Memory T cells persisting in the brain following MCMV infection induce long-term microglial activation via interferon-?.
ABSTRACT: Murine cytomegalovirus (MCMV) brain infection stimulates microglial cell-driven proinflammatory chemokine production which precedes the presence of brain-infiltrating systemic immune cells. Here, we show that in response to MCMV brain infection, antigen-specific CD8(+) T cells migrated into the brain and persisted as long-lived memory cells. The role of these persistent T cells in the brain is unclear because most of our understanding of antimicrobial T cell responses comes from analyses of lymphoid tissue. Strikingly, memory T cells isolated from the brain exhibited an effector phenotype and produced IFN-? upon restimulation with viral peptide. Furthermore, we observed time-dependent and long-term activation of resident microglia, indicated by chronic MHC class II up-regulation and TNF-? production. The immune response in this immunologically restricted site persisted in the absence of active viral replication. Lymphocyte infiltrates were detected until 30 days post-infection (p.i.), with CD8(+) and CD4(+) T cells present at a 3:1 ratio, respectively. We then investigated the role of IFN-? in chronic microglial activation by using IFN-?-knockout (GKO) mice. At 30 days p.i., GKO mice demonstrated a similar phenotypic brain infiltrate when compared to wild-type mice (Wt), however, MHC class II expression on microglia isolated from these GKO mice was significantly lower compared to Wt animals. When IFN-? producing CD8(+) T cells were reconstituted in GKO mice, MHC class II up-regulation on microglial cells was restored. Taken together, these results suggest that MCMV brain infection results in long-term persistence of antigen-specific CD8(+) T cells which produce IFN-? and drive chronic microglial cell activation. This response was found to be dependent on IFN-? production by viral Ag-specific T cells during the chronic phase of disease.
Project description:MicroRNA (miR) miR-155 modulates microglial activation and polarization, but its role in activation of microglia during bacterial brain infection is unclear. We studied miR-155 expression in brains of C57BL/6 (B6.WT) mice infected i.p. with the neuro-invasive bacterial pathogen Listeria monocytogenes (L. monocytogenes). Infected mice were treated with ampicillin starting 2 days (d) post-infection (p.i.) and analyzed 3d, 7d, and 14d p.i. Virulent L. monocytogenes strains EGD and 10403s upregulated miR-155 in whole brain 7 d p.i. whereas infection with avirulent, non-neurotropic ?hly or ?actA L. monocytogenes mutants did not. Similarly, infection with virulent but not mutated bacteria upregulated IFN-? mRNA in the brain at 7 d p.i. Upregulation of miR-155 in microglia was confirmed by qPCR of flow cytometry-sorted CD45intCD11bpos brain cells. Subsequently, brain leukocyte influxes and gene expression in sorted microglia were compared in L. monocytogenes-infected B6.WT and B6.Cg-Mir155tm1.1Rsky/J (B6.miR-155-/-) mice. Brain influxes of Ly-6Chigh monocytes and upregulation of IFN-related genes in microglia were similar to B6.WT mice at 3 d p.i. In contrast, by d 7 p.i. expressions of microglial IFN-related genes, including markers of M1 polarization, were significantly lower in B6.miR-155-/- mice and by 14 d p.i., influxes of activated T-lymphocytes were markedly reduced. Notably, CD45highCD11bpos brain cells from B6.miR-155-/- mice isolated at 7 d p.i. expressed 2-fold fewer IFN-? transcripts than did cells from B6.WT mice suggesting reduced IFN-? stimulation contributed to dampened gene expression in B6.miR-155-/- microglia. Lastly, in vitro stimulation of 7 d p.i. brain cells with heat-killed L. monocytogenes induced greater production of TNF in B6.miR-155-/- microglia than in B6.WT microglia. Thus, miR-155 affects brain inflammation by multiple mechanisms during neuroinvasive L. monocytogenes infection. Peripheral miR-155 promotes brain inflammation through its required role in optimal development of IFN-?-secreting lymphocytes that enter the brain and activate microglia. Microglial miR-155 promotes M1 polarization, and also inhibits inflammatory responses to stimulation by heat-killed L. monocytogenes, perhaps by targeting Tab2.
