Depleting myeloid-biased haematopoietic stem cells rejuvenates aged immunity.

Ageing of the immune system is characterized by decreased lymphopoiesis and adaptive immunity, and increased inflammation and myeloid pathologies1,2. Age-related changes in populations of self-renewing haematopoietic stem cells (HSCs) are thought to underlie these phenomena3. During youth, HSCs with balanced output of lymphoid and myeloid cells (bal-HSCs) predominate over HSCs with myeloid-biased output (my-HSCs), thereby promoting the lymphopoiesis required for initiating adaptive immune responses, while limiting the production of myeloid cells, which can be pro-inflammatory4. Ageing is associated with increased proportions of my-HSCs, resulting in decreased lymphopoiesis and increased myelopoiesis3,5,6. Transfer of bal-HSCs results in abundant lymphoid and myeloid cells, a stable phenotype that is retained after secondary transfer; my-HSCs also retain their patterns of production after secondary transfer5. The origin and potential interconversion of these two subsets is still unclear. If they are separate subsets postnatally, it might be possible to reverse the ageing phenotype by eliminating my-HSCs in aged mice. Here we demonstrate that antibody-mediated depletion of my-HSCs in aged mice restores characteristic features of a more youthful immune system, including increasing common lymphocyte progenitors, naive T cells and B cells, while decreasing age-related markers of immune decline. Depletion of my-HSCs in aged mice improves primary and secondary adaptive immune responses to viral infection. These findings may have relevance to the understanding and intervention of diseases exacerbated or caused by dominance of the haematopoietic system by my-HSCs.

A bivalent Adenovirus-Vectored Vaccine induces a robust humoral response, but does not protect cynomolgus macaques against a lethal challenge with Sudan virus.

The most recent Sudan virus (SUDV) outbreak in Uganda was first detected in September 2022 and resulted in 164 laboratory-confirmed cases and 77 deaths. There are no approved vaccines against SUDV. Here, we investigated the protective efficacy of ChAdOx1-biEBOV in cynomolgus macaques using a prime or a prime-boost regimen. ChAdOx1-biEBOV is a replication-deficient simian adenovirus vector encoding SUDV and Ebola virus (EBOV) glycoproteins (GPs). Intramuscular vaccination induced SUDV and EBOV GP-specific IgG responses and neutralizing antibodies. Upon challenge with SUDV, vaccinated animals showed signs of disease like those observed in control animals, and no difference in survival outcomes were measured among all three groups. Viral load in blood samples and in tissue samples obtained after necropsy were not significantly different between groups. Overall, this study highlights the importance of evaluating vaccines in multiple animal models and demonstrates the importance of understanding protective efficacy in both animal models and human hosts.

Intranasal ChAdOx1 nCoV-19/AZD1222 vaccination reduces viral shedding after SARS-CoV-2 D614G challenge in preclinical models.

ChAdOx1 nCoV-19/AZD1222 is an approved adenovirus-based vaccine for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) currently being deployed globally. Previous studies in rhesus macaques revealed that intramuscular vaccination with ChAdOx1 nCoV-19/AZD1222 provided protection against pneumonia but did not reduce shedding of SARS-CoV-2 from the upper respiratory tract. Here, we investigated whether intranasally administered ChAdOx1 nCoV-19 reduces detection of virus in nasal swabs after challenging vaccinated macaques and hamsters with SARS-CoV-2 carrying a D614G mutation in the spike protein. Viral loads in swabs obtained from intranasally vaccinated hamsters were decreased compared to control hamsters, and no viral RNA or infectious virus was found in lung tissue after a direct challenge or after direct contact with infected hamsters. Intranasal vaccination of rhesus macaques resulted in reduced virus concentrations in nasal swabs and a reduction in viral loads in bronchoalveolar lavage and lower respiratory tract tissue. Intranasal vaccination with ChAdOx1 nCoV-19/AZD1222 reduced virus concentrations in nasal swabs in two different SARS-CoV-2 animal models, warranting further investigation as a potential vaccination route for COVID-19 vaccines.

The liver X receptor agonist LXR 623 restricts flavivirus replication.

