vaccine design.

Pathogenic Autoimmunity in Atherosclerosis Evolves From Initially Protective Apolipoprotein B100-Reactive CD4+ T-Regulatory Cells.

BACKGROUND: Throughout the inflammatory response that accompanies atherosclerosis, autoreactive CD4+ T-helper cells accumulate in the atherosclerotic plaque. Apolipoprotein B100 (apoB), the core protein of low-density lipoprotein, is an autoantigen that drives the generation of pathogenic T-helper type 1 (TH1) cells with proinflammatory cytokine secretion. Clinical data suggest the existence of apoB-specific CD4+ T cells with an atheroprotective, regulatory T cell (Treg) phenotype in healthy individuals. Yet, the function of apoB-reactive Tregs and their relationship with pathogenic TH1 cells remain unknown. METHODS: To interrogate the function of autoreactive CD4+ T cells in atherosclerosis, we used a novel tetramer of major histocompatibility complex II to track T cells reactive to the mouse self-peptide apo B978-993 (apoB+) at the single-cell level. RESULTS: We found that apoB+ T cells build an oligoclonal population in lymph nodes of healthy mice that exhibit a Treg-like transcriptome, although only 21% of all apoB+ T cells expressed the Treg transcription factor FoxP3 (Forkhead Box P3) protein as detected by flow cytometry. In single-cell RNA sequencing, apoB+ T cells formed several clusters with mixed TH signatures that suggested overlapping multilineage phenotypes with pro- and anti-inflammatory transcripts of TH1, T helper cell type 2 (TH2), and T helper cell type 17 (TH17), and of follicular-helper T cells. ApoB+ T cells were increased in mice and humans with atherosclerosis and progressively converted into pathogenic TH1/TH17-like cells with proinflammatory properties and only a residual Treg transcriptome. Plaque T cells that expanded during progression of atherosclerosis consistently showed a mixed TH1/TH17 phenotype in single-cell RNA sequencing. In addition, we observed a loss of FoxP3 in a fraction of apoB+ Tregs in lineage tracing of hyperlipidemic Apoe-/- mice. In adoptive transfer experiments, converting apoB+ Tregs failed to protect from atherosclerosis. CONCLUSIONS: Our results demonstrate an unexpected mixed phenotype of apoB-reactive autoimmune T cells in atherosclerosis and suggest an initially protective autoimmune response against apoB with a progressive derangement in clinical disease. These findings identify apoB autoreactive Tregs as a novel cellular target in atherosclerosis.

Structure of human cytomegalovirus UL144, an HVEM orthologue, bound to the B and T cell lymphocyte attenuator.

Human cytomegalovirus (HCMV) is a ß-herpesvirus that has co-evolved with the host immune system to establish lifelong persistence. HCMV encodes many immunomodulatory molecules, including the glycoprotein UL144. UL144 is a structural mimic of the tumor necrosis factor receptor superfamily member HVEM (herpesvirus entry mediator), which binds to the various ligands LIGHT, LTα, BTLA, CD160, and gD. However, in contrast to HVEM, UL144 only binds BTLA, inhibiting T-cell activation. Here, we report the crystal structure of the UL144-BTLA complex, revealing that UL144 utilizes residues from its N-terminal cysteine-rich domain 1 (CRD1) to interact uniquely with BTLA. The shorter CRD2 loop of UL144 also alters the relative orientation of BTLA binding with both N-terminal CRDs. By employing structure-guided mutagenesis, we have identified a mutant of BTLA (L123A) that interferes with HVEM binding but preserves UL144 interactions. Furthermore, our results illuminate structural differences between UL144 and HVEM that explain its binding selectivity and highlight it as a suitable scaffold for designing superior, immune inhibitory BTLA agonists.

Cytomegalovirus Evades TRAIL-Mediated Innate Lymphoid Cell 1 Defenses.

