In vivo application of recombinant interleukin 2 in the immunotherapy of established cytomegalovirus infection

We have shown in a murine model system for cytomegalovirus (CMV) disease in the immunocompromised host that in vivo application of recombinant human IL-2 (rhIL-2) can enhance the antiviral effect of a limited number of CD8+T lymphocytes, not only in prophylaxis, but also in therapy, when virus has already colonized host tissues. The observed net effect of IL-2 was consistent with the assumption of daily effector population doublings. The prospects for IL-2-supported immunotherapy of established CMV infection depend upon the tissues involved in disease. It appears that the prospects for controlling established CMV adrenalitis are less promising than for a therapy of interstitial CMV pneumonia

The cytolytic T lymphocyte response to the murine cytomegalovirus

Limiting dilution (LD) analysis with two modifications, the expansion and the restimulation LD assay, led to the detection and quantification of two distinct in vivo maturation stages within the lineage of virus- specific self-restricted CTL after infection of mice with the murine cytomegalovirus (MCMV). A low frequency set, representing an average of 15% of the specifically activated CTL-P in a draining lymph node, generated virus-specific lytic activity in the absence of antigen, solely under expansion conditions provided by growth and differentiation interleukins. These cells were considered to be active and were denoted antigen-independent or interleukin-receptive CTL-P (IL- CTL-P). A high frequency set required additional antigen in vitro to generate functionally active clones, and therefore the cells were termed antigen-dependent. Both sets are present in vivo simultaneously at the peak of the acute immune response and represent antigen- activated cells because their existence strictly depends on a preceding priming event. IL-CTL-P disappear quickly after acute infection and are absent during the memory state. It is proposed that the isolation of IL- CTL-P could serve to detect viral antigen expression during persistent and/or recurrent herpes virus infections

Host immune response to cytomegalovirus

To confirm that immediate-early (IE) genes of murine cytomegalovirus (MCMV) give rise to antigens recognized by specific cytolytic T lymphocytes (CTL), a 10.8-kilobase fragment of MCMV DNA which is abundantly transcribed at IE times was transfected into L cells expressing the Ld class I major histocompatibility glycoprotein. The viral genome fragment contains sequences of the three IE transcription units of MCMV: ie1, ie2, and ie3. In the transfected cell lines, only the predominant 2.75-kilobase transcript of ie1 and its translation product pp89 could be detected. The transfectants were analyzed for membrane expression of an IE antigen by employing clone IE1, an IE-specific CTL clone, as the probe. Only cells that expressed both the MCMV IE gene(s) and the Ld gene were recognized by the CTL clone

Studies on the Morphogenesis of Murine Cytomegalovirus

Two modes of assembly of murine cytomegalovirus (MCMV) were observed in cultured mouse embryo fïbroblasts, generating two morphologically different types of viral particles: monocapsid virions and multicapsid virions. The assembly of nucleocapsids appeared to be the same for both types of morphogenesis. Three successive stages of intranuclear capsid formation could be distinguished: capsids with electron-lucent cores, coreless capsids, and capsids with dense cores. Some of the capsids were enveloped at the inner nuclear membrane to form monocapsid virions, which were first detectable in the perinuclear cisterna. Other capsids left the nucleus via nuclear pores and usually entered cytoplasmic capsid aggregates that received an envelope by budding into extended cytoplasmic vacuoles, thereby forming multicapsid virions. Since the formation of multicapsid virions is not restricted to cell culture conditions and also occurs in vivo in immunosuppressed mice, multicapsid virions may play a role in the pathogenesis of cytomegalovirus infection

Rescue of myeloid lineage-committed preprogenitor cells from cytomegalovirus-infected bone marrow stroma

The effect of murine cytomegalovirus on myelopoiesis was studied in long-term bone marrow culture to find an in vitro correlate for the lethal virus interference with bone marrow reconstitution (W. Mutter, M. J. Reddehase, F. W. Busch, H.-J. Bühring, and U. H. Koszinowski, J. Exp. Med. 167:1645-1658, 1988). The in vitro generation of granulocyte-monocyte progenitors (CFU-GM) discontinued after infection of the stromal cell layer, whereas the proliferation and differentiation of CFU-GM to granulocyte-monocyte colonies remained unaffected. A protocol was established to probe the functional integrity of earlier hematopoietic cells. Pre-CFU-GM (the progenitors of the CFU-GM) could be recovered from an infected bone marrow donor culture by transfer onto an inductive recipient stromal cell layer. Thus, at least in vitro, infection of bone marrow stroma appears to be the only cause of the defect in myelopoiesis

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

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

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

Presentation of CMV immediate-early antigen to cytolytic T lymphocytes is selectively prevented by viral genes expressed in the early phase

The regulation of antigen processing and presentation to MHC class I-restricted cytolytic T lymphocytes was studied in cells infected with murine cytomegalovirus. Recognition by cytolytic T lymphocytes of the phosphoprotein pp89, the immunodominant viral antigen expressed in the immediate-early phase of infection, was selectively prevented during the subsequent expression of viral early genes. The surface expression of MHC class I glycoproteins and their capacity to present externally added pp89-derived antigenic peptides were not affected. Because recognition of several other antigens occurred during the early phase, a general failure in processing and presentation was excluded. Since neither rate of synthesis, amount, stability, nor nuclear transport of pp89 was modified, the failure in recognition indicates a selective interference with pp89 antigen processing and presentation

II. Detection of an antigen on resting T cells down-regulated after activation

The expression of an antigen on porcine T lymphocytes detected by murine monoclonal antibody (mAb) 8/1 was investigated by functional studies and dual-parameter immunofluorescence. mAb 8/1 reacts with greater than 95% of thymocytes and in peripheral blood with all T lymphocytes and with cells of the monocyte/macrophage lineage, but not with B cells, erythrocytes, and platelets. Pretreatment of peripheral blood lymphocytes with mAb 8/1 plus complement abrogated the proliferative response in vitro to mitogen, soluble antigen, and MHC determinants. Dual-parameter immunofluorescence revealed that resting porcine T8+ as well as T4+ lymphocytes express the 8/1 antigen, whereas after in vitro activation, cell surface expression of the antigen was low or absent in both T cell subsets. Thus, the 8/1 antigen represents a marker that discriminates between resting and activated T lymphocytes. Distribution and functional criteria indicate that 8/1 represents a novel marker not described before for any other mammalian species