ORCID Profile
0000-0002-2097-6428
Current Organisations
Peter MacCallum Cancer Centre
,
Massey University
,
Monash University
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Publisher: Springer Science and Business Media LLC
Date: 04-02-2019
DOI: 10.1038/S41598-018-37883-Y
Abstract: Both targeted therapy and immunotherapy have been used successfully to treat melanoma, but the development of resistance and poor response rates to the in idual therapies has limited their success. Designing rational combinations of targeted therapy and immunotherapy may overcome these obstacles, but requires assessment in preclinical models with the capacity to respond to both therapeutic classes. Herein, we describe the development and characterization of a novel, immunogenic variant of the Braf V600E Cdkn2a −/− Pten −/− YUMM1.1 tumor model that expresses the immunogen, ovalbumin (YOVAL1.1). We demonstrate that, unlike parental tumors, YOVAL1.1 tumors are immunogenic in vivo and can be controlled by immunotherapy. Importantly, YOVAL1.1 tumors are sensitive to targeted inhibitors of BRAF V600E and MEK, responding in a manner consistent with human BRAF V600E melanoma. The YOVAL1.1 melanoma model is transplantable, immunogenic and sensitive to clinical therapies, making it a valuable platform to guide strategic development of combined targeted therapy and immunotherapy approaches in BRAF V600E melanoma.
Publisher: Wiley
Date: 04-11-2008
DOI: 10.1038/ICB.2008.79
Abstract: With new imaging technologies and fluorescent probes, live imaging of cells in vitro has revolutionized many aspects of cell biology. A key goal now is to develop systems to optimize in vitro imaging, which do not compromise the physiological relevance of the study. We have developed a methodology that contains non-adherent cells within the field of view. 'Cell paddocks' are created by generating an array of microgrids using polydimethylsiloxane. Each microgrid is up to 250 x 250 microm(2) with a height of 60 microm. Overlayed cells settle into the grids and the walls restrict their lateral movement, but a contiguous supply of medium between neighboring microgrids facilitates the exchange of cytokines and growth factors. This allows culture over at least 6 days with no impact upon viability and proliferation. Adaptations of the microgrids have enabled imaging and tracking of lymphocyte ision through multiple generations of long-term interactions between T lymphocytes and dendritic cells, and of thymocyte-stromal cell interactions.
Publisher: MDPI AG
Date: 14-01-2020
DOI: 10.3390/CELLS9010207
Abstract: One of the hallmarks of cancer cells is their ability to evade cell death via apoptosis. The inhibitor of apoptosis proteins (IAPs) are a family of proteins that act to promote cell survival. For this reason, upregulation of IAPs is associated with a number of cancer types as a mechanism of resistance to cell death and chemotherapy. As such, IAPs are considered a promising therapeutic target for cancer treatment, based on the role of IAPs in resistance to apoptosis, tumour progression and poor patient prognosis. The mitochondrial protein smac (second mitochondrial activator of caspases), is an endogenous inhibitor of IAPs, and several small molecule mimetics of smac (smac-mimetics) have been developed in order to antagonise IAPs in cancer cells and restore sensitivity to apoptotic stimuli. However, recent studies have revealed that smac-mimetics have broader effects than was first attributed. It is now understood that they are key regulators of innate immune signalling and have wide reaching immuno-modulatory properties. As such, they are ideal candidates for immunotherapy combinations. Pre-clinically, successful combination therapies incorporating smac-mimetics and oncolytic viruses, as with chimeric antigen receptor (CAR) T cell therapy, have been reported, and clinical trials incorporating smac-mimetics and immune checkpoint blockade are ongoing. Here, the potential of IAP antagonism to enhance immunotherapy strategies for the treatment of cancer will be discussed.
Publisher: Hindawi Limited
Date: 2012
DOI: 10.1155/2012/417485
Abstract: Polarity refers to the asymmetric distribution of different cellular components within a cell and is central to many cell functions. In T-cells, polarity regulates the activation, migration, and effector function of cytotoxic T-cells (CTLs) during an immune response. The regulation of asymmetric cell ision by polarity proteins may also dictate CTL effector and memory differentiation following antigen presentation. Small GTPases, along with their associated polarity and adaptor proteins, are critical for mediating the polarity changes necessary for T-cell activation and function, and in turn, are regulated by guanine exchange factors (GEFS) and GTPase activating proteins (GAPS). For ex le, a novel GEF, dedicator of cytokinesis 8 (DOCK8) was recently identified as a regulator of immune cell function and mutations in DOCK8 have been detected in patients with severe combined immunodeficiency. Both B and T-cells from DOCK8 mutant mice form defective immunological synapses and have abnormal functions, in addition to impaired immune memory development. This paper will discuss the interplay between polarity proteins and GTPases, and their role in T-cell function.