Cytomegalovirus (CMV) establishes a lifelong infection facilitated, in part, by circumventing immune defenses mediated by tumor necrosis factor (TNF)-family cytokines. An example of this is the mouse CMV (MCMV) m166 protein, which restricts expression of the TNF-related apoptosis-inducing ligand (TRAIL) death receptors, promoting early-phase replication. We show here that replication of an MCMV mutant lacking m166 is also severely attenuated during viral persistence in the salivary glands (SG). Depleting group I innate lymphoid cells (ILCs) or infecting Trail-/- mice completely restored persistent replication of this mutant. Group I ILCs are comprised of two subsets, conventional natural killer cells (cNK) and tissue-resident cells often referred to as innate lymphoid type I cells (ILC1). Using recently identified phenotypic markers to discriminate between these two cell types, their relative expression of TRAIL and gamma interferon (IFN-γ) was assessed during both early and persistent infection. ILC1 were found to be the major TRAIL expressers during both of these infection phases, with cNK expressing very little, indicating that it is ILC1 that curtail replication via TRAIL in the absence of m166-imposed countermeasures. Notably, despite high TRAIL expression by SG-resident ILC1, IFN-γ production by both ILC1 and cNK was minimal at this site of viral persistence. Together these results highlight TRAIL as a key ILC1-utilized effector molecule that can operate in defense against persistent infection at times when other innate control mechanisms may be muted and highlight the importance for the evolution of virus-employed countermeasures.IMPORTANCE Cytomegalovirus (a betaherpesvirus) is a master at manipulating immune responses to promote its lifelong persistence, a result of millions of years of coevolution with its host. Using a one-of-a-kind MCMV mutant unable to restrict expression of the TNF-related apoptosis-inducing ligand death receptors (TRAIL-DR), we show that TRAIL-DR signaling significantly restricts both early and persistent viral replication. Our results also reveal that these defenses are employed by TRAIL-expressing innate lymphoid type I cells (ILC1) but not conventional NK cells. Overall, our results are significant because they show the key importance of viral counterstrategies specifically neutralizing TRAIL effector functions mediated by a specific, tissue-resident subset of group I ILCs.

Restriction of Human Cytomegalovirus Infection by Galectin-9.

Human cytomegalovirus (HCMV) is a ubiquitous human herpesvirus. While HCMV infection is generally asymptomatic in the immunocompetent, it can have devastating consequences in those with compromised or underdeveloped immune systems, including transplant recipients and neonates. Galectins are a widely expressed protein family that have been demonstrated to modulate both antiviral immunity and regulate direct host-virus interactions. The potential for galectins to directly modulate HCMV infection has not previously been studied, and our results reveal that galectin-9 (Gal-9) can potently inhibit HCMV infection. Gal-9-mediated inhibition of HCMV was dependent upon its carbohydrate recognition domains and thus dependent on glycan interactions. Temperature shift studies revealed that Gal-9 specific inhibition was mediated primarily at the level of virus-cell fusion and not binding. Additionally, we found that during reactivation of HCMV in hematopoietic stem cell transplant (HSCT) patients soluble Gal-9 is upregulated. This study provides the first evidence for Gal-9 functioning as a potent antiviral defense effector molecule against HCMV infection and identifies it as a potential clinical candidate to restrict HCMV infections.IMPORTANCE Human cytomegalovirus (HCMV) continues to cause serious and often life-threatening disease in those with impaired or underdeveloped immune systems. This virus is able to infect and replicate in a wide range of human cell types, which enables the virus to spread to other individuals in a number of settings. Current antiviral drugs are associated with a significant toxicity profile, and there is no vaccine; these factors highlight a need to identify additional targets for the development of anti-HCMV therapies. We demonstrate for the first time that secretion of a member of the galectin family of proteins, galectin-9 (Gal-9), is upregulated during natural HCMV-reactivated infection and that this soluble cellular protein possesses a potent capacity to block HCMV infection by inhibiting virus entry into the host cell. Our findings support the possibility of harnessing the antiviral properties of Gal-9 to prevent HCMV infection and disease.

Cytomegalovirus: Shape-Shifting the Immune System.

