J A Trapani

Peter MacCallum Cancer Centre, Melbourne, Victoria, Australia

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Publications (241)1560.03 Total impact

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    ABSTRACT: The cytolytic protein perforin is a key component of the immune response and is implicated in a number of human pathologies and therapy-induced conditions. A novel series of small molecule inhibitors of perforin function have been developed as potential immunosuppressive agents. The pharmacokinetics and metabolic stability of a series of 16 inhibitors of perforin was evaluated in male CD1 mice following intravenous administration. The compounds were well tolerated 6 h after dosing. After intravenous administration at 5 mg/kg, maximum plasma concentrations ranged from 532 ± 200 to 10,061 ± 12 ng/mL across the series. Plasma concentrations were greater than the concentrations required for in vitro inhibitory activity for 11 of the compounds. Following an initial rapid distribution phase, the elimination half-life values for the series ranged from 0.82 ± 0.25 to 4.38 ± 4.48 h. All compounds in the series were susceptible to oxidative biotransformation. Following incubations with microsomal preparations, a tenfold range in in vitro half-life was observed across the series. The data suggests that oxidative biotransformation was not singularly responsible for clearance of the compounds and no direct relationship between microsomal clearance and plasma clearance was observed. Structural modifications however, do provide some information as to the relative microsomal stability of the compounds, which may be useful for further drug development.
    European Journal of Drug Metabolism and Pharmacokinetics 08/2014; · 1.31 Impact Factor
  • I. Voskoboinik, K. Thia, J.A. Trapani
    Internal Medicine Journal 08/2014; 44(8). · 1.82 Impact Factor
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    ABSTRACT: Cytotoxic lymphocytes eliminate virally infected or neoplastic cells through the action of cytotoxic proteases (granzymes). The pore-forming protein perforin is essential for delivery of granzymes into the cytoplasm of target cells, however the mechanism of this delivery is incompletely understood. Perforin contains a membrane attack complex / perforin (MACPF) domain and oligomerises to form an aqueous pore in the plasma membrane, therefore the simplest (and best supported) model suggests that granzymes passively diffuse through the perforin pore into the cytoplasm of the target cell. Here we demonstrate that perforin preferentially delivers cationic molecules while anionic and neutral cargoes are delivered inefficiently. Furthermore, another distantly related pore-forming MACPF protein, pleurotolysin (from the oyster mushroom), also favours the delivery of cationic molecules, and efficiently delivers human granzyme B. We propose that this facilitated diffusion is due to conserved features of oligomerised MACPF proteins, which may include an anionic lumen.
    Journal of Biological Chemistry 02/2014; · 4.65 Impact Factor
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    ABSTRACT: CpG oligodeoxynucleotides (CpG) and IL-21 are two promising agents for the treatment of B-cell chronic lymphocytic leukemia (B-CLL). Recently, we reported that the combination of CpG and IL-21 (CpG/IL-21) can induce granzyme B (GrB)-dependent apoptosis in B-CLL cells. Here, we demonstrate that treatment of B-CLL cells with CpG and IL-21 results in the development of antigen-presenting cell (APC)-like cells with cytotoxic features. These properties eventually give rise to B-CLL cell apoptosis, independently of their cytogenetic phenotype, whereas normal B-cell survival is not negatively affected by CpG/IL-21. APC- and CTL-typical molecules found to be up-regulated in CpG/IL-21-stimulated B-CLL cells include GrB, perforin, T-bet, monokine-induced by IFN-γ and IFN-γ-inducible protein 10 (IP-10), as well as molecules important for cell adhesion, antigen cross-presentation and costimulation. Also induced are molecules involved in GrB induction, trafficking and processing, whereas the GrB inhibitor Serpin B9 [formerly proteinase inhibitor-9 (PI-9)] is down-modulated by CpG/IL-21. In conclusion, CpG/IL-21-stimulated B-CLL cells acquire features that are reminiscent of killer dendritic cells, and which result in enhanced immunogenicity, cytotoxicity and apoptosis. Our results provide novel insights into the aberrant immune state of B-CLL cells and may establish a basis for the development of an innovative cellular vaccination approach in B-CLL.
