Joseph A Trapani

Peter MacCallum Cancer Centre, Melbourne, Victoria, Australia

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Publications (191)1395.72 Total impact

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    ABSTRACT: The anionic proteoglycan serglycin is a major constituent of secretory granules in CTL/NK cells, and is proposed to promote the safe storage of the mostly cationic granule toxins, granzymes and perforin. Despite the extensive defects of mast cell function reported in serglycin gene-disrupted mice, no comprehensive study of physiologically relevant CTL/NK cell populations has ever been reported. We show that the cytotoxicity of serglycin-deficient CTL and NK cells is severely compromised, but can be partly compensated in both cell types when they become activated. Reduced intracellular granzyme B levels were noted, particularly in CD27+CD11b+ mature NK cells while serglycin-/- TCR-transgenic (OTI) CD8 T cells also had reduced perforin stores. Culture supernatants from serglycin-/- OTI T cells and IL-2-activated NK contained increased granzyme B, linking reduced storage with heightened export. By contrast, granzyme A was not significantly reduced in cells lacking serglycin, pointing to differentially regulated trafficking and/or storage for the two granzymes. A quantitative analysis of different granule classes by transmission EM showed a selective loss of dense-core granules in serglycin-/- CD8+ CTLs, but other granule types were maintained quantitatively. Our findings show that serglycin plays a critical role in the maturation of dense-core cytotoxic granules in cytotoxic lymphocytes and the trafficking and storage of perforin and granzyme B, whereas granzyme A is unaffected. The skewed retention of cytotoxic effector molecules markedly reduces CTL/NK cell cytotoxicity, but this is partly compensated by activating the cells by physiological means. This article is protected by copyright. All rights reserved.
    No preview · Article · Jan 2016 · FEBS Journal
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    ABSTRACT: Evolution from a furan-containing high-throughput screen (HTS) hit (1) resulted in isobenzofuran-1(3H)-one (2) as a potent inhibitor of the function of both isolated perforin protein and perforin delivered in situ by intact KHYG-1 NK cells. In the current study, structure-activity relationship (SAR) development towards a novel series of diarylthiophene analogues has continued through the use of substituted-benzene and -pyridyl moieties as bioisosteres for 2-thioxoimidazolidin-4-one (A) on a thiophene (B) -isobenzofuranone (C) scaffold. The resulting compounds were tested for their ability to inhibit perforin lytic activity in vitro. Carboxamide (23) shows a 4-fold increase over (2) in lytic activity against isolated perforin and provides good rationale for continued development within this class.
    Preview · Article · Dec 2015 · Bioorganic & medicinal chemistry letters
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    ABSTRACT: Perforin is an essential component in the cytotoxic lymphocyte-mediated cell death pathway. The traditional view holds that perforin monomers assemble into pores in the target cell membrane via a calcium-dependent process and facilitate translocation of cytotoxic proteases into the cytoplasm to induce apoptosis. Although many studies have examined the structure and role of perforin, the mechanics of pore assembly and granzyme delivery remain unclear. Here we have employed quartz crystal microbalance with dissipation monitoring (QCM-D) to investigate binding and assembly of perforin on lipid membranes, and show that perforin monomers bind to the membrane in a cooperative manner. We also found that cholesterol influences perforin binding and activity on intact cells and model membranes. Finally, contrary to current thinking, perforin efficiently binds membranes in the absence of calcium. When calcium is added to perforin already on the membrane, the QCM-D response changes significantly, indicating that perforin becomes membranolytic only after calcium binding.
    No preview · Article · Nov 2015 · Journal of Biological Chemistry

  • No preview · Article · Oct 2015
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    ABSTRACT: Natural killer (NK) cells and cytotoxic T-lymphocytes (CTL) deploy perforin (Prf) and granzymes to kill infected host cells. Perforin, secreted by immune cells, binds target membranes to form pores that deliver pro-apoptotic granzymes into the target cell. A crucial first step in this process is interaction of its C2 domain with target cell membranes, which is a calcium-dependent event. Some aspects of this process are understood, but many molecular details remain unclear. To address this, we investigated the mechanism of Ca(2+) and lipid binding to the C2 domain by NMR spectroscopy and X-ray crystallography. Calcium titrations, together with dodecylphosphocholine (DPC) micelle experiments confirmed that multiple Ca(2+) ions bind within the calcium binding regions (CBR), activating perforin with respect to membrane binding. We have also determined the affinities of several of these binding sites and shown that this interaction causes a significant structural rearrangement in CBR1. Thus, it is proposed that Ca(2+)-binding at the weakest affinity site triggers changes in the C2 domain that facilitate its interaction with lipid membranes. Copyright © 2015, The American Society for Biochemistry and Molecular Biology.
