In vivo elimination of MHC-I-deficient lymphocytes by activated natural killer cells is independent of granzymes A and B

NK cells kill target cells mainly via exocytosis of granules containing perforin (perf) and granzymes (gzm). In vitro, gzm delivery into the target cell cytosol results in apoptosis, and induction of apoptosis is severely impaired in the absence of gzm A and B. However, their importance for in vivo cytotoxicity by cytotoxic T cells has been questioned. We used an in vivo NK cytotoxicity assay, in which splenocytes from wild-type and β(2)microglobulin-deficient (MHC-I(neg)) mice are co-injected into recipients whose NK cells were activated by virus infection or synthetic Toll-like receptor ligands. Elimination of adoptively transferred MHC-I(neg) splenocytes was unimpaired in the absence of gzmA and gzmB, but dependent on perforin. This target cell rejection was NK cell dependent, since NK cell depletion abrogated it. Furthermore, target cell elimination in vivo was equally rapid in both wild-type and gzmAxB-deficient recipients, with the majority of specific target cells lost from lymphoid tissue within less than one to two hours after transfer. Thus, similar to T cell cytotoxicity, the contribution of gzmA and B to in vivo target cell elimination remains unresolved.This work was supported by the Australian National Health and Medical Research Council and by a government block grant to the John Curtin School of Medical Research

MHC Class II–Alpha Chain Knockout Mice Support Increased Viral Replication That Is Independent of Their Lack of MHC Class II Cell Surface Expression and Associated Immune Function Deficiencies

MHCII molecules are heterodimeric cell surface proteins composed of an α and β chain. These molecules are almost exclusively expressed on thymic epithelium and antigen presenting cells (APCs) and play a central role in the development and function of CD4 T cells. Various MHC-II knockout mice have been generated including MHC-IIAα(-/-) (I-Aα(-/-)), MHC-IIAβ(-/-) (I-β(-/-)) and the double knockout (I-Aαxβ(-/-)). Here we report a very striking observation, namely that alphaviruses including the avirulent strain of Semliki Forest virus (aSFV), which causes asymptomatic infection in wild-type C57BL6/J (B6) mice, causes a very acute and lethal infection in I-Aα(-/-), but not in I-β(-/-) or I-Aαxβ(-/-), mice. This susceptibility to aSFV is associated with high virus titres in muscle, spleen, liver, and brain compared to B6 mice. In addition, I-Aα(-/-) mice show intact IFN-I responses in terms of IFN-I serum levels and IFN-I receptor expression and function. Radiation bone marrow chimeras of B6 mice reconstituted with I-Aα(-/-) bone marrow expressed B6 phenotype, whereas radiation chimeras of I-Aα(-/-) mice reconstituted with B6 bone marrow expressed the phenotype of high viral susceptibility. Virus replication experiments both in vivo and in vitro showed enhanced virus growth in tissues and cell cultures derived form I-Aα(-/-) compared to B6 mice. This enhanced virus replication is evident for other alpha-, flavi- and poxviruses and may be of great benefit to producers of viral vaccines. In conclusion, I-Aα(-/-) mice exhibit a striking susceptibility to virus infections independent of their defective MHC-II expression. Detailed genetic analysis will be carried out to characterise the underlining genetic defects responsible for the observed phenomenon.This work was supported by institutional research support to Prof Mullbacher laboratory at the John Curtin School of Medical Research. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Apoptotic pathways are selectively activated by granzyme A and/or granzyme B in CTL-mediated target cell lysis

Purified cytolytic T lymphocyte (CTL) proteases granzyme (gzm)A and gzmB with sublytic dose of perforin (perf) initiate distinct proapoptotic pathways. Their physiological relevance in CTL-mediated target cell apoptosis is elusive. Using ex vivo virus-immune CD8+ T cells from mice deficient in perf, gzmA and/or gzmB, and the Fas-resistant EL4.F15 tumor target cell, we show that (a) CTL from gzmA−/− or gzmB−/− mice similarly induced early proapoptotic features, such as phosphatidyl serine (PS) exposure on plasma membrane, ΔΨm loss, and reactive oxygen radical generation, though with distinct kinetics; (b) CTL from gzmA−/− but not from gzmB−/− mice activate caspase 3 and 9; (c) PS exposure induced by CTL from gzmA−/− or gzmB−/− mice is prevented, respectively, by caspase inhibitors or by reactive oxygen scavengers without interfering with target cell death; and (d) all gzm-induced apoptotic features analyzed depend critically on perf. Thus, perf is the principal regulator in CTL-mediated and gzm-facilitated intracellular processes. The ability of gzmA and gzmB to induce multiple independent cell death pathways may be the hosts response to circumvent evasion strategies of pathogens and tumors

