Prolonged E55+ retrovirus expression in aged mice is associated with a decline in the anti-virus immune response.

E55+ murine leukemia retrovirus (E55+ MuLV) infection of young and aged C57BL/6 (B6) mice was used to investigate the relationship between increased incidences of infection and decreased immune responsiveness of elderly individuals. Young mice decreased E55+ MuLV burden to below detectable levels by 8 weeks postinfection (p.i.). In contrast, virus burden in aged mice did not reach undetectable levels until 20 weeks p.i. A significant T cell proliferative response to E55+ MuLV was detected from 2 to 12 weeks p.i. in young mice, but was never observed in aged mice. Both age groups demonstrated significant E55+ MuLV-specific T-cell-mediated cytotoxic responses at 3 and 4 weeks p.i. and virus neutralizing antibody titers at 2, 4, 8, and 12 weeks p.i. In both cases, responses were consistently higher in young mice (P < 0.04 and P < 0.02, respectively). These results demonstrate that the observed delay in E55+ MuLV clearance by aged mice is associated with an age-related decrease in the immune response to the virus.

Rational design of cytotoxic T-cell inhibitors.

This study describes the use of the CD8/major histocompatibility complex (MHC) class I crystal structure as a template for the de novo design of low-molecular-weight surface mimetics. The analogs were designed from a local surface region on the CD8 alpha-chain directly adjacent to the bound MHC class I, to block the protein associations in the T-cell activation cluster that occur upon stimulation of the cytotoxic T lymphocytes (CTLs). One small conformationally restrained peptide showed dose-dependent inhibition of a primary allogeneic CTL assay while having no effect on the CD4-dependent mixed lymphocyte reaction (MLR). The analog's activity could be modulated through subtle changes in its side chain composition. Administration of the analog prevented CD8-dependent clearance of a murine retrovirus in BALB/c mice. In C57BL/6 mice challenged with the same retrovirus, the analog selectively inhibited the antiviral CTL responses without affecting the ability of the CTLs to generate robust allogeneic responses.

Genetic regulation of long-term nonprogression in E-55+ murine leukemia virus infection in mice.

Certain inbred mouse strains display progression to lymphoma development after infection with E-55+ murine leukemia virus (E-55+ MuLV), while others demonstrate long-term nonprogression. This difference in disease progression occurs despite the fact that E-55+ MuLV causes persistent infection in both immunocompetent BALB/c-H-2(k) (BALB.K) progressor (P) and C57BL/10-H-2(k) (B10.BR) long-term nonprogressor (LTNP) mice. In contrast to immunocompetent mice, immunosuppressed mice from both P and LTNP strains develop lymphomas about 2 months after infection, indicating that the LTNP phenotype is determined by the immune response of the infected mouse. In this study, we used bone marrow chimeras to demonstrate that the LTNP phenotype is associated with the genotype of donor bone marrow and not the recipient microenvironment. In addition, we have mapped a genetic locus that may be responsible for the LTNP trait. Microsatellite-based linkage analysis demonstrated that a non-major histocompatibility complex gene on chromosome 15 regulates long-term survival and is located in the same region as the Rfv3 gene. Rfv3 is involved in recovery from Friend virus-induced leukemia and has been demonstrated to regulate neutralizing virus antibody titers. In our studies, however, both P and LTNP strains produce similar titers of neutralizing and cytotoxic anti-E-55+ MuLV. Therefore, while it is possible that Rfv3 influences the course of E-55+ MuLV infection, it is more likely that the LTNP phenotype in E-55+ MuLV-infected mice is regulated by a different, closely linked gene.

Strain-dependent migration of lymphocytes to the vaginal mucosa after peripheral immunization.

We have previously demonstrated a genetic predisposition among mice regarding their ability to be protected against vaginal candidiasis after peripheral immunization. Both BALB/c and (BALB/cx C57BL/6) F1 mice are protected against vaginal candidiasis after subcutaneous immunization with Candida albicans extract and C57BL/6 mice are not protected by this immunization. In the present study, the ability of F1-derived immune cells to transfer protection to naive parental strains was observed in BALB/c recipient mice, but not apparent in B6 recipient mice. This result is highly suggestive that the microenvironment of the B6 mouse is responsible for the susceptible phenotype. Genetic studies using (BALB/cx C57BL/6)F1x C57BL/6 backcross mice demonstrated that two genes appeared to regulate the protective effect of peripheral immunization to vaginal challenge. Microsatellite mapping indicated that candidate loci involved in controlling the immune response to vaginal candidiasis after peripheral immunization included the intercellular adhesion molecule-1 (ICAM-1), the Icam-1 related sequence 1, and the Fc epsilon RII (P<0.01). Thus, the ability of cells to bind to vaginal endothelial cells may play an important role in protection against vaginal candidiasis mediated by peripheral immunization.