The vector-borne flaviviruses (VBFVs) are well known for causing great misery and death in humans worldwide. The VBFVs include those transmitted by mosquitos, such as Zika virus (ZIKV), dengue virus; and those transmitted by ticks including the tick-borne flavivirus serocomplex and Powassan virus (POWV). Two of our recent reports showed that intracranial POWV infection in the reservoir host, Peromyscus leucopus, was restricted and caused no overt clinical disease. Several modes of analyses suggested activation of the LXR pathway. Activation of the LXR pathway leads to increased efflux of cholesterol from cells and consequent disturbances in membrane biogenesis. Because VBFV replication is dependent on membrane biogenesis, we evaluated the effect of an LXR agonist (LXR623) on POWV and ZIKV infection and observed that the compound impaired permissive replication of both viruses in a human neuroblastoma SK-N-SH cell line. The LXR agonist resulted in failure of the viruses to induce ER expansion and elaborate vesicle formation, suggesting that the efflux of cholesterol was part of the antiviral mechanism. We also observed that the LXR agonist contributed to the mechanism of virus suppression by increased expression of mRNAs encoding for the antiviral cytokines CXCL10, RANTES and IFN1ß. In sharp contrast, a LXR antagonist (GSK2033) had no significant effect on VBFV replication. We conclude that LXR623 impairs flavivirus replication by stimulating cellular antiviral factors.

Mastomys natalensis Has a Cellular Immune Response Profile Distinct from Laboratory Mice.

The multimammate mouse (Mastomys natalensis; M. natalensis) has been identified as a major reservoir for multiple human pathogens including Lassa virus (LASV), Leishmania spp., Yersinia spp., and Borrelia spp. Although M. natalensis are related to well-characterized mouse and rat species commonly used in laboratory models, there is an absence of established assays and reagents to study the host immune responses of M. natalensis. As a result, there are major limitations to our understanding of immunopathology and mechanisms of immunological pathogen control in this increasingly important rodent species. In the current study, a large panel of commercially available rodent reagents were screened to identify their cross-reactivity with M. natalensis. Using these reagents, ex vivo assays were established and optimized to evaluate lymphocyte proliferation and cytokine production by M. natalensis lymphocytes. In contrast to C57BL/6J mice, lymphocytes from M. natalensis were relatively non-responsive to common stimuli such as phytohaemagglutinin P and lipopolysaccharide. However, they readily responded to concanavalin A stimulation as indicated by proliferation and cytokine production. In summary, we describe lymphoproliferative and cytokine assays demonstrating that the cellular immune responses in M. natalensis to commonly used mitogens differ from a laboratory-bred mouse strain.

Upregulation of CD47 Is a Host Checkpoint Response to Pathogen Recognition.

It is well understood that the adaptive immune response to infectious agents includes a modulating suppressive component as well as an activating component. We now show that the very early innate response also has an immunosuppressive component. Infected cells upregulate the CD47 "don't eat me" signal, which slows the phagocytic uptake of dying and viable cells as well as downstream antigen-presenting cell (APC) functions. A CD47 mimic that acts as an essential virulence factor is encoded by all poxviruses, but CD47 expression on infected cells was found to be upregulated even by pathogens, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), that encode no mimic. CD47 upregulation was revealed to be a host response induced by the stimulation of both endosomal and cytosolic pathogen recognition receptors (PRRs). Furthermore, proinflammatory cytokines, including those found in the plasma of hepatitis C patients, upregulated CD47 on uninfected dendritic cells, thereby linking innate modulation with downstream adaptive immune responses. Indeed, results from antibody-mediated CD47 blockade experiments as well as CD47 knockout mice revealed an immunosuppressive role for CD47 during infections with lymphocytic choriomeningitis virus and Mycobacterium tuberculosis Since CD47 blockade operates at the level of pattern recognition receptors rather than at a pathogen or antigen-specific level, these findings identify CD47 as a novel potential immunotherapeutic target for the enhancement of immune responses to a broad range of infectious agents.IMPORTANCE Immune responses to infectious agents are initiated when a pathogen or its components bind to pattern recognition receptors (PRRs). PRR binding sets off a cascade of events that activates immune responses. We now show that, in addition to activating immune responses, PRR signaling also initiates an immunosuppressive response, probably to limit inflammation. The importance of the current findings is that blockade of immunomodulatory signaling, which is mediated by the upregulation of the CD47 molecule, can lead to enhanced immune responses to any pathogen that triggers PRR signaling. Since most or all pathogens trigger PRRs, CD47 blockade could be used to speed up and strengthen both innate and adaptive immune responses when medically indicated. Such immunotherapy could be done without a requirement for knowing the HLA type of the individual, the specific antigens of the pathogen, or, in the case of bacterial infections, the antimicrobial resistance profile.