    International Immunology 02/2014; · 3.14 Impact Factor
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    ABSTRACT: The human lymphocyte toxins granzyme B (hGrzB) and perforin cooperatively induce apoptosis of virus-infected or transformed cells: perforin pores enable entry of the serine protease hGrzB into the cytosol, where it processes Bid to selectively activate the intrinsic apoptosis pathway. Truncated Bid (tBid) induces Bax/Bak-dependent mitochondrial outer membrane permeability and the release of cytochrome c and Smac/Diablo. To identify cellular proteins that regulate perforin/hGrzB-mediated Bid cleavage and subsequent apoptosis, we performed a gene-knockdown (KD) screen using a lentiviral pool of short hairpin RNAs embedded within a miR30 backbone (shRNAmiR). We transduced HeLa cells with a lentiviral pool expressing shRNAmiRs that target 1213 genes known to be involved in cell death signaling and selected cells with acquired resistance to perforin/hGrzB-mediated apoptosis. Twenty-two shRNAmiRs were identified in the positive-selection screen including two, PCAF and ADA3, whose gene products are known to reside in the same epigenetic regulatory complexes. Small interfering (si)RNA-mediated gene-KD of PCAF or ADA3 also conferred resistance to perforin/hGrzB-mediated apoptosis providing independent validation of the screen results. Mechanistically, PCAF and ADA3 exerted their pro-apoptotic effect upstream of mitochondrial membrane permeabilization, as indicated by reduced cytochrome c release in PCAF-KD cells exposed to perforin/hGrzB. While overall levels of Bid were unaltered, perforin/hGrzB-mediated cleavage of Bid was reduced in PCAF-KD or ADA3-KD cells. We discovered that PCAF-KD or ADA3-KD resulted in reduced expression of PACS2, a protein implicated in Bid trafficking to mitochondria and importantly, targeted PACS2-KD phenocopied the effect of PCAF-KD or ADA3-KD. We conclude that PCAF and ADA3 regulate Bid processing via PACS2, to modulate the mitochondrial cell death pathway in response to hGrzB.Cell Death and Differentiation advance online publication, 24 January 2014; doi:10.1038/cdd.2013.203.
    Cell death and differentiation 01/2014; · 8.24 Impact Factor
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    ABSTRACT: Granzymes are generally recognized for their capacity to induce various pathways of perforin-dependent target cell death. Within this serine protease family, Granzyme M (GrzM) is unique owing to its preferential expression in innate effectors such as natural killer (NK) cells. During Listeria monocytogenes infection, we observed markedly reduced secretion of macrophage inflammatory protein-1 alpha (MIP-1α) in livers of GrzM-deficient mice, which resulted in significantly impaired NK cell recruitment. Direct stimulation with IL-12 and IL-15 demonstrated that GrzM was required for maximal secretion of active MIP-1α. This effect was not due to reduced protein induction but resulted from heightened intracellular accumulation of MIP-1α, with reduced release. These results demonstrate that GrzM is a critical mediator of innate immunity that can regulate chemotactic networks and has an important role in the initiation of immune responses and pathogen control.
    Cell Death & Disease 01/2014; 5:e1115. · 6.04 Impact Factor
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    ABSTRACT: Following its secretion from cytotoxic lymphocytes into the immune synapse, perforin binds to target cell membranes through its Ca2+-dependent C2 domain. Membrane-bound perforin then forms pores that allow passage of pro-apoptopic granzymes into the target cell. Here, structural and biochemical studies reveal that Ca2+ binding triggers a conformational change in the C2 domain that permits four key hydrophobic residues to interact with the plasma membrane. However, contrary to previous suggestions, these movements and membrane binding do not trigger irreversible conformational changes in the pore-forming MACPF domain, indicating that subsequent monomer-monomer interactions at the membrane surface are required for perforin pore formation.