    Preview · Article · Aug 2015 · Journal of Biological Chemistry
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    ABSTRACT: Granzyme B (GzmB) is a serine protease best known for inducing target cell apoptosis when released by cytotoxic T lymphocytes (CTLs) or natural killer cells with pore-forming perforin. As a result, GzmB detected in the serum of virus-infected individuals has typically been attributed to these sources. Here, we show that patients with recently diagnosed infectious mononucleosis caused by Epstein-Barr virus (EBV) have high circulating levels of GzmB that may be derived from infected B cells early in course of disease. We recently reported that human B cells from healthy donors secrete active GzmB when stimulated in vitro through B-cell receptor (BCR) ligation and interleukin (IL)-21. We found that infecting B cells with EBV greatly amplified GzmB secretion in response to the same stimuli, but the expression was terminated once the infection had become latent. Our results represent a rare instance of GzmB expression by non-CTL/natural killer cells in the context of infection with a human pathogen.
    Full-text · Article · Jun 2015
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    ABSTRACT: Chimeric Antigen Receptor (CAR) T cells have enjoyed unprecedented clinical success for haematological malignancies in recent years. However several aspects of CAR T cell biology remain unknown. We compared CAR and TCR killing in the same effector cell and showed that CAR T cells can not only efficiently kill single tumor targets, they can also kill multiple tumor targets in a sequential manner. Single and serial killing events were not sustained over the long term due to CAR down-regulation after 20 hours.
    Full-text · Article · Jun 2015 · OncoImmunology
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    ABSTRACT: There is increasing evidence that some cancer therapies can promote tumor immunogenicity to boost the endogenous antitumor immune response. In this study, we used the novel combination of agonistic anti-TRAIL-R1 antibody (mapatumumab, Mapa) with low dose bortezomib (LDB) for this purpose. The combination induced profound myeloma cell apoptosis, greatly enhanced the uptake of myeloma cell apoptotic bodies by dendritic cell (DC) and induced anti-myeloma cytotoxicity by both CD8(+) T cells and NK cells. Cytotoxic lymphocyte expansion was detected within 24 h of commencing therapy and was maximized when myeloma-pulsed DC were co-treated with low dose bortezomib and mapatumumab (LDB+Mapa) in the presence of NK cells. This study shows that Mapa has two distinct but connected modes of action against multiple myeloma (MM). First, when combined with LDB, Mapa produced powerful myeloma cell apoptosis; secondly, it promoted DC priming and an NK cell-mediated expansion of anti-myeloma cytotoxic lymphocyte (CTL). Overall, this study indicates that Mapa can be used to drive potent anti-MM immune responses.
    No preview · Article · May 2015 · OncoImmunology
  • Joseph A. Trapani · Ilia Voskoboinik · Misty R. Jenkins
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    ABSTRACT: Failure of natural killer cells or cytotoxic T-lymphocytes to kill cognate target cells results in cytokine/chemokine hypersecretion and markedly delayed killer/target cell detachment. With congenital perforin deficiency, fatal cytokine storm results. In cancer cells, where corrupted apoptotic signaling frequently delays apoptosis, we propose that failed death may alter the tumor microenvironment and skew immune infiltrates, even when perforin is delivered normally.
