T-cell-mediated control of poxvirus infection in mice.
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Animals↗
Biomedical subjects
A Müllbacher
Publications and source records attributed to A Müllbacher.
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Australia↗
Antiviral cytotoxic T cells cross-reactively recognize disparate peptide determinants from related viruses but ignore more similar self- and foreign determinants.
We have investigated the reactivities of cytotoxic T (Tc) cells against the two immunodominant, H-2K(k)-restricted determinants from the FLAVIVIRUS: Murray Valley encephalitis virus (MVE), MVE(1785) (REHSGNEI) and MVE(1971) (DEGEGRVI). The respective Tc cell populations cross-reactively lysed target cells pulsed with determinants from the MVE(1785)- and MVE(1971)-corresponding positions of six other flaviviruses, despite low sequence homology in some cases. Notably, anti-MVE(1785) Tc cells recognized a determinant (TDGEERVI) that shares with the determinant used for stimulation only the carboxyl-terminal amino acid residue, one of two H-2K(k) anchor residues. These reactivity patterns were also observed in peptide-dependent IFN-gamma production and the requirements for in vitro restimulation of memory Tc cells. However, the broad cross-reactivity appeared to be limited to flavivirus-derived determinants, as none of a range of determinants from endogenous mouse-derived sequences, similar to the MVE-determinants, were recognized. Neither were cells infected with a number of unrelated viruses recognized. These results raise the paradox that virus-immune Tc cell responses, which are mostly directed against only a few "immunodominant" viral determinants, are remarkably peptide cross-reactive.
Animals↗
Effector cytolotic function but not IFN-gamma production in cytotoxic T cells triggered by virus-infected target cells in vitro.
Cytolysis and interferon(IFN)-gamma production are two independent effector functions of activated cytotoxic T (Tc) cells. We have used the Tc-cell response against the flavivirus, Murray Valley encephalitis virus (MVE), to investigate the requirements for inducing these two functions with regard to antigen-concentration and CD8 coreceptor involvement. Cognate peptide-pulsed target cells triggered cytolysis by primary ex vivo MVE-immune as well as in vitro peptide-restimulated splenocytes at lower peptide concentrations than IFNgamma-production (100-fold lower in the case of primary ex vivo effectors). Little difference was observed in CD8 dependency. Importantly, neither of the effector populations were triggered to produce IFN-gamma by virus-infected target cells, although cytolysis occurred. This result raises the possibility that the levels of presentation of cognate antigen on virus-infected cells in vivo may be below the threshold required for the IFN-gamma production.
Animals↗
Cell death mediated by alloreactive cytotoxic T cells via the granule exocytosis or the Fas pathway is independent of p34cdc2 kinase: Fas dependent killing of cells arrested in the cell cycle.
Inappropriate activation of p34cdc2 kinase has been shown to occur during apoptosis induced by cytotoxic T-cell derived perforin and fragmentin. We analysed the effect of two inhibitors of p34cdc2 kinase on alloreactive Tc-cell-mediated lysis and DNA fragmentation of P815 and L1210 target cells. Olomoucine, a specific inhibitor of cyclin dependent kinases, did not affect DNA fragmentation in the target cells. Lysis of olomoucine-treated target cells as assessed by 51Cr release over a typical 8-h period was also unaffected. We also examined the effects of thapsigargin on target cell death. This toxin causes increased intracellular calcium rises that then result in irreversible inhibition of cyclin dependent kinases, including p34cdc2 kinase. The same extent of specific cell lysis was induced by cytotoxic T cells from perforin(-/-), granzyme B(-/-), granzyme A(-/-), perforin(-/-) X granzymeB(-/-) X granzymeA(-/-) KO mice or normal mice in untreated target cells or target cells treated with either olomoucine or thapsigargin. Similarly DNA fragmentation measured by release of tritiated DNA was also unaffected. Thus inhibition of p34cdc2 kinase affects neither the Fas nor the perforin/granzyme pathways of alloreactive cytotoxic T-cell killing as measured by DNA fragmentation or chromium release. P815 cells treated with olomoucine were arrested in the cell cycle after 12-16 h exposure to the toxin. After cell cycle arrest, target cells now showed enhanced 51Cr release induced by effector cytotoxic T cells (CTL) derived from perforin(-/-) mice compared to untreated cells. This lysis was accompanied by an increase in cell surface Fas expression. Olomoucine induced cell cycle arrest and expression of Fas was reversible and when cells re-entered the cell cycle, surface expression of Fas was lost.
Animals↗
Virulence of mousepox virus is independent of serpin-mediated control of cellular cytotoxicity.