    Biochemical Journal 09/2013; · 4.65 Impact Factor
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    ABSTRACT: The effective engagement of cytotoxic lymphocytes (CLs) with their target cells is essential for the removal of virus-infected and malignant cells from the body. The spatiotemporal properties that define CL engagement and killing of target cells remain largely uncharacterized due to a lack of biological reporters. We have used a novel live cell microscopy technique to visualize the engagement of primary human and mouse CL with their targets and the subsequent delivery of the lethal hit. Extensive quantitative real-time analysis of individual effector-target cell conjugates demonstrated that a single effector calcium flux event was sufficient for the degranulation of human CLs, resulting in the breach of the target cell membrane by perforin within 65-100 s. In contrast, mouse CLs demonstrated distinct calcium signaling profiles leading to degranulation: whereas mouse NKs required a single calcium flux event, CD8(+) T cells typically required several calcium flux events before perforin delivery. Irrespective of their signaling profile, every target cell that was damaged by perforin died by apoptosis. To our knowledge, we demonstrate for the first time that perforin pore delivery is unidirectional, occurring exclusively on the target cell membrane, but sparing the killer cell. Despite this, the CTL membrane was not intrinsically perforin resistant, as intact CTLs presented as targets to effector CTLs were capable of being killed by perforin-dependent mechanisms. Our results highlight the remarkable efficiency and specificity of perforin pore delivery by CLs.
    The Journal of Immunology 07/2013; · 5.52 Impact Factor
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    ABSTRACT: In a phase I study of autologous chimeric antigen receptor (CAR) anti-LeY T cell therapy of acute myeloid leukemia (AML), we examined the safety and post-infusion persistence of adoptively transferred T cells. Following fludarabine-containing pre-conditioning four patients received up to 1.3 x 10(9) total T cells, of which 14% to 38% expressed the CAR. Grade 3 or 4 toxicity was not observed. One patient achieved a cytogenetic remission, whilst another with active leukemia had a reduction in peripheral blood blasts and a third showed a protracted remission. Using an aliquot of (111)In-labeled CAR-T cells we demonstrated trafficking to the bone marrow in those patients with the greatest clinical benefit. Furthermore, in a patient with leukemia cutis, CAR-T cells infiltrated proven sites of disease. Serial PCR of peripheral blood and bone marrow for the LeY transgene demonstrated that infused CAR-T cells persisted for up to 10 months. Our study supports the feasibility and safety of CAR-T cell therapy in high-risk AML, and demonstrates durable in vivo persistence.Molecular Therapy (2013); doi:10.1038/mt.2013.154.
    Molecular Therapy 07/2013; · 7.04 Impact Factor
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    ABSTRACT: Human and mouse granzyme (Gzm)B both induce target cell apoptosis in concert with pore-forming perforin (Pfp); however the mechanisms by which other Gzms induce non-apoptotic death remain controversial and poorly characterised. We used timelapse microscopy to document, quantitatively and in real time, the death of target cells exposed to primary natural killer (NK) cells from mice deficient in key Gzms. We found that in the vast majority of cases, NK cells from wild-type mice induced classic apoptosis. However, NK cells from syngeneic Gzm B-deficient mice induced a novel form of cell death characterised by slower kinetics and a pronounced, writhing, 'worm-like' morphology. Dying cells initially contracted but did not undergo membrane blebbing, and annexin-V staining was delayed until the onset of secondary necrosis. As it is different from any cell death process previously reported, we tentatively termed this cell death 'athetosis'. Two independent lines of evidence showed this alternate form of death was due to Gzm A: first, cell death was revealed in the absence of Gzm B, but was completely lost when the NK cells were deficient in both Gzm A and B; second, the athetotic morphology was precisely reproduced when recombinant mouse Gzm A was delivered by an otherwise innocuous dose of recombinant Pfp. Gzm A-mediated athetosis did not require caspase activation, early mitochondrial disruption or generation of reactive oxygen species, but did require an intact actin cytoskeleton and was abolished by latrunculin B and mycalolide B. This work defines an authentic role for mouse Gzm A in granule-induced cell death by cytotoxic lymphocytes.Cell Death and Differentiation advance online publication, 7 June 2013; doi:10.1038/cdd.2013.59.