    No preview · Article · May 2015 · OncoImmunology
  • Ilia Voskoboinik · James C Whisstock · Joseph A Trapani
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    ABSTRACT: A defining property of cytotoxic lymphocytes is their expression and regulated secretion of potent toxins, including the pore-forming protein perforin and serine protease granzymes. Until recently, mechanisms of pore formation and granzyme transfer into the target cell were poorly understood, but advances in structural and cellular biology have now begun to unravel how synergy between perforin and granzymes brings about target cell death. These and other advances are demonstrating the surprisingly broad pathophysiological roles of the perforin-granzyme pathway, and this has important implications for understanding immune homeostasis and for developing immunotherapies for cancer and other diseases. In particular, we are beginning to define and understand a range of human diseases that are associated with a failure to deliver active perforin to target cells. In this Review, we discuss the current understanding of the structural, cellular and clinical aspects of perforin and granzyme biology.
    No preview · Article · May 2015 · Nature Reviews Immunology
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    ABSTRACT: The production and delivery of functional perforin (PRF; PRF1 gene) by cytotoxic lymphocytes maintains immune homeostasis and tumour immune surveillance. In humans, inheritance of the common PRF1 polymorphism, p.A91V, (c.272C>T) found in 8-9% of the Caucasian population, with another mutated allele resulting in reduced PRF function or trafficking, has been shown to result in hyperinflammatory diseases and/or haematological cancers. In this study, we sought to investigate the function of p.A91V on a wild-type (WT) perforin background. We first developed an assay that distinguishes the relative levels of transcription of individual PRF1 alleles, including p.A91V. The p.A91V allele was seen to be expressed at similar levels as the WT allele in primary human natural killer (NK) cells, ruling out that allelic expression imbalance influenced their function. We then demonstrated that the p.A91V mutation results in protein misfolding and an appreciable reduction in NK-cell cytotoxicity in healthy carriers of p.A91V. We propose that this level of cytotoxic dysfunction may readily account for the predisposition to immune-mediated disease in individuals homozygous for p.A91V. Also, the fact that monoallelic mutations of PRF1 decrease NK-cell cytotoxicity should be considered in individuals presenting with the manifestations of immune deficiency states that impinge on NK-cell cytotoxicity.Immunology and Cell Biology advance online publication, 17 March 2015; doi:10.1038/icb.2015.1.
    Full-text · Article · Mar 2015 · Immunology and Cell Biology
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    ABSTRACT: Failure of cytotoxic T lymphocytes (CTLs) or natural killer (NK) cells to kill target cells by perforin (Prf)/granzyme (Gzm)-induced apoptosis causes severe immune dysregulation. In familial hemophagocytic lymphohistiocytosis, Prf-deficient infants suffer a fatal "cytokine storm" resulting from macrophage overactivation, but the link to failed target cell death is not understood. We show that prolonged target cell survival greatly amplifies the quanta of inflammatory cytokines secreted by CTLs/NK cells and that interferon-γ (IFN-γ) directly invokes the activation and secondary overproduction of proinflammatory IL-6 from naive macrophages. Furthermore, using live cell microscopy to visualize hundreds of synapses formed between wild-type, Prf-null, or GzmA/B-null CTLs/NK cells and their targets in real time, we show that hypersecretion of IL-2, TNF, IFN-γ, and various chemokines is linked to failed disengagement of Prf- or Gzm-deficient lymphocytes from their targets, with mean synapse time increased fivefold, from ∼8 to >40 min. Surprisingly, the signal for detachment arose from the dying target cell and was caspase dependent, as delaying target cell death with various forms of caspase blockade also prevented their disengagement from fully competent CTLs/NK cells and caused cytokine hypersecretion. Our findings provide the cellular mechanism through which failed killing by lymphocytes causes systemic inflammation involving recruitment and activation of myeloid cells. © 2015 Jenkins et al.
