Biomedical subjects

P G Stevenson

Publications and source records attributed to P G Stevenson.

At least 19 recordsLinked to original sources

PubMed2002

K3-mediated evasion of CD8(+) T cells aids amplification of a latent gamma-herpesvirus.

The murine gamma-herpesvirus-68 (MHV-68) K3 protein, like that of the Kaposi's sarcoma associated herpesvirus, down-regulates major histocompatibility complex (MHC) class I expression. However, how this contributes to viral replication in vivo is unclear. After intranasal MHV-68 infection, K3 was transcribed both during acute lytic infection in the lung and during latency establishment in lymphoid tissue. K3-deficient viruses were not cleared more rapidly from the lung, but the number of latently infected spleen cells was reduced and the frequency of virus-specific CD8(+) cytotoxic T lymphocytes (CTLs) was increased. CTL depletion reversed the viral latency deficit. Thus, a major function of K3 appears to be CTL evasion during viral latency expansion.

3T3 Cells↗

PubMed2002

Uncoupling of virus-induced inflammation and anti-viral immunity in the brain parenchyma.

Non-neuroadapted influenza virus confined to the brain parenchyma does not induce antigen-specific immunity. Nevertheless, infection in this site upregulated major histocompatibility complex (MHC) class I and MHC class II expression and recruited lymphocytes to a perivascular compartment. T cells recovered from the brain had an activated/memory phenotype but did not respond to viral antigens. In contrast, T cells recovered from the brain after infection in a lateral cerebral ventricle, which is immunogenic, showed virus-specific responses. As with infectious virus, influenza virus-infected dendritic cells elicited virus-specific immunity when inoculated into the cerebrospinal fluid but not when inoculated into the brain parenchyma. Thus, inflammation and dendritic cell function were both uncoupled from immune priming in the microenvironment of the brain parenchyma and neither was sufficient to overcome immunological privilege.

Animals↗

PubMed2001

A secreted chemokine binding protein encoded by murine gammaherpesvirus-68 is necessary for the establishment of a normal latent load.

Herpesviruses encode a variety of proteins with the potential to disrupt chemokine signaling, and hence immune organization. However, little is known of how these might function in vivo. The B cell-tropic murine gammaherpesvirus-68 (MHV-68) is related to the Kaposi's sarcoma-associated herpesvirus (KSHV), but whereas KSHV expresses small chemokine homologues, MHV-68 encodes a broad spectrum chemokine binding protein (M3). Here we have analyzed the effect on viral pathogenesis of a targeted disruption of the M3 gene. After intranasal infection, an M3 deficiency had surprisingly little effect on lytic cycle replication in the respiratory tract or the initial spread of virus to lymphoid tissues. However, the amplification of latently infected B cells in the spleen that normally drives MHV-68-induced infectious mononucleosis failed to occur. Thus, there was a marked reduction in latent virus recoverable by in vitro reactivation, latency-associated viral tRNA transcripts detectable by in situ hybridization, total viral DNA load, and virus-driven B cell activation. In vivo CD8(+) T cell depletion largely reversed this deficiency, suggesting that the chemokine neutralization afforded by M3 may function to block effective CD8(+) T cell recruitment into lymphoid tissue during the expansion of latently infected B cell numbers. In the absence of M3, MHV-68 was unable to establish a normal latent load.

Animals↗

PubMed2001

Dissecting the host response to a gamma-herpesvirus.

The murine gamma-herpesvirus 68 (MHV-68) provides a unique experimental model for dissecting immunity to large DNA viruses that persist in B lymphocytes. The analysis is greatly facilitated by the availability of genetically disrupted (-/-) mice that lack key host-response elements, and by the fact that MHV-68 is a lytic virus that can readily be manipulated for mutational analysis. The mutant virus strategy is being used, for example, to characterize the part played in vivo by an MHV-68-encoded chemokine-binding protein that may ultimately find an application in human therapeutics. Experiments with various -/- mice and monoclonal antibody depletion protocols have shown very clearly that type I interferons (IFNs) are essential for the early control of MHV-68 replication, while CD4+ T cells producing IFN-gamma function to limit the consequences of viral persistence. Virus-specific CD8+ effectors acting in the absence of the CD4+ subset seem initially to control the lytic phase in the lung following respiratory challenge, but are then unable to prevent the reactivation of replicative infection in epithelia and the eventual death of CD4+ T-cell-deficient mice. This could reflect the fact that the interaction between the CD8+ T cells and the virus-infected targets is partially compromised by the MHV-68 K3 protein, which inhibits antigen presentation by MHC class I glycoproteins. Immunization strategies focusing on the CD8+ T-cell response to epitopes expressed during the lytic phase of MHV-68 infection can limit virus replication, but are unable to prevent the establishment of latency. Other experiments with mutant viruses also suggest that there is a disconnection between lytic MHV-68 infection and latency. The massive nonspecific immunoglobulin response and the dramatic expansion of Vbeta4+ CD8+ T cells, which is apparently MHC independent, could represent some sort of 'smoke screen' used by MHV-68 to subvert immunity. Although MHV-68 is neither Epstein-Barr virus nor human herpesvirus-8, the results generated from this system suggest possibilities that may usefully be addressed with these human pathogens. Perhaps the main lesson learned to date is that all the components of immunity are likely to be important for the control of these complex viruses.