Project description:Vasoactive intestinal peptide (VIP) is a neuropeptide hormone that suppresses Th1-mediated cellular immunity. We previously reported that VIP-knockout (VIP-KO) mice have enhanced cellular immune responses and increased survival following murine cytomegalovirus (mCMV) infection in C57BL/6 mice. In this study, we tested whether treatment with a VIP receptor antagonistic peptide protects C57BL/6 and BALB/c mice from mCMV-infection. One week of daily subcutaneous injections of VIPhyb was non-toxic and did not alter frequencies of immune cell subsets in non-infected mice. VIPhyb administration to mCMV-infected C57BL/6 and BALB/c mice markedly enhanced survival, viral clearance, and reduced liver and lung pathology compared with saline-treated controls. The numbers of effector/memory CD8+ T-cells and mature NK cells were increased in VIPhyb-treated mice compared with PBS-treated groups. Pharmacological blockade of VIP-receptor binding or genetic blockade of VIP-signaling prevented the up-regulation of PD-L1 and PD-1 expression on DC and activated CD8+ T-cells, respectively, in mCMV-infected mice, and enhanced CD80, CD86, and MHC-II expression on conventional and plasmacytoid DC. VIPhyb-treatment increased type-I IFN synthesis, numbers of IFN-?- and TNF-?-expressing NK cells and T-cells, and the numbers of mCMV-M45 epitope-peptide-MHC-I tetramer CD8+ T-cells following mCMV infection. VIP-treatment lowered the percentage of Treg cells in spleens compared with PBS-treated WT mice following mCMV infection, while significantly decreasing levels of serum VEGF induced by mCMV-infection. The mice in all treated groups exhibited similar levels of anti-mCMV antibody titers. Short-term administration of a VIP-receptor antagonist represents a novel approach to enhance innate and adaptive cellular immunity in a murine model of CMV infection.
Project description:Engagement of the programmed death (PD)-1 receptor on activated cells by its ligand (PD-L1) is a mechanism for suppression of activated T-lymphocytes. Microglia, the resident inflammatory cells of the brain, are important for pathogen detection and initiation of innate immunity, however, a novel role for these cells as immune regulators has also emerged. PD-L1 on microglia has been shown to negatively regulate T-cell activation in models of multiple sclerosis and acute viral encephalitis. In this study, we investigated the role of glial cell PD-L1 in controlling encephalitogenic CD8(+) T-lymphocytes, which infiltrate the brain to manage viral infection, but remain to produce chronic neuroinflammation. Using a model of chronic neuroinflammation following murine cytomegalovirus (MCMV)-induced encephalitis, we found that CD8(+) T-cells persisting within the brain expressed PD-1. Conversely, activated microglia expressed PD-L1. In vitro, primary murine microglia, which express low basal levels of PD-L1, upregulated the co-inhibitory ligand on IFN-?-treatment. Blockade of the PD-1: PD-L1 pathway in microglial: CD8(+) T-cell co-cultures increased T-cell IFN-? and interleukin (IL)-2 production. We observed a similar phenomenon following blockade of this co-inhibitory pathway in astrocyte: CD8(+) T-cell co-cultures. Using ex vivo cultures of brain leukocytes, including microglia and CD8(+) T-cells, obtained from mice with MCMV-induced chronic neuroinflammation, we found that neutralization of either PD-1 or PD-L1 increased IFN-? production from virus-specific CD8(+) T-cells stimulated with MCMV IE1168-176 peptide. These data demonstrate that microglia and astrocytes control antiviral T-cell responses and suggest a therapeutic potential of PD1: PD-L1 modulation to manage the deleterious consequences of uncontrolled neuroinflammation.