    Full-text · Article · Mar 2015 · Journal of Experimental Medicine
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    ABSTRACT: Adoptive therapy with chimeric antigen receptor (CAR) T cells shows great promise clinically. However, there are important aspects of CAR-T cell biology which have not been explored particularly with respect to the kinetics of: activation, immune synapse formation and tumor cell killing. Moreover, the effects of signaling via the endogenous TCR or CAR on killing kinetics are unclear. To address these issues, we developed a novel transgenic mouse (designated CAR.OT-I), in which CD8+ T cells co-expressed the clonogenic OT-I T cell receptor, recognizing the H-2Kb presented ovalbumin peptide SIINFEKL, and a scFv specific for human HER2. Primed CAR.OT-I T cells were mixed with SIINFEKL-pulsed or HER2-expressing tumor cells and visualized in real time using time-lapse microscopy. We found that engagement via CAR or TCR did not affect cell death kinetics, except that the time from degranulation to CAR-T cell detachment was faster when CAR was engaged. We showed, for the first time, that individual CAR.OT-I cells can kill multiple tumor cells ('serial killing'), irrespective of the mode of recognition. At low E:T ratios, tumor cell killing rate was similar via TCR or CAR ligation over the first 20 hours of co-incubation. However, from 20-50 hours, tumor cell death mediated through CAR became attenuated, due to CAR downregulation throughout the timecourse. Our study provides important insights into CAR-T/tumor cell interactions, with implications for single- or dual-receptor-focused T cell therapy. Copyright © 2015, American Association for Cancer Research.
    Full-text · Article · Feb 2015
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    ABSTRACT: Membrane attack complex/perforin-like (MACPF) proteins comprise the largest superfamily of pore-forming proteins, playing crucial roles in immunity and pathogenesis. Soluble monomers assemble into large transmembrane pores via conformational transitions that remain to be structurally and mechanistically characterised. Here we present an 11 Å resolution cryo-electron microscopy (cryo-EM) structure of the two-part, fungal toxin Pleurotolysin (Ply), together with crystal structures of both components (the lipid binding PlyA protein and the pore-forming MACPF component PlyB). These data reveal a 13-fold pore 80 Å in diameter and 100 Å in height, with each subunit comprised of a PlyB molecule atop a membrane bound dimer of PlyA. The resolution of the EM map, together with biophysical and computational experiments, allowed confident assignment of subdomains in a MACPF pore assembly. The major conformational changes in PlyB are a ∼70° opening of the bent and distorted central β-sheet of the MACPF domain, accompanied by extrusion and refolding of two α-helical regions into transmembrane β-hairpins (TMH1 and TMH2). We determined the structures of three different disulphide bond-trapped prepore intermediates. Analysis of these data by molecular modelling and flexible fitting allows us to generate a potential trajectory of β-sheet unbending. The results suggest that MACPF conformational change is triggered through disruption of the interface between a conserved helix-turn-helix motif and the top of TMH2. Following their release we propose that the transmembrane regions assemble into β-hairpins via top down zippering of backbone hydrogen bonds to form the membrane-inserted β-barrel. The intermediate structures of the MACPF domain during refolding into the β-barrel pore establish a structural paradigm for the transition from soluble monomer to pore, which may be conserved across the whole superfamily. The TMH2 region is critical for the release of both TMH clusters, suggesting why this region is targeted by endogenous inhibitors of MACPF function.
    Full-text · Article · Feb 2015 · PLoS Biology
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    ABSTRACT: Many immune response genes are highly polymorphic, consistent with the selective pressure imposed by pathogens over evolutionary time, and the need to balance infection control with the risk of auto-immunity. Epidemiological and genomic studies have identified many genetic variants that confer susceptibility or resistance to pathogenic micro-organisms. While extensive polymorphism has been reported for the granzyme B (GzmB) gene, its relevance to pathogen immunity is unexplored. Here, we describe the biochemical and cytotoxic functions of a common allele of GzmB (GzmBW) common in wild mouse. While retaining 'Asp-ase' activity, GzmBW has substrate preferences that differ considerably from GzmBP, which is common to all inbred strains. In vitro, GzmBW preferentially cleaves recombinant Bid, whereas GzmBP activates pro-caspases directly. Recombinant GzmBW and GzmBP induced equivalent apoptosis of uninfected targets cells when delivered with perforin in vitro. Nonetheless, mice homozygous for GzmBW were unable to control murine cytomegalovirus (MCMV) infection, and succumbed as a result of excessive liver damage. Although similar numbers of anti-viral CD8 T cells were generated in both mouse strains, GzmBW-expressing CD8 T cells isolated from infected mice were unable to kill MCMV-infected targets in vitro. Our results suggest that known virally-encoded inhibitors of the intrinsic (mitochondrial) apoptotic pathway account for the increased susceptibility of GzmBW mice to MCMV. We conclude that different natural variants of GzmB have a profound impact on the immune response to a common and authentic viral pathogen.