Differences in the immune response during the acute phase of E-55+ murine leukemia virus infection in progressor BALB and long term nonprogressor C57BL mice.

E-55+ murine leukemia virus infection of both progressor (BALB) and long term nonprogressor (C57BL) mouse strains is characterized by an acute and a persistent phase of infection. During the acute phase, progressor strains require CD8+ T cells to decrease virus burden, whereas the long term nonprogressor strains do not. In the present studies the immune response in BALB and C57BL mice during the acute phase of E-55+ murine leukemia virus infection was examined. The results demonstrate that BALB mice produce both IL-4 and IFN-gamma, in contrast to C57BL mice, which produce only IFN-gamma. In BALB mice, IL-4 production results in the absolute requirement for CD8+ T cells to reduce the virus burden during the acute phase of infection. The anti-virus immune response in these mice is IFN-gamma dependent. On the other hand, C57BL mice do not produce IL-4 and, in the absence of both CD8+ T cells and IFN-gamma, still generate an effective anti-virus immune response. Genetic studies suggest that these distinct immune responses are regulated by more than one non-MHC-linked gene. Two candidate regions that may encode this gene(s), located on chromosomes 7 and 19, respectively, were identified by recombinant inbred strain linkage analysis.

Genetic basis for protection against experimental vaginal candidiasis by peripheral immunization.

In these studies, significant protection against experimental vaginal candidiasis after a subcutaneous immunization with Candida albicans extract was achieved in BALB/c mice but not in C57BL/6 (B6) mice. Protection from vaginal candidiasis was transferred to naive BALB/c mice by a population of spleen cells derived from immunized BALB/c mice. Removal of CD3 or CD4 but not CD8 T cells before transfer completely abrogated resistance to vaginal candidiasis. Recombinant inbred (RI) strains of mice derived from BALB/c and B6 strains were used for mapping loci that might be responsible for regulating vaginal protection after subcutaneous immunization. Linkage analysis using microsatellite-based genome mapping in these RI strains revealed four candidate loci on chromosomes 3, 7, 8, and 18 that exhibit statistically significant linkage to the strain distribution pattern. These results may contribute to the understanding of host genetic factors controlling the immune response to vaginal infections.

Role of T-cell subsets in acute and persistent E-55+ murine leukemia virus infection in susceptible progressor and resistant long-term nonprogressor mouse strains. Women and Infants Transmission Study.

Previous studies from this laboratory have demonstrated that E-55+MuLV-infected BALB/c-H-2k (BALB.K) mice progress to develop thymic lymphoma about 7 months after infection whereas infected C57BL/10-H-2k (B10.BR) mice are long-term nonprogressors that fail to develop disease even after 2 years of infection. Both resistant long-term nonprogressor (B10.BR) and progressor (BALB.K) mice generate an early immune response that results in a dramatic decrease in the number of virus-infected cells. Despite this early immune response, mice from both strains become persistently infected. However, resistant B10.BR mice also demonstrate a late T-cell-mediated response that may be causally related to long-term nonprogression whereas susceptible BALB.K mice fail to demonstrate this late T-cell response. In the present studies, the T-cell subsets involved in the effective early immune response in both B10.BR and BALB.K mice as well as the late T-cell response in B10.BR mice were determined by in vivo antibody-mediated depletion. Results from these studies demonstrate that during the early acute phase of infection, elimination of CD4+ T cells ablated the ability of both BALB.K and B10.BR mice to decrease the burden of virus-infected cells. However, elimination of CD8+ T cells ablated this result in BALB.K but not B10.BR mice. Thus, despite the fact that both immunocompetent B10.BR and BALB.K mice are able to decrease the number of virus-infected cells during the early acute phase of infection, there is a difference in the T-cell subsets that mediate this effect in these strains of mice. In addition, characterization of the late immune response that keeps virus at very low levels during the persistent stage of virus infection in resistant B10.BR mice demonstrated that simultaneous elimination of both CD4+ and CD8+ T cells allowed the emergence of virus-infected cells whereas the elimination of either subset alone showed no effect compared to untreated control mice that are immunologically intact. Since B10.BR and BALB.K are identical with respect to their H-2k-haplotypes, it appears that the differences between these strains with respect to the generation of effective early and late anti-virus immune responses are regulated by a non-H-2-linked gene(s).