Project description:Vasoactive intestinal peptide (VIP) induces regulatory dendritic cells (DC) in vitro that inhibit cellular immune responses. We tested the role of physiological levels of VIP on immune responses to murine CMV (mCMV) using VIP-knockout (VIP-KO) mice and radiation chimeras engrafted with syngenic VIP-KO hematopoietic cells. VIP-KO mice had less weight loss and better survival following mCMV infection compared with wild-type (WT) littermates. mCMV-infected VIP-KO mice had lower viral loads, faster clearance of virus, with increased numbers of IFN-?(+) NK and NKT cells, and enhanced cytolytic activity of NK cells. Adaptive antiviral cellular immunity was increased in mCMV-infected VIP-KO mice compared with WT mice, with more Th1/Tc1-polarized T cells, fewer IL-10(+) T cells, and more mCMV-M45 epitope peptide MHC class I tetramer(+) CD8(+) T cells (tetramer(+) CD8 T cells). mCMV-immune VIP-KO mice had enhanced ability to clear mCMV peptide-pulsed target cells in vivo. Enhanced antiviral immunity was also seen in WT transplant recipients engrafted with VIP-KO hematopoietic cells, indicating that VIP synthesized by neuronal cells did not suppress immune responses. Following mCMV infection there was a marked upregulation of MHC-II and CD80 costimulatory molecule expression on DC from VIP-KO mice compared with DC from WT mice, whereas programmed death-1 and programmed death ligand-1 expression were upregulated in activated CD8(+) T cells and DC, respectively, in WT mice, but not in VIP-KO mice. Because the absence of VIP in immune cells increased innate and adaptive antiviral immunity by altering costimulatory and coinhibitory pathways, selective targeting of VIP signaling represents an attractive therapeutic target to enhance antiviral immunity.
Project description:Horizontal transmission of cytomegaloviruses (CMV) occurs via prolonged excretion from mucosal surfaces. We used murine CMV (MCMV) infection to investigate the mechanisms of immune control in secretory organs. CD4 T cells were crucial to cease MCMV replication in the salivary gland (SG) via direct secretion of IFN? that initiated antiviral signaling on non-hematopoietic cells. In contrast, CD4 T cell helper functions for CD8 T cells or B cells were dispensable. Despite SG-resident MCMV-specific CD8 T cells being able to produce IFN?, the absence of MHC class I molecules on infected acinar glandular epithelial cells due to viral immune evasion, and the paucity of cross-presenting antigen presenting cells (APCs) prevented their local activation. Thus, local activation of MCMV-specific T cells is confined to the CD4 subset due to exclusive presentation of MCMV-derived antigens by MHC class II molecules on bystander APCs, resulting in IFN? secretion interfering with viral replication in cells of non-hematopoietic origin.
Project description:MHC class I (MHC I) is essential to NK- and T-cell effector and surveillance functions. However, it is unknown whether MHC I polymorphism influences adaptive immunity through NK cells. Previously, we found that MHC I D(k), a cognate ligand for the Ly49G2 inhibitory receptor, was essential to NK control of murine (M)CMV infection. Here we assessed the significance of NK inhibitory receptor recognition of MCMV on CD8 T cells in genetically defined MHC I D(k) disparate mice. We observed that D(k)-licensed Ly49G2? NK cells stabilized and then enhanced conventional dendritic cells (cDCs) recovery after infection. Furthermore, licensed NK support of cDC recovery was essential to enhance the tempo, magnitude, and effector activity of virus-specific CD8 T cells. Minimal cDC and CD8 T-cell number differences after low-dose MCMV in D(k) disparate animals further implied that licensed NK recognition of MCMV imparted qualitative cDC changes to enhance CD8 T-cell priming.
Project description:<h4>Background</h4>Cytomegalovirus establishes lifelong persistency in the host and leads to life threatening situations in immunocompromised patients. FoxP3+ T regulatory cells (Tregs) critically control and suppress innate and adaptive immune responses. However, their specific role during MCMV infection, especially pertaining to their interaction with NK cells, remains incompletely defined.<h4>Methods</h4>To understand the contribution of Tregs on NK cell function during acute MCMV infection, we infected Treg depleted and undepleted DEREG mice with WT MCMV and examined Treg and NK cell frequency, number, activation and effector function in vivo.<h4>Results</h4>Our results reveal an increased frequency of activated Tregs within the CD4+ T cell population shortly after MCMV infection. Specific depletion of Tregs in DEREG mice under homeostatic conditions leads to an increase in NK cell number as well as to a higher activation status of these cells as compared with non-depleted controls. Interestingly, upon infection this effect on NK cells is completely neutralized in terms of cell frequency, CD69 expression and functionality with respect to IFN-? production. Furthermore, composition of the NK cell population with regard to Ly49H expression remains unchanged. In contrast, absence of Tregs still boosts the general T cell response upon infection to a level comparable to the enhanced activation seen in uninfected mice. CD4+ T cells especially benefit from Treg depletion exhibiting a two-fold increase of CD69+ cells 40 h and IFN-?+ cells 7 days p.i. while, MCMV infection per se induces robust CD8+ T cell activation which is also further augmented in Treg-depleted mice. Nevertheless, the viral burden in the liver and spleen remain unaltered upon Treg ablation during the course of infection.<h4>Conclusions</h4>Thus, MCMV infection abolishes Treg suppressing effects on NK cells whereas T cells benefit from their absence during acute infection. This study provides novel information in understanding the collaborative interaction between NK cells and Tregs during a viral infection and provides further knowledge that could be adopted in therapeutic setups to improve current treatment of organ transplant patients where modulation of Tregs is envisioned as a strategy to overcome transplant rejection.