Placental Myeloid Cells Protect against Zika Virus Vertical Transmission in a Rag1-Deficient Mouse Model.

The ability of Zika virus (ZIKV) to cross the placenta and infect the fetus is a key mechanism by which ZIKV causes microcephaly. How the virus crosses the placenta and the role of the immune response in this process remain unclear. In the current study, we examined how ZIKV infection affected innate immune cells within the placenta and fetus and whether these cells influenced virus vertical transmission (VTx). We found myeloid cells were elevated in the placenta of pregnant ZIKV-infected Rag1-/- mice treated with an anti-IFNAR Ab, primarily at the end of pregnancy as well as transiently in the fetus several days before birth. These cells, which included maternal monocyte/macrophages, neutrophils, and fetal myeloid cells contained viral RNA and infectious virus, suggesting they may be infected and contributing to viral replication and VTx. However, depletion of monocyte/macrophage myeloid cells from the dam during ZIKV infection resulted in increased ZIKV infection in the fetus. Myeloid cells in the fetus were not depleted in this experiment, likely because of an inability of liposome particles containing the cytotoxic drug to cross the placenta. Thus, the increased virus infection in the fetus was not the result of an impaired fetal myeloid response or breakdown of the placental barrier. Collectively, these data suggest that monocyte/macrophage myeloid cells in the placenta play a significant role in inhibiting ZIKV VTx to the fetus, possibly through phagocytosis of virus or virus-infected cells.

Effects of Friend Virus Infection and Regulatory T Cells on the Antigen Presentation Function of B Cells.

Friend virus (FV) is a naturally occurring mouse retrovirus that infects dividing cells of the hematopoietic lineage, including antigen-presenting cells (APCs). The infection of APCs by viruses often induces their dysfunction, and it has been shown that FV infection reduces the ability of dendritic cells (DCs) to prime critical CD8+ T cell responses. Nonetheless, mice mount vigorous CD8+ T cell responses, so we investigated whether B cells might serve as alternative APCs during FV infection. Direct ex vivo analysis of B cells from FV-infected mice revealed that infected but not uninfected B cells upregulated expression of the costimulatory molecules CD80, CD86, and CD40, as well as major histocompatibility complex class II (MHC-II) molecules. Furthermore, in vitro studies showed that, compared to uninfected B cells from the same mice, the FV-infected B cells had significantly enhanced APC function, as measured by their capacity to prime CD8+ T cell activation and proliferation. Thus, in contrast to DCs, infection of B cells with FV enhanced their APC capacity and ability to stimulate the CD8+ T cell responses essential for virus control. FV infections also induce the activation and expansion of regulatory T cells (Tregs), so it was of interest to determine the impact of Tregs on B cell activation. The upregulation of costimulatory molecule expression and APC function of B cells was even more strongly enhanced by in vivo depletion of regulatory T cells than infection. Thus, Tregs exert potent homeostatic suppression of B cell activation that is partially overcome by FV infection.IMPORTANCE The primary role of B cells in immunity is considered the production of pathogen-specific antibodies, but another, less-well-studied, function of B cells is to present foreign antigens to T cells to stimulate their activation and proliferation. Dendritic cells (DCs) are considered the most important antigen-presenting cells (APCs) for CD8+ T cells, but DCs lose APC function when infected with Friend virus (FV), a model retrovirus of mice. Interestingly, B cells were better able to stimulate CD8+ T cell responses when they were infected with FV. We also found that the activation status of B cells under homeostatic conditions was potently modulated by regulatory T cells. This study illustrates an important link between B cell and T cell responses and illustrates an additional mechanism by which regulatory T cells suppress critical T cell responses during viral infections.

Adaptive Evolution of MERS-CoV to Species Variation in DPP4.