In vitro- and ex vivo-derived cytolytic leukocytes from granzyme A x B double knockout mice are defective in granule-mediated apoptosis but not lysis of target cells

Granzyme (gzm) A and gzmB have been implicated in Fas-independent nucleolytic and cytolytic processes exerted by cytotoxic T (Tc) cells, but the underlying mechanism(s) remains unclear. In this study, we compare the potential of Tc and natural killer (NK) cells of mice deficient in both gzmA and B (gzmAxB-/-) with those from single knockout mice deficient in gzmA (-/-), gzmB (-/-), or perforin (-/-) to induce nuclear damage and lysis in target cells. With the exception of perforin-/-, all in vitro- and ex vivo-derived Tc and NK cell populations from the mutant strains induced 51Cr-release in target cells at levels and with kinetics similar to those of normal mice. This contrasts with their capacity to induce apoptotic nuclear damage in target cells. In gzmAxB-/- mice, Tc/NK-mediated target cell DNA fragmentation was not observed, even after extended incubation periods (10 h), but was normal in gzmA-deficient and only impaired in gzmB-deficient mice in short-term (2-4 h), but not long-term (4-10 h), nucleolytic assays. This suggests that gzmA and B are critical for Tc/NK granule- mediated nucleolysis, with gzmB being the main contributor, while target cell lysis is due solely to perforin and independent of both proteases

Intranasal Flu Vaccine Protective against Seasonal and H5N1 Avian Influenza Infections

Background Influenza A (flu) virus causes significant morbidity and mortality worldwide, and current vaccines require annual updating to protect against the rapidly arising antigenic variations due to antigenic shift and drift. In fact, current subunit or split flu vaccines rely exclusively on antibody responses for protection and do not induce cytotoxic T (Tc) cell responses, which are broadly cross-reactive between virus strains. We have previously reported that γ-ray inactivated flu virus can induce cross-reactive Tc cell responses. Methodology/Principal Finding Here, we report that intranasal administration of purified γ-ray inactivated human influenza A virus preparations (γ-Flu) effectively induces heterotypic and cross-protective immunity. A single intranasal administration of γ-A/PR8[H1N1] protects mice against lethal H5N1 and other heterotypic infections. Conclusions/Significance Intranasal γ-Flu represents a unique approach for a cross-protective vaccine against both seasonal as well as possible future pandemic influenza A virus infections.Mohammed Alsharifi, Yoichi Furuya, Timothy R. Bowden, Mario Lobigs, Aulikki Koskinen, Matthias Regner, Lee Trinidad, David B. Boyle and Arno Müllbache

Gamma-Irradiated Influenza Virus Uniquely Induces IFN-I Mediated Lymphocyte Activation Independent of the TLR7/MyD88 Pathway

Background: We have shown previously in mice, that infection with live viruses, including influenza/A and Semliki Forest virus (SFV), induces systemic partial activation of lymphocytes, characterized by cell surface expression of CD69 and CD86, but not CD25. This partial lymphocytes activation is mediated by type-I interferons (IFN-I). Importantly, we have shown that c-irradiated SFV does not induce IFN-I and the associated lymphocyte activation. Principal Findings: Here we report that, in contrast to SFV, c-irradiated influenza A virus elicits partial lymphocyte activation in vivo. Furthermore, we show that when using influenza viruses inactivated by a variety of methods (UV, ionising radiation and formalin treatment), as well as commercially available influenza vaccines, only c-irradiated influenza virus is able to trigger IFN-I-dependent partial lymphocyte activation in the absence of the TLR7/MyD88 signalling pathways. Conclusions: Our data suggest an important mechanism for the recognition of c-irradiated influenza vaccine by cytosolic receptors, which correspond with the ability of c-irradiated influenza virus to induce cross-reactive and cross-protective cytotoxic T cell responses.Yoichi Furuya, Jennifer Chan, En-Chi Wan, Aulikki Koskinen, Kerrilyn R. Diener, John D. Hayball, Matthias Regner, Arno Müllbacher, Mohammed Alsharif

The mitochondrial protein Bak is pivotal for gliotoxin-induced apoptosis and a critical host factor of Aspergillus fumigatus virulence in mice

