MCMV Infection Research Articles

Interferon lambda is required for interferon gamma-expressing NK cell responses but does not afford antiviral protection during acute and persistent murine cytomegalovirus infection.

Interferon lambda (IFNλ) is a group of cytokines that belong to the IL-10 family. They exhibit antiviral activities against certain viruses during infection of the liver and mucosal tissues. Here we report that IFNλ restricts in vitro replication of the β-herpesvirus murine cytomegalovirus (mCMV). However, IFNλR1-deficient (Ifnλr1-/-) mice were not preferentially susceptible to mCMV infection in vivo during acute infection after systemic or mucosal challenge, or during virus persistence in the mucosa. Instead, our studies revealed that IFNλ influences NK cell responses during mCMV infection. Ifnλr1-/- mice exhibited defective development of conventional interferon-gamma (IFNγ)-expressing NK cells in the spleen during mCMV infection whereas accumulation of granzyme B-expressing NK cells was unaltered. In vitro, development of splenic IFNγ+ NK cells following stimulation with IL-12 or, to a lesser extent, IL-18 was abrogated by IFNλR1-deficiency. Thus, IFNλ regulates NK cell responses during mCMV infection and restricts virus replication in vitro but is redundant in the control of acute and persistent mCMV replication within mucosal and non-mucosal tissues.

Cytomegalovirus blockade of TRAIL-mediated type 1 ILC defenses regulates viral persistence

Abstract Tumor necrosis factor related apoptosis-inducing ligand (TRAIL) regulates cellular apoptosis and inflammation, and growing evidence indicates a key role in antiviral defense. Cytomegalovirus (CMV, a b-herpesvirus) establishes a lifelong persistent/latent infection that depends upon viral subversion of host innate and adaptive defenses. We have shown that both human and mouse CMV inhibit signaling by the TRAIL death receptors (DR) through the use of specific viral proteins. MCMV m166 blocks TRAIL-DR expression and promotes early viral replication by evading TRAIL-mediated natural killer (NK) cell defenses. After one week of systemic replication, MCMV spreads to the salivary glands (SG), the major mucosal organ responsible for horizontal transmission of the virus. We show that a MCMVΔm166 mutant replicates at lower absolute levels in the SG, but, surprisingly, persists for much longer times. Recent evidence suggests a role of type 1 Innate Lymphoid cells (ILC1) in control/clearance of MCMV. We investigated the role of the 2 main ILC1 populations (conventional and tissue resident (tr) NK cells) in controlling MCMV SG persistence. Depleting ILC1 prior to infection, or infection of TRAIL−/− mice, restored MCMVΔm166 SG replication to normal levels. In addition, SG trNK cells display a unique phenotype and express high levels of TRAIL during MCMV infection when compared to their conventional counterparts. Together our data reveal a key role for m166 inhibition of TRAIL-DR expression in promoting viral persistence by blocking ILC1 effector functions mediated by TRAIL, highlighting this TNF-family cytokine as an important factor regulating innate defenses in mucosal tissues.

Tissue-specific control of latent CMV reactivation by regulatory T cells.

Cytomegalovirus (CMV) causes a persistent, lifelong infection. CMV persists in a latent state and undergoes intermittent subclinical viral reactivation that is quelled by ongoing T cell responses. While T cells are critical to maintain control of infection, the immunological factors that promote CMV persistence remain unclear. Here, we investigated the role of regulatory T cells (Treg) in a mouse model of latent CMV infection using Foxp3-diphtheria toxin receptor (Foxp3-DTR) mice. Eight months after infection, MCMV had established latency in the spleen, salivary gland, lung, and pancreas, which was accompanied by an increased frequency of Treg. Administration of diphtheria toxin (DT) after establishment of latency efficiently depleted Treg and drove a significant increase in the numbers of functional MCMV-specific CD4+ and CD8+ T cells. Strikingly, Treg depletion decreased the number of animals with reactivatable latent MCMV in the spleen. Unexpectedly, in the same animals, ablation of Treg drove a significant increase in viral reactivation in the salivary gland that was accompanied with augmented local IL-10 production by Foxp3-CD4+T cells. Further, neutralization of IL-10 after Treg depletion significantly decreased viral load in the salivary gland. Combined, these data show that Treg have divergent control of MCMV infection depending upon the tissue. In the spleen, Treg antagonize CD8+ effector function and promote viral persistence while in the salivary gland Treg prevent IL-10 production and limit viral reactivation and replication. These data provide new insights into the organ-specific roles of Treg in controlling the reactivation of latent MCMV infection.