Middle East Respiratory Syndrome Coronavirus (MERS-CoV) likely originated in bats and passed to humans through dromedary camels; however, the genetic mechanisms underlying cross-species adaptation remain poorly understood. Variation in the host receptor, dipeptidyl peptidase 4 (DPP4), can block the interaction with the MERS-CoV spike protein and form a species barrier to infection. To better understand the species adaptability of MERS-CoV, we identified a suboptimal species-derived variant of DPP4 to study viral adaption. Passaging virus on cells expressing this DPP4 variant led to accumulation of mutations in the viral spike which increased replication. Parallel passages revealed distinct paths of viral adaptation to the same DPP4 variant. Structural analysis and functional assays showed that these mutations enhanced viral entry with suboptimal DPP4 by altering the surface charge of spike. These findings demonstrate that MERS-CoV spike can utilize multiple paths to rapidly adapt to novel species variation in DPP4.

Cutting Edge: CCR2 Is Not Required for Ly6Chi Monocyte Egress from the Bone Marrow but Is Necessary for Migration within the Brain in La Crosse Virus Encephalitis.

Inflammatory monocyte (iMO) recruitment to the brain is a hallmark of many neurologic diseases. Prior to entering the brain, iMOs must egress into the blood from the bone marrow through a mechanism, which for known encephalitic viruses, is CCR2 dependent. In this article, we show that during La Crosse Virus-induced encephalitis, egress of iMOs was surprisingly independent of CCR2, with similar percentages of iMOs in the blood and brain of heterozygous and CCR2-/- mice following infection. Interestingly, CCR2 was required for iMO trafficking from perivascular areas to sites of virus infection within the brain. Thus, CCR2 was not essential for iMO trafficking to the blood or the brain but was essential for trafficking within the brain parenchyma. Analysis of other orthobunyaviruses showed that Jamestown Canyon virus also induced CCR2-independent iMO egress to the blood. These studies demonstrate that the CCR2 requirement for iMO egress to the blood is not universal for all viruses.

Ebola Virus Replication and Disease Without Immunopathology in Mice Expressing Transgenes to Support Human Myeloid and Lymphoid Cell Engraftment.

The study of Ebola virus (EBOV) pathogenesis in vivo has been limited to nonhuman primate models or use of an adapted virus to cause disease in rodent models. Herein we describe wild-type EBOV (Makona variant) infection of mice engrafted with human hematopoietic CD34+ stem cells (Hu-NSG™-SGM3 mice; hereafter referred to as SGM3 HuMice). SGM3 HuMice support increased development of myeloid immune cells, which are primary EBOV targets. In SGM3 HuMice, EBOV replicated to high levels, and disease was observed following either intraperitoneal or intramuscular inoculation. Despite the high levels of viral antigen and inflammatory cell infiltration in the liver, the characteristic histopathology of Ebola virus disease was not observed, and this absence of severe immunopathology may have contributed to the recovery and survival of some of the animals. Future investigations into the underlying mechanisms of the atypical disease presentation in SGM3 HuMice will provide additional insights into the immunopathogenesis of severe EBOV disease.

Replication of Salmonella enterica Serovar Typhimurium in Human Monocyte-Derived Macrophages.

Salmonella enterica serovar Typhimurium is a common cause of food-borne gastrointestinal illness, but additionally it causes potentially fatal bacteremia in some immunocompromised patients. In mice, systemic spread and replication of the bacteria depend upon infection of and replication within macrophages, but replication in human macrophages is not widely reported or well studied. In order to assess the ability of Salmonella Typhimurium to replicate in human macrophages, we infected primary monocyte-derived macrophages (MDM) that had been differentiated under conditions known to generate different phenotypes. We found that replication in MDM depends greatly upon the phenotype of the cells, as M1-skewed macrophages did not allow replication, while M2a macrophages and macrophages differentiated with macrophage colony-stimulating factor (M-CSF) alone (termed M0) did. We describe how additional conditions that alter the macrophage phenotype or the gene expression of the bacteria affect the outcome of infection. In M0 MDM, the temporal expression of representative genes from Salmonella pathogenicity islands 1 and 2 (SPI1 and SPI2) and the importance of the PhoP/Q two-component regulatory system are similar to what has been shown in mouse macrophages. However, in contrast to mouse macrophages, where replication is SPI2 dependent, we observed early SPI2-independent replication in addition to later SPI2-dependent replication in M0 macrophages. Only SPI2-dependent replication was associated with death of the host cell at later time points. Altogether, our results reveal a very nuanced interaction between Salmonella and human macrophages.