Systems-based based approaches have begun to shed light on extrinsic factors that contribute to immune system variation. Among these, CMV (HHV-5, a ß-herpesvirus) imposes a surprisingly profound impact. Most of the world's population is CMV+, and the virus goes through three distinct infection phases en route to establishing lifelong détente with its host. Immune control of CMV in each phase recruits unique arms of host defense, and in turn the virus employs multiple immune-modulatory strategies that help facilitate the establishment of lifelong persistence. In this review, we explain how CMV shapes immunity and discuss the impact it may have on overall health.

Crystal structure of murine 4-1BB and its interaction with 4-1BBL support a role for galectin-9 in 4-1BB signaling.

4-1BB (CD137) is a TNF receptor superfamily (TNFRSF) member that is thought to undergo receptor trimerization upon binding to its trimeric TNF superfamily ligand (4-1BBL) to stimulate immune responses. 4-1BB also can bind to the tandem repeat-type lectin galectin-9 (Gal-9), and signaling through mouse (m)4-1BB is reduced in galectin-9 (Gal-9)-deficient mice, suggesting a pivotal role of Gal-9 in m4-1BB activation. Here, using sulfur-SAD phasing, we determined the crystal structure of m4-1BB to 2.2-Å resolution. We found that similar to other TNFRSFs, m4-1BB has four cysteine-rich domains (CRDs). However, the organization of CRD1 and the orientation of CRD3 and CRD4 with respect to CRD2 in the m4-1BB structure distinctly differed from those of other TNFRSFs. Moreover, we mapped two Asn residues within CRD4 that are N-linked glycosylated and mediate m4-1BB binding to Gal-9. Kinetics studies of m4-1BB disclosed a very tight nanomolar binding affinity to m4-1BBL with an unexpectedly strong avidity effect. Both N- and C-terminal domains of Gal-9 bound m4-1BB, but with lower affinity compared with m4-1BBL. Although the TNF homology domain (THD) of human (h)4-1BBL forms non-covalent trimers, we found that m4-1BBL formed a covalent dimer via 2 cysteines absent in h4-1BBL. As multimerization and clustering is a prerequisite for TNFR intracellular signaling, and as m4-1BBL can only recruit two m4-1BB monomers, we hypothesize that m4-1BBL and Gal-9 act together to aid aggregation of m4-1BB monomers to efficiently initiate m4-1BB signaling.

Cytomegalovirus-Specific CD4 T Cells Are Cytolytic and Mediate Vaccine Protection.

UNLABELLED: CD4 T cells provide protection against cytomegalovirus (CMV) and other persistent viruses, and the ability to quantify and characterize epitope-specific responses is essential to gain a more precise understanding of their effector roles in this regard. Here, we report the first two I-A(d)-restricted CD4 T cell responses specific for mouse CMV (MCMV) epitopes and use a major histocompatibility complex class II (MHC-II) tetramer to characterize their phenotypes and functions. We demonstrate that MCMV-specific CD4 T cells can express high levels of granzyme B and kill target cells in an epitope- and organ-specific manner. In addition, CD4 T cell epitope vaccination of immunocompetent mice reduced MCMV replication in the same organs where CD4 cytotoxic T lymphocyte (CTL) activity was observed. Together, our studies show that MCMV epitope-specific CD4 T cells have the potential to mediate antiviral defense by multiple effector mechanisms in vivo. IMPORTANCE: CD4 T cells mediate immune protection by using their T cell receptors to recognize specific portions of viral proteins, called epitopes, that are presented by major histocompatibility complex class II (MHC-II) molecules on the surfaces of professional antigen-presenting cells (APCs). In this study, we discovered the first two epitopes derived from mouse cytomegalovirus (MCMV) that are recognized by CD4 T cells in BALB/c mice, a mouse strain commonly used to study the pathogenesis of this virus infection. Here, we report the sequences of these epitopes, characterize the CD4 T cells that recognize them to fight off MCMV infection, and show that we can use the epitopes to vaccinate mice and protect against MCMV.

cGAS-STING Signaling Regulates Initial Innate Control of Cytomegalovirus Infection.