Expression pattern of CD11c on lung immune cells after disseminated murine cytomegalovirus infection

BackgroundCytomegalovirus (CMV) infection occurs frequently and is widespread globally. Numerous studies have shown that various types of immune cells play roles in mediating the response to CMV infection. CD11c, a commonly used dendritic cell (DC) marker, is expressed by other immune cells as well, such as T cells. This study analyzed the immune cells that express CD11c and monitored the expression level of their specific cell surface markers in the lung following a disseminated murine (M)CMV infection.MethodsMouse models of disseminated MCMV infection were used; uninfected and lipopolysaccharide (LPS)-treated mice were used as controls. At 1, 3 and 7 days following infection, single cell suspensions prepared from freshly digested lung tissue were stained for CD11c, CD86 and MHC II. Stained cells were analyzed using flow cytometry. Peripheral blood and single cell suspensions from spleen were sorted as well. Then these cells were subjected to analyze the CD11c expression pattern on natural killer (NK) cells and T cells.ResultsThis assay showed that after MCMV infection, the expression of CD86 on pulmonary CD11chiMHC-IIhi cells (encompassing conventional DCs) was higher at 3 days post-infection than at 1 or 7 days post-infection, accompanied by a downregulation of MHC II. In addition, expression of CD11c was greatly increased in the MCMV infection group at 7 days post infection. This study also detected a large population of cells displaying an intermediate level of expression of CD11c (CD11cint); these cells were in the MCMV groups exclusively, and were subsequently identified as CD8+ T cells. In lung, spleen and blood, different proportions of CD11cint cells among the NK cell and T cell populations were observed between the BALB/c and C57BL/6 mice with or without MCMV infection. The expression level of NKp46 in NK cells dropped to a lower level after MCMV infection.ConclusionsThe findings collectively indicate that CD11cintCD8+ T cells might play a key role in anti-MCMV adaptive immune response in lungs, as well as in spleen and blood. B220+CD11cint NK cells might be a more effective type of NK cell, participating in anti-MCMV infection. The downregulation of NKp46, in particular, might be linked with the immune evasion of MCMV.

Determination of suitable reference genes for RT-qPCR analysis of murine Cytomegalovirus in vivo and in vitro

Reverse transcription quantitative PCR (RT-qPCR) is the most commonly used method to evaluate gene expression. Reliable qPCR results are highly dependent on accurate normalization using suitable reference genes. We investigated expression of commonly used reference genes during murine Cytomegalovirus (mCMV) infection and latency to determine those genes least perturbed by infection. Following mCMV infection in BALB/c mice, lung, salivary gland, liver, spleen and kidney were evaluated. Liver sinusoidal endothelial cells and NIH-3T3 cells were also evaluated. RT-qPCR was performed during acute and latent mCMV infection for 11 commonly used reference genes with comparisons made to uninfected samples. Normfinder, BestKeeper, GeNorm and the comparative delta CT method produced comparable analyses that were combined in RefFinder to generate an overall ranking. Ppia, B2m and Gapdh are the most stable reference genes for in vitro infection studies. For in vivo studies the most suitable reference genes were highly tissue and cell type dependent. Comparing infected and uninfected groups revealed viral influence on transcription of some genes. We provide reference gene guidelines for investigations of gene expression for mCMV Smith strain infection of Balb/cJ mice or NIH-3T3 cells. These results also suggest careful consideration of reference genes for different host tissues evaluated.

TLR3-independent activation of mast cells by cytomegalovirus contributes to control of pulmonary infection.

