Impaired T cell immunity in B cell-deficient mice following viral central nervous system infection.

CD8(+) T cells are required to control acute viral replication in the CNS following infection with neurotropic coronavirus. By contrast, studies in B cell-deficient (muMT) mice revealed Abs as key effectors in suppressing virus recrudescence. The apparent loss of initial T cell-mediated immune control in the absence of B cells was investigated by comparing T cell populations in CNS mononuclear cells from infected muMT and wild-type mice. Following viral recrudescence in muMT mice, total CD8(+) T cell numbers were similar to those of wild-type mice that had cleared infectious virus; however, virus-specific T cells were reduced at least 3-fold by class I tetramer and IFN-gamma ELISPOT analysis. Although overall T cell recruitment into the CNS of muMT mice was not impaired, discrepancies in frequencies of virus-specific CD8(+) T cells were most severe during acute infection. Impaired ex vivo cytolytic activity of muMT CNS mononuclear cells, concomitant with reduced frequencies, implicated IFN-gamma as the primary anti viral factor early in infection. Reduced virus-specific CD8(+) T cell responses in the CNS coincided with poor peripheral expansion and diminished CD4(+) T cell help. Thus, in addition to the lack of Ab, limited CD8(+) and CD4(+) T cell responses in muMT mice contribute to the ultimate loss of control of CNS infection. Using a model of virus infection restricted to the CNS, the results provide novel evidence for a role of B cells in regulating T cell expansion and differentiation into effector cells.

MHV infection of the CNS: mechanisms of immune-mediated control.

Mice infected with neurotropic strains of mouse hepatitis virus (MHV) clear infectious virus; nevertheless, viral persistence in the central nervous system (CNS) is associated with ongoing primary demyelination. Acute infection induces a potent regional CD8+ T-cell response. The high prevalence of virus specific T cells correlates with ex vivo cytolytic activity, interferon-gamma (IFN-gamma) secretion and efficient reduction in virus. Viral clearance from most cell types is controlled by a perforin dependent mechanism. However, IFN-gamma is essential for controlling virus replication in oligodendrocytes. Furthermore, CD4+ T cells enhance CD8+ T-cell survival and effectiveness. Clearance of infectious virus is associated with a gradual decline of CNS T cells; nevertheless, activated T cells are retained within the CNS. The loss of cytolytic activity, but retention of IFN-gamma secretion during viral clearance suggests stringent regulation of CD8+ T-cell effector function, possibly as a means to minimize CNS damage. However, similar CD8+ T-cell responses to demyelinating and non demyelinating JHMV variants support the notion that CD8+ T cells do not contribute to the demyelinating process. Although T-cell retention is tightly linked to the presence of persisting virus, contributions to regulating the latent state are unknown. Studies in B-cell-deficient mice suggest that antibodies are required to prevent virus recrudescence. Although acute JHMV infection is thus primarily controlled by CD8+ T cells, both CD4+ T cells and B cells make significant contributions in maintaining the balance between viral replication and immune control, thus allowing host and pathogen survival.

Role of viral persistence in retaining CD8(+) T cells within the central nervous system.

The continued presence of virus-specific CD8(+) T cells within the central nervous system (CNS) following resolution of acute viral encephalomyelitis implicates organ-specific retention. The role of viral persistence in locally maintaining T cells was investigated by infecting mice with either a demyelinating, paralytic (V-1) or nonpathogenic (V-2) variant of a neurotropic mouse hepatitis virus, which differ in the ability to persist within the CNS. Class I tetramer technology revealed more infiltrating virus-specific CD8(+) T cells during acute V-1 compared to V-2 infection. However, both total and virus-specific CD8(+) T cells accumulated at similar peak levels in spinal cords by day 10 postinfection (p.i.). Decreasing viral RNA levels in both brains and spinal cords following initial virus clearance coincided with an overall progressive loss of both total and virus-specific CD8(+) T cells. By 9 weeks p.i., T cells had largely disappeared from brains of both infected groups, consistent with the decline of viral RNA. T cells also completely disappeared from V-2-infected spinal cords coincident with the absence of viral RNA. By contrast, a significant number of CD8(+) T cells which contained detectable viral RNA were recovered from spinal cords of V-1-infected mice. The data indicate that residual virus from a primary CNS infection is a vital component in mediating local retention of both CD8(+) and CD4(+) T cells and that once minimal thresholds of stimuli are lost, T cells within the CNS cannot survive in an autonomous fashion.