Inhibition of T cell activation by an autoantibody induced by murine retrovirus infection.

This report describes a murine monoclonal IgM antibody, 6E3.C4, induced by retrovirus infection of BALB/ c-H-2b mice which inhibits mitogen stimulation of both mouse and human lymphocytes in vitro. The molecule bound by this antibody appears to be an activation antigen since binding is upregulated by mitogen stimulation. Analysis of the epitope bound by mAb 6E3.C4 revealed that it is associated with a 52-kDa protein with a pI of approximately 5.7 as determined by Western blot analysis. A protein expressing this or a cross-reactive epitope was isolated and determined to be alpha-tubulin by amino acid sequencing. Reactivity with purified alpha-tubulin confirms this identification. These findings suggest a potential role for a cell surface molecule that is either alpha-tubulin or a cross-reactive molecule in the activation processes of T cells.

Environmental lighting alters the infection process in an animal model of AIDS.

In this study, we examined the effects of altered environmental lighting on the infection process of a murine leukemia virus, E-55(+), which induces a thymic lymphoma/leukemia in 100% of BALB.K mice inoculated as adults. One to two weeks after inoculation, high levels of proviral DNA are usually found. This is followed by an asymptomatic period of many weeks during which proviral DNA becomes essentially undetectable. Leukemia develops approximately 28 weeks postinoculation. In this experiment, one group of mice was exposed a consistent 10L: 14D cycle while a second was maintained in constant light (LL). A third group was exposed to a rotating cycle characterized by phase shifting a 10L: 14D cycle every three 24-h days (rLD). All cycles began 2 weeks prior to inoculation and were maintained thereafter. Animals were sacrificed at 1, 5, 10, and 15 weeks, and hematopoietic tissue was examined for proviral DNA content. At 1 week, LL- and rLD-exposed animals showed considerably less proviral DNA in bone marrow and spleen compared with controls. At 15 weeks, thymuses from controls were showing signs of infection whereas tissue from LL and rLD mice remained at background levels. We conclude that environmental lighting does alter the infective pattern displayed by this retrovirus, although whether this effect is mediated by changes in the target stem cells or through immunoenhancement has not yet been determined.

Effect of non-H-2-linked genes on anti-virus immune responses and long-term survival in mice persistently infected with E-55+ murine leukemia virus.

We have previously demonstrated that BALB/c-H-2k (BALB.K) mice are susceptible to the development of thymic lymphoma induced by E-55+ murine leukemia virus (MuLV). In the present studies, C57BL/10-H-2k (B10.BR) mice were found to be resistant to E-55+ MuLV-induced lymphoma despite the fact that these mice become persistently infected. This resistance to lymphomagensis is mediated by the anti-virus immune response since immunosuppressed mice progress to develop disease. The protective immune response in B10.BR mice is bimodal with respect to time after virus infection. The early immune response results in a dramatic decrease in the number of virus-infected cells within 4-8 weeks after infection. This decrease in virus-infected cells occurs in immunocompetent mice from strains that are either resistant (B10.BR) or susceptible (BALB.K) to E-55+ MuLV-induced disease. Subsequently, susceptible mice develop an increase in infected cells, whereas no increase in infected cells occurs in resistant mice despite the fact that they are persistently infected. This later phase of resistance in B10.BR appears to be mediated by T cells. Since B10.BR and BALB.K both express the H-2k haplotype, resistance appears to be mediated by a non-H-2-linked gene(s). (BALB.K x B10.BR)F1 mice are resistant to disease development, indicating resistance is a dominant trait.

A replication-competent, endogenous retrovirus from an aged DBA/2 mouse contains the complete env from Emv-3 and a novel gag partially related to AKT-8.