Interstitial pneumonia is a life-threatening clinical manifestation of human cytomegalovirus (hCMV) infection. In particular, it can be deadly in patients with hematopoietic malignancies who undergo hematopoietic cell transplantation (HCT) in whom a ‘window of risk’, which is defined by transient immunodeficiency, occurs between hematoablative therapeutic treatment and immunological reconstitution. As few clinical studies have addressed the underlying mechanisms for this phenomenon, a mouse model of HCT and murine cytomegalovirus (mCMV) infection has been established and has revealed a key role for antiviral CD8+ T cells in controlling pulmonary infections. Using this mouse model, recent studies have provided evidence for the role of mast cells (MC) in more efficiently recruiting antiviral CD8+ T cells to CMV-infected lungs in a process that involves the release of CCL5, a chemokine. MC were found to be direct cellular targets of murine CMV infection and could be activated and degranulated by two mechanisms: the rapid degranulation requiring TLR3/TRIF signaling in non-MC cells and a delayed TLR3/TRIF-independent mechanism. Here, we provide evidence showing that TLR3/TRIF-independent MC activation by CMV is the dominant mechanism in controlling pulmonary infection.

Crucial role of FOXO1 in inflationary CD8+ T-cell responses during persistent viral infection

Abstract Cytomegalovirus (CMV) establish persistent infection that induce the accumulation of virus-specific T-cells over time in a process called memory inflation. Although persistence of antigen (Ag) is considered essential, the molecular pathway for the inflationary T-cell development is poorly understood. Mainly, inflationary CD8 T-cells are KLRG1hi CD27−, but a low percentage of them are KLRG1lo CD27+, which are considered as their memory precursors. To study the role of FOXO1 in CD8 T cells during MCMV infection, we analyzed mouse bone marrow chimeras in which FOXO1 was specifically deleted in CD8 T cells. These mice were infected with MCMV-Δm157 strain, and the expansion and phenotype of inflationary was examined. The results revealed that the IE3 and M38 CD8 T-cell responses do not inflate in absence of FOXO1. Furthermore, with an absence of FOXO1, there were fewer Ag-specific CD8 T cells that produced both IFNγ and TNFα. Consistent with this lack of CD8 effector cells the mice were less able to control the virus in the spleen and the liver at day 6 after infection. Moreover, the FOXO1 KO Ag-specific CD8 T cells fail to up-regulate the memory associated transcription factors, TCF-1 and EOMES, correlating with a lower percentage of memory precursors. Then, we found that FOXO1 KO Ag-specific CD8 T cells display an anergic state and are prone to apoptosis. Finally, we delete FOXO1 at day 40 after infection, we showed that the FOXO1 KO Ag-specific CD8 T cells survive less efficiently and lose their memory characteristics as well as their recall capacities. Collectively, these results demonstrate an intrinsic role for FOXO1 in establishing and maintaining the inflationary memory program that is essential to forming long-lived effector and memory cells.

Tissue-resident liver NK cell responses to MCMV infection

Abstract Natural Killer (NK) cells belong to the ILC1 subgroup, which contains conventional NK (cNK) cells, ILC1 and tissue-resident NK (trNK) cells. The murine liver is home to both cNK cells and trNK cells. In this organ, cNK cells and trNK cells can be distinguished based on the expression of the transcription factors Eomes and T-bet. cNK cells are double positive for Eomes and T-bet while trNK cells do not express Eomes. During viral infection, the fate of the activated cNK cell population is relatively well defined. However, the fate of the trNK cell population remains unexplored. Using the MCMV model of infection, we found that a contraction phase precedes a replenishment of the trNK cell subset, which inversely correlates with cNK cell kinetics. We sought to determine whether the trNK cell response was a direct result of MCMV infection or an indirect response to inflammatory milieu. In order to help distinguish between these two possibilities, mice were injected with the iNKT ligand a-galactosylceramide (a-GalCer). We found that treatment with a-GalCer lead to an initial contraction phase of liver trNK cells, indicating that the phenotype observed is independent of MCMV infection. Altogether, these data suggest that liver cNK and trNK cell subsets, which arise from distinct developmental pathways, perform different functions in this organ.