    Cell death and differentiation 06/2013; · 8.24 Impact Factor
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    ABSTRACT: In mammals the 67 kDa pore-forming protein perforin is essential to the granule exocytosis pathway used by cytotoxic lymphocytes to eliminate virally infected and malignant cells. There is indirect evidence that this pathway exists in lower vertebrates such as teleost fish and birds, although in genome databases for the chicken and other bird species the perforin gene is incomplete and no full length expressed sequence tag has been reported. We present here the full gene and transcript sequence of chicken perforin. The inferred protein product contains an extended C-terminus that is at least 90 amino acids longer than any mammalian perforin, which is also evident in partial genomic sequences from other birds. To determine whether this extension is present in the translated protein, we raised two polyclonal antisera. The antisera identified a protein of just less than 80 kDa in both transfected COS-1 cells and concanavalin A stimulated chicken splenocytes, indicating that the extended C-terminus is present in the mature protein. Our findings confirm that perforin exists in birds, and show that it is considerably longer than perforin of non-avian vertebrates.
    Developmental and comparative immunology 05/2013; · 3.29 Impact Factor
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    ABSTRACT: Loss-of-function mutations in the gene coding for perforin (PRF1) markedly reduce the ability of cytotoxic T lymphocytes and natural killer cells to kill target cells, causing immunosuppression and impairing immune regulation. In humans, nearly half of the cases of type 2 familial hemophagocytic lymphohistiocytosis are due to bi-allelic PRF1 mutations. The partial inactivation of PRF1 due to mutations that promote protein misfolding or the common hypomorphic allele coding for the A91V substitution have been associated with lymphoid malignancies in childhood and adolescence. To investigate whether PRF1 mutations also predispose adults to cancer, we genotyped 566 individuals diagnosed with melanoma (101), lymphoma (65), colorectal carcinoma (30) or ovarian cancer (370). The frequency of PRF1 genotypes was similar in all disease groups and 424 matched controls, indicating that the PRF1 status is not associated with an increased susceptibility to these malignancies. However, four out of 15 additional individuals diagnosed with melanoma and B-cell lymphoma during their lifetime expressed either PRF1(A91V) or the rare pathogenic PRF1(R28C) variant (p = 0.04), and developed melanoma relatively early in life. Both PRF1(A91V)- and PRF1(R28C)-expressing lymphocytes exhibited severely impaired but measurable cytotoxic function. Our results suggest that defects in human PRF1 predispose individuals to develop both melanoma and lymphoma. However, these findings require validation in larger patient cohorts.
    Oncoimmunology. 04/2013; 2(4):e24185.
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    ABSTRACT: Cytotoxic lymphocytes serve a key role in immune homeostasis by eliminating virus-infected and transformed target cells through the perforin-dependent delivery of pro-apoptotic granzymes. However, the mechanism of granzyme entry into cells remains unresolved. Using biochemical approaches combined with time-lapse microscopy of human primary cytotoxic lymphocytes engaging their respective targets, we defined the time course of perforin pore formation in the context of the physiological immune synapse. We show that upon recognition of targets, calcium influx into the lymphocyte led to perforin exocytosis and target cell permeabilisation in as little as 30-sec. Within the synaptic cleft, target cell permeabilisation by perforin resulted in the rapid diffusion of extracellular milieu derived granzymes. Repair of these pores was initiated within 20-sec and completed within 80-sec, thus limiting granzyme diffusion. Remarkably, even such a short timeframe was sufficient for the delivery of lethal amounts of granzymes into the target cell. Rapid initiation of apoptosis was evident from caspase-dependent target cell rounding within 2-min of perforin permeabilisation. The current study defines the final sequence of events controlling cytotoxic lymphocyte immune defence, in which perforin pores assemble on the target cell plasma membrane, sufficient to ensure efficient delivery of lethal granzymes.