Neuropeptide Y Negatively Influences Monocyte Recruitment to the Central Nervous System during Retrovirus Infection.

UNLABELLED: Monocyte infiltration into the CNS is a hallmark of several viral infections of the central nervous system (CNS), including retrovirus infection. Understanding the factors that mediate monocyte migration in the CNS is essential for the development of therapeutics that can alter the disease process. In the current study, we found that neuropeptide Y (NPY) suppressed monocyte recruitment to the CNS in a mouse model of polytropic retrovirus infection. NPY(-/-) mice had increased incidence and kinetics of retrovirus-induced neurological disease, which correlated with a significant increase in monocytes in the CNS compared to wild-type mice. Both Ly6C(hi) inflammatory and Ly6C(lo) alternatively activated monocytes were increased in the CNS of NPY(-/-) mice following virus infection, suggesting that NPY suppresses the infiltration of both cell types. Ex vivo analysis of myeloid cells from brain tissue demonstrated that infiltrating monocytes expressed high levels of the NPY receptor Y2R. Correlating with the expression of Y2R on monocytes, treatment of NPY(-/-) mice with a truncated, Y2R-specific NPY peptide suppressed the incidence of retrovirus-induced neurological disease. These data demonstrate a clear role for NPY as a negative regulator of monocyte recruitment into the CNS and provide a new mechanism for suppression of retrovirus-induced neurological disease. IMPORTANCE: Monocyte recruitment to the brain is associated with multiple neurological diseases. However, the factors that influence the recruitment of these cells to the brain are still not well understood. In the current study, we found that neuropeptide Y, a protein produced by neurons, affected monocyte recruitment to the brain during retrovirus infection. We show that mice deficient in NPY have increased influx of monocytes into the brain and that this increase in monocytes correlates with neurological-disease development. These studies provide a mechanism by which the nervous system, through the production of NPY, can suppress monocyte trafficking to the brain and reduce retrovirus-induced neurological disease.

Activated CD8+ T cells induce expansion of Vß5+ regulatory T cells via TNFR2 signaling.

Vß5(+) regulatory T cells (Tregs), which are specific for a mouse endogenous retroviral superantigen, become activated and proliferate in response to Friend virus (FV) infection. We previously reported that FV-induced expansion of this Treg subset was dependent on CD8(+) T cells and TNF-α, but independent of IL-2. We now show that the inflammatory milieu associated with FV infection is not necessary for induction of Vß5(+) Treg expansion. Rather, it is the presence of activated CD8(+) T cells that is critical for their expansion. The data indicate that the mechanism involves signaling between the membrane-bound form of TNF-α on activated CD8(+) T cells and TNFR2 on Tregs. CD8(+) T cells expressing membrane-bound TNF-α but no soluble TNF-α remained competent to induce strong Vß5(+) Treg expansion in vivo. In addition, Vß5(+) Tregs expressing only TNFR2 but no TNFR1 were still responsive to expansion. Finally, treatment of naive mice with soluble TNF-α did not induce Vß5(+) Treg expansion, but treatment with a TNFR2-specific agonist did. These results reveal a new mechanism of intercellular communication between activated CD8(+) T cell effectors and Tregs that results in the activation and expansion of a Treg subset that subsequently suppresses CD8(+) T cell functions.

Age-related differences in neuroinflammatory responses associated with a distinct profile of regulatory markers on neonatal microglia.