Animals↗

PubMed2001

MHC class I ubiquitination by a viral PHD/LAP finger protein.

The murine gamma-herpesvirus-68 K3 (MK3) is a PHD/LAP finger protein that downregulates major histocompatibility complex (MHC) class I expression. In transfected cell lines, MK3 was expressed in the endoplasmic reticulum (ER) membrane, where it bound the cytoplasmic tail of newly synthesized H-2D(b) glycoproteins and targeted them for degradation. Proteasome inhibitors blocked the degradation and led to an accumulation of ubiquitinated H-2D(b). Because this retained its native conformation, ubiquitination preceded any denaturation or dislocation to the cytosol. The PHD/LAP finger of MK3 was not required for H-2D(b) binding but was essential for its ubiquitination and degradation. Thus, gamma-herpesviruses have adapted the cellular PHD/LAP motif to immune evasion, apparently for the catalysis of MHC class I ubiquitination.

Amino Acid Motifs↗

PubMed2000

Contemporary analysis of MHC-related immunodominance hierarchies in the CD8+ T cell response to influenza A viruses.

Early studies of influenza virus-specific CD8+ T cell-mediated immunity indicated that the level of CTL activity associated with H2Db is greatly diminished in mice that also express H2Kk. Such MHC-related immunodominance hierarchies are of some interest, as they could lead to variable outcomes for peptide-based vaccination protocols in human populations. The influence of H2Kk on the H2Db-restricted response profile has thus been looked at again using a contemporary, quantitative, IFN-gamma-based flow cytometric assay. The depressive effect of H2Kk was very apparent for the influenza DbPA224 epitope and was also reproduced when CTL activity was measured for H2Db-expressing targets pulsed with the immunodominant NP366 peptide. The secondary CD8+IFN-gamma+ DbNP366-specific response was much greater in parental H2b than in H2kxbF1 mice, but the sizes of the CD8+ sets specific for KkNP50 and DbNP366 were essentially equivalent in the F1 animals. Thus, although the immunodominance profile associated with DbNP366 is lost when H2Kk is also present, the response is still substantial. A further, MHC-related effect was also identified for the KkNS1152 epitope, which was consistently associated with a greater CD8+IFN-gamma+ response in H2KkDb recombinant than in (H2KkDk x H2KbDb)F1 mice. The diminished DbPA224 response in H2kxbF1 mice was characterized by loss of a prominent Vbeta7 TCR responder phenotype, supporting the idea that TCR deletion during ontogeny shapes the available repertoire. The overall conclusion is that these MHC-related immunodominance hierarchies are more subtle than the early CTL assays suggested and, although inherently unpredictable, are unlikely to cause a problem for peptide-based vaccine strategies.

Animals↗

PubMed2000

Inhibition of MHC class I-restricted antigen presentation by gamma 2-herpesviruses.

The gamma-herpesviruses, in contrast to the alpha- and beta-herpesviruses, are not known to inhibit antigen presentation to CD8(+) cytotoxic T lymphocytes (CTLs) during lytic cycle replication. However, murine gamma-herpesvirus 68 causes a chronic lytic infection in CD4(+) T cell-deficient mice despite the persistence of a substantial CTL response, suggesting that CTL evasion occurs. Here we show that, distinct from host protein synthesis shutoff, gamma-herpesvirus 68 down-regulates surface MHC class I expression on lytically infected fibroblasts and inhibits their recognition by antigen-specific CTLs. The viral K3 gene, encoding a zinc-finger-containing protein, dramatically reduced the half-life of nascent class I molecules and the level of surface MHC class I expression and was by itself sufficient to block antigen presentation. The homologous K3 and K5 genes of the related Kaposi's sarcoma-associated virus also inhibited antigen presentation and decreased cell surface expression of HLA class I antigens. Thus it appears that an immune evasion strategy shared by at least two gamma-herpesviruses allows continued lytic infection in the face of strong CTL immunity.