The immune environment dictates the dominance of licensed versus unlicensed natural killer cell subsets during viral infection

Abstract Natural Killer (NK) cells are innate lymphocytes involved in anti-viral and anti-cancer responses. They can be functionally and phenotypically divided based on expression of inhibitory receptors capable of binding to self-MHC class I, a concept termed licensing/education. NK cells capable of binding to self-MHC (licensed) have been shown to be more functionally competent compared to those that cannot (unlicensed). It is still unclear which subset is more critical during an anti-viral response. Using two models of murine cytomegalovirus infection (MCMV), we show distinct subsets of NK cells that can both directly and indirectly contribute to the anti-viral response. We have previously shown that after hematopoietic stem cell transplantation (HSCT), the licensed effector NK population was predominantly responsible for resistance to MCMV, which was in part due to the absence of T cells present immediately after transplant. However, MCMV infection in non-HSCT mice yielded an opposite pattern with the unlicensed NK subset augmenting resistance to MCMV. We found preferential localization patterns of the different NK subsets which correlated with increased antigen specific T cell expansion and viral clearance. Interestingly, by depleting the different NK subsets in HSCT vs non-HSCT mice, we demonstrated differences in subset roles. The unlicensed NK cells, which promote antigen specific T cells, are more critical during normal conditions, while the licensed NK cells serve a better direct anti-viral role following HSCT where T cells take longer to repopulate. Thus, the contributions of these different NK subsets and their dependence on the immune environment should be taken into consideration when applying NK cells therapeutically.

The role of SHP-2 on NK cell development and functions

Abstract The SH2 domain-containing tyrosine phosphatase SHP-2 (PTPN11) has been shown to play essential roles in immune cell signaling. Several inhibitory receptors expressed on natural killer (NK) cells associate with SHP-2 to exert downstream functions. Here we examined the influence of SHP-2 on NK cell development and effector activity. Because SHP-2 is required for embryonic development in mice, we generated mice conditionally deficient for SHP-2 in NK cells. SHP-2fl/fl mice were crossed to NKp46-Cre mice in order to inactivate Ptpn11 in NKp46+ cells. These mice were also crossed to R26R-EYFP reporter mice to track cells with active Cre recombinase. We found an increase in NK cell frequency and number in these mice compared to littermate controls. The frequency of mature NK cells (CD11b+CD27−) was also increased in the periphery while decreased in the bone marrow. In addition, the homeostatic proliferation of SHP-2-deficient NK cells was impaired. However, effector functions were not affected significantly. These included IFN-γ production at the peak of the NK cell response during MCMV infection, rejection of β2-microglobulin deficient splenocytes, and degranulation via Ly49H using M157-transfected cells as targets. Taken together, our data suggest that SHP-2 is dispensable for NK cell effector functions and education, but is important for NK cell maturation.

The D-form of a novel heparan binding peptide decreases cytomegalovirus infection in vivo and in vitro

Human cytomegalovirus (HCMV) infection in utero can lead to congenital sensory neural hearing loss and mental retardation. Reactivation or primary infection can increase the morbidity and mortality in immune suppressed transplant recipients and AIDS patients. The current standard of care for HCMV disease is nucleoside analogs, which can be nephrotoxic. In addition resistance to current treatments is becoming increasingly common. In an effort to develop novel CMV treatments, we tested the effectiveness of the D-form of a novel heparan sulfate binding peptide, p5RD, at reducing infection of ganciclovir (GCV) resistant HCMVs in vitro and MCMV in vivo. HCMV infection was reduced by greater than 90% when cells were pretreated with p5RD. Because p5RD acts by a mechanism unrelated to those used by current antivirals, it was effective at reducing GCV resistant HCMVs by 85%. We show that p5RD is resistant to common proteases and serum inactivation, which likely contributed to its ability to significantly reduced infection of peritoneal exudate cells and viral loads in the spleen and the lungs in vivo. The ability of p5RD to reduce HCMV infectivity in vitro including GCV resistant HCMVs and MCMV infection in vivo suggests that this peptide could be a novel anti-CMV therapeutic.

Siglec-H protects from virus-triggered severe systemic autoimmunity.