    Blood 02/2013; · 9.06 Impact Factor
  • Joseph A Trapani, Ilia Voskoboinik
    Blood 01/2013; 121(4):568-70. · 9.06 Impact Factor
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    Ilia Voskoboinik, Joseph A Trapani
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    ABSTRACT: Congenital perforin deficiency is considered a rare cause of human immunopathology and immune dysregulation, and classically presents as a fatal illness early in infancy. However, we propose that a group of related disorders in which killer lymphocytes deliver only partially active perforin or a reduced quantum of wild-type perforin to the immune synapse should be considered part of an extended syndrome with overlapping but more variable clinical features. Apart from the many rare mutations scattered over the coding sequences, up to 10% of Caucasians carry the severely hypomorphic PRF1 allele C272 > T (leading to A91V mutation) and the overall prevalence of the homozygous state for A91V is around 1 in 600 individuals. We therefore postulate that the partial loss of perforin function and its clinical consequences may be more common then currently suspected. An acute clinical presentation is infrequent in A91V heterozygous individuals, but we postulate that the partial loss of perforin function may potentially be manifested in childhood or early adulthood as "idiopathic" inflammatory disease, or through increased cancer susceptibility - either hematological malignancy or multiple, independent primary cancers. We suggest the new term "perforinopathy" to signify the common functional endpoints of all the known consequences of perforin deficiency and failure to deliver fully functional perforin.
    Frontiers in Immunology 01/2013; 4:441.
  • O Susanto, J A Trapani, D Brasacchio
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    ABSTRACT: Granzymes (Grz) are a family of serine proteases found in the granules of cytotoxic lymphocytes and are emerging as an important group of proteins involved in immune function and surveillance. Grz have both cytotoxic and more recently reported non-cytotoxic roles, however these functions are still subject to thorough investigation. The significance of the cytotoxic and importantly the non-cytotoxic roles of Grz will be discussed in this review, detailing accepted and controversial functions.
    Tissue Antigens 12/2012; 80(6):477-87. · 2.93 Impact Factor
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    ABSTRACT: Considerable progress has been made in understanding how cytotoxic lymphocytes use the highly toxic pore-forming protein perforin to eliminate dangerous cells, while remaining refractory to lysis. At least two mechanisms jointly preserve the killer cell: the C-terminal residues of perforin dictate its rapid export from the endoplasmic reticulum (ER), whose milieu otherwise favours pore formation; perforin is then stored in secretory granules whose acidity prevent its oligomerisation. Following exocytosis, perforin delivers the proapoptotic protease, granzyme B, into the target cell by disrupting its plasma membrane. Although the precise mechanism of perforin/granzyme synergy remains controversial, the recently defined crystal structure of the perforin monomer and cryo-electron microscopy (EM) of the entire pore suggest that passive transmembrane granzyme diffusion is the dominant proapoptotic mechanism.
    Trends in Immunology 05/2012; 33(8):406-12. · 9.49 Impact Factor
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    ABSTRACT: The pore-forming protein perforin is central to the granule-exocytosis pathway used by cytotoxic lymphocytes to kill abnormal cells. Although this mechanism of killing is conserved in bony vertebrates, cytotoxic cells are present in other chordates and invertebrates, and their cytotoxic mechanism has not been elucidated. In order to understand the evolution of this pathway, here we characterize the origins and evolution of perforin. We identified orthologs and homologs of human perforin in all but one species analysed from Euteleostomi, and present evidence for an earlier ortholog in Gnathostomata but not in more primitive chordates. In placental mammals perforin is a single copy gene, but there are multiple perforin genes in all lineages predating marsupials, except birds. Our comparisons of these many-to-one homologs of human perforin show that they mainly arose from lineage-specific gene duplications in multiple taxa, suggesting acquisition of new roles or different modes of regulation. We also present evidence that perforin arose from duplication of the ancient MPEG1 gene, and that it shares a common ancestor with the functionally related complement proteins. The evolution of perforin in vertebrates involved a complex pattern of gene, as well as intron, gain and loss. The primordial perforin gene arose at least 500 million years ago, at around the time that the major histocompatibility complex-T cell receptor antigen recognition system was established. As it is absent from primitive chordates and invertebrates, cytotoxic cells from these lineages must possess a different effector molecule or cytotoxic mechanism.