Publisher: American Association for Cancer Research (AACR)
Date: 04-04-2023
DOI: 10.1158/2326-6066.22544828.V1
Abstract: Cell lines used in this study.
Publisher: American Society for Microbiology
Date: 06-2000
DOI: 10.1128/IAI.68.6.3337-3343.2000
Abstract: Experiments were performed using the standardized murine model of Helicobacter pylori infection to determine the immunogenicity of H. pylori outer membrane vesicles in immune protection. These vesicles, which are naturally shed from the surface of the bacterium, induce a protective response when administered intragastrically to mice in the presence of cholera holotoxin, despite the absence of the urease enzyme and associated Hsp54 chaperonin. Immunoblotting identified a specific serum immunoglobulin G (IgG) response to an 18-kDa outer membrane protein in a significant number of immunized animals. This commonly expressed, immunodominant protein was subsequently identified as lipoprotein 20 (Lpp20). Hybridoma backpacks secreting an IgG1 subclass monoclonal antibody to Lpp20 were generated in H. pylori -infected mice and were found to significantly reduce bacterial numbers, providing evidence that this surface-exposed antigen is a true vaccine candidate and not merely an antigenic marker for successful, protective immunization.
Publisher: The American Association of Immunologists
Date: 12-2014
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.
Publisher: The American Association of Immunologists
Date: 15-01-2014
Abstract: DNAX accessory molecule 1 (DNAM-1) is expressed on all CD8+ T cells and promotes their activation and effector function. DNAM-1 interacts with LFA-1, a critical molecule for immunological synapse formation between T cells and APCs, and for cytotoxic killing of target cells. Mice that lack DNAM-1 display abnormal T cell responses and antitumor activity however, the mechanism involved is unclear. In this article, we show that DNAM-1 deficiency results in reduced proliferation of CD8+ T cells after Ag presentation and impaired cytotoxic activity. We also demonstrate that DNAM-1–deficient T cells show reduced conjugations with tumor cells and decreased recruitment of both LFA-1 and lipid rafts to the immunological synapse, which correlates with reduced tumor cell killing in vitro. This synapse defect may explain why DNAM-1–deficient mice cannot clear tumors in vivo, and highlights the importance of DNAM-1 and the immunological synapse in T cell–mediated antitumor immunity.
Publisher: American Association for Cancer Research (AACR)
Date: 04-04-2023
DOI: 10.1158/2326-6066.22543848.V1
Abstract: Supplementary Table S1
Publisher: American Association for Cancer Research (AACR)
Date: 14-05-2021
DOI: 10.1158/2159-8290.CD-20-1554
Abstract: Immunologic memory is critical for sustained antitumor immunity. Our discovery that CDK4/6 inhibitors drive T-cell memory fate commitment sheds new light on their clinical activity, which is essential for the design of clinical trial protocols incorporating these agents, particularly in combination with immunotherapy, for the treatment of cancer. This article is highlighted in the In This Issue feature, p. 2355
Publisher: Elsevier BV
Date: 09-2019
DOI: 10.1016/J.CELREP.2019.08.017
Abstract: Despite the clinical success of cancer immunotherapies, the majority of patients fail to respond or develop resistance through disruption of pathways that promote neo-antigen presentation on MHC I molecules. Here, we conducted a series of unbiased, genome-wide CRISPR/Cas9 screens to identify genes that limit natural killer (NK) cell anti-tumor activity. We identified that genes associated with antigen presentation and/or interferon-γ (IFN-γ) signaling protect tumor cells from NK cell killing. Indeed, Jak1-deficient melanoma cells were sensitized to NK cell killing through attenuated NK cell-derived IFN-γ-driven transcriptional events that regulate MHC I expression. Importantly, tumor cells that became resistant to T cell killing through enrichment of MHC I-deficient clones were highly sensitive to NK cell killing. Taken together, we reveal the genes targeted by tumor cells to drive checkpoint blockade resistance but simultaneously increase their vulnerability to NK cells, unveiling NK cell-based immunotherapies as a strategy to antagonize tumor immune escape.