It is controversial whether virus infections can contribute to the development of autoimmune diseases. Type I interferons (IFNs) are critical antiviral cytokines during virus infections and have also been implicated in the pathogenesis of systemic lupus erythematosus. Type I IFN is mainly produced by plasmacytoid dendritic cells (pDCs). The secretion of type I IFN of pDCs is modulated by Siglec-H, a DAP12-associated receptor on pDCs. In this study, we show that Siglec-H-deficient pDCs produce more of the type I IFN, IFN-α, in vitro and that Siglec-H knockout (KO) mice produce more IFN-α after murine cytomegalovirus (mCMV) infection in vivo. This did not impact control of viral replication. Remarkably, several weeks after a single mCMV infection, Siglec-H KO mice developed a severe form of systemic lupus-like autoimmune disease with strong kidney nephritis. In contrast, uninfected aging Siglec-H KO mice developed a mild form of systemic autoimmunity. The induction of systemic autoimmune disease after virus infection in Siglec-H KO mice was accompanied by a type I IFN signature and fully dependent on type I IFN signaling. These results show that Siglec-H normally serves as a modulator of type I IFN responses after infection with a persistent virus and thereby prevents induction of autoimmune disease.

Glial cell activation, recruitment, and survival of B-lineage cells following MCMV brain infection

BackgroundChemokines produced by reactive glia drive migration of immune cells and previous studies from our laboratory have demonstrated that CD19+ B cells infiltrate the brain. In this study, in vivo and in vitro experiments investigated the role of reactive glial cells in recruitment and survival of B-lineage cells in response to (murine cytomegalovirus) MCMV infection.MethodsFlow cytometric analysis was used to assess chemokine receptor expression on brain-infiltrating B cells. Real-time RT-PCR and ELISA were used to measure chemokine levels. Dual-immunohistochemical staining was used to co-localize chemokine production by reactive glia. Primary glial cell cultures and migration assays were used to examine chemokine-mediated recruitment. Astrocyte: B cell co-cultures were used to investigate survival and proliferation.ResultsThe chemokine receptors CXCR3, CXCR5, CCR5, and CCR7 were detected on CD19+ cells isolated from the brain during MCMV infection. In particular, CXCR3 was found to be elevated on an increasing number of cells over the time course of infection, and it was the primary chemokine receptor expressed at 60 days post infection Quite different expression kinetics were observed for CXCR5, CCR5, and CCR7, which were elevated on the highest number of cells early during infection and decreased by 14, 30, and 60 days post infection Correspondingly, elevated levels of CXCL9, CXCL10, and CXCL13, as well as CCL5, were found within the brains of infected animals, and only low levels of CCL3 and CCL19 were detected. Differential expression of CXCL9/CXCL10 and CXCL13 between microglia and astrocytes was apparent, and B cells moved towards supernatants from MCMV-infected microglia, but not astrocytes. Pretreatment with neutralizing Abs to CXCL9 and CXCL10 inhibited this migration. In contrast, neutralizing Abs to the ligand of CXCR5 (i.e., CXCL13) did not significantly block chemotaxis. Proliferation of brain-infiltrating B cells was detected at 7 days post infection and persisted through the latest time tested (60 days post infection). Finally, astrocytes produce BAFF (B cell activating factor of the TNF family) and promote proliferation of B cells via cell-to-cell contact.ConclusionsCXCR3 is the primary chemokine receptor on CD19+ B cells persisting within the brain, and migration to microglial cell supernatants is mediated through this receptor. Correspondingly, microglial cells produce CXCL9 and CXCL10, but not CXCL13. Reactive astrocytes promote B cell proliferation.

Tregs Modulate Lymphocyte Proliferation, Activation, and Resident-Memory T-Cell Accumulation within the Brain during MCMV Infection.