    Full-text · Article · Dec 2014 · PLoS Pathogens
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    Magdalena Hagn · Vivien R Sutton · Joseph A Trapani
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    ABSTRACT: The serine protease Granzyme B (GzmB) mediates target cell apoptosis when released by cytotoxic T lymphocytes (CTL) or natural killer (NK) cells. GzmB is the most studied granzyme in humans and mice and therefore, researchers need specific and reliable tools to study its function and role in pathophysiology. This especially necessitates assays that do not recognize proteases such as caspases or other granzymes that are structurally or functionally related. Here, we apply GzmB's preference for cleavage after aspartic acid residues in a colorimetric assay using the peptide thioester Boc-Ala-Ala-Asp-S-Bzl. GzmB is the only mammalian serine protease capable of cleaving this substrate. The substrate is cleaved with similar efficiency by human, mouse and rat GzmB, a property not shared by other commercially available peptide substrates, even some that are advertised as being suitable for this purpose. This protocol is demonstrated using unfractionated lysates from activated NK cells or CTL and is also suitable for recombinant proteases generated in a variety of prokaryotic and eukaryotic systems, provided the correct controls are used. This assay is a highly specific method to ascertain the potential pro-apoptotic activity of cytotoxic molecules in mammalian lymphocytes, and of their recombinant counterparts expressed by a variety of methodologies.
    Preview · Article · Nov 2014 · Journal of Visualized Experiments
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    ABSTRACT: Immunosuppressive pretransplantation conditioning is essential for donor cell engraftment in allogeneic bone marrow transplantation (BMT). The role of residual postconditioning recipient immunity in determining engraftment is poorly understood. We examined the role of recipient perforin in the kinetics of donor cell engraftment. MHC-mismatched BMT mouse models demonstrated that both the rate and proportion of donor lymphoid cell engraftment and expansion of effector memory donor T cells in both spleen and BM were significantly increased within 5 to 7 days post-BMT in perforin-deficient (pfn−/−) recipients, compared with wild-type. In wild-type recipients, depletion of natural killer (NK) cells before BMT enhanced donor lymphoid cell engraftment to that seen in pfn−/− recipients. This demonstrated that a perforin-dependent, NK-mediated, host-versus-graft (HVG) effect limits the rate of donor engraftment and T cell activation. Radiation-resistant natural killer T (NKT) cells survived in the BM of lethally irradiated mice and may drive NK cell activation, resulting in the HVG effect. Furthermore, reduced pretransplant irradiation doses in pfn−/− recipients permitted long-term donor lymphoid cell engraftment. These findings suggest that suppression of perforin activity or selective depletion of recipient NK cells before BMT could be used to improve donor stem cell engraftment, in turn allowing for the reduction of pretransplant conditioning.
    No preview · Article · Nov 2014 · Biology of Blood and Marrow Transplantation
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    ABSTRACT: Cytotoxic lymphocytes destroy pathogen-infected and transformed cells through the cytotoxic granule exocytosis death pathway, which is dependent on the delivery of proapoptotic granzymes into the target cell cytosol by the pore-forming protein, perforin. Despite the importance of mouse models in understanding the role of cytotoxic lymphocytes in immune-mediated disease and their role in cancer immune surveillance, no reliable intracellular detection method exists for mouse perforin. Consequently, rapid, flow-based assessment of cytotoxic potential has been problematic, and complex assays of function are generally required. In this study, we have developed a novel method for detecting perforin in primary mouse cytotoxic T lymphocytes by immunofluorescence and flow cytometry. We used this new technique to validate perforin colocalization with granzyme B in cytotoxic granules polarized to the immunological synapse, and to assess the expression of perforin in cytotoxic T lymphocytes at various stages of activation. The sensitivity of this technique also allowed us to distinguish perforin levels in Prf1(+/+) and Prf1(+/-) mice. This new methodology will have broad applications and contribute to advances within the fields of lymphocyte biology, infectious disease, and cancer.
