CD28(-/-) mice show defects in cellular and humoral immunity but are able to control infection with murine gammaherpesvirus 68

The role of CD28-dependent costimulatory interactions in the development and maintenance of antiviral immune responses was investigated in a mouse model of gammaherpesvirus infection. CD28(-/-) mice could clear a productive infection with murine gammaherpesvirus 68 (MHV-68), although early lung viral titers were significantly increased. Both CD28(-/-) and CD28(+/+) mice maintained effective long-term control of MHV-68. Gamma interferon responses appeared to develop more slowly in CD28(-/-) mice, while cytotoxic T-cell activity was similar to that in wild-type mice. Splenomegaly developed normally in CD28(-/-) mice, whereas virus-specific antibody responses were significantly reduced and aberrant class switching was observed. This work demonstrates that costimulatory interactions involving CD28 are not an absolute requirement for the control of infection with MHV-68.     

COX-2 induction during murine gammaherpesvirus 68 infection leads to enhancement of viral gene expression

The murine gammaherpesvirus 68 (MHV-68 or gammaHV-68) model provides many advantages for studying virus-host interactions involved in gammaherpesvirus replication, including the role of cellular responses to infection. We examined the effects of cellular cyclooxygenase-2 (COX-2) and its by-product prostaglandin E(2) (PGE(2)) on MHV-68 gene expression and protein production following de novo infection of cultured cells. Western blot analyses revealed an induction of COX-2 protein in MHV-68-infected cells but not in cells infected with UV-irradiated MHV-68. Luciferase reporter assays demonstrated activation of the COX-2 promoter during MHV-68 replication. Two nonsteroidal anti-inflammatory drugs, a COX-2-specific inhibitor (NS-398) and a COX-1-COX-2 inhibitor (indomethacin), substantially reduced MHV-68 protein production in infected cells. Inhibition of viral protein expression and virion production by NS-398 was reversed in the presence of exogenous PGE(2). Global gene expression analysis using an MHV-68 DNA array showed that PGE(2) increased production of multiple viral gene products, and NS-398 inhibited production of many of the same genes. These studies suggest that COX-2 activity and PGE(2) production may play significant roles during MHV-68 de novo infection.     

Gamma interferon is not essential for recovery from acute infection with murine gammaherpesvirus 68.

Murine gammaherpesvirus 68 (MHV-68) when administered intranasally induces high levels of gamma interferon (IFN-gamma) in the lymphoid tissues of infected mice. In order to investigate the role of this cytokine in the immune response to MHV-68, mice which were congenitally deficient in the IFN-gamma gene (IFN-gamma knockout mice) were infected with the virus. Comparison of the courses of the disease in wild-type control and IFN-gamma knockout mice revealed surprisingly little difference. Both groups of mice had cleared infectious virus from the lungs 15 days after infection, although there did appear to be a slight delay in viral clearance in the IFN-gamma knockout mice. In addition, after the initial phase of viral clearance, the lungs of both groups remained clear of replicating virus throughout the course of the experiment, which concluded 34 days after infection. Consistent with these observations, cytotoxic T-cell activities were similar in the two groups of mice. Levels of latent virus were comparable in wild-type and knockout mice over the time course studied. Furthermore, analysis of the numbers, types, and activation status of cells in the lungs, lymph nodes, and spleens of control and knockout mice revealed no striking difference. This suggests that IFN-gamma is not essential for regulating the cell recruitment or proliferation that normally occurs during this viral infection. Apart from the expected lack of IFN-gamma, cytokine profiles were not dramatically altered in IFN-gamma knockout mice, demonstrating that IFN-gamma did not suppress the proliferation or differentiation of Th2 cells during MHV-68 infection. These observations indicate that IFN-gamma plays a nonessential or redundant role in the control of acute infection with MHV-68.     