Accumulation and retention of regulatory T-cells (Tregs) has been reported within post viral-encephalitic brains, however, the full extent to which these cells modulate neuroinflammation is yet to be elucidated. Here, we used Foxp3-DTR (diphtheria toxin receptor) knock-in transgenic mice, which upon administration of low dose diphtheria toxin (DTx) results in specific deletion of Tregs. We investigated the proliferation status of various immune cell subtypes within inflamed central nervous system (CNS) tissue. Depletion of Tregs resulted in increased proliferation of both CD8+ and CD4+ T-cell subsets within the brain at 14 d post infection (dpi) when compared to Treg-sufficient animals. At 30 dpi, while proliferation of CD8+ T-cells was controlled within brains of both Treg-depleted and undepleted mice, proliferation of CD4+ T-cells remained significantly enhanced with DTx-treatment. Previous studies have demonstrated that Treg numbers within the brain rebound following DTx treatment to even higher numbers than in untreated animals. Despite this rebound, CD8+ and CD4+ T-cells proliferated at a higher rate when compared to that of Treg-sufficient mice, thus maintaining sustained neuroinflammation. Furthermore, at 30 dpi we found the majority of CD8+ T-cells were CD127hi KLRG1- indicating that the cells were long lived memory precursor cells. These cells showed marked elevation of CD103 expression, a marker of tissue resident-memory T-cells (TRM) in the CNS, in untreated animals when compared to DTx-treated animals suggesting that generation of TRM is impaired upon Treg depletion. Moreover, the effector function of TRM as indicated by granzyme B production in response to peptide re-stimulation was found to be more potent in Treg-sufficient animals. Taken together, our findings demonstrate that Tregs limit neuroinflammatory responses to viral infection by controlling cell proliferation and may direct a larger proportion of lymphocytes within the brain to be maintained as TRM cells.

TLR5 Agonist Recombinant Flagellin, CBLB502 Increases Anti-CMV Immunity By Increasing T-Bet and Eomes Expressing T Cells after MCMV Infection

We have previously shown that highly purified cGMP grade recombinant flagellin, CBLB502, a TLR5 agonist, protected allo-HSCT recipients from GvHD without compromising anti-CMV immunity of donor T cells in vivo. A single prophylactic dose (25mg/mouse i.p) of CBLB502 in wild type C57BL/6 (B6) mice also enhanced anti-CMV immunity of NK cells against CMV. CMV is a potential opportunistic infectious agent and most often causes severe life-threatening clinical complications in immunocompromized patients, particularly in cancer patients treated with allogenic bone marrow transplantation (allo-BMT). A mechanism by which CBLB502 protected host against CMV infection has not been well investigated. Transcription factors ROR gammaT, T-bet and EOMES are known to control immune responses of innate and adaptive immunity in vivo. In this study, we have explored the transcription factors ROR gammaT, T-bet and EOMES expressing NK and T cells in CBLB502-treated and MCMV-infected B6 mice. Total numbers of spleen cells increased by 1.7-fold within 48 hours of CBLB502 treatment (day 0 MCMV infection) and by 2.3-fold on day 3 after MCMV infection compared with PBS-treated MCMV-infected mice. T-bet expressing CD8+ T cells (not CD4+ T cells) increased significantly on day 0 of mCMV infection, 48hrs after CBLB502 treatment (CD4, p=0.1; CD8, p<0.001) and both CD4+ and CD8+ T cells increased significantly on day 3 (CD4, p<0.001; CD8, p<0.01) after MCMV infection. CBLB502 treatment had no effect on the ROR gammaT expression on T cells. Interestingly, although EOMES expressing CD4+ and CD8+ T cells increased significantly on day 0 (48 hrs after CBLB502 treatment) (CD4, p<0.01; CD8, p<0.01) and day 3 (CD4, p<0.001; CD8, p<0.001) in the spleens of CBLB502-treated mice compared with the PBS-treated mice, EOMES expression on CMV-specific tetramer+ CD8+ T cells rapidly decreased by day 10 after infection in both CBLB502 and PBS treated mice. However, >90% of CMV-specific CD8+ T cells had persistent expression of T-bet even after 30 days post CMV infection. In contrast, CBLB502 treatment did not have a significant effect on the numbers of ROR gammaT, T-bet and EOMES expressing NK cells compared with PBS-treated mice. These data suggest that 1) CBLB502-induced anti-CMV immunity of T cells is persistently maintained through increased T-bet expression but not by EOMES expression; and 2) CBLB502-induced anti-viral immune regulation is not maintained through transcription factor ROR gammaT. 3) CBLB502-induced increased anti-CMV immunity of NK cells is independent of transcription factors ROR gammaT, T-bet and EOMES. Taken together, these data support the early peri-transplant use of TLR5 agonists as a novel method to enhance antigen-specific anti-viral immunity in recipients of allo-BMT. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Absence of Vasoactive Intestinal Peptide Signaling Significantly Enhances T Cell Survival Following MCMV Infection Via Increased Expression of Bcl-2 and Bcl-XL