UNLABELLED: Several innate sensing pathways contribute to the control of early cytomegalovirus (CMV) infection, leading to a multiphasic type I interferon (IFN-I) response that limits viral replication and promotes host defenses. Toll-like receptor (TLR)-dependent pathways induce IFN-I production in CMV-infected plasmacytoid dendritic cells; however, the initial burst of IFN-I that occurs within the first few hours in vivo is TLR independent and emanates from stromal cells. Here we show that primary human endothelial cells mount robust IFN-I responses to human CMV that are dependent upon cyclic GMP-AMP synthase (cGAS), STING, and interferon regulatory factor 3 (IRF3) signaling. Disruption of STING expression in endothelial cells by clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 revealed that it is essential for the induction of IFN-I and restriction of CMV replication. Consistently, STING was necessary to mount the first phase of IFN-I production and curb CMV replication in infected mice. Thus, DNA sensing through STING is critical for primary detection of both human and mouse CMV in nonhematopoietic cells and drives the initial wave of IFN-I that is key for controlling early viral replication in vivo. IMPORTANCE: Cytomegalovirus (CMV) is one of the most common viral pathogens, with the majority of people contracting the virus in their lifetime. Although acute infection is mostly asymptomatic in healthy persons, significant pathology is observed in immunocompromised individuals, and chronic CMV infection may exacerbate a myriad of inflammatory conditions. Here we show that primary human endothelial cells mount robust IFN-I responses against CMV via a cGAS/STING/IRF3 pathway. Disruption of STING expression by CRISPRs revealed an essential role in eliciting IFN-I responses and restricting CMV replication. Consistently, in mice, STING is necessary for the first phase of IFN-I production that limits early CMV replication. Our results demonstrate a pivotal role for the cGAS-STING pathway in the initial detection of CMV infection.

Cardif (MAVS) Regulates the Maturation of NK Cells.

Cardif, also known as IPS-1, VISA, and MAVS, is an intracellular adaptor protein that functions downstream of the retinoic acid-inducible gene I family of pattern recognition receptors. Cardif is required for the production of type I IFNs and other inflammatory cytokines after retinoic acid-inducible gene I-like receptors recognize intracellular antigenic RNA. Studies have recently shown that Cardif may have other roles in the immune system in addition to its role in viral immunity. In this study, we find that the absence of Cardif alters normal NK cell development and maturation. Cardif(-/-) mice have a 35% loss of mature CD27(-)CD11b(+) NK cells in the periphery. In addition, Cardif(-/-) NK cells have altered surface marker expression, lower cytotoxicity, decreased intracellular STAT1 levels, increased apoptosis, and decreased proliferation compared with wild-type NK cells. Mixed chimeric mice revealed that the defective maturation and increased apoptotic rate of peripheral Cardif(-/-) NK cells is cell intrinsic. However, Cardif(-/-) mice showed enhanced control of mouse CMV (a DNA ß-herpesvirus) by NK cells, commensurate with increased activation and IFN-γ production by these immature NK cell subsets. These results indicate that the skewed differentiation and altered STAT expression of Cardif(-/-) NK cells can result in their hyperresponsiveness in some settings and support recent findings that Cardif-dependent signaling can regulate aspects of immune cell development and/or function distinct from its well-characterized role in mediating cell-intrinsic defense to RNA viruses.

Inhibition of the TRAIL death receptor by CMV reveals its importance in NK cell-mediated antiviral defense.

TNF-related apoptosis inducing ligand (TRAIL) death receptors (DR) regulate apoptosis and inflammation, but their role in antiviral defense is poorly understood. Cytomegaloviruses (CMV) encode many immune-modulatory genes that shape host immunity, and they utilize multiple strategies to target the TNF-family cytokines. Here we show that the m166 open reading frame (orf) of mouse CMV (MCMV) is strictly required to inhibit expression of TRAIL-DR in infected cells. An MCMV mutant lacking m166 expression (m166stop) is severely compromised for replication in vivo, most notably in the liver, and depleting natural killer (NK) cells, or infecting TRAIL-DR-/- mice, restored MCMV-m166stop replication completely. These results highlight the critical importance for CMV to have evolved a strategy to inhibit TRAIL-DR signaling to thwart NK-mediated defenses.

Distinct requirements for activation of NKT and NK cells during viral infection.