Chemokine binding protein M3 of murine gammaherpesvirus 68 modulates the host response to infection in a natural host

Murine γ-herpesvirus 68 (MHV-68) infection of Mus musculus-derived strains of mice is an attractive model of γ-herpesvirus infection. Surprisingly, however, ablation of expression of MHV-68 M3, a secreted protein with broad chemokine-binding properties in vitro, has no discernable effect during experimental infection via the respiratory tract. Here we demonstrate that M3 indeed contributes significantly to MHV-68 infection, but only in the context of a natural host, the wood mouse (Apodemus sylvaticus). Specifically, M3 was essential for two features unique to the wood mouse: virus-dependent inducible bronchus-associated lymphoid tissue (iBALT) in the lung and highly organized secondary follicles in the spleen, both predominant sites of latency in these organs. Consequently, lack of M3 resulted in substantially reduced latency in the spleen and lung. In the absence of M3, splenic germinal centers appeared as previously described for MHV-68-infected laboratory strains of mice, further evidence that M3 is not fully functional in the established model host. Finally, analyses of M3's influence on chemokine and cytokine levels within the lungs of infected wood mice were consistent with the known chemokine-binding profile of M3, and revealed additional influences that provide further insight into its role in MHV-68 biology.     

Mature B cells are required for acute splenic infection, but not for establishment of latency, by murine gammaherpesvirus 68.

Murine gammaherpesvirus 68 (gamma HV-68; also referred to as MHV-68) is a gammaherpesvirus which infects murid rodents. Previous studies showed that CD8 T cells are important for controlling gamma HV-68 replication during the first 2 weeks of infection and suggested a role for B cells in latent or persistent gamma HV-68 infection. To further define the importance of B cells and CD8 T cells during acute and chronic gamma HV-68 infection, we examined splenic infection in mice with null mutations in the transmembrane domain of the mu-heavy-chain constant region (MuMT; B-cell and antibody deficient) or in the beta2-microglobulin gene (beta2 -/-; CD8 deficient). Immunocompetent mice infected intraperitoneally with gamma HV-68 demonstrated peak splenic titers 9 to 10 days postinfection, cleared infectious virus 15 to 20 days postinfection, and harbored low levels of latent virus at 6 weeks postinfection. Beta2-/- mice showed peak splenic gamma HV-68 titers similar to those of normal mice but were unable to clear infectious virus completely from the spleen, demonstrating persistent infectious virus 6 weeks postinfection. These data indicate that CD8 T cells are important for clearing infectious gamma HV-68 from the spleen. Infected MuMT mice did not demonstrate detectable infectious gamma HV-68 in the spleen at any time after infection, indicating that mature B lymphocytes are necessary for acute splenic infection by gamma HV-68. Despite the lack of measurable acute infection, MuMT spleen cells harbored latent virus 6 weeks postinfection at a level about 100-fold higher than that in normal mice. These data demonstrate establishment of latency by a herpesvirus in an organ in the absence of acute viral replication in that organ. In addition, they demonstrate that gamma HV-68 can establish latency in a cell type other than mature B lymphocytes.     

Murine gammaherpesvirus 68 infection is associated with lymphoproliferative disease and lymphoma in BALB beta2 microglobulin-deficient mice

Human gammaherpesvirus infections are associated with development of lymphoproliferative disease. Understanding of the mechanisms of gammaherpesvirus lymphomagenesis during chronic infection in a natural host has been limited by the exquisite species specificity of human gammaherpesviruses and the expense of primates. Murine gammaherpesvirus gammaHV68 is genetically and biologically related to human gammaherpesviruses and herpesvirus saimiri and has been reported to be associated with lymphoproliferative disease in mice (N. P. Sunil-Chandra, J. Arno, J. Fazakerley, and A. A. Nash, Am. J. Pathol. 145:818-826, 1994). We report the development of an animal model of gammaHV68 lymphomagenesis in BALB/c beta2 microglobulin-deficient mice (BALB beta2m-/-). GammaHV68 infection induced two lymphoproliferative lesions: B-cell lymphoma and atypical lymphoid hyperplasia (ALH). ALH lesion histology resembled lesions of Epstein-Barr virus-associated posttransplant lymphoproliferative disease and was characterized by the abnormal infiltration of the white pulp with cells expressing the plasma cell marker CD138. Lymphomas observed in gammaHV68-infected animals were B220+/CD3- large-cell lymphomas. GammaHV68-infected cells were common in ALH lesions as measured by in situ hybridization with a probe specific for viral tRNAs (vtRNAs), but they were scarce in gammaHV68-infected spleens with normal histology. Unlike ALH lesions, gammaHV68 vtRNA-positive cells were rare in lymphomas. GammaHV68 infection of BALB beta2m-/- mice results in lymphoproliferation and lymphoma, providing a valuable tool for identifying viral and host genes involved in gammaherpesvirus-associated malignancies. Our findings suggest that gammaHV68 induces lymphomas via hit-and-run oncogenesis, paracrine effects, or stimulation of chronic inflammation.     