Publisher: Wiley
Date: 07-09-2010
DOI: 10.1016/J.FEBSLET.2010.09.003
Abstract: CD46 is a cell surface protein that regulates complement activity and is utilized as a receptor by numerous viral and bacterial pathogens that infect humans. CD46 is not just an entry site for pathogens, but can affect various cellular activities in response to pathogen binding that can have profound consequences for the host response to infection. The study of CD46 signaling in T cells has emerged as an exciting area of research that is shedding new light on how pathogens might manipulate the host immune response. This review will focus on our current understanding of CD46 signaling in T cell polarity and how this might influence disease outcome.
Publisher: American Association for Cancer Research (AACR)
Date: 04-04-2023
DOI: 10.1158/2326-6066.22544807
Abstract: Supp Video 4
Publisher: Wiley
Date: 22-09-2015
DOI: 10.1038/ICB.2015.82
Abstract: Lethal giant larvae-1 (Lgl-1) is an evolutionary conserved protein that regulates cell polarity in erse lineages however, the role of Lgl-1 in the polarity and function of immune cells remains to be elucidated. To assess the role of Lgl-1 in T cells, we generated chimeric mice with a hematopoietic system deficient for Lgl-1. Lgl-1 deficiency did not impair the activation or function of peripheral CD8(+) T cells in response to antigen presentation in vitro, but did skew effector and memory T-cell differentiation. When challenged with antigen-expressing virus or tumor, Lgl-1-deficient mice displayed altered T-cell responses. This manifested in a stronger antiviral and antitumor effector CD8(+) T-cell response, the latter resulting in enhanced control of MC38-OVA tumors. These results reveal a novel role for Lgl-1 in the regulation of virus-specific T-cell responses and antitumor immunity.
Publisher: Public Library of Science (PLoS)
Date: 27-01-2014
Publisher: American Association for Cancer Research (AACR)
Date: 04-04-2023
DOI: 10.1158/2326-6066.22543851.V1
Abstract: Supplementary Figures S1-S5
Publisher: Informa UK Limited
Date: 27-06-2016
Publisher: American Association for Cancer Research (AACR)
Date: 04-04-2023
DOI: 10.1158/2326-6066.22544825.V1
Abstract: Supplementary Video Legends and Supplementary Figures 1-10
Publisher: The American Association of Immunologists
Date: 07-2010
Abstract: Asymmetric cell ision is a potential means by which cell fate choices during an immune response are orchestrated. Defining the molecular mechanisms that underlie asymmetric ision of T cells is paramount for determining the role of this process in the generation of effector and memory T cell subsets. In other cell types, asymmetric cell ision is regulated by conserved polarity protein complexes that control the localization of cell fate determinants and spindle orientation during ision. We have developed a tractable, in vitro model of naive CD8+ T cells undergoing initial ision while attached to dendritic cells during Ag presentation to investigate whether similar mechanisms might regulate asymmetric ision of T cells. Using this system, we show that direct interactions with APCs provide the cue for polarization of T cells. Interestingly, the immunological synapse disseminates before ision even though the T cells retain contact with the APC. The cue from the APC is translated into polarization of cell fate determinants via the polarity network of the Par3 and Scribble complexes, and orientation of the mitotic spindle during ision is orchestrated by the partner of inscuteable/G protein complex. These findings suggest that T cells have selectively adapted a number of evolutionarily conserved mechanisms to generate ersity through asymmetric cell ision.
Publisher: American Association for Cancer Research (AACR)
Date: 04-04-2023
DOI: 10.1158/2326-6066.22544813
Abstract: Supp Video 3
Publisher: American Association for Cancer Research (AACR)
Date: 02-2021
DOI: 10.1158/2326-6066.CIR-20-0401
Abstract: Combined inhibition of BRAF, MEK, and CDK4/6 is currently under evaluation in clinical trials for patients with melanoma harboring a BRAFV600 mutation. While this triple therapy has potent tumor-intrinsic effects, the impact of this combination on antitumor immunity remains unexplored. Here, using a syngeneic BrafV600ECdkn2a−/−Pten−/− melanoma model, we demonstrated that triple therapy promoted durable tumor control through tumor-intrinsic mechanisms and promoted immunogenic cell death and T-cell infiltration. Despite this, tumors treated with triple therapy were unresponsive to immune checkpoint blockade (ICB). Flow cytometric and single-cell RNA sequencing analyses of tumor-infiltrating immune populations revealed that triple therapy markedly depleted proinflammatory macrophages and cross-priming CD103+ dendritic cells, the absence of which correlated with poor overall survival and clinical responses to ICB in patients with melanoma. Indeed, immune populations isolated from tumors of mice treated with triple therapy failed to stimulate T-cell responses ex vivo. While combined BRAF, MEK, and CDK4/6 inhibition demonstrates favorable tumor-intrinsic activity, these data suggest that collateral effects on tumor-infiltrating myeloid populations may impact antitumor immunity. These findings have important implications for the design of combination strategies and clinical trials that incorporate BRAF, MEK, and CDK4/6 inhibition with immunotherapy for the treatment of patients with melanoma.