NK cells are key regulators of innate defense against mouse CMV (MCMV). Like NK cells, NKT cells also produce high levels of IFN-γ rapidly after MCMV infection. However, whether similar mechanisms govern activation of these two cell types, as well as the significance of NKT cells for host resistance, remain unknown. In this article, we show that, although both NKT and NK cells are activated via cytokines, their particular cytokine requirements differ significantly in vitro and in vivo. IL-12 is required for NKT cell activation in vitro but is not sufficient, whereas NK cells have the capacity to be activated more promiscuously in response to individual cytokines from innate cells. In line with these results, GM-CSF-derived dendritic cells activated only NK cells upon MCMV infection, consistent with their virtual lack of IL-12 production, whereas Flt3 ligand-derived dendritic cells produced IL-12 and activated both NK and NKT cells. In vivo, NKT cell activation was abolished in IL-12(-/-) mice infected with MCMV, whereas NK cells were still activated. In turn, splenic NK cell activation was more IL-18 dependent. The differential requirements for IL-12 and IL-18 correlated with the levels of cytokine receptor expression by NK and NKT cells. Finally, mice lacking NKT cells showed reduced control of MCMV, and depleting NK cells further enhanced viral replication. Taken together, our results show that NKT and NK cells have differing requirements for cytokine-mediated activation, and both can contribute nonredundantly to MCMV defense, revealing that these two innate lymphocyte subsets function together to fine-tune antiviral responses.

Structure of human cytomegalovirus UL141 binding to TRAIL-R2 reveals novel, non-canonical death receptor interactions.

The TRAIL (TNF-related apoptosis inducing ligand) death receptors (DRs) of the tumor necrosis factor receptor superfamily (TNFRSF) can promote apoptosis and regulate antiviral immunity by maintaining immune homeostasis during infection. In turn, human cytomegalovirus (HCMV) expresses immunomodulatory proteins that down-regulate cell surface expression of TNFRSF members as well as poliovirus receptor-related proteins in an effort to inhibit host immune effector pathways that would lead to viral clearance. The UL141 glycoprotein of human cytomegalovirus inhibits host defenses by blocking cell surface expression of TRAIL DRs (by retention in ER) and poliovirus receptor CD155, a nectin-like Ig-fold molecule. Here we show that the immunomodulatory function of HCMV UL141 is associated with its ability to bind diverse proteins, while utilizing at least two distinct binding sites to selectively engage TRAIL DRs or CD155. Binding studies revealed high affinity interaction of UL141 with both TRAIL-R2 and CD155 and low affinity binding to TRAIL-R1. We determined the crystal structure of UL141 bound to TRAIL-R2 at 2.1 Å resolution, which revealed that UL141 forms a homodimer that engages two TRAIL-R2 monomers 90° apart to form a heterotetrameric complex. Our structural and biochemical data reveal that UL141 utilizes its Ig-domain to facilitate non-canonical death receptor interactions while UL141 partially mimics the binding site of TRAIL on TRAIL-R2, which we found to be distinct from that of CD155. Moreover, UL141 also binds to an additional surface patch on TRAIL-R2 that is distinct from the TRAIL binding site. Therefore, the breadth of UL141-mediated effects indicates that HCMV has evolved sophisticated strategies to evade the immune system by modulating multiple effector pathways.

CD160 activation by herpesvirus entry mediator augments inflammatory cytokine production and cytolytic function by NK cells.

Lymphocyte activation is regulated by costimulatory and inhibitory receptors, of which both B and T lymphocyte attenuator (BTLA) and CD160 engage herpesvirus entry mediator (HVEM). Notably, it remains unclear how HVEM functions with each of its ligands during immune responses. In this study, we show that HVEM specifically activates CD160 on effector NK cells challenged with virus-infected cells. Human CD56(dim) NK cells were costimulated specifically by HVEM but not by other receptors that share the HVEM ligands LIGHT, Lymphotoxin-α, or BTLA. HVEM enhanced human NK cell activation by type I IFN and IL-2, resulting in increased IFN-γ and TNF-α secretion, and tumor cell-expressed HVEM activated CD160 in a human NK cell line, causing rapid hyperphosphorylation of serine kinases ERK1/2 and AKT and enhanced cytolysis of target cells. In contrast, HVEM activation of BTLA reduced cytolysis of target cells. Together, our results demonstrate that HVEM functions as a regulator of immune function that activates NK cells via CD160 and limits lymphocyte-induced inflammation via association with BTLA.