CD40 engagement on dendritic cells, but not on B or T cells, is required for long-term control of murine gammaherpesvirus 68

CD4 T cells are not essential for primary clearance of replicating murine gammaherpesvirus 68 (MHV-68) but are required for effective long-term control. The virus reactivates in the lungs of major histocompatibility complex class II-deficient (CII-/-) mice that lack functional CD4 T cells. CD40 ligand (CD40L) is upregulated on activated CD4 T cells, and it is thought that CD40-CD40L interactions are an important component of CD4 T-cell help. Our previous studies have shown that agonistic antibodies to CD40 can substitute for CD4 T-cell function in the long-term control of MHV-68. In the present study, we sought to identify the CD40-positive cell type mediating this effect. To address this question, we adoptively transferred MHV-68 peptide-pulsed CII(-/-) dendritic cells (DC) that had been treated with an agonistic antibody to CD40 into MHV-68-infected CII(-/-) recipients. Viral reactivation was significantly lower in mice injected with anti-CD40-treated DC than in those injected with control DC or in mice that did not receive any DC. However, in similar experiments with B cells, anti-CD40 treatment had no effect. We also investigated the requirement for CD40 expression on T cells by adoptive transfer of T cells from CD40(+/+) or CD40(-/-) mice into T-cell-deficient recipients that were subsequently infected with MHV-68. The results showed that CD40 expression on T cells is not necessary for preventing viral reactivation. Taken together, our data suggest that CD40 engagement on DC, but not on T or B cells, is essential for effective long-term control of MHV-68.     

Characterization of cytotoxic cells from reovirus-infected SCID mice: activated cells express natural killer- and lymphokine-activated killer-like activity but fail to clear infection.

Severe combined immune deficient (SCID) mice infected orally with reovirus type 1/L die of hepatitis. Leukocytes bearing the cell surface antigens Thy-1.2 and asialoGM-1 (AsGM1) accumulate in the livers of infected animals. These cells display lytic activity toward natural killer-sensitive (YAC-1) and -resistant (P815) cell lines and murine hepatoma line Hepa 1/A1. Although these cells have the capacity to lyse infected hepatoma targets, they cannot clear the virus.     

NK cell-deficient mice develop a Th1-like response but fail to mount an efficient antigen-specific IgG2a antibody response.

NK cells have been shown to play a role in the modulation of B cell differentiation and Ab production. Using a novel murine model of NK cell deficiency, we analyzed the in vivo role of NK cells in the regulation of Ag-specific Ab production. After immunization with OVA or keyhole limpet hemocyanin in CFA, NK cell-deficient (NK-T+) mice developed an efficient Th1 response and produced significant levels of IFN-gamma but displayed markedly reduced or absent Ag-specific IgG2a production. There were no differences in the levels of Ag-specific IgG, IgG1, and IgG2b between NK-T+ and NK+T+ mice. Furthermore, NK cell-reconstituted, NK+T+ (tgepsilon26Y) mice produced significant amounts of Ag-specific IgG2a after immunization with OVA. These results indicate that NK cells are involved in the induction of Ag-specific IgG2a production in vivo. Moreover, they also demonstrate that the lack of Ag-specific IgG2a Ab production in NK-T+ mice is not associated with the impaired Th1 response and IFN-gamma production.     