We have investigated whether the differential virulence seen of two Ectromelia (Ect) strains, EctMoscow and ECtHampstead egg, is due to mutation or differential regulation of their serpins (SPI). Poxvirus encoded serine proteinase inhibitors (serpins) have been shown to interfere with cytolytic activity of leukocytes and can also determine virulence. We show that the deduced amino acid sequences of SPI-1, 2, and 3 are identical for the highly virulent EctMoscow and the low virulent EctHampstead strains and that the two viruses express similar potential to inhibit T-cell cytotoxicity, in particular, Fas-mediated target cell lysis, by allorective effectors. Virus titres in wild type B6 mice were effectively controlled very early after inoculation with EctHampstead as compared with EctMoscow, but lack of perforin renders B6 mice similarly susceptible to both virus strains. The data demonstrate that in Ect infection the perforin-mediated cytolytic pathway is not the primary target of serpins and suggest that the apparent attenuation of EctHampstead seen in B6 mice is due to control elements distinct from SPI-1, 2, and 3.
Amino Acid Sequence↗
Immunogenicity of two peptide determinants in the cytolytic T-cell response to flavivirus infection: inverse correlation between peptide affinity for MHC class I and T-cell precursor frequency.
We used the CD8+ cytotoxic T (Tc) cell immune response against the flavivirus, Murray Valley encephalitis virus (MVE), restricted by the H-2Kk major histocompatibility complex (MHC) class I molecule, to investigate immunodominance. Split-clone limiting dilution analysis revealed almost exclusive recognition of two peptides, MVE1785 and MVE1971, derived from the viral NS3 protein. The precursor frequency of MVE-reactive Tc cells was determined by limiting dilution analysis for cytotoxic function and intracellular staining for interferon-gamma; the latter gave a 100-fold higher estimate of MVE-reactive Tc cell precursors. MHC class I cell surface stabilization assays revealed that affinity for H-2Kk as well as halflives of the peptide-H-2Kk-complexes were markedly different for the two peptides. However, a kinetic study of antigen presentation showed that both peptides are presented for recognition by Tc cells with a comparable kinetics during the latent period of virus infection. Nevertheless, the lower affinity peptide MVE1785 elicited roughly twofold more Tc cell clones than the high-affinity peptide MVE1971. While the cytolytic activity against both determinants was similar after in vitro restimulation at the peak of the primary response, the smaller pool of memory anti-MVE1971 Tc cells correlated with an impaired memory response against that determinant, suggesting that the available T-cell repertoire is a major factor influencing the establishment of T-cell memory.
Animals↗
Modulation of transporter associated with antigen processing (TAP)-mediated peptide import into the endoplasmic reticulum by flavivirus infection.
In contrast to many other viruses that escape the cellular immune response by downregulating major histocompatibility complex (MHC) class I molecules, flavivirus infection can upregulate their cell surface expression. Previously we have presented evidence that during flavivirus infection, peptide supply to the endoplasmic reticulum is increased (A. Müllbacher and M. Lobigs, Immunity 3:207-214, 1995). Here we show that during the early phase of infection with different flaviviruses, the transport activity of the peptide transporter associated with antigen processing (TAP) is augmented by up to 50%. TAP expression is unaltered during infection, and viral but not host macromolecular synthesis is required for enhanced peptide transport. This study is the first demonstration of transient enhancement of TAP-dependent peptide import into the lumen of the endoplasmic reticulum as a consequence of a viral infection. We suggest that the increased supply of peptides for assembly with MHC class I molecules in flavivirus-infected cells accounts for the upregulation of MHC class I cell surface expression with the biological consequence of viral evasion of natural killer cell recognition.
ATP-Binding Cassette Transporters↗
Creation of killer poxvirus could have been predicted.
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Animals↗
Concerted action of the FasL/Fas and perforin/granzyme A and B pathways is mandatory for the development of early viral hepatitis but not for recovery from viral infection.
Cytotoxic T lymphocytes (CTL) play a major role in the recovery from primary viral infections and the accompanying tissue injuries. However, it is unclear to what extent the two main cytolytic pathways, perforin-granzyme A and B exocytosis and Fas ligand (FasL)-Fas interaction, contribute to these processes. Here we have employed mouse strains with either spontaneous mutations or targeted gene defects in one or more components of either of the two cytolytic pathways to analyze the molecular basis of viral clearance and induction of hepatitis during lymphocytic choriomeningitis virus infection. Our results reveal that viral clearance is solely dependent on perforin but that virus-induced liver damage only occurs when both the FasL/Fas and the perforin pathways, including granzymes A and B, are simultaneously activated. The finding that development of hepatitis but not viral clearance is dependent on the concomitant activation of FasL-Fas and perforin-granzymes may be helpful in designing novel strategies to prevent hepatic failures during viral infections.