Maintenance of CD8(+) T-cell memory following infection with recombinant sindbis and vaccinia viruses.

CD8(+) T-cell memory is critical for protection against pathogens poorly controlled by humoral immunity. To characterize two distinct vaccine vectors, the acute and memory CD8(+) T-cell responses to an HIV-1 epitope (p18) expressed by recombinant vaccinia (vp18) and Sindbis (SINp18) viruses were compared. Whereas 9 to 13% of CD8(+) splenocytes were p18 specific during the acute response to vp18, 4% were induced by SINp18 as revealed by class I tetramer staining. Increased T-cell activation by vp18 was confirmed by higher numbers of both p18-specific IFN-gamma-secreting splenocytes and activated CD8(+) and CD4(+) T cells. Although higher frequencies of p18-specific CD8(+) T cells during primary responses correlated with higher frequencies during memory, the overall decline was only two- to threefold during the transition to memory, demonstrating equally efficient maintenance of memory in SINp18- as in vp18-immune mice. Despite modest in vivo activation, SINp18-induced CD4(+) T cells secreted substantial amounts of IFN-gamma and IL-2, potentially contributing to sustained CD8(+) memory. Collectively the data indicate that Sindbis virus recombinants provide effective vaccines for inducing protective memory CD8(+) T cells in the absence of the extensive inflammation and replication associated with vaccinia virus.

Contributions of Fas-Fas ligand interactions to the pathogenesis of mouse hepatitis virus in the central nervous system.

The pathogenesis of the neurotropic strain of mouse hepatitis virus in Fas-deficient mice suggested that Fas-mediated cytotoxicity may be required during viral clearance after the loss of perforin-mediated cytotoxicity. The absence of both Fas- and perforin-mediated cytolysis resulted in an uncontrolled infection, suggesting a redundancy of cytolytic pathways to control virus replication.

Contributions of CD8+ T cells and viral spread to demyelinating disease.

Acute and chronic demyelination are hallmarks of CNS infection by the neurotropic JHM strain of mouse hepatitis virus. Although infectious virus is cleared by CD8+ T cells, both viral RNA and activated CD8+ T cells remain in the CNS during persistence potentially contributing to pathology. To dissociate immune from virus-mediated determinants initiating and maintaining demyelinating disease, mice were infected with two attenuated viral variants differing in a hypervariable region of the spike protein. Despite similar viral replication and tropism, one infection was marked by extensive demyelination and paralysis, whereas the other resulted in no clinical symptoms and minimal neuropathology. Mononuclear cells from either infected brain exhibited virus specific ex vivo cytolytic activity, which was rapidly lost during viral clearance. As revealed by class I tetramer technology the paralytic variant was superior in inducing specific CD8+ T cells during the acute disease. However, after infectious virus was cleared, twice as many virus-specific IFN-gamma-secreting CD8+ T cells were recovered from the brains of asymptomatic mice compared with mice undergoing demyelination, suggesting that IFN-gamma ameliorates rather than perpetuates JHM strain of mouse hepatitis virus-induced demyelination. The present data thus indicate that in immunocompetent mice, effector CD8+ T cells control infection without mediating either clinical disease or demyelination. In contrast, demyelination correlated with early and sustained infection of the spinal cord. Rapid viral spread, attributed to determinants within the spike protein and possibly perpetuated by suboptimal CD8+ T cell effector function, thus ultimately leads to the process of immune-mediated demyelination.

Microglia exhibit clonal variability in eliciting cytotoxic T lymphocyte responses independent of class I expression.

