CMV late phase-induced mTOR activation is essential for efficient virus replication in polarized human macrophages.

Human cytomegalovirus (CMV) remains one of the most important pathogens following solid-organ transplantation. Mounting evidence indicates that mammalian target of rapamycin (mTOR) inhibitors may decrease the incidence of CMV infection in solid-organ recipients. Here we aimed at elucidating the molecular mechanisms of this effect by employing a human CMV (HCMV) infection model in human macrophages, since myeloid cells are the principal in vivo targets of HCMV. We demonstrate a highly divergent host cell permissiveness for HCMV with optimal infection susceptibility in M2 but not M1 polarized macrophages. Employing an ultrahigh purified HCMV stock we observed rapamycin-independent viral entry and induction of IFN-ß transcripts, but no proinflammatory cytokines or mitogen-activated protein kinases and mTOR activation early after infection. However, in the late infection phase, sustained mTOR activation was observed in HCMV-infected cells and was required for efficient viral protein synthesis including the viral late phase proteins pUL-44 and pp65. Accordingly, rapamycin strongly suppressed CMV replication 3 and 5 days postinfection in macrophages. In conclusion, these data indicate that mTOR is essential for virus replication during late phases of the viral cycle in myeloid cells and might explain the potent anti-CMV effects of mTOR inhibitors after organ transplantation.

Cytolytic T lymphocyte recognition of the murine cytomegalovirus nonstructural immediate-early protein pp89 expressed by recombinant vaccinia virus.

The murine immediate-early (IE) protein pp89 is a nonstructural virus-encoded phosphoprotein residing in the nucleus of infected cells, where it acts as transcriptional activator. Frequency analysis has shown that in BALB/c mice the majority of virus-specific CTL recognize IE antigens. The present study was performed to assess whether pp89 causes membrane antigen expression detected by IE-specific CTL. Site-directed mutagenesis has been used to delete the introns from gene ieI, encoding pp89, for subsequent integration of the continuous coding sequence into the vaccinia virus genome. After infection with the vaccinia recombinant, the authentic pp89 was expressed in cells that became susceptible to lysis by an IE-specific CTL clone. Priming of mice with the vaccinia recombinant sensitized polyclonal CTL that recognized MCMV-infected cells and transfected cells expressing pp89. Thus, a herpesviral IE polypeptide with essential function in viral transcriptional regulation can also serve as a dominant antigen for the specific CTL response of the host.

Site-restricted persistent cytomegalovirus infection after selective long-term depletion of CD4+ T lymphocytes.

We have established a murine model system for exploring the ability of a CD4 subset-deficient host to cope with cytomegalovirus infection, and reported three findings. First, an antiviral response of the CD8 subset of T lymphocytes could be not only initiated but also maintained for a long period of time despite a continued absence of the CD4 subset, whereas the production of antiviral antibody proved strictly dependent upon help provided by the CD4 subset. Second, no function in the defense against infection could be ascribed as yet to CD4-CD8- T lymphocytes, which were seen to accumulate to a new subset as a result of depletion of the CD4 subset. This newly arising subset did not substitute for CD4+ T lymphocytes in providing help to B lymphocytes, and was also not effective in controlling the spread of virus in host tissues. As long as a function of these cells in the generation and maintenance of a CD8 subset-mediated response is not disproved, caution is indicated with concern to an autonomy of the CD8 subset. Third, even though with delay, the CD8+ effector cells raised in the CD4 subset-deficient host were able of clear vital tissues from productive infection and to restrict asymptomatic, persistent infection to acinar glandular epithelial cells in salivary gland tissue.

The conditions of primary infection define the load of latent viral genome in organs and the risk of recurrent cytomegalovirus disease.

Recurrence of cytomegalovirus (CMV) from latency is a frequent cause of disease in immunocompromised patients. To date, there is no explanation for the diversity in the clinical manifestations. Primary infection can occur perinatally or later in life, and inevitably results in latent infection. Seropositivity for antibodies against CMV is indicative of latent infection, but is insufficient as a predictor for the risk of recurrence. As a model for this important medical problem, we compared the risks of murine CMV recurrence from latency established after neonatal primary infection and after infection at adult age. The risk of CMV recurrence was high only after neonatal infection. The copy number of latent viral genome in tissues was identified as the key parameter that determines the overall and organ-specific risks of recurrence. Latent CMV burden and risk of recurrence were related to the extent of virus multiplication during primary infection. The presence of latent CMV in multiple organs provides the molecular basis for stochastic events of recurrence in single organs or in any combination thereof. These findings are discussed as a concept of multifocal CMV latency and recurrence. It provides a rationale for the diversity in the clinical outcome of CMV disease.