Lymphoid-tissue stromal cells coordinate innate defense to cytomegalovirus.

During mouse cytomegalovirus (MCMV) infection, the first wave of type I interferon (IFN-I) production peaks at ≈ 8 h. This IFN-I emanates from splenic stromal cells located in the marginal zone (MZ) and requires B cells that express lymphotoxin. The amount of IFN-I produced at these initial times is at least equivalent in magnitude to that produced later by dendritic cells (≈ 36 to 48 h), but the relative roles of these two IFN-I sources in regulating MCMV defense remain unclear. Here we show that IFN-I produced by MZ stromal cells dramatically restricts the first measurable burst of viral production, which occurs at ≈ 32 h. This primary innate control by IFN-I is partially mediated through the activation of natural killer (NK) cells, which produce gamma interferon in an IFN-I-dependent fashion, and is independent of Ly49H. Strikingly, MCMV production in the spleens of immunocompetent mice never increases at times after 32 h. These results highlight the critical importance of lymphoid-tissue stromal cells in orchestrating the earliest phase of innate defense to MCMV infection, capping replication levels, and blocking spread until infection is ultimately controlled.

CD27-CD70 costimulation controls T cell immunity during acute and persistent cytomegalovirus infection.

Cytomegaloviruses (CMVs) establish lifelong infections that are controlled in part by CD4(+) and CD8(+) T cells. To promote persistence, CMVs utilize multiple strategies to evade host immunity, including modulation of costimulatory molecules on infected antigen-presenting cells. In humans, CMV-specific memory T cells are characterized by the loss of CD27 expression, which suggests a critical role of the costimulatory receptor-ligand pair CD27-CD70 for the development of CMV-specific T cell immunity. In this study, the in vivo role of CD27-CD70 costimulation during mouse CMV infection was examined. During the acute phase of infection, the magnitudes of CMV-specific CD4(+) and CD8(+) T cell responses were decreased in mice with abrogated CD27-CD70 costimulation. Moreover, the accumulation of inflationary memory T cells during the persistent phase of infection and the ability to undergo secondary expansion required CD27-CD70 interactions. The downmodulation of CD27 expression, however, which occurs gradually and exclusively on inflationary memory T cells, is ligand independent. Furthermore, the IL-2 production in both noninflationary and inflationary CMV-specific T cells was dependent on CD27-CD70 costimulation. Collectively, these results highlight the importance of the CD27-CD70 costimulation pathway for the development of CMV-specific T cell immunity during acute and persistent infection.

Cytomegalovirus exploits IL-10-mediated immune regulation in the salivary glands.

The salivary glands represent a major site of cytomegalovirus replication and transmission to other hosts. Despite control of viral infection by strong T cell responses in visceral organs cytomegalovirus replication continues in the salivary glands of mice, suggesting that the virus exploits the mucosal microenvironment. Here, we show that T cell immunity in the salivary glands is limited by the induction of CD4 T cells expressing the regulatory cytokine interleukin (IL)-10. Blockade of IL-10 receptor (IL-10R) with an antagonist antibody dramatically reduced viral load in the salivary glands, but not in the spleen. The mucosa-specific protection afforded by IL-10R blockade was associated with an increased accumulation of CD4 T cells expressing interferon gamma, suggesting that IL-10R signaling limits effector T cell differentiation. Consistent with this, an agonist antibody targeting the tumor necrosis factor receptor superfamily member OX40 (TNFRSF4) enhanced effector T cell differentiation and increased the number of interferon gamma-producing T cells, thus limiting virus replication in the salivary glands. Collectively, the results indicate that modulating effector T cell differentiation can counteract pathogen exploitation of the mucosa, thus limiting persistent virus replication and transmission.