Animals↗
Cytotoxic T cells specifically induce Fas on target cells, thereby facilitating exocytosis-independent induction of apoptosis.
Cytotoxic T (Tc) cells deficient in perforin lyse Fas-negative targets after lengthy incubation periods. This process is independent of granzymes, and killing occurs via the Fas pathway for the following reasons. Interaction of perforin-deficient Tc cells with Fas-negative targets leads to an up-regulation of Fas that is dependent on Ag recognition, de novo synthesis, and transport of proteins to the target cell surface. Treatment of effectors with brefeldin A, but not with the exocytosis inhibitor concanamycin, inhibited this process. Lysis of targets is inhibited by anti-Fas Abs, soluble mouse Fas-Fc, and the caspase-cascade inhibitor, crm-A. Targets from Fas-mutant lpr mice are refractory to lysis, and Tc cells from mice deficient in Fas- and perforin-mediated lysis do not lyse Fas-negative targets. The possible relevance of this exocytosis-independent cytolytic process in the regulation of T cell activity and control of pathogens is discussed.
Animals↗
Murine cytomegalovirus replication in salivary glands is controlled by both perforin and granzymes during acute infection.
The course of mouse cytomegalovirus (MCMV) infection was compared between wild-type and mutant C57BL / 6 (B6) mice deficient in either RAG-2, perforin, granzyme A, granzyme B or combinations thereof at two time points post infection (p. i.). At day 15 p. i., virus titers were similarly elevated in salivary glands of all mutant, but not wild-type B6 mice and undetectable in lung and spleen tissues of any of the mouse strains. Significant pathological alterations were only seen in salivary glands and spleen from RAG2(- / -), but not in those from other mice whereas few inflammatory foci were observed in lung tissues of all mice except B6. At day 30 p. i., elevated virus titers were observed only in salivary glands, lung and spleen from RAG2(- / -), but in none of the other mice, and were accompanied by extended pathological alterations in all three organs. The data extend previous reports on the critical role of NK / CD8(+) T cells in the early control of MCMV infection by showing that both perforin and granzymes A / B contribute to viral elimination in salivary glands; however, neither of the three molecules alone seem to be indispensable for the final control of infection.
Animals↗
Proteolytic processing of peptides in the lumen of the endoplasmic reticulum for antigen presentation by major histocompatibility class I.
We have tested the hypothesis that MHC class I molecules are actively involved as protease in the production of natural MHC class I ligands. First, the structure of a class I molecule was analyzed for homology with catalytic sites of known proteases. While several clusters of amino acids in the restriction element resembled protease active sites, structural discrepancies and the influence of nearby residues suggest that these sites are unlikely to have protease activity. Second, we have tested the presentation of viral cytotoxic T cell determinants with affinity for the same restriction element (H-2K(d) or K(k)), when targeted as tandem peptides into the endoplasmic reticulum. Peptide transporter-defective cells were used to exclude cleavage of the tandem peptides by cytosolic proteases. Cleavage by signal peptidase of the tandem peptides was ascertained. The C-terminal peptides in the tandem arrays were almost exclusively presented, suggesting that an aminopeptidase in the endoplasmic reticulum degraded the N-terminally positioned peptides. This result is inconsistent with an MHC class I-catalyzed cleavage following binding of longer peptides in the cleft of the restriction elements. Finally, we conclusively show that an aminopeptidase in the endoplasmic reticulum is also involved in antigen presentation in cells with a functional peptide transporter.
Amino Acid Sequence↗
Granzymes are the essential downstream effector molecules for the control of primary virus infections by cytolytic leukocytes.
Analysis of perforin-deficient mice has identified the cytolytic pathway and perforin as the preeminent effector molecule in T cell-mediated control of virus infections. In this paper, we show that mice lacking both granzyme A (gzmA) and granzyme B (gzmB), which are, beside perforin, key constituents of cytolytic vesicles, are as incapable as are perforin-deficient mice of controlling primary infections by the natural mouse pathogen ectromelia, a poxvirus. Death of gzmAxgzmB double knockout mice occurred in a dose-dependent manner, despite the expression of functionally active perforin and the absence of an intrinsic defect to generate splenic cytolytic T cells. These results establish that both gzmA and gzmB are indispensable effector molecules acting in concert with perforin in granule exocytosis-mediated host defense against natural viral pathogens.
Animals↗
Adenovirus-induced liver pathology is mediated through TNF receptors I and II but is independent of TNF or lymphotoxin.