Antibodies are not essential for the resolution of primary cytomegalovirus infection but limit dissemination of recurrent virus.

Virus shedding from the epithelial cells of the serous acini of salivary glands is a major source for the horizontal transmission of cytomegalovirus. These cells are, different to other tissues, exempt from CD8 T lymphocyte control. CD4 T lymphocytes are essential to terminate the productive infection. Here, we prove that T-B cooperation and the production of antibodies are not required for this process. For the infection with murine cytomegalovirus, mutant mice were used which do not produce antibodies because of a disrupted membrane exon of the immunoglobulin mu chain gene. Also, in these mice the virus clearance from salivary glands is a function of CD4 T lymphocytes. However, these mice clear the virus and establish viral latency with a kinetics that is distinguishable from normal mice. Reactivation from virus latency is the only stage at which the absence of antibodies alters the phenotype of infection. In immunoglobulin-deficient mice, virus recurrence results in higher virus titers. The adoptive serum transfer proved that antibody is the limited factor that prevents virus dissemination in the immunodeficient host.

The immunoevasive function encoded by the mouse cytomegalovirus gene m152 protects the virus against T cell control in vivo.

Cytomegaloviruses encode numerous functions that inhibit antigen presentation in the major histocompatibility complex (MHC) class I pathway in vitro. One example is the mouse cytomegalovirus (MCMV) glycoprotein gp40, encoded by the m152 gene, which selectively retains murine but not human MHC class I complexes in the endoplasmic reticulum-Golgi intermediate compartment/cis-Golgi compartment (Ziegler, H., R. Thäle, P. Lucin, W. Muranyi, T. Flohr, H. Hengel, H. Farrell, W. Rawlinson, and U.H. Koszinowski. 1997. Immunity. 6:57-66). To investigate the in vivo significance of this gene function during MCMV infection of the natural host, we constructed recombinants of MCMV in which the m152 gene was deleted, as were the corresponding virus revertants. We report on the following findings: Deletion of the m152 gene has no effect on virus replication in cell culture, whereas after infection of mice, the m152-deficient virus replicates to significantly lower virus titers. This attenuating effect is lifted by reinsertion of the gene into the mutant. Mutants and revertants grow to the same titer in animals deprived of the function targeted by the viral gene function, namely in mice deficient in beta2-microglobulin, mice deficient in the CD8 molecule, and mice depleted of T cells. Upon adoptive transfer of naive lymphocytes into infected mice, the absence of the m152 gene function sensitizes the virus to primary lymphocyte control. These results prove that MHC-reactive functions protect CMVs against attack by CD8(+) T lymphocytes in vivo.

Hierarchical and redundant lymphocyte subset control precludes cytomegalovirus replication during latent infection.

Reactivation from latent cytomegalovirus (CMV) infection is often associated with conditions of immunosuppression and can result in fatal disease. Whether the maintenance of systemic CMV latency is mainly governed by factors of the infected cell or by immune control functions is unknown. Likewise, the putative immune control mechanisms which could prevent the induction and spread of recurrent CMV infection are not clearly identified. We took advantage of latently infected B cell-deficient mice and a sensitive method for virus detection to study CMV reactivation after ablation of lymphocyte subsets. A crucial role of both T lymphocytes and natural killer (NK) cells was demonstrated. Within 5 d after depletion of lymphocytes, productive infection occurred in 50% of mice, and 14 d later 100% of mice exhibited recurrent infection. A hierarchy of immune control functions of CD8(+), NK, and CD4(+) cells was established. Reactivation was rare if only one of the lymphocyte subsets was depleted, but was evident after removal of a further subset, indicating a functional redundancy of control mechanisms. The salivary glands were identified as the site of most rapid virus shedding, followed by the detection of recurrent virus in the lungs, and eventually in the spleen. Our findings document a previously unknown propensity of latent CMV genomes to enter productive infection immediately and with a high frequency after immune cell depletion. The data indicate that only the sustained cellular immune control prevents CMV replication and restricts the viral genome to a systemic state of latency.

The role of CD4 and CD8 T cells in viral infections.