3T3 Cells↗

PubMed2000

Postexposure vaccination massively increases the prevalence of gamma-herpesvirus-specific CD8+ T cells but confers minimal survival advantage on CD4-deficient mice.

Mice that lack CD4(+) T cells remain clinically normal for more than 60 days after respiratory challenge with the murine gamma-herpesvirus 68 (gammaHV-68), then develop symptoms of a progressive wasting disease. The gammaHV-68-specific CD8(+) T cells that persist in these I-A(b-/-) mice are unable to prevent continued, but relatively low level, virus replication. Postexposure challenge with recombinant vaccinia viruses expressing gammaHV-68 lytic cycle epitopes massively increased the magnitude of the gammaHV-68-specific CD8(+) population detectable by staining with tetrameric complexes of MHC class I glycoprotein + peptide, or by interferon-gamma production subsequent to in vitro restimulation with peptide. The boosting effect was comparable for gammaHV-68-infected I-A(b-/-) and I-A(b+/+) mice within 7 days of challenge, and took more than 110 days to return to prevaccination levels in the I-A(b+/+) controls. Although the life-span of the I-A(b-/-) mice was significantly increased, there was no effect on long-term survival. A further boost with a recombinant influenza A virus failed to improve the situation. Onset of weight loss was associated with a decline in gammaHV-68-specific CD8(+) T cell numbers, though it is not clear whether this was a cause or an effect of the underlying pathology. Even very high levels of virus-specific CD8(+) T cells thus provide only transient protection against the uniformly lethal consequences of gammaHV-68 infection under conditions of CD4(+) T cell deficiency.

Animals↗

PubMed1999

A gamma-herpesvirus sneaks through a CD8(+) T cell response primed to a lytic-phase epitope.

To determine whether established CD8(+) T cell memory to an epitope prominent during the replicative phase of a gamma-herpesvirus infection protects against subsequent challenge, mice were primed with a recombinant vaccinia virus expressing the p56 peptide and then boosted by intranasal exposure to an influenza A virus incorporating p56 in the neuraminidase protein. Clonally expanded populations of functional, p56-specific CD8(+) T cells were present at high frequency in both the lung and the lymphoid tissue 1 month later, immediately before respiratory challenge with gammaHV-68. This prime-and-boost regime led to a massive reduction of productive gammaHV-68 infection in the respiratory tract and, initially, to much lower levels of latency in both the regional lymph nodes and the spleen. The CD8(+) T cell response to another epitope (p79) was diminished, there was less evidence of B cell activation, and the onset of the CD4(+) T cell-dependent splenomegaly was delayed. Within 3-4 weeks of the gammaHV-68 challenge, however, the extent of latent infection in the lymph nodes and spleen was equivalent, and both groups developed the prominent infectious mononucleosis-like syndrome that is characteristic of this infection. The reverse protocol (influenza then vaccinia) seemed to be slightly less effective. Even though immune CD8(+) T cells may be present at the time and site of virus challenge, establishing a high level of CD8(+) T cell memory to lytic-phase epitopes alone does not protect against the longer-term consequences of this gammaHV infection.

Animals↗

PubMed1999

Changing patterns of dominance in the CD8+ T cell response during acute and persistent murine gamma-herpesvirus infection.

The murine gamma-herpesvirus MHV-68 causes an acute, transient pneumonitis, followed by an infectious mononucleosis (IM)-like illness with splenomegaly, widespread latent infection of B lymphocytes and an expansion of Vbeta4+ CD8+ T cells. CD8+ T cells specific for an H-2Db-restricted epitope were prominent during the acute respiratory infection, but their prevalence declined rapidly during the mononucleosis. In contrast, CD8+ T cells specific for an H-2Kb-restricted epitope, apparently expressed by virus-infected B lymphocytes, were most numerous during the mononucleosis illness and were maintained at relatively high frequencies thereafter. The prevalence of all peptide-specific CD8+ T cells decreased during the expansion of the Vbeta4+ CD8+ population, which did not recognize any peptide epitopes identified and was apparent also in an MHC class I-deficient environment. The CD8+ T cell population recognizing productively infected epithelial cells thus differed substantially from that responding during the IM illness.