B cell-deficient mice are highly resistant to Leishmania donovani infection, but develop neutrophil-mediated tissue pathology

Resolution of Leishmania infection is T cell-dependent, and B lymphocytes have been considered to play a minimal role in host defense. In this study, the contribution of B lymphocytes to the response against Leishmania donovani was investigated using genetically modified IgM transmembrane domain (muMT) mutant mice, which lack mature B lymphocytes. When compared with wild-type mice, muMT mice cleared parasites more rapidly from the liver, and infection failed to establish in the spleen. The rapid clearance of parasites in muMT mice was associated with accelerated and more extensive hepatic granuloma formation compared with wild-type mice. However, the liver of infected muMT mice also showed signs of destructive pathology, associated with the presence of increased numbers of neutrophils. The role of neutrophils in controlling parasite growth in the viscera was determined by depletion with the mAb RB6-8C5. This treatment led to a dramatic enhancement of parasite growth in both the liver and spleen of muMT and wild-type mice. As assessed by transfer of both normal and chronic-infection serum, Ig protects microMT mice from destructive hepatic pathology, but minimally alters their resistance compared with wild-type mice. However, adoptive transfer of CD4+ and CD8+ T cells into recombinase activating gene 1 (RAG1-/-) recipients, suggested that T cell function was not altered by maturation in a B cell-deficient environment. Taken together, these data suggest an inhibitory role for B lymphocytes in resistance to L. donovani unrelated to the presence or absence of Ig. However, Ig protects muMT mice from the exaggerated pathology that occurs during infection.     

Establishment of B-cell lines latently infected with reactivation-competent murine gammaherpesvirus 68 provides evidence for viral alteration of a DNA damage-signaling cascade

Gammaherpesvirus 68 (γHV68, or MHV68) is a naturally occurring rodent pathogen that replicates to high titer in cell culture and is amenable to in vivo experimental evaluation of viral and host determinants of gammaherpesvirus disease. However, the inability of MHV68 to transform primary murine B cells in culture, the absence of a robust cell culture latency system, and the paucity of MHV68-positive tumor cell lines have limited an understanding of the molecular mechanisms by which MHV68 modulates the host cell during latency and reactivation. To facilitate a more complete understanding of viral and host determinants that regulate MHV68 latency and reactivation in B cells, we generated a recombinant MHV68 virus that encodes a hygromycin resistance protein fused to enhanced green fluorescent protein as a means to select cells in culture that harbor latent virus. We utilized this virus to infect the A20 murine mature B-cell line and evaluate reactivation competence following treatment with diverse stimuli to reveal viral gene expression, DNA replication, and production of progeny virions. Comparative analyses of parental and infected A20 cells indicated a correlation between infection and alterations in DNA damage signaling following etoposide treatment. The data described in this study highlight the potential utility of this new cell culture-based system to dissect molecular mechanisms that regulate MHV68 latency and reactivation, as well as having the potential of illuminating biochemical alterations that contribute to gammaherpesvirus pathogenesis. In addition, such cell lines may be of value in evaluating targeted therapies to gammaherpesvirus-related tumors.     

A gammaherpesvirus 68 gene 50 null mutant establishes long-term latency in the lung but fails to vaccinate against a wild-type virus challenge

The gammaherpesvirus immediate-early genes are critical regulators of virus replication and reactivation from latency. Rta, encoded by gene 50, serves as the major transactivator of the lytic program and is highly conserved among all the gammaherpesviruses, including Epstein-Barr virus, Kaposi's sarcoma-associated herpesvirus, and murine gammaherpesvirus 68 (gammaHV68). Introduction of a translation stop codon in gammaHV68 gene 50 (gene 50.stop gammaHV68) demonstrated that Rta is essential for virus replication in vitro. To investigate the role that virus replication plays in the establishment and maintenance of latency, we infected mice with gene 50.stop gammaHV68. Notably, the gene 50.stop virus established a long-term infection in lung B cells following intranasal infection of mice but was unable to establish latency in the spleen. This complete block in the establishment of latency in the spleen was also seen when lytic virus production was inhibited by treating mice infected with wild-type virus with the antiviral drug cidofovir, implicating virus replication and not an independent function of Rta in the establishment of splenic latency. Furthermore, we showed that gene 50.stop gammaHV68 was unable to prime the immune system and was unable to protect against a challenge with wild-type gammaHV68, despite its ability to chronically infect lung B cells. These data indicate gammaherpesviruses that are unable to undergo lytic replication in vivo may not be viable vaccine candidates despite the detection of cells harboring viral genome at late times postinfection.     