Microglia are important immunoregulatory cells within the central nervous system (CNS). Viral infection of primary microglia and splenic macrophage clones revealed that both exhibited a heterogeneous, but relatively low, sensitivity to cytolysis mediated by CD8(+) cytotoxic T lymphocytes (CTL). The majority of clones were poor in processing and presenting epitopes, despite triggering lysis when coated with peptide. These characteristics were retained by stable microglia lines. Reduced lysis did not correlate with class I expression and IFN-gamma treatment only partially enhanced recognition. In contrast, targeting the epitope into the endoplasmic reticulum restored cytolysis to levels achieved with exogenous peptide. An inherent resistance to cytolysis was revealed by efficient engagement of T cells in competition assays and the inability of saturating peptide to enhance cytolysis. These data suggest that microglia heterogeneity in antigen processing, in addition to low sensitivity to CTL lysis, contributes to limited CD8(+) T cell responses and viral CNS persistence.

Efficient processing of the immunodominant, HLA-A*0201-restricted human immunodeficiency virus type 1 cytotoxic T-lymphocyte epitope despite multiple variations in the epitope flanking sequences.

Immune escape from cytotoxic T-lymphocyte (CTL) responses has been shown to occur not only by changes within the targeted epitope but also by changes in the flanking sequences which interfere with the processing of the immunogenic peptide. However, the frequency of such an escape mechanism has not been determined. To investigate whether naturally occurring variations in the flanking sequences of an immunodominant human immunodeficiency virus type 1 (HIV-1) Gag CTL epitope prevent antigen processing, cells infected with HIV-1 or vaccinia virus constructs encoding different patient-derived Gag sequences were tested for recognition by HLA-A*0201-restricted, p17-specific CTL. We found that the immunodominant p17 epitope (SL9) and its variants were efficiently processed from minigene expressing vectors and from six HIV-1 Gag variants expressed by recombinant vaccinia virus constructs. Furthermore, SL9-specific CTL clones derived from multiple donors efficiently inhibited virus replication when added to HLA-A*0201-bearing cells infected with primary or laboratory-adapted strains of virus, despite the variability in the SL9 flanking sequences. These data suggest that escape from this immunodominant CTL response is not frequently accomplished by changes in the epitope flanking sequences.

Inverted immunodominance and impaired cytolytic function of CD8+ T cells during viral persistence in the central nervous system.

Mice infected with the neurotropic JHM strain of mouse hepatitis virus (JHMV) clear infectious virus; nevertheless, virus persists in the CNS as noninfectious RNA, resulting in ongoing primary demyelination. Phenotypic and functional analysis of CNS infiltrating cells during acute infection revealed a potent regional CD8+ T cell response comprising up to 50% virus-specific T cells. The high prevalence of virus-specific T cells correlated with ex vivo cytolytic activity and efficient reduction in viral titers. Progressive viral clearance coincided with the loss of cytolytic activity, but retention of IFN-gamma secretion and increased expression of the early activation marker CD69, indicating differential regulation of effector function. Although the total number of infiltrating T cells declined following clearance of infectious virus, CD8+ T cells, both specific for the dominant viral epitopes and of unknown specificity, were retained within the CNS, suggesting an ongoing T cell response during persistent CNS infection involving a virus-independent component. Reversed immunodominance within the virus-specific CD8+ T cell population further indicated epitope-specific regulation, supporting ongoing T cell activation. Even in the absence of infectious virus, the CNS thus provides an environment that maintains both unspecific and Ag-specific CD8+ T cells with restricted effector function. Chronic T cell stimulation may thus play a role in preventing viral recrudescence, while increasing the risk of pathological conditions, such as demyelination.

Antibody prevents virus reactivation within the central nervous system.