Mice infected with an adenovirus mutant in which the E3 region is deleted, including TNF-resistance genes, develop fatal liver pathology within 3-4 days after infection. At least 10-fold more wild-type virus was needed to cause comparable pathology. These results indicate that the E3 region is critically involved in modulating the pathogenesis of adenovirus infection and that TNF may play a role in liver damage. To explore the latter possibility, the course of disease was examined in infected mice lacking TNFR-I and/or TNFRII, TNF only, or both TNF and lymphotoxin-alpha. Only mice lacking both TNFRI and TNFRII were protected from the lethal affects of the mutant adenovirus. Mice deficient in TNF or TNF and lymphotoxin-alpha displayed the fatal pathology. This outcome is consistent with the existence of another related ligand that binds TNFRI/II to mediate liver damage during infection with this mutant.
Adenoviridae Infections↗
Poxvirus-encoded serpins do not prevent cytolytic T cell-mediated recovery from primary infections.
Previous observations that the highly conserved poxvirus-encoded serpins inhibit cytotoxic activities of alloreactive CTL via granule and/or Fas-mediated pathways was taken to indicate their involvement in immune evasion by poxviruses. We now show that interference with 51Cr release from target cells by ectromelia and cowpoxvirus is limited to alloreactive but not MHC-restricted CTL. The data are in support of the paramount importance of CTL and its effector molecule perforin in the recovery from primary ectromelia virus infection and question the role of serpins in the evasion of poxviruses from killing by CTL. Further analysis of poxvirus interference with target cell lysis by alloreactive CTL revealed that suppression primarily affects the Fas-mediated, and to a lesser extent, the granule exocytosis pathway. Serpin-2 is the main contributor to suppression for both killing pathways. In addition, inhibition of lysis was shown to be both target cell type- and MHC allotype-dependent. We hypothesize that differences in TCR affinities and/or state of activation between alloreactive and MHC-restricted CTL as well as the quality (origin) of target cells are responsible for the observed phenomenon.
Animals↗
Spontaneous mutation at position 114 in H-2Kd affects cytotoxic T cell responses to influenza virus infection.
Vaccinia virus (VV)-encoded MHC class I Kd molecules which differ by a single amino acid change from glutamine (Kdw, wild type) to histidine (Kdm, mutant) at position 114 located in the floor of the peptide binding groove were compared in terms of peptide binding and cytotoxic T (Tc) cell recognition. Most anti-viral Tc cells were not affected or only marginally affected. However, the Kdm molecule did not detectably present the immunodominant peptide (NPP147-155) of influenza virus nucleoprotein (NP), encoded by the full-length NP gene either in influenza A virus or recombinant VV. This defect could be overcome by using exogenous synthetic NPP147-155 or translation from a minigene encoding NPP147-155 in VV. Kdw presented NPP147-155 encoded by the full-length NP gene, but Kdw-NPP147-155 complexes were at least 100-fold less abundant than after translation from a minigene.
Animals↗
Antigen presentation in syrian hamster cells: substrate selectivity of TAP controlled by polymorphic residues in TAP1 and differential requirements for loading of H2 class I molecules.
Expression of mouse major histocompatibility complex (MHC) class I molecules in different cell lines derived from Syrian hamsters has revealed antigen presentation deficiencies of some H2 allelic products in two cell lines (BHK and NIL-2) which were overcome by transient expression of the rat transporter associated with antigen processing (TAP; Lobigs et al. 1995). Here we show that in both cell lines the endogenous MHC class I cell surface expression was completely down-regulated. Lymphokine treatment induced endogenous and recombinant mouse MHC class I cell surface expression to levels similar to that in other Syrian hamster cell lines competent for antigen presentation through transduced H2 molecules. Accordingly, constitutive downregulation of expression of accessory molecules of the MHC class I pathway can reveal differences between H2 class I alleles in antigen presentation not encountered when the expression levels are augmented. In addition to the differential expression of MHC class I pathway genes, two cell lines representing competent (FF) and defective (BHK) antigen presentation phenotypes for mouse class I MHC restriction elements demonstrated substantial sequence polymorphism in Tap1 but not Tap2. Cytokine-treated FF or BHK cells and human TAP-deficient T2 cells transfected with FF or BHK TAP1 in combination with FF TAP2 differed in their preference for C-terminal peptide residues, as shown by an in vitro peptide transport assay. Thus, polymorphic residues in TAP1 can influence the substrate selectivity of the Syrian hamster peptide transporter.
ATP Binding Cassette Transporter, Subfamily B, Mem↗