NADPH phagocyte oxidase knockout mice control Trypanosoma cruzi proliferation, but develop circulatory collapse and succumb to infection

^•NO is considered to be a key macrophage-derived cytotoxic effector during Trypanosoma cruzi infection. On the other hand, the microbicidal properties of reactive oxygen species (ROS) are well recognized, but little importance has been attributed to them during in vivo infection with T. cruzi. In order to investigate the role of ROS in T. cruzi infection, mice deficient in NADPH phagocyte oxidase (gp91^{phox −/−} or phox KO) were infected with Y strain of T. cruzi and the course of infection was followed. phox KO mice had similar parasitemia, similar tissue parasitism and similar levels of IFN-γ and TNF in serum and spleen cell culture supernatants, when compared to wild-type controls. However, all phox KO mice succumbed to infection between day 15 and 21 after inoculation with the parasite, while 60% of wild-type mice were alive 50 days after infection. Further investigation demonstrated increased serum levels of nitrite and nitrate (NOx) at day 15 of infection in phox KO animals, associated with a drop in blood pressure. Treatment with a NOS2 inhibitor corrected the blood pressure, implicating NOS2 in this phenomenon. We postulate that superoxide reacts with ^•NO in vivo, preventing blood pressure drops in wild type mice. Hence, whilst superoxide from phagocytes did not play a critical role in parasite control in the phox KO animals, its production would have an important protective effect against blood pressure decline during infection with T. cruzi.     

Mice lacking the nuclear pore complex protein ALADIN show female infertility but fail to develop a phenotype resembling human triple A syndrome

Triple A syndrome is a human autosomal recessive disorder characterized by adrenal insufficiency, achalasia, alacrima, and neurological abnormalities affecting the central, peripheral, and autonomic nervous systems. In humans, this disease is caused by mutations in the AAAS gene, which encodes ALADIN, a protein that belongs to the family of WD-repeat proteins and localizes to nuclear pore complexes. To analyze the function of the gene in the context of the whole organism and in an attempt to obtain an animal model for human triple A syndrome, we generated mice lacking a functional Aaas gene. The Aaas-/- animals were found to be externally indistinguishable from their wild-type littermates, although their body weight was on the average lower than that of wild-type mice. Histological analysis of various tissues failed to reveal any differences between Aaas-/- and wild-type mice. Aaas-/- mice exhibit unexpectedly mild abnormal behavior and only minor neurological deficits. Our data show that the lack of ALADIN in mice does not lead to a triple A syndrome-like disease. Thus, in mice either the function of ALADIN differs from that in humans, its loss can be readily compensated for, or additional factors, such as environmental conditions or genetic modifiers, contribute to the disease.     

Lymphotoxin alpha-/- mice develop functionally impaired CD8+ T cell responses and fail to contain virus infection of the central nervous system

Recent observations have indicated that viral persistence and tumor spreading could occur because of effector function-defective CD8(+) T cells. Although chronic exposure to Ag, lack of CD4 help, and epitope dominance are suggested to interfere with CTL differentiation, mechanisms underlying the defective effector function remain obscure. We demonstrate in this report that lymphotoxin alpha-deficient mice develop CD8(+) T cells at normal frequencies when infected with HSV or immunized with OVA Ag but show impaired cytotoxic and cytokine-mediated effector functions resulting in enhanced susceptibility to HSV-induced encephalitis. Although these cells display near normal levels of perforin and Fas ligand, they remain largely at a naive state as judged by high expression of CD62 ligand and failure to up-regulate activation or memory markers. In particular, these CD8(+) T cells revealed inadequate expression of the IL-12 receptor, thus establishing a link between CTL differentiation and LTalpha possibly through regulation of IL-12 receptor. Viruses and tumors could evade immunity by targeting the same pathway.     