BackgroundThe nervous system's influence on immune responses has become increasingly appreciated following the identification of a variety of immunomodulatory products secreted from neurons. One such product, vasoactive intestinal peptide (VIP), has been identified as an anti-inflammatory mediator exerting effects on both the innate and adaptive arms of the immune system. Interference with this pathway through blocking the VIP receptors VPAC1 and VPAC2 has been demonstrated to enhance T cell responses to viral infection as well as cancer. VIP antagonism results in increased numbers of antigen-specific cells in the peripheral blood and a shift in the balance of co-stimulatory and co-inhibitory molecules towards a pro-inflammatory phenotype. The consequences of such effects are a reduction in viral load in a model of MCMV infection and a reduced tumor burden and increased survival in a model of lymphoma.MethodsIn order to better understand the molecular mechanisms underlying the effects of VIP absence or antagonism, we used the MCMV infection model. Male VIP WT and VIP KO mice were infected with 105 pfu MCMV ip and sacrificed at various time points. Spleens were harvested and T cells were FACS sorted based on the expression of activation markers. Expression of pro and anti-apoptotic molecules was assessed via Western blot. Expression of VIP and its receptor VPAC1 was determined using qRT-PCR of extracted mRNA. In vitro T cell proliferation was assessed using CFSE dilution of splenocytes stimulated with CD3/CD28 beads and IL-2.ResultsWe first examined levels of VIP and VPAC1 over the course of MCMV infection via qRT-PCR of extracted mRNA. Expression of VIP and VPAC1 was found to change in response to infection with an initial decrease in the levels of VPAC1 followed by an increase back to baseline levels. The pattern of VIP expression was opposite with an initial increase followed by a decrease. In order to gain more mechanistic insight into the effects of VIP signaling in T cells during MCMV infection, activated splenic CD8 T cells from WT mice and mice lacking VIP were FACS sorted at various time points. Activated CD69+CD8+ T cells from KO mice expressed higher levels of the anti-apoptotic protein Bcl-XL both during the initial response and at the peak by Western blot (Figure 1). Intracellular staining revealed an increased frequency of Bcl-2-expressing CD8 T cells in KO mice at the peak of the response (Figure 1). Proliferation of CFSE-labeled CD8 T cells in response to in vitro CD3/CD28 stimulation was found to be identical in wild type and VIP knockout cells suggesting modified T cell survival rather than proliferation. The negative influence of VIP on T cell survival resulted in significantly higher T cell frequency and a significant increase in the absolute numbers of antigen-specific splenic CD8 T cells in knockout mice (Figure 2). These results provide further support for VIP antagonism as a viable option for th e enhancement of CD8 T cell-mediated immunity. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

ID: 200: IL-27 enhances MCMV chronicity by promoting the development of immune suppressive virus-specific IL-10+ CD4+ T-cells

Human cytomegalovirus (HCMV) is an important viral pathogen in the immune suppressed and immunologically immature. HCMV encodes a functional homologue of the immune modulatory cytokine interleukin-10 (IL-10), implying the importance of this pathway in moderating antiviral immunity. Using the murine model of CMV infection, we now report that virus-specific CD4 T-cells produce IL-10 in response to infection in an epitope-restricted manner. Using CD4creIL-10flox mice we demonstrated that CD4+ cell-derived IL-10 promoted viral persistence by inhibiting antiviral T-cell responses. We sought to investigate the governing factors of IL-10 production by CD4+ T-cells during infection. We found that IL-10 production by CD4+ T-cells during the early stages of MCMV infection was driven by IL-27. Using WSX1−/− (IL-27R−/−) mice, we found that impaired early IL-10 expression by virus-specific T cells in the periphery permits a greater functional anti-viral mucosal response during virus chronicity. Thus specific targeting and abrogation of IL-10-producing CD4+ T-cells via inhibition of IL-27R signalling will enable the development of effective anti-CMV T-cell immunity in mucosal surfaces.