Presentation of viral antigens to T cells does not require uptake by 'professional' antigen-presenting cells. Viruses have specialized to enter the cells in which they replicate. Virus entry, uncoating and new viral protein synthesis can load both the cytosolic and the endosomal pathway of antigen processing, resulting in viral peptide presentation to CD8 and CD4 T cells by MHC class I and II molecules, respectively. Although a role of CD8 T cells in the control of viral infection has been well documented, current research interest centers on the contribution of the different CD4 T-cell subsets.

Molecular analysis of herpesviral gene products recognized by protective cytolytic T lymphocytes.

The infection of the mouse with murine cytomegalovirus (MCMV) served as a model system to understand the biology of human CMV infection. The contribution of cytolytic T lymphocytes (CTL) to the recovery from infection was studied. Protection against lethal MCMV disease could be conferred on immunodepleted hosts by adoptive transfer of lymphocytes. The antiviral effect was mediated by specifically sensitized T lymphocytes of the CD8+ subset. These cells limited viral spread, prevented tissue destruction by viral cytopathic effects, and protected from lethal disease. Transferred cells have protective therapeutic function even when the virus has already colonized host tissues. CD8+ cells do not require the contribution of CD4+ cells for in vivo function. Selective expression of immediate-early (IE) phase genes in target cells allowed the detection of the immunodominant IE antigen recognized by CTL. The major IE gene ieI encodes a non-structural viral phosphoprotein, pp89, which resides in the nucleus of infected cells where it acts as transcriptional regulator. Expression of gene ieI is under temporal control, and membrane presentation of the protein domain detected by CTL is down-regulated by MCMV early-phase products. A recombinant vaccinia virus expressing gene ieI induced immunity that protected mice against a subsequent challenge with a lethal dose of MCMV. The protective effect was entirely mediated by CD8+ T lymphocytes. Thus, an experimental vaccine expressing a single nonstructural herpesvirus protein can induce a protective cellular immune response.

Immune responsiveness in offspring of adoptively immunized mothers.

The rate of appearance of cells forming 19S hemolytic antibody (PFC) in the spleens of F1 newborn mice after adoptive anti-paternal immunization of fetuses, newborns and/or their mothers during pregnancy has been studied. An increase in the number of PFC was found at the age of 6 to 11 days in offspring of adoptively immunized mothers. These newborns, even when foster nursed by untreated mothers, still had a significantly higher number of PFC in comparison to the controls. In contrast, adoptive immunization of the newborns themselves resulted in a decrease of PFC during the second week after birth. Several possible explanations for the obtained results are discussed including the putative transplacental passage of immunocompetent cells.

Ganglioside expression in tissues of mice lacking the tumor necrosis factor receptor 1.

This study presents a comparative analysis of gangliosides from lymphoid (spleen and thymus) and other tissues (brain, liver, lung, muscle) of C57BL/6 mice homozygous (-/-) and heterozygous (+/-) for the tumor necrosis factor receptor 1 (TNFRp55). Quantitative and qualitative differences in the expression of the lipid-bound N-acetylneuraminic (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc) and of various ganglioside biosynthesis pathways were detected between the tissues of the TNFRp55 -/- and the control TNFRp55 +/- mice. Sialic acid profiles showed a strong decrease in the absolute amount of sialic acids (Neu5Ac + Neu5Gc) in the lungs and thymus of homozygous (1.41 and 0.3 ng/mg wet weight, respectively) compared with control heterozygous animals (7.18 and 2.05 ng/mg wet weight, respectively). Considerable differences of Neu5Ac/Neu5Gc ratios in the lungs, muscle, spleen, and thymus were also detected. The gangliosides GM3(Neu5Ac) and GM3(Neu5Gc) were the dominant gangliosides in the lungs of the control animals, whereas the knockout mice almost completely lacked these structures in this organ. Reduced expression of GM1b-type gangliosides (GM1b and GalNAc-GM1b) was also found in the lungs, spleen, and thymus of the TNFRp55 knockout mice. On the other hand, neolacto-series gangliosides were more abundant in the lungs, brain, and muscle of the knockout mice, whereas their expression in the liver, spleen, and thymus was similar in both groups of animals. This study provides in vivo evidence that TNF signaling via the TNFRp55 is involved in the acquisition of a distinct ganglioside assembly in different mouse organs. TNFRp55 signaling seems to be especially important for the activation of the GM1b-type ganglioside biosynthetic pathway that is a unique characteristic of the mouse lymphoid tissues.

Increase in the number of plaque-forming cells in the spleen of newborns from adoptively immunized mothers.