Slc25a13-knockout mice harbor metabolic deficits but fail to display hallmarks of adult-onset type II citrullinemia

Adult-onset type II citrullinemia (CTLN2) is an autosomal recessive disease caused by mutations in SLC25A13, the gene encoding the mitochondrial aspartate/glutamate carrier citrin. The absence of citrin leads to a liver-specific, quantitative decrease of argininosuccinate synthetase (ASS), causing hyperammonemia and citrullinemia. To investigate the physiological role of citrin and the development of CTLN2, an Slc25a13-knockout (also known as Ctrn-deficient) mouse model was created. The resulting Ctrn-/- mice were devoid of Slc25a13 mRNA and citrin protein. Liver mitochondrial assays revealed markedly decreased activities in aspartate transport and the malate-aspartate shuttle. Liver perfusion also demonstrated deficits in ureogenesis from ammonia, gluconeogenesis from lactate, and an increase in the lactate-to-pyruvate ratio within hepatocytes. Surprisingly, Ctrn-/- mice up to 1 year of age failed to show CTLN2-like symptoms due to normal hepatic ASS activity. Serological measures of glucose, amino acid, and ammonia metabolism also showed no significant alterations. Nitrogen-loading treatments produced only minor changes in the hepatic ammonia and amino acid levels. These results suggest that citrin deficiency alone may not be sufficient to produce a CTLN2-like phenotype in mice. These observations are compatible, however, with the variable age of onset, incomplete penetrance, and strong ethnic bias seen in CTLN2 where additional environmental and/or genetic triggers are now suspected.     

B cell immunodeficiency fails to develop in CD4-deficient mice infected with BM5: murine AIDS as a multistep disease

The immunodeficiency syndrome murine AIDS (MAIDS), caused by the BM5 retrovirus preparation, involves the activation, division, and subsequent anergy of the entire CD4(+) T cell population as well as extensive B cell hyperproliferation and hypergammaglobulinemia, resulting in splenomegaly and lymphadenopathy, followed many weeks later by death. The development of MAIDS requires CD4(+) T cells and MHC class II expression by the infected host, supporting a role for T-B interaction in disease development or progression. To explore this possibility, we examined development of MAIDS in mice deficient in CD4 (CD4 knockout), in which T-B interactions are compromised. We find that in CD4 knockout hosts, BM5 causes T cell immunodeficiency in the remaining T cells but has only a limited ability to induce B cell phenotypic changes, hyperproliferation, hypergammaglobulinemia, or splenomegaly. There is also delayed death of infected mice. This implies that CD4 dependent T-B interaction is needed to induce the B cell aspects of disease and supports a multistep mechanism of disease in which B cell changes follow and are caused by CD4(+) T cell effects.     

Pathological changes in the spleens of gamma interferon receptor-deficient mice infected with murine gammaherpesvirus: a role for CD8 T cells.

Murine gammaherpesvirus is a natural rodent pathogen which causes a primary infection in the lungs and establishes a persistent infection in B lymphocytes. During the primary infection, large amounts of gamma interferon (IFN-gamma) are produced by spleen, mediastinal, and cervical lymph node cells. To investigate the role of IFN-gamma in control of the virus infection, mice lacking the cellular receptor for IFN-gamma (IFN-gamma R-/- mice) were infected with murine gammaherpesvirus 68 (MHV68). IFN-gamma R-/- mice showed no difference from wild-type mice in the titers of infectious virus in the lungs or in the rate of clearance of the lung infection. In the spleen, however, clear differences were observed. By 14 days postinfection, spleens from IFN-gamma R-/- mice were pale, shrunken, and fibrous. Histological examination showed that there was an early (day 10) infiltration of granulocytes followed by widespread destruction of splenic architecture (days 14 to 17). A marked decrease in the number of splenic B cells and CD4+ and CD8+ T cells occurred. These changes were accompanied by a 10- to 100-fold greater load of latently infected cells in IFN-gamma R-/- mice than in wild-type mice at 14 to 17 days postinfection, but this was reduced to the levels found in wild-type mice by 21 days postinfection. Treatment of the mice with the antiviral drug 2'-deoxyl-5-ethyl-beta-4'-thiouridine from 6 days postinfection did not prevent the occurrence of these changes. The changes were, however, completely reversed by depletion of CD8+ T cells prior to and during the primary infection. Depletion of CD4+ T cells also reversed the major pathological and virological changes, although in this case there was evidence of some histological changes. Thus, the lack of IFN-gamma receptor had profound consequences in spleens of MHV68-infected mice. The possible mechanisms involved in these changes are discussed.