Project description:There is an imperative need for effective preventive vaccines against human cytomegalovirus as it poses a significant threat to the immunologically immature, causing congenital disease, and to the immune compromised including transplant recipients. In this study, we examined the efficacy of synthetic long peptides (SLPs) as a CD4+ and CD8+ T cell-eliciting preventive vaccine approach against mouse CMV (MCMV) infection. In addition, the use of agonistic OX40 antibodies to enhance vaccine efficacy was explored. Immunocompetent C57BL/6 mice were vaccinated in a prime-boost vaccination regiment with SLPs comprising various MHC class I- and II-restricted peptide epitopes of MCMV-encoded antigens. Enforced OX40 stimulation resulted in superior MCMV-specific CD4+ as CD8+ T cell responses when applied during booster SLP vaccination. Vaccination with a mixture of SLPs containing MHC class II epitopes and OX40 agonistic antibodies resulted in a moderate reduction of the viral titers after challenge with lytic MCMV infection. Markedly, the combination of SLP vaccines containing both MHC class I and II epitopes plus OX40 activation during booster vaccination resulted in polyfunctional (i.e., IFN-?+, TNF+, IL-2+) CD4+ and CD8+ T cell responses that were even higher in magnitude when compared to those induced by the virus, and this resulted in the best containment of virus dissemination. Our results show that the induction of strong T cell responses can be a fundamental component in the design of vaccines against persistent viral infections.
Project description:The role of non-classical T cells during viral infection remains poorly understood. Using the well-established murine model of CMV infection (MCMV) and mice deficient in MHC class Ia molecules, we found that non-classical CD8+ T cells robustly expand after MCMV challenge, become highly activated effectors, and are capable of forming durable memory. Interestingly, although these cells are restricted by MHC class Ib molecules, they respond similarly to conventional T cells. Remarkably, when acting as the sole component of the adaptive immune response, non-classical CD8+ T cells are sufficient to protect against MCMV-induced lethality. We also demonstrate that the MHC class Ib molecule Qa-1 (encoded by H2-T23) restricts a large, and critical, portion of this population. These findings reveal a potential adaptation of the host immune response to compensate for viral evasion of classical T cell immunity.
Project description:Fc? receptors (Fc?Rs) for IgG couple innate and adaptive immunity through activation of effector cells by antigen-antibody complexes. We investigated relative levels of activating and inhibitory Fc?Rs on brain-resident microglia following murine cytomegalovirus (MCMV) infection. Flow cytometric analysis of microglial cells obtained from infected brain tissue demonstrated that activating Fc?Rs were expressed maximally at 5?d post-infection (dpi), while the inhibitory receptor (Fc?RIIB) remained highly elevated during both acute and chronic phases of infection. The highly induced expression of activating Fc?RIV during the acute phase of infection was also noteworthy. Furthermore, in vitro analysis using cultured primary microglia demonstrated the role of interferon (IFN)? and interleukin (IL)-4 in polarizing these cells towards a M1 or M2 phenotype, respectively. Microglial cell-polarization correlated with maximal expression of either Fc?RIV or Fc?RIIB following stimulation with IFN? or IL-4, respectively. Finally, we observed a significant delay in polarization of microglia towards an M2 phenotype in the absence of Fc?Rs in MCMV-infected Fcer1g and FcgR2b knockout mice. These studies demonstrate that neuro-inflammation following viral infection increases expression of activating Fc?Rs on M1-polarized microglia. In contrast, expression of the inhibitory Fc?RIIB receptor promotes M2-polarization in order to shut-down deleterious immune responses and limit bystander brain damage.