Joseph A Trapani

Peter MacCallum Cancer Centre, Melbourne, Victoria, Australia

Are you Joseph A Trapani?

Claim your profile

Publications (255)1655.47 Total impact

  • [Show abstract] [Hide abstract]
    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.
    Immunology and Cell Biology 03/2015; DOI:10.1038/icb.2015.1 · 4.21 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    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.
    Journal of Experimental Medicine 03/2015; 212(3). DOI:10.1084/jem.20140964 · 13.91 Impact Factor
  • [Show abstract] [Hide abstract]
    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.
    02/2015; DOI:10.1158/2326-6066.CIR-15-0048
  • Source
    [Show abstract] [Hide abstract]
    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.
    PLoS Biology 02/2015; 13(2):e1002049. DOI:10.1371/journal.pbio.1002049 · 11.77 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    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.
    PLoS Pathogens 12/2014; 10:e1004526. DOI:10.1371/journal.ppat.1004526 · 8.14 Impact Factor
  • [Show abstract] [Hide abstract]
    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.
    Biology of Blood and Marrow Transplantation 11/2014; 21(2). DOI:10.1016/j.bbmt.2014.11.003 · 3.35 Impact Factor
  • [Show abstract] [Hide abstract]
    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.
  • [Show abstract] [Hide abstract]
    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. © 2014 S. Karger AG, Basel.
    International Archives of Allergy and Immunology 10/2014; 165(1):68-74. DOI:10.1159/000368403 · 2.43 Impact Factor
  • [Show abstract] [Hide abstract]
    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.
    Immunity 10/2014; 41(4):646-56. DOI:10.1016/j.immuni.2014.09.013 · 19.75 Impact Factor
  • [Show abstract] [Hide abstract]
    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; DOI:10.1007/s13318-014-0220-y · 1.31 Impact Factor
  • I. Voskoboinik, K. Thia, J.A. Trapani
    Internal Medicine Journal 08/2014; 44(8). DOI:10.1111/imj.12493 · 1.70 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    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 03/2014; 5:e1115. DOI:10.1038/cddis.2014.74 · 5.18 Impact Factor
  • [Show abstract] [Hide abstract]
    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; 289(13). DOI:10.1074/jbc.M113.544890 · 4.60 Impact Factor
  • [Show abstract] [Hide abstract]
    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; DOI:10.1093/intimm/dxu001 · 3.18 Impact Factor
  • [Show abstract] [Hide abstract]
    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; DOI:10.1038/cdd.2013.203 · 8.39 Impact Factor
  • Magdalena Hagn, Vivien R Sutton, Joseph A Trapani
    [Show abstract] [Hide abstract]
    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.
    Journal of Visualized Experiments 01/2014; DOI:10.3791/52419
  • Source
    Ilia Voskoboinik, Joseph A Trapani
    [Show abstract] [Hide abstract]
    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 12/2013; 4:441. DOI:10.3389/fimmu.2013.00441
  • [Show abstract] [Hide abstract]
    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; DOI:10.1042/BJ20130999 · 4.78 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    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; 191(5). DOI:10.4049/jimmunol.1301205 · 5.36 Impact Factor
  • [Show abstract] [Hide abstract]
    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; DOI:10.1038/mt.2013.154 · 6.43 Impact Factor

Publication Stats

12k Citations
1,655.47 Total Impact Points

Institutions

  • 2000–2015
    • Peter MacCallum Cancer Centre
      • • Peter MacCallum Cancer Center
      • • Cancer Immunology Program
      • • Cancer Cell Death Laboratory
      Melbourne, Victoria, Australia
  • 1985–2014
    • University of Melbourne
      • • Sir Peter MacCallum Department of Oncology
      • • Department of Pathology
      Melbourne, Victoria, Australia
  • 2011
    • The Walter and Eliza Hall Institute of Medical Research
      • Division of Immunology
      Melbourne, Victoria, Australia
  • 2007
    • Washington University in St. Louis
      • Department of Pathology and Immunology
      San Luis, Missouri, United States
  • 2006
    • Saint Vincent's Institute
      Melbourne, Victoria, Australia
    • University of Freiburg
      • Institute of Molecular Medicine and Cell Research
      Freiburg, Baden-Württemberg, Germany
  • 1997–2006
    • University of Vic
      Vic, Catalonia, Spain
    • University of Manitoba
      • Manitoba Institute of Cell Biology
      Winnipeg, Manitoba, Canada
  • 2005
    • Monash University (Australia)
      • Department of Biochemistry and Molecular Biology
      Melbourne, Victoria, Australia
  • 1988–2002
    • Royal Melbourne Hospital
      Melbourne, Victoria, Australia
  • 1999
    • Australian National University
      • Molecular Bioscience Department
      Canberra, Australian Capital Territory, Australia
  • 1998
    • Box Hill Institute
      Box Hill, Victoria, Australia
    • Eastern Virginia Medical School
      Norfolk, Virginia, United States
  • 1992–1997
    • Austin Health
      Melbourne, Victoria, Australia
  • 1996
    • Queensland Institute of Medical Research
      Brisbane, Queensland, Australia
  • 1990
    • The Rockefeller University
      • Laboratory of Cellular Physiology and Immunology
      New York City, NY, United States
  • 1988–1990
    • Memorial Sloan-Kettering Cancer Center
      • Human Immunogenetics Laboratory
      New York City, New York, United States
  • 1989
    • Roswell Park Cancer Institute
      Buffalo, New York, United States
  • 1983
    • Victoria University Melbourne
      Melbourne, Victoria, Australia