Acute Disease↗

PubMed1999

Immunological control of a murine gammaherpesvirus independent of CD8+ T cells.

Adult thymectomized C57 BL/6J mice were depleted of T cell subsets by MAb treatment either prior to, or after, respiratory challenge with murine gammaherpesvirus-68. Protection against acute infection was maintained when either the CD4+ or the CD8+ T cell population was greatly diminished, whereas the concurrent removal of both T cell subsets proved invariably fatal. The same depletions had little effect on mice with established infection. The results indicate firstly that both CD4+ and CD8+ T cells play a significant part in dealing with the acute infection, and secondly that virus-specific antibody contributes to controlling persistent infection with this gammaherpesvirus.

Animals↗

PubMed1999

Non-antigen-specific B-cell activation following murine gammaherpesvirus infection is CD4 independent in vitro but CD4 dependent in vivo.

The murine gammaherpesvirus MHV-68 multiplies in the respiratory epithelium after intranasal inoculation, then spreads to infect B cells in lymphoid germinal centers. Exposing B cells to MHV-68 in vitro caused an increase in cell size, up-regulation of the CD69 activation marker, and immunoglobulin M (IgM) production. The infectious process in vivo was also associated with increased CD69 expression on B cells in the draining lymph nodes and spleen, together with a rise in total serum Ig. However, whereas the in vitro effect on B cells was entirely T-cell independent, evidence of in vivo B-cell activation was minimal in CD4(+) T-cell-deficient (I-Ab-/-) or CD4(+) T-cell-depleted mice. Furthermore, the Ig present at high levels in serum was predominantly of the IgG class. Surprisingly, the titer of influenza virus-specific serum IgG in previously immunized mice fell following MHV-68 infection, suggesting that there was relatively little activation of memory B cells. Thus, CD4(+) T cells seemed both to amplify a direct viral activation of B cells in lymphoid tissue and to promote new Ig class switching despite a lack of obvious cognate antigen.

Animals↗

PubMed1998

Virus-specific CD8(+) T cell numbers are maintained during gamma-herpesvirus reactivation in CD4-deficient mice.

The murine gamma-herpesvirus 68 replicates in epithelial sites after intranasal challenge, then persists in various cell types, including B lymphocytes. Mice that lack CD4(+) T cells (I-Ab-/-) control the acute infection, but suffer an ultimately lethal recrudescence of lytic viral replication in the respiratory tract. The consequences of CD4(+) T cell deficiency for the generation and maintenance of murine gamma-herpesvirus 68-specific CD8(+) set now have been analyzed by direct staining with viral peptides bound to major histocompatibility complex class I tetramers and by a spectrum of functional assays. Both acutely and during viral reactivation, the CD8(+) T cell responses in the I-Ab-/- group were no less substantial than in the I-Ab+/+ controls. Indeed, virus-specific CD8(+) T cell numbers were increased in the lymphoid tissue of clinically compromised I-Ab-/- mice, although relatively few of the potential cytotoxic T lymphocyte effectors were recruited back to the site of pathology in the lung. Thus the viral reactivation that occurs in the absence of CD4(+) T cells was not associated with any exhaustion of the virus-specific cytotoxic T lymphocyte response. It seems that CD8(+) T cells alone are insufficient to maintain long-term control of this persistent gamma-herpesvirus.

Animals↗

PubMed1998

Long-term persistence of activated cytotoxic T lymphocytes after viral infection of the central nervous system.

Mice intranasally inoculated with influenza A/X-31 are protected against a subsequent intracerebral challenge with the neurovirulent influenza A/WSN and this heterotypic protection is mediated by CD8(+) cytotoxic T lymphocytes. We have studied the kinetics of this secondary immune response and found that despite the elimination of replication-competent virus by day 10, we were able to recover activated influenza-specific cytotoxic T lymphocytes (CTLs) that killed freshly ex vivo from the brains of mice for at least 320 d after the intracerebral inoculation. The activated antiviral CTLs expressed high levels of the early activation marker CD69, suggesting continuing TCR signaling despite a lack of viral protein and major histocompatibility complex staining by immunohistochemistry in the brain parenchyma and barely detectable levels of viral nucleic acid by single and two-step reverse transcription PCR. Local persistence of activated lymphocytes may be important for efficient long-term responses to viruses prone to recrudesce in sites of relative immune privilege.