BACKGROUND: The perinatal period is one in which the mammalian brain is particularly vulnerable to immune-mediated damage. Early inflammation in the central nervous system (CNS) is linked with long-term impairment in learning and behavior, necessitating a better understanding of mediators of neuroinflammation. We therefore directly examined how age affected neuroinflammatory responses to pathogenic stimuli. METHODS: In mice, susceptibility to neurological damage changes dramatically during the first few weeks of life. Accordingly, we compared neuroinflammatory responses to pathogen associated molecular patterns (PAMPs) of neonatal (two day-old) and weanling (21 day-old) mice. Mice were inoculated intracerebrally with PAMPs and the cellular and molecular changes in the neuroinflammatory response were examined. RESULTS: Of the 12 cytokines detected in the CNS following toll-like receptor 4 (TLR4) stimulation, ten were significantly higher in neonates compared with weanling mice. A similar pattern of increased cytokines in neonates was also observed with TLR9 stimulation. Analysis of cellular responses indicated a difference in microglial activation markers in the CNS of neonatal mice and increased expression of proteins known to modulate cellular activation including CD11a, F4/80 and CD172a. We also identified a new marker on microglia, SLAMF7, which was expressed at higher levels in neonates compared with weanlings. CONCLUSIONS: A unique neuroinflammatory profile, including higher expression of several proinflammatory cytokines and differential expression of microglial markers, was observed in brain tissue from neonates following TLR stimulation. This increased neuroinflammatory response to PAMPs may explain why the developing brain is particularly sensitive to infection and why infection or stress during this time can lead to long-term damage in the CNS.

Glycosylphosphatidylinositol anchoring directs the assembly of Sup35NM protein into non-fibrillar, membrane-bound aggregates.

In prion-infected hosts, PrPSc usually accumulates as non-fibrillar, membrane-bound aggregates. Glycosylphosphatidylinositol (GPI) anchor-directed membrane association appears to be an important factor controlling the biophysical properties of PrPSc aggregates. To determine whether GPI anchoring can similarly modulate the assembly of other amyloid-forming proteins, neuronal cell lines were generated that expressed a GPI-anchored form of a model amyloidogenic protein, the NM domain of the yeast prion protein Sup35 (Sup35(GPI)). We recently reported that GPI anchoring facilitated the induction of Sup35(GPI) prions in this system. Here, we report the ultrastructural characterization of self-propagating Sup35(GPI) aggregates of either spontaneous or induced origin. Like membrane-bound PrPSc, Sup35(GPI) aggregates resisted release from cells treated with phosphatidylinositol-specific phospholipase C. Sup35(GPI) aggregates of spontaneous origin were detergent-insoluble, protease-resistant, and self-propagating, in a manner similar to that reported for recombinant Sup35NM amyloid fibrils and induced Sup35(GPI) aggregates. However, GPI-anchored Sup35 aggregates were not stained with amyloid-binding dyes, such as Thioflavin T. This was consistent with ultrastructural analyses, which showed that the aggregates corresponded to dense cell surface accumulations of membrane vesicle-like structures and were not fibrillar. Together, these results showed that GPI anchoring directs the assembly of Sup35NM into non-fibrillar, membrane-bound aggregates that resemble PrPSc, raising the possibility that GPI anchor-dependent modulation of protein aggregation might occur with other amyloidogenic proteins. This may contribute to differences in pathogenesis and pathology between prion diseases, which uniquely involve aggregation of a GPI-anchored protein, versus other protein misfolding diseases.

Tick-borne flaviviruses antagonize both IRF-1 and type I IFN signaling to inhibit dendritic cell function.

Tick-borne encephalitis virus (TBEV), a member of the Flaviviridae family, is a leading cause of viral encephalitis in Europe and Asia. Dendritic cells (DCs), as early cellular targets of infection, provide an opportunity for flaviviruses to inhibit innate and adaptive immune responses. Flaviviruses modulate DC function, but the mechanisms underpinning this are not defined. We examined the maturation phenotype and function of murine bone marrow-derived DCs infected with Langat virus (LGTV), a naturally attenuated member of the TBEV serogroup. LGTV infection failed to induce DC maturation or a cytokine response. Treatment with LPS or LPS/IFN-γ, strong inducers of inflammatory cytokines, resulted in enhanced TNF-α and IL-6 production, but suppressed IL-12 production in infected DCs compared with uninfected "bystander" cells or mock-infected controls. LGTV-mediated antagonism of type I IFN (IFN-I) signaling contributed to inhibition of IL-12p40 mRNA expression at late time points after stimulation. However, early suppression was still observed in DCs lacking the IFN-I receptor (Ifnar(-/-)), suggesting that additional mechanisms of antagonism exist. The early IFN-independent inhibition of IL-12p40 was nearly abolished in DCs deficient in IFN regulatory factor-1 (IRF-1), a key transcription factor required for IL-12 production. LGTV infection did not affect Irf-1 mRNA expression, but rather diminished IRF-1 protein levels and nuclear localization. The effect on IRF-1 was also observed in DCs infected with the highly virulent Sofjin strain of TBEV. Thus, antagonism of IRF-1 is a novel mechanism that synergizes with the noted ability of flaviviruses to suppress IFN-α/ß receptor-dependent signaling, resulting in the orchestrated evasion of host innate immunity.