This report is an analysis of the rate of appearance of cells forming 19S haemolytic antibody in the spleen of newborns after adoptive immunization of their mothers during pregnancy. The findings are discussed as the result of several possible mechanisms including the putative transplacental exchange of immunocompetent cells.

Surgical removal of mouse salivary glands.

Surgical removal of the salivary glands is a simple procedure aimed at providing glandular tissues for studies in histopathology, immunohistology, DNA and RNA analysis, cytokine production, and virus detection and isolation. This unit describes the surgical removal of the salivary glands in mice, but a similar protocol is applicable to rats and other rodents.

Monoclonal antibodies reactive with swine lymphocytes. I. Antibodies to membrane structures that define the cytolytic T lymphocyte subset in the swine.

A panel of monoclonal antibodies (mAb) with specificity for swine leukocytes was prepared by somatic cell hybridization with the use of spleen cells from mice immunized with swine thymocytes. The reactivity of two mAb (295/33 and 122/28), which both immunoprecipitated from the surface of swine leukocytes an antigen termed S-L2 with an apparent m.w. of 33 to 35 kilodaltons under reducing and 65 to 70 kilodaltons under nonreducing conditions, was investigated in detail. These mAb were reactive in indirect immunofluorescence with 50 to 60% of thymocytes, 35% of peripheral blood lymphocytes, and 55% of E rosette-positive cells; they were nonreactive with bone marrow cells, Ig+ B cells, nonrosetting lymphocytes, granulocytes, and monocytes. In functional studies, the elimination of S-L2+ cells partially reduced the proliferative response to concanavalin A and pokeweed mitogen but not to Staphylococcus aureus and lipopolysaccharide. The S-L2- subset proliferated well to alloantigens. Both cytolytic T effector cells and precursor cells carried the antigen S-L2 and could be depleted from heterogeneous cell populations by both antibodies in the presence of complement. These data suggest that the mAb 295/33 and 122/28 recognize a specific polypeptide present on the surface of swine cytolytic T cells. These antibodies will be useful in studies on the swine immune system.

Systematic excision of vector sequences from the BAC-cloned herpesvirus genome during virus reconstitution.

Recently the mouse cytomegalovirus (MCMV) genome was cloned as an infectious bacterial artificial chromosome (BAC) (M. Messerle, I. Crnkovic, W. Hammerschmidt, H. Ziegler, and U. H. Koszinowski, Proc. Natl. Acad. Sci. USA 94:14759-14763, 1997). The virus obtained from this construct is attenuated in vivo due to deletion of viral sequences and insertion of the BAC vector. We reconstituted the full-length MCMV genome and flanked the BAC vector with identical viral sequences. This new construct represents a versatile basis for construction of MCMV mutants since virus generated from the construct loses the bacterial sequences and acquires wild-type properties.

Distribution of T-lymphocyte subsets in porcine lymphoid tissues.

The distribution of the functional subsets of porcine T cells, the cytolytic/suppressor (Tc/s) and the helper/inducer (Th/i) cells was studied in cryostat sections of thymus, lymph nodes, tonsils, Peyer's patches, spleen and liver using the indirect immunoperoxidase technique. Three murine monoclonal antibodies (mAb) were used. The mAb 8/1 reacts with an antigen present on all T cells and on cells of the myeloid lineage; the antigen has not yet been characterized biochemically. The mAb 295/33 (anti-T8) binds to the porcine T8 antigen and defines the Tc/s subset, while mAb PT-4 (anti-T4) detects the porcine T4 antigen and defines the Th/i subset. Practically all thymocytes were stained by mAb 8/1. The majority of cortical thymocytes apparently co-expressed T8 and T4, whereas distinct fractions of medullary cells were labelled by either anti-T8 or anti-T4. In peripheral lymphoid organs all three mAb reacted with cells in the thymus-dependent areas and with cells scattered in the lymphoid follicles. In lymph nodes, tonsils and Peyer's patches, anti-T8 and anti-T4 each labelled approximately half of the cells stained by mAb 8/1. In the periarteriolar lymphoid sheath of the spleen, anti-T4 labelled more cells than did anti-T8. The reactivity of mAb 8/1 with the Kupffer cells of the liver demonstrated the expression of the 8/1 antigen on cells of the monocyte lineage. The T8 and T4 antigens could not be detected in acetone-fixed and paraffin-embedded sections, while the antigen recognized by mAb 8/1 remained preserved. Altogether, despite an inverted microanatomical structure of porcine lymph nodes, the frequency and distribution of T8+ and T4+ cells in thymus-dependent areas proved to be similar to those found in other species.