    No preview · Article · Oct 2014 · The Journal of Immunology
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    ABSTRACT: Background: Many of the functions attributed to mast cells depend on the various pro-inflammatory mediators that are secreted upon mast cell activation. These include a panel of mast cell-specific proteases. In addition, recent studies have indicated that murine mast cells also express granzyme D, a protease previously thought to be confined to cytotoxic lymphocytes. Here, we address the human relevance of the latter findings by investigating whether human mast cells express granzyme H, the granzyme that may represent the functional counterpart to murine granzyme D. Methods: Cord blood-derived mast cells, LAD2 cells and skin mast cells in situ were evaluated for their expression of granzymes using quantitative PCR, Western blot analysis and immunostaining. Mast cells were activated by either calcium ionophore stimulation or IgE receptor cross-linking. Results: Cord blood-derived mast cells and LAD2 cells were shown to express granzyme H and B mRNA, while granzyme A, K and M expression was undetectable. Mast cell activation by either calcium ionophore or IgE receptor cross-linking caused down-regulated expression of granzyme H. In contrast, granzyme B expression was up-regulated by the same stimuli. Granzyme H expression was also confirmed at the protein level, as shown by both Western blot analysis and confocal microscopy. Further, we show that granzyme H is expressed by human skin mast cells in situ. Conclusions: The present findings implicate granzyme H as a novel protease expressed by human mast cells and support earlier findings obtained in natural killer cells suggesting that granzymes B and H are reciprocally regulated.
    No preview · Article · Oct 2014 · International Archives of Allergy and Immunology
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    ABSTRACT: Natural killer (NK) cells have been reported to control adaptive immune responses that occur in lymphoid organs at the early stages of immune challenge. The physiological purpose of such regulatory activity remains unclear, because it generally does not confer a survival advantage. We found that NK cells specifically eliminated activated CD4(+) T cells in the salivary gland during chronic murine cytomegalovirus (MCMV) infection. This was dependent on TNF-related apoptosis inducing ligand (TRAIL) expression by NK cells. Although NK cell-mediated deletion of CD4(+) T cells prolonged the chronicity of infection, it also constrained viral-induced autoimmunity. In the absence of this activity, chronic infection was associated with a Sjogren's-like syndrome characterized by focal lymphocytic infiltration into the glands, production of autoantibodies, and reduced saliva and tear secretion. Thus, NK cells are an important homeostatic control that balances the efficacy of adaptive immune responses with the risk of developing autoimmunity.
    No preview · Article · Oct 2014 · Immunity

Publication Stats

10k Citations
1,395.72 Total Impact Points

Institutions

  • 2000-2016
    • Peter MacCallum Cancer Centre
      • • Killer Cell Biology Laboratory
      • • Cancer Immunology Program
      • • Cancer Cell Death Laboratory
      Melbourne, Victoria, Australia
  • 1985-2015
    • University of Melbourne
      • • Sir Peter MacCallum Department of Oncology
      • • Department of Microbiology and Immunology
      • • Department of Pathology
      Melbourne, Victoria, Australia
  • 2011
    • The Walter and Eliza Hall Institute of Medical Research
      • Division of Immunology
      Melbourne, Victoria, Australia
  • 2005
    • Monash University (Australia)
      • Department of Biochemistry and Molecular Biology
      Melbourne, Victoria, Australia
  • 2002-2003
    • Royal Melbourne Hospital
      Melbourne, Victoria, Australia
  • 1998
    • University of Virginia
      Charlottesville, Virginia, United States
    • Box Hill Institute
      Box Hill, Victoria, Australia
  • 1997
    • University of Manitoba
      • Manitoba Institute of Cell Biology
      Winnipeg, Manitoba, Canada
  • 1992-1997
    • Austin Health
      • Department of Pathology
      Melbourne, Victoria, Australia
  • 1989-1990
    • Memorial Sloan-Kettering Cancer Center
      • Human Immunogenetics Laboratory
      New York City, New York, United States
    • Roswell Park Cancer Institute
      Buffalo, New York, United States
  • 1983
    • Victoria University Melbourne
      Melbourne, Victoria, Australia