We previously described an endogenous murine retrovirus, rv-DBA/2aged, isolated from an aged DBA/2 mouse. The previous report showed that a recombination which resulted in the replacement of Emv-3 gag sequences with gag sequences homologous to those found in the AKT-8 virus had taken place. This recombination allowed production of a competent virus from the defective Emv-3 locus. However, the extent of replacement of Emv-3 gag was not known. We report here the entire sequence for the gag gene of rv-DBA/2aged as well as the previously unsequenced 3' end of the Emv-3 gag gene. These data demonstrate that while sequences homologous to the entire gag gene fragment found in AKT-8 are represented in rv-DBA/2aged, the remainder of rv-DBA/2aged gag is not derived from Emv-3 but is a unique gag sequence. Furthermore, a complete comparison of env sequences shows that the env of rv-DBA/2aged is derived entirely from Emv-3. Additional data suggest that the recombination which led to production of the rv-DBA/2aged virus may be a common event in aging DBA/2 mice. Finally, comparison of the new sequences of Emv-3 with those of the Akv virus (also designated AKR-623 and Emv-11) and Emv-1 shows that this endogenous virus locus is very closely related to the other Emv loci at the nucleotide sequence level.

A murine leukemia virus expressed in aged DBA/2 mice is derived by recombination of the Emv-3 locus and another endogenous gag sequence.

Although most inbred strains of mice contain endogenous retroviral sequences, these sequences are usually not capable of producing infectious retroviruses. In some cases, the retroviral sequences are small fragments of viral genomes. In a few cases the sequences for complete retroviruses exist, but contain small defects which prevent the production of infectious virus. While the ability of reversions of these defects to produce retroviruses has been studied by site-directed mutagenesis and chemical-mediated mutagenesis, the presence of spontaneously occurring reversions has not been completely evaluated. We characterized an infectious ecotropic retrovirus spontaneously expressed in aged DBA/2 mice, which carries the complete but defective Emv-3. Although this endogenously produced retrovirus was related to the endogenous Emv-3 sequence, it had undergone recombination with another retroviral sequence to correct the defect which resided in the gag of Emv-3. Thus, recombination of endogenous ecotropic retroviral sequences may be a mechanism to produce infectious retroviruses in adult animals that contributes to pathologic disease.

Differences between young and aged mice in susceptibility to Friend virus.

Friend virus (FV) is a murine leukemia virus that infects progenitor red blood cells and causes an erythroleukemia in susceptible mouse strains, resulting in splenomegaly. Several genetic loci of the host have been identified that affect erythroleukemia development, differentiation status of target cells and virus replication. Since age may change expression of these loci, age may affect FV disease. To explore this possibility, FV expression in four genetically diverse strains of mice of different ages was examined. Extent of viral replication and of disease were evaluated by measuring spleen focus forming units (SFFU), spleen weight and reverse transcriptase (RT) activity in target organs. Young DBA/2 and (C57BL/6 x DBA/2)F1 mice exhibited a greater level of virus expression than their aged counterparts in all parameters investigated. Young CBA/Ca mice had slightly higher spleen weights and SFFU values than aged CBA/Ca mice, but a definitive age-related change was not observed in the RT activity of the target organs. C57BL/6 mice, which are genetically resistant to the development of FV-induced erythroleukemia, exhibited a limited degree of virus replication that was not effected by the age of the animal. Our results indicate that the age of the mouse, as well as the genetic background, can contribute to the level of susceptibility to FV.

Intracerebral hemorrhages and syncytium formation induced by endothelial cell infection with a murine leukemia virus.

The mechanisms of endothelial cell damage that lead to cerebral hemorrhage are not completely understood. In this study, a cloned murine retrovirus, TR1.3, that uniformly induced stroke in neonatal BALB/c mice is described. Restriction digest mapping suggests that TR1.3 is part of the Friend murine leukemia virus (FMuLV) family. However, unlike mice exposed to other FMuLVs, mice infected with TR1.3 virus developed tremors and seizures within 8 to 18 days postinoculation. This was uniformly followed by paralysis and death within 1 to 2 days. Postmortem examination of TR1.3-inoculated mice revealed edematous brain tissue with large areas of intracerebral hemorrhage. Histologic analysis revealed prominent small vessel pathology including syncytium formation of endothelial cells. Immunohistochemical analysis of frozen brain sections using double fluorescence staining demonstrated that TR1.3 virus specifically infected small vessel endothelial cells. Although infection of vessel endothelial cells was detected in several organs, only brain endothelial cells displayed viral infection associated with hemorrhage. The primary determinant of TR1.3-induced neuropathogenicity was found to reside within a 3.0-kb fragment containing the 3' end of the pol gene, the env gene, and the U3 region of the long terminal repeat. The restricted tropism and acute pathogenicity of this cloned murine retrovirus provide a model for studying virus-induced stroke and for elucidating the mechanisms involved in syncytium formation by retroviruses in vivo.