    BMC Evolutionary Biology 05/2012; 12:59. · 3.29 Impact Factor
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    ABSTRACT: Recently, it has been reported that human B cells express and secrete the cytotoxic protease granzyme B (GrB) after stimulation with IL-21 and BCR cross-linking. To date, there are few clues on the function of GrB in B cell biology. As experimental transgenic murine systems should provide insights into these issues, we assayed for GrB in C57BL/6 B cells using an extensive array of physiologically relevant stimuli but were unable to detect either GrB expression or its proteolytic activity, even when Ag-specific transgenic BCRs were engaged. Similar results were also obtained with B cells from DBA/2, CBA, or BALB/c mice. In vivo, infection with either influenza virus or murine γ-herpesvirus induced the expected expression of GrB in CTLs, but not in B cell populations. We also investigated a possible role of GrB on the humoral immune response to the model Ag 4-hydroxy-3-nitrophenylacetyl-keyhole limpet hemocyanin, but GrB-deficient mice produced normal amounts of Ab with typical affinity maturation and a heightened secondary response, demonstrating conclusively the redundancy of GrB for Ab responses. Our results highlight the complex evolutionary differences that have shaped the immune systems of mice and humans. The physiological consequences of GrB expression in human B cells remain unclear, and the current study suggests that experimental mouse models will not be helpful in addressing this issue.
    The Journal of Immunology 03/2012; 188(8):3886-92. · 5.52 Impact Factor
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    Sabine Hoves, Vivien R Sutton, Joseph A Trapani
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    ABSTRACT: The cytotoxic properties of granzymes are well established, though recent publications suggest additional roles for granzymes in immunity. We demonstrated that granzymes can act as regulators of cross-presentation by dendritic cells by inducing critical "eat-me" signals on the dying tumor cell, resulting in efficient phagocytosis of cell-associated tumor antigen.
    Oncoimmunology. 03/2012; 1(2):219-221.

Publication Stats

9k Citations
1,560.03 Total Impact Points

Institutions

  • 2000–2014
    • Peter MacCallum Cancer Centre
      • • Cancer Immunology Program
      • • Gene Regulation Laboratory
      • • Cancer Cell Death Laboratory
      Melbourne, Victoria, Australia
  • 1996–2013
    • Monash University (Australia)
      • • Department of Biochemistry and Molecular Biology
      • • Department of Medicine
      Melbourne, Victoria, Australia
  • 1985–2013
    • University of Melbourne
      • • Sir Peter MacCallum Department of Oncology
      • • Department of Microbiology and Immunology
      • • Department of Pathology
      Melbourne, Victoria, Australia
  • 2012
    • Ludwig-Maximilian-University of Munich
      • Department of Clinical Pharmacology
      München, Bavaria, Germany
    • University of Auckland
      • Faculty of Medical and Health Sciences
      Auckland, Auckland, New Zealand
  • 2006–2012
    • Saint Vincent's Institute
      Fitzroy, Victoria, Australia
  • 2011
    • The Walter and Eliza Hall Institute of Medical Research
      • Division of Immunology
      Melbourne, Victoria, Australia
  • 2009
    • University of Zaragoza
      Caesaraugusta, Aragon, Spain
  • 1996–2001
    • Australian National University
      • Molecular Bioscience Department
      Canberra, Australian Capital Territory, Australia
  • 1997–1998
    • Box Hill Institute
      Box Hill, Victoria, Australia
  • 1992–1998
    • Austin Health
      Melbourne, Victoria, Australia
  • 1988–1990
    • Memorial Sloan-Kettering Cancer Center
      • Human Immunogenetics Laboratory
      New York City, New York, United States
  • 1983
    • Royal Melbourne Hospital
      Melbourne, Victoria, Australia
    • Victoria University Melbourne
      Melbourne, Victoria, Australia