IL-2-independent and TNF-α-dependent expansion of Vß5+ natural regulatory T cells during retrovirus infection.

Friend virus infection of mice induces the expansion and activation of regulatory T cells (Tregs) that dampen acute immune responses and promote the establishment and maintenance of chronic infection. Adoptive transfer experiments and the expression of neuropilin-1 indicate that these cells are predominantly natural Tregs rather than virus-specific conventional CD4(+) T cells that converted into induced Tregs. Analysis of Treg TCR Vß chain usage revealed a broadly distributed polyclonal response with a high proportionate expansion of the Vß5(+) Treg subset, which is known to be responsive to endogenous retrovirus-encoded superantigens. In contrast to the major population of Tregs, the Vß5(+) subset expressed markers of terminally differentiated effector cells, and their expansion was associated with the level of the antiviral CD8(+) T cell response rather than the level of Friend virus infection. Surprisingly, the expansion and accumulation of the Vß5(+) Tregs was IL-2 independent but dependent on TNF-α. These experiments reveal a subset-specific Treg induction by a new pathway.

Tissue-specific abundance of regulatory T cells correlates with CD8+ T cell dysfunction and chronic retrovirus loads.

Infection of mice with Friend virus induces the activation of CD4(+) regulatory T cells (Tregs) that suppress virus-specific CD8(+) T cells. This suppression leads to incomplete virus clearance and the establishment of virus persistence. We now show that Treg-mediated suppression of CD8(+) T cells is tissue specific, occurring in the spleen but not the liver. Regardless of infection status, there was a 5-fold lower proportion of Tregs in the liver than in the spleen, much lower absolute cell numbers, and the relatively few Tregs present expressed less CD25. Results indicated that reduced expression of CD25 on liver Tregs was due to microenvironmental factors including low levels of IL-2 production by CD4(+) Th cells in that tissue. Low CD25 expression on liver Tregs did not impair their ability to suppress CD8(+) T cells in vitro. Correlating with the decreased proportion of Tregs in the liver was a significantly increased proportion of virus-specific CD8(+) T cells compared with the spleen. The virus-specific CD8(+) T cells from the liver did not appear suppressed given that they produced both IFN-gamma and granzyme B, and they also showed evidence of recent cytolytic activity (CD107a(+)). The functional phenotype of the virus-specific CD8(+) T cells correlated with a 10-fold reduction of chronic Friend virus levels in the liver compared with the spleen. Thus, suppression of CD8(+) T cells by virus-induced Tregs occurs in a tissue-specific manner and correlates with profound effects on localized levels of chronic infection.

CD137 costimulation of CD8+ T cells confers resistance to suppression by virus-induced regulatory T cells.

Chronic viral infections cause high levels of morbidity and mortality worldwide, making the development of effective therapies a high priority for improving human health. We have used mice infected with Friend virus as a model to study immunotherapeutic approaches to the cure of chronic retroviral infections. In chronic Friend virus infections CD4(+) T regulatory (Treg) cells suppress CD8(+) T cell effector functions critical for virus clearance. In this study, we demonstrate that immunotherapy with a combination of agonistic anti-CD137 Ab and virus-specific, TCR-transgenic CD8(+) T cells produced greater than 99% reductions of virus levels within 2 wk. In vitro studies indicated that the CD137-specific Ab rendered the CD8(+) T cells resistant to Treg cell-mediated suppression with no direct effect on the suppressive function of the Treg cells. By 2 weeks after transfer, the adoptively transferred CD8(+) T cells were lost, likely due to activation-induced cell death. The highly focused immunological pressure placed on the virus by the single specificity CD8(+) T cells led to the appearance of escape variants, indicating that broader epitope specificity will be required for long-term virus control. However, the results demonstrate a potent strategy to potentiate the function of CD8(+) T cells in the context of immunosuppressive Treg cells.