The neurotropic JHM strain of mouse hepatitis virus (JHMV) produces an acute CNS infection characterized by encephalomyelitis and demyelination. The immune response cannot completely eliminate virus, resulting in persistence associated with chronic ongoing CNS demyelination. The contribution of humoral immunity to viral clearance and persistent infection was investigated in mice homozygous for disruption of the Ig mu gene (IgM-/-). Acute disease developed with equal kinetics and severity in IgM-/- and syngeneic C57BL/6 (wt) mice. However, clinical disease progressed in IgM-/- mice, while wt mice recovered. Viral clearance during acute infection was similar in both groups, supporting a primary role of cell-mediated immunity in viral clearance. In contrast to wt mice, in which infectious virus was reduced to below detection following acute infection, increasing infectious virus was recovered from the CNS of the IgM-/- mice following initial clearance. No evidence was obtained for selection of variant viruses nor was there an apparent loss of cell-mediated immunity in the absence of Ab. Passive transfer of anti-JHMV Ab following initial clearance prevented reactivation of infectious virus within the CNS of IgM-/- mice. These data demonstrate the clearance of infectious virus during acute disease by cell-mediated immunity. However, immunologic control is not maintained in the absence of anti-viral Ab, resulting in recrudescence of infectious virus. These data suggest that humoral immunity plays no role in controlling virus during acute infection, but plays an important role in establishing and maintaining CNS viral persistence.

Selection of CD8+ T cells with highly focused specificity during viral persistence in the central nervous system.

The relationships between T cell populations during primary viral infection and persistence are poorly understood. Mice infected with the neurotropic JHMV strain of mouse hepatitis virus mount potent regional CTL responses that effectively reduce infectious virus; nevertheless, viral RNA persists in the central nervous system (CNS). To evaluate whether persistence influences Ag-specific CD8+ T cells, functional TCR diversity was studied in spleen and CNS-derived CTL populations based on differential recognition of variant peptides for the dominant nucleocapsid epitope. Increased specificity of peripheral CTL from persistently infected mice for the index epitope compared with immunized mice suggested T cell selection during persistence. This was confirmed with CD8+ T cell clones derived from the CNS of either acutely (CTLac) or persistently (CTLper) infected mice. Whereas CTLac clones recognized a broad diversity of amino acid substitutions, CTLper clones exhibited exquisite specificity for the wild-type sequence. Highly focused specificity was CD8 independent but correlated with longer complementarity-determining regions 3 characteristic of CTLper clonotypes despite limited TCR alpha/beta-chain heterogeneity. Direct ex vivo analysis of CNS-derived mononuclear cells by IFN-gamma enzyme-linked immunospot assay confirmed the selection of T cells with narrow Ag specificity during persistence at the population level. These data suggest that broadly reactive CTL during primary infection are capable of controlling potentially emerging mutations. By contrast, the predominance of CD8+ T cells with dramatically focused specificity during persistence at the site of infection and in the periphery supports selective pressure driven by persisting Ag.

Enhanced cytotoxic T cell activity in IL-4-deficient mice.

CD8+ effectors are critical components of type 1 responses against viral infections as well as for antiviral vaccines. IL-4 plays a clear role as an inhibitor of CD4+ Th1 cells; however, its role in CD8+ T cell regulation appears to be more complex. Thus, IL-4 may augment CD8+ T cell growth, but also limit effector function. Moreover, abundant IL-4 is inhibitory for viral clearance, but the lack of IL-4 appears not to affect CTL-mediated immunity. This report investigates these disparate roles of IL-4 in CD8+ T lymphocyte regulation by comparing T cell responses specific for a single HIV-IIIIB gp120-derived epitope in BALB/c mice deficient in IL-4 to those in wild-type controls. CTL activation was monitored during the acute and memory phases following immunization with recombinant vaccinia virus. Similar frequencies of gp120-specific CTL precursors in splenocytes from both groups indicated that IL-4 plays no significant role in either CTL priming or the establishment of memory. However, cytolytic activity in cultures derived from IL-4-deficient mice developed earlier and was strikingly enhanced following in vitro restimulation, an effect exhibited by both primary and memory T cells. Secretion of IL-2 and IFN-gamma by CD8+ T cells from IL-4-deficient mice was also elevated, reflecting their enhanced activation. Thus, IL-4 appears to limit the activation, expansion, and differentiation of CD8+ T cells with high cytolytic potential.

IFN-gamma is required for viral clearance from central nervous system oligodendroglia.