Animals↗

PubMed1998

Kinetic analysis of the specific host response to a murine gammaherpesvirus.

Respiratory infection of BALB/c mice with the murine gammaherpesvirus 68 (MHV-68) induces the clonal expansion of virus-specific cytotoxic T-lymphocyte (CTL) precursors (CTLp) in the regional, mediastinal lymph nodes (MLN). Some of these CTLps differentiate to become fully functional CTL effectors, which can be detected in both the lymphoid tissue and in the site of pathology in the lung. Though the lymph nodes and spleen harbor substantial populations of latently infected B cells for life, the level of virus-specific CTL activity decreases rapidly in all sites. The CD8+ CTLp numbers fall to background levels in the MLN within several months of the termination of the productive phase of MHV-68 infection in the respiratory epithelium but are maintained at relatively low frequency in the spleen. The continued presence of a gamma interferon-producing, MHV-68-specific CD4+ set can also be demonstrated in cultured spleen cells. The virus-specific immunoglobulin G (IgG) response is slow to develop, with serum neutralizing antibody and enzyme-linked immunosorbent assay titers continuing to rise for several months. The level of total serum IgG increases dramatically within 2 weeks of infection, probably as a consequence of polyclonal B-cell activation, and remains high. The immune response profile is clearly influenced by the persistence of this DNA virus.

Animals↗

PubMed1997

Virus dissemination through the brain parenchyma without immunologic control.

After inoculation into the cerebrospinal fluid, the neurovirulent influenza virus A/WSN caused a rapidly progressive encephalitis that was uniformly fatal within 8 days. After inoculation into the brain parenchyma, the same virus replicated for 7 to 20 days without causing clinical illness, but when infection reached the cerebrospinal fluid, encephalitis was lethal within a further 6 days. As the virus spread through the brain parenchyma, there was intense intracerebral inflammation, with up-regulation of MHC class I and MHC class II expression and recruitment of CD44(high) CD49d(high) T cells. However, this was not associated with antiviral Ab production, and the infiltrating cells, unlike primed A/WSN-specific T cells, did not eliminate the virus in vivo or show evidence of virus recognition in vitro. Thus, a neurovirulent virus was able to disseminate widely through the brain parenchyma and induce considerable intracerebral inflammation without eliciting protective immunity.

Animals↗

PubMed1997

Protection against influenza virus encephalitis by adoptive lymphocyte transfer.

CD8+ cytotoxic T lymphocytes (CTL) have an established role in anti-viral immunity, but whether CTL function efficiently in the brain remains unclear. In particular, virus-infected neurons, which express only low levels of MHC class I antigens and are resistant to the induction of apoptosis, could constitute a relatively intractable CTL target. We have used immune lymphocytes adoptively transferred into the CSF to protect naive mice against an intracerebral infection with influenza A/WSN, a virus that infects neurons in the brain parenchyma and causes a lethal encephalitis. After in vitro restimulation, heterotypically immune spleen cells protected against A/WSN encephalitis in an H-2-restricted, CD8-dependent, CD4-independent manner. Adoptively transferred CTL clones were also protective. Homotypically immune spleen cells additionally mediated CD8-independent, H-2-unrestricted protection, probably due to the generation of A/WSN-specific plasma cells from memory B cells during in vitro restimulation. Thus after in vitro restimulation, either CTL or B cells adoptively transferred into the CSF protected against an acutely lethal intracerebral virus infection.

Animals↗

PubMed1997

Recruitment, activation and proliferation of CD8+ memory T cells in an immunoprivileged site.

The capacity of a memory cytotoxic T lymphocyte (CTL) population to protect against viral infections is well established, but the processes underlying this protection are less well understood. We have used heterotypic intranasal immunization with influenza A/X31 (H3N2) to protect against a subsequent infection with the neurovirulent influenza A/WSN (H1N1) in either the cerebrospinal fluid or the immunoprivileged brain parenchyma. Viral clearance from both sites was associated with a local infiltration and proliferation of A/WSN-specific CD8+ T cells. Infection in the cerebrospinal fluid elicited a proliferative response in the draining lymph nodes, an anti-H1N1 serum antibody response and an increase in the extracerebral A/WSN-specific CTL precursor frequency. In contrast, infection in the brain parenchyma elicited no lymph node proliferative response or serum antibody response and caused a transient decrease in the extracerebral CTL precursor frequency. Thus the memory CTL population protected against an intracerebral viral infection independent of any immune response occurring in systemic lymphoid tissue.

Animals↗