Sir Peter MacCallum Department of Oncology - Research Publications

Permanent URI for this collection

http://hdl.handle.net/11343/274

Filters

2013 - 2015 3
3
Reset filters

Settings
Statistics
Citations
Author: Trapani, Joseph × Author: Jenkins, Misty-Rayna × Author: Lopez, Jamie ×

Search Results

Now showing 1 - 3 of 3
  • Item
    No Preview Available
    Perforin forms transient pores on the target cell plasma membrane to facilitate rapid access of granzymes during killer cell attack
    Lopez, JA ; Susanto, O ; Jenkins, MR ; Lukoyanova, N ; Sutton, VR ; Law, RHP ; Johnston, A ; Bird, CH ; Bird, PI ; Whisstock, JC ; Trapani, JA ; Saibil, HR ; Voskoboinik, I (AMER SOC HEMATOLOGY, 2013-04-04)
    Cytotoxic lymphocytes serve a key role in immune homeostasis by eliminating virus-infected and transformed target cells through the perforin-dependent delivery of proapoptotic 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, on recognition of targets, calcium influx into the lymphocyte led to perforin exocytosis and target cell permeabilization in as little as 30 seconds. Within the synaptic cleft, target cell permeabilization by perforin resulted in the rapid diffusion of extracellular milieu-derived granzymes. Repair of these pores was initiated within 20 seconds and was completed within 80 seconds, thus limiting granzyme diffusion. Remarkably, even such a short time frame 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 minutes of perforin permeabilization. This study defines the final sequence of events controlling cytotoxic lymphocyte immune defense, in which perforin pores assemble on the target cell plasma membrane, ensuring efficient delivery of lethal granzymes.
  • Item
    Thumbnail Image
    Failed CTL/NK cell killing and cytokine hypersecretion are directly linked through prolonged synapse time
    Jenkins, MR ; Rudd-Schmidt, JA ; Lopez, JA ; Ramsbottom, KM ; Mannering, SI ; Andrews, DM ; Voskoboinik, I ; Trapani, JA (ROCKEFELLER UNIV PRESS, 2015-03)
    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.
  • Item
    No Preview Available
    Rapid and Unidirectional Perforin Pore Delivery at the Cytotoxic Immune Synapse
    Lopez, JA ; Jenkins, MR ; Rudd-Schmidt, JA ; Brennan, AJ ; Danne, JC ; Mannering, SI ; Trapani, JA ; Voskoboinik, I (AMER ASSOC IMMUNOLOGISTS, 2013-09-01)
    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.