Adoptive immunotherapy of murine cytomegalovirus adrenalitis in the immunocompromised host: CD4-helper-independent antiviral function of CD8-positive memory T lymphocytes derived from latently infected donors.

The ability of memory T lymphocytes derived from latently infected mice to control murine cytomegalovirus disease in the immunocompromised host was studied by adoptive transfer experiments. At a stage of pathogenesis when virus had already colonized target tissues, a therapeutic antiviral function could be ascribed to the CD8+ subset. This in vivo function was not restricted to sites in which intravenously infused lymphocytes usually are trapped or home in, such as the lungs or the spleen, respectively, but was also evident in the adrenal glands, a site to which antiviral effector cells have to specifically migrate. Specific infiltration of adrenal gland cortical tissue by donor-derived CD8+ memory T lymphocytes was demonstrated. CD4+ memory T lymphocytes had no antiviral effect by themselves and also were not required for the function of the CD8+ effector cells in this short-term immunotherapy model. These findings should help settle the debate about which subset of T lymphocytes comprises the effector cells that can directly control cytomegalovirus infection in the murine model system.

A nonstructural viral protein expressed by a recombinant vaccinia virus protects against lethal cytomegalovirus infection.

The nonstructural immediate-early protein pp89 of murine cytomegalovirus (MCMV) is the first viral protein synthesized after infection and has a regulatory function in viral gene expression. Despite its localization in the nucleus of infected cells, pp89 is also the dominant antigen recognized by MCMV-specific cytolytic T lymphocytes. The recombinant vaccinia virus MCMV-ieI-VAC, which expresses pp89, was used to study the capacity of this protein to induce protective immunity in BALB/c mice. Vaccination with MCMV-ieI-VAC induced a long-lasting immunity that protected mice against challenge with a lethal dose of MCMV but did not prevent infection and morbidity. In vivo depletion of CD8+ T lymphocytes before challenge completely abrogated the protective immunity. CD8+ T lymphocytes derived from MCMV-ieI-VAC-primed donors and adoptively transferred into sublethally irradiated and MCMV-infected recipients were found to limit viral replication in host tissues, whereas CD4+ T lymphocytes and pp89-specific antiserum had no protective effect. The data demonstrate for the first time that a single nonstructural viral protein can confer protection against a lethal cytolytic infection and that this immunity is entirely mediated by the CD8+ subpopulation of T lymphocytes.

Restoration of cytomegalovirus antigen presentation by gamma interferon combats viral escape.

An immediate-early protein of murine cytomegalovirus (MCMV), pp89, elicits an immunodominant and protective major histocompatibility complex (MHC) class I Ld-restricted CD8+ T-lymphocyte response. Remarkably, presentation of the naturally processed peptide of pp89, the nonapeptide YPHFMPTNL, is abolished during permissive MCMV infection in vitro. This defect in pp89 presentation is due to the expression of MCMV early gene functions that specifically block the transport of peptide-charged MHC class I complexes to the cell surface (M. Del Val, H. Hengel, H. Häcker, U. Hartlaub, T. Ruppert, P. Lucin, and U. H. Koszinowski, J. Exp. Med. 176:729-738, 1992). Here, we demonstrate that MCMV-specific CD8+ T lymphocytes can reconstitute pp89 presentation in a parakrine fashion. The lymphocytes mediate the restoration of antigen presentation by MCMV-infected cells by releasing gamma interferon (IFN-gamma). IFN-gamma has no effect on synthesis and stability of the viral antigen pp89 nor does it interfere with the expression of viral early genes and their inhibitory effect on MHC class I molecular maturation. IFN-gamma results in a 25-fold increase in the synthesis of MHC class I molecules and a similar increase in the abundance of pp89-derived peptide. Many of the MHC molecules remain retained by the viral effect, but a surplus of MHC molecules escapes the effect and provides the effective surface presentation of the peptide. Adoptive cell transfer studies demonstrate the IFN-gamma dependence of CD8+ T-lymphocyte function in vivo. Altogether, these data reconcile the paradoxical findings of an impaired pp89 presentation in vitro in parallel with pp89-specific CD8+ T-cell protection in vivo. The results also imply a role of IFN-gamma in the T-lymphocyte-mediated control of cytomegalovirus infection. The known propensity of cytomegalovirus to cause serious disease in the immunocompromised host is discussed in the light of these findings.