Infection of the central nervous system (CNS) by the JHM strain of mouse hepatitis virus (JHMV) is a rodent model of the human demyelinating disease multiple sclerosis. The inability of effective host immune responses to eliminate virus from the CNS results in a chronic infection associated with ongoing recurrent demyelination. JHMV infects a variety of CNS cell types during the acute phase of infection including ependymal cells, astrocytes, microglia, oligodendroglia, and rarely in neurons. Replication within the majority of CNS cell types is controlled by perforin-dependent virus-specific CTL. However, inhibition of viral replication in oligodendroglia occurs via a perforin-independent mechanism(s). The potential role for IFN-gamma as mediator controlling JHMV replication in oligodendroglia was examined in mice deficient in IFN-gamma secretion (IFN-gamma0/0 mice). IFN-gamma0/0 mice exhibited increased clinical symptoms and mortality associated with persistent virus, demonstrating an inability to control replication. Neither antiviral Ab nor CTL responses were diminished in the absence of IFN-gamma, although increased IgG1 was detected in IFN-gamma0/0 mice. Increased virus Ag in the absence of IFN-gamma localized almost exclusively to oligodendroglia and was associated with increased CD8+ T cells localized within white matter. These data suggest that although perforin-dependent CTL control virus replication within astrocytes and microglia, which constitute the majority of infected CNS cells, IFN-gamma is critical for control of viral replication in oligodendroglia. Therefore, different mechanisms are used by the host defenses to control virus replication within the CNS, dependent upon the phenotype of the targets of virus replication.

Apoptosis of JHMV-specific CTL in the CNS in the absence of CD4+ T cells.

The role of CD4+ T cells in altering the activity of cytotoxic T lymphocytes (CTL) during infection of the central nervous system (CNS) by the neuroptropic JHMV strain of mouse hepatitis virus was examined. Adoptive transfer of in vitro activated CTL into CD4-depleted and control recipients showed that CTL were not effective in reducing JHMV replication within the CNS. The distribution of CD4+ and CD8+ T cells within the CNS during JHMV infection showed that the CD4+ T cells remained in perivascular and subarachnoid spaces and few entered the parenchyma. By contrast approximately half of the CD8+ T cells entered the parenchyma. In CD4-depleted mice the trafficking of CD8+ T cells was not inhibited; however, the majority of the cells were found to be apoptotic. These data suggested that CD4+ T cells were not required for CTL induction but were required for the maintenance of CTL viability. The limited role of CD4+ T cells in CTL induction was confirmed by comparison of CTL activity from CD4-depleted and control mice.

Viral evolution and CTL epitope stability during JHMV infection in the central nervous system.

The JHM strain of mouse hepatitis virus (JHMV) establishes a persistent infection in the murine central nervous system (CNS) associated with chronic ongoing demyelination in the absence of detectable virus. To distinguish between immune and replication associated mechanisms of persistence, brains from acutely and persistently infected mice were analyzed for viral RNA mutations in the encapsidation sequence (ECS) and regions encoding either the transmembrane domains of the matrix (M) protein or a protective CTL epitope in the nucleocapsid (N) protein. Detection of the ECS to 120 days post infection (p.i.) indicated low levels of replication. The ECS remained stable whereas the fragment encoding the CTL epitope revealed extensive diversity with mutation frequencies in the order of 2.0 per 1000 nts. The M gene also remained stable despite random mutations during the acute phase. Mutations in the N gene were random and not selected for during persistence, with the exception of a single prominent Pro363 to Ser substitution in a region not associated with any known regulatory function or immune response. Mutations within the CTL epitope affecting CTL recognition were found early in responder BALB/c mice (H-2d), but also in non-responder C57BL/6 (H-2b) mice, suggesting that CTL escape variants play no significant role in establishing persistence.

Recombinant Sindbis viruses expressing a cytotoxic T-lymphocyte epitope of a malaria parasite or of influenza virus elicit protection against the corresponding pathogen in mice.

Subcutaneous administration in mice of recombinant Sindbis viruses expressing a class I major histocompatibility complex-restricted 9-mer epitope of the Plasmodium yoelii circumsporozoite protein or the nucleoprotein of influenza virus induces a large epitope-specific CD8(+) T-cell response. This immunization also elicits a high degree of protection against infection with malaria or influenza A virus.