MAPKAP kinase 2 regulates IL-10 expression and prevents formation of intrahepatic myeloid cell aggregates during cytomegalovirus infections

The kinase p38(MAPK) and its downstream target MAPKAP kinase (MK) 2 are critical regulators of inflammatory responses towards pathogens. To date, the relevance of MK2 for regulating IL-10 expression and other cytokine responses towards cytomegalovirus (CMV) infection and the impact of this pathway on viral replication in vitro and in vivo is unknown and the subject of this study. The effect of MK2, interferon-α receptor (IFNAR)1, tristetraprolin (TTP) and IL-10 on mouse (M)CMV virus titres, cytokine expression, signal transduction, transcript stability, liver enzymes release, immune cell recruitment and aggregation in response to MCMV infection were studied ex vivo in hepatocytes and macrophages, as well as in vivo. MK2 is critical for MCMV-induced production of IL-10, IFN-α2 and 4, IFN-β, IL-6, and TNF-α but not for IFN-γ. The MCMV-induced IL-10 production requires activation of IFNAR1 and is further regulated by MK2 and TTP-dependent stabilization of IL-10 transcripts. MK2(-/-) mice are able to control acute MCMV replication, despite deregulated cytokine production. This may be related to the observation that MCMV-infected MK2(-/-) mice show enhanced formation of focal intrahepatic lymphocyte infiltrates resembling intrahepatic myeloid cell aggregates of T cell expansion (iMATEs), which were also observed in MCMV-infected IL-10(-/-) mice but are almost absent in MCMV-infected wild-type controls. The data suggest that MK2 is critical for regulating cytokine responses towards acute MCMV infection, including that of IL-10 via IFNARI-mediated circuits. MCMV stimulates expression of MK2-dependent cytokines, in particular IL-10 and thereby prevents enhanced formation of intrahepatic iMATE-like cellular aggregates.

Salivary gland lymphocyte response to murine cytomegalovirus (INC1P.344)

Abstract The β-herpes virus MCMV, a homologue of HCMV, is a well-characterized animal model of viral infection that results in a non-replicative, chronic infection of an immune-competent animal. In most organs, MCMV is cleared within days by cytotoxic lymphocytes. However, the virus persists in the submandibular salivary glands (SMG) for several weeks, before establishing latency for the life of the host. NK cells are crucial for the early containment of MCMV before T cells can mount an effector response. Recent work from our laboratory has shown that SMG NK cells are hyporesponsive during MCMV infection. Here we show that SMG NK cells regain normal effector functions against MCMV when adoptively transferred into different tissue environments. This indicates that the hyporesponsive phenotype observed is tissue specific, and not cell-intrinsic. In addition, using E4BP4 deficient animals, we determined that two CD3-NK1.1+ subsets reside within the salivary glands. One subset is derived from the conventional NK cell population and relies on E4BP4 for development, while the other is E4BP4 independent. When the E4BP4 independent NK cells are transferred into different tissue environments, they produce IFN-γ in response to MCMV infection. The SMGs of E4BP4-/- mice also contain a novel population of CD3+NK1.1+NKp46+ cells that produce IFN-γ during MCMV infection. Altogether, our data show that the SMG tissue environment shapes a unique repertoire of NK1.1+ cells with distinctive phenotypes.

Gata3 regulates the maturation program required for bone marrow exit and proliferation in Natural Killer cells (INM1P.435)

Abstract Natural killer (NK) cells are innate lymphocytes capable of killing virally infected cells or transformed cells. NK cells differentiate in the bone marrow before they migrate out to peripheral organs. The developmental program leading to functionally mature NK cells has been studied in the context of several transcription factors. However, the role of GATA-3 in NK cell development has not been completely understood. Using NK-cell specific Gata3 knockout mice (NK-Gata3-/-), we demonstrated that GATA-3 is required for the NK cell maturation beyond the CD27 single positive stage and for efficient IFN-g production. The frequencies of NK cells from NK-Gata3-/- mice were found higher in the bone marrow but lower in peripheral organs compared to control littermates, indicating that GATA-3 controls the maturation program required for bone marrow egress. Despite this, upon MCMV infection, NK cells from NK-Gata3-/- mice were able to expand vigorously, achieving NK cell frequencies surpassing those in controls, and therefore provided comparable protection. The enhanced proliferation of GATA-3-deficient NK cells was associated with enhanced upregulation of CD25 expression. Thus, the NK-Gata3-/- mice serve as a model to study the NK cell developmental process linked to cell migration, effector functions and proliferation.