Protection against murine disseminated candidiasis mediated by a Candida albicans-specific T-cell line.

The role of T lymphocytes in disseminated candidiasis in a mouse model of irradiation-induced immunosuppression was investigated. A continuously cultured Candida albicans-specific T-cell line mediated protection of sublethally irradiated mice from disseminated candidiasis as measured by both the fungal load in the kidneys and mortality. These results are the first to demonstrate directly a role for antigen-specific T cells in the protective immune response against murine disseminated candidiasis.

Pentoxifylline- and caffeine-induced modulation of major histocompatibility complex class I expression on murine tumor cell lines.

The methylxanthines, pentoxifylline (PTX) and caffeine, modulated major histocompatibility complex class I expression on three constitutively class I-positive murine T cell lymphoma lines. On two cell lines, PTX or caffeine treatment enhanced H-2K and H-2D expression. Treatment with PTX and either interferon-gamma, interferon-alpha/beta, tumor necrosis factor, or lymphotoxin increased the levels of K and D expression above those observed following treatment with either PTX or cytokines alone. On the third cell line, PTX or caffeine treatment enhanced D expression and reduced K expression. Treatment with PTX and any of the cytokines resulted in a level of D expression greater than that seen following treatment with either PTX or cytokines alone. However, PTX inhibited the cytokine-induced enhancement of K expression. PTX and caffeine did not induce class I expression on three constitutively class I-negative murine T cell lymphoma lines. Dibutyryl cAMP modulated class I expression in the same manner as PTX and caffeine. The PTX- and caffeine-mediated enhancement of class I expression was at least partially blocked by an inhibitor of cAMP-dependent protein kinase A. These results demonstrate that PTX and caffeine are able to regulate class I expression and that this regulation involves a cAMP-dependent mechanism.

Loss of antigenic epitopes as the result of env gene recombination in retrovirus-induced leukemia in immunocompetent mice.

The murine leukemia virus, E-55+ virus, induces a thymic lymphoma/leukemia in 100% of BALB.K mice infected as adults after a latent period of 4 months or more (Pozsgay et al., Virology 173, 330-334, 1989). Two molecular clones of virus designated E-55+ and E-55- based on their ability to encode the E-55 epitope detected by the monoclonal antibody 55 (mAb 55) were isolated from a leukemic BALB.K mouse inoculated with a biologically cloned E-55+ virus (Chesebro et al., Virology 112, 131-144, 1981). Env gene sequence analysis of E-55+ and E-55- clones showed that the E-55- virus was generated from the E-55+ virus as the result of a recombination between E-55+ virus and the endogenous ecotropic virus, emv-1, carried in the genome of the BALB.K mouse strain. The recombinant E-55- virus is replication competent. This recombination event and the consequential expression of E-55- virus consistently occur in immunocompetent BALB.K mice inoculated with the E-55+ virus and appear to play a role in the loss of epitopes recognized by virus neutralizing antibodies. The loss of these epitopes apparently allows the virus to evade the host immune response.

Murine leukemia virus infection in immunocompetent adult mice.

The course of infection of a chronic murine leukemia virus (MuLV), E-55+, which induces thymic lymphoma/leukemia in 100% of mice infected as adults, was followed after inoculation of virus into adult mice which generate both cytotoxic T cell and neutralizing antibody responses (Blank 1976). DNA from the tissues of these infected mice was analyzed using a polymerase chain reaction assay to detect integrated provirus. Infection in adult immunocompetent BALB.K mice results in an acute phase characterized by a high number of cells containing provirus DNA followed by a period in which provirus DNA in most tissues reaches background levels until the development of thymic lymphoma/leukemia at 4-7 months. This pattern of infection is different from what has been observed in immunosuppressed mice in which levels of virus-infected cells remain high throughout the preleukemic and leukemic phases.