Aspergillus fumigatus infections cause high levels of morbidity and mortality in immunocompromised patients. Gliotoxin (GT), a secondary metabolite, is cytotoxic for mammalian cells, but the molecular basis and biological relevance of this toxicity remain speculative. We show that GT induces apoptotic cell death by activating the proapoptotic Bcl-2 family member Bak, but not Bax, to elicit the generation of reactive oxygen species, the mitochondrial release of apoptogenic factors, and caspase-3 activation. Activation of Bak by GT is direct, as GT triggers in vitro a dose-dependent release of cytochrome c from purified mitochondria isolated from wild-type and Bax- but not Bak-deficient cells. Resistance to A. fumigatus of mice lacking Bak compared to wild-type mice demonstrates the in vivo relevance of this GT-induced apoptotic pathway involving Bak and suggests a correlation between GT production and virulence. The elucidation of the molecular basis opens new strategies for the development of therapeutic regimens to combat A. fumigatus and related fungal infections

Caspase-Dependent Inhibition of Mousepox Replication by gzmB

BACKGROUND: Ectromelia virus is a natural mouse pathogen, causing mousepox. The cytotoxic T (Tc) cell granule serine-protease, granzyme B, is important for its control, but the underlying mechanism is unknown. Using ex vivo virus immune Tc cells, we have previously shown that granzyme B is able to activate several independent pro-apoptotic pathways, including those mediated by Bid/Bak/Bax and caspases-3/-7, in target cells pulsed with Tc cell determinants. METHODS AND FINDINGS: Here we analysed the physiological relevance of those pro-apoptotic pathways in ectromelia infection, by incubating ectromelia-immune ex vivo Tc cells from granzyme A deficient (GzmB(+) Tc cells) or granzyme A and granzyme B deficient (GzmAxB(-/-) Tc cell) mice with ectromelia-infected target cells. We found that gzmB-induced apoptosis was totally blocked in ectromelia infected or peptide pulsed cells lacking caspases-3/-7. However ectromelia inhibited only partially apoptosis in cells deficient for Bid/Bak/Bax and not at all when both pathways were operative suggesting that the virus is able to interfere with apoptosis induced by gzmB in case not all pathways are activated. Importantly, inhibition of viral replication in vitro, as seen with wild type cells, was not affected by the lack of Bid/Bak/Bax but was significantly reduced in caspase-3/-7-deficient cells. Both caspase dependent processes were strictly dependent on gzmB, since Tc cells, lacking both gzms, neither induced apoptosis nor reduced viral titers. SIGNIFICANCE: Out findings present the first evidence on the biological importance of the independent gzmB-inducible pro-apoptotic pathways in a physiological relevant virus infection model

MHC class II-alpha chain knockout mice support increased viral replication that is independent of their lack of MHC class II cell surface expression and associated immune function deficiencies

MHCII molecules are heterodimeric cell surface proteins composed of an α and β chain. These molecules are almost exclusively expressed on thymic epithelium and antigen presenting cells (APCs) and play a central role in the development and function of CD4 T cells. Various MHC-II knockout mice have been generated including MHC-IIAα-/- (I-Aα-/-), MHC-IIAβ-/- (I-β-/-) and the double knockout (I-Aαxβ-/-). Here we report a very striking observation, namely that alphaviruses including the avirulent strain of Semliki Forest virus (aSFV), which causes asymptomatic infection in wild-type C57BL6/J (B6) mice, causes a very acute and lethal infection in I-Aα-/-, but not in I-β-/- or I-Aαxβ-/-, mice. This susceptibility to aSFV is associated with high virus titres in muscle, spleen, liver, and brain compared to B6 mice. In addition, I-Aα-/- mice show intact IFN-I responses in terms of IFN-I serum levels and IFN-I receptor expression and function. Radiation bone marrow chimeras of B6 mice reconstituted with I-Aα-/- bone marrow expressed B6 phenotype, whereas radiation chimeras of I-Aα-/- mice reconstituted with B6 bone marrow expressed the phenotype of high viral susceptibility. Virus replication experiments both in vivo and in vitro showed enhanced virus growth in tissues and cell cultures derived form I-Aα-/- compared to B6 mice. This enhanced virus replication is evident for other alpha-, flavi- and poxviruses and may be of great benefit to producers of viral vaccines. In conclusion, I-Aα-/- mice exhibit a striking susceptibility to virus infections independent of their defective MHC-II expression. Detailed genetic analysis will be carried out to characterise the underlining genetic defects responsible for the observed phenomenon.Mohammed Alsharifi, Aulikki Koskinen, Danushka K. Wijesundara, Jayaram Bettadapura, Arno Müllbache