Publisher: Rockefeller University Press
Date: 14-09-2015
Abstract: During mammalian T cell development, the requirement for expansion of many in idual T cell clones, rather than merely expansion of the entire T cell population, suggests a possible role for asymmetric cell ision (ACD). We show that ACD of developing T cells controls cell fate through differential inheritance of cell fate determinants Numb and α-Adaptin. ACD occurs specifically during the β-selection stage of T cell development, and subsequent isions are predominantly symmetric. ACD is controlled by interaction with stromal cells and chemokine receptor signaling and uses a conserved network of polarity regulators. The disruption of polarity by deletion of the polarity regulator, Scribble, or the altered inheritance of fate determinants impacts subsequent fate decisions to influence the numbers of DN4 cells arising after the β-selection checkpoint. These findings indicate that ACD enables the thymic microenvironment to orchestrate fate decisions related to differentiation and self-renewal.
Publisher: Springer Science and Business Media LLC
Date: 30-06-2017
DOI: 10.1038/CDD.2017.94
Publisher: Elsevier BV
Date: 12-2002
DOI: 10.1016/S0378-1135(02)00223-7
Abstract: Pathogens have developed different strategies to survive and multiply within their host. Among them is the ability to control phagocyte apoptosis while another is to affect the expression of cytokines which is necessary for a normal protective function of the immune response. To establish themselves and cause chronic disease in humans and animals, Brucella spp. invade and proliferate within monocytic phagocytes. We have established that in humans, Brucella suis impairs the apoptosis of monocytes and macrophages, thus preventing its host cell elimination. In mice, which are not naturally colonized by the bacteria, Brucella infection results in Type1 (Th1) cellular immune response which promotes a clearance of the bacterial organism. The development of this response is under the control of major cytokines like TNF-alpha, IFN-gamma and IL-12 produced at the onset of infection. We have observed that in humans, B. suis-infected macrophages which produce IL-1, IL-6, IL-10 and several chemokines including IL-8, do not secrete TNF-alpha. By constructing null mutants, we demonstrated that this inhibition involves the outer membrane protein Omp25 of Brucella, however the mechanism regulating the inhibition has not yet been clearly defined. It is likely that the Omp25-induced effect on TNF-alpha production assists bacterial evasion of antimicrobial defences at different levels. Firstly, by preventing the autocrine activation of macrophages thus inhibiting innate immunity and secondly by impairing the production of IL-12 and the development of a Th1 type specific immunity. In addition to the central role of the macrophage in Brucella infection, others cells of the innate immune response are recruited and influenced by the interactions between bacteria and host. For instance, human Vgamma9Vdelta2 T-cells play an important role in the early response to infection with intracellular pathogens. Evidence has been presented that their number dramatically increased in the peripheral blood of patients with acute brucellosis. We have shown that human Vgamma9Vdelta2 T-cells can be specifically activated by non-peptidic low molecular weight compound(s) from B. suis lysate or by soluble factors produced by B. suis-infected macrophages. Under these conditions, they produce TNF-alpha and IFN-gamma and reduce the bacterial multiplication inside infected autologous macrophages. This impairment of B. suis multiplication is due to both soluble factors released from activated gammadeltaT-cells (including TNF-alpha and IFN-gamma) and to a contact-dependent cytotoxicity directed against the infected cells. The interactions between the bacteria and these cells can counteract the intramacrophagic development of the bacteria and finally influence the further development of the host defense. We hypothesize that the chronicity or the elimination of the infection will depend on the balance between contradictory effects induced by the bacteria which favor either the host or the pathogen. Moreover, the interrelationship between the different cells must be taken into account in the analysis of the virulence of the bacteria and in the development of in vitro models of human macrophage infection.
Publisher: American Association for the Advancement of Science (AAAS)
Date: 19-02-2021
Abstract: A new technology, SUGAR-seq, enables simultaneous detection of surface glycans, epitopes, transcripts, and TCR repertoire.
Publisher: American Association for Cancer Research (AACR)
Date: 04-04-2023
DOI: 10.1158/2326-6066.22544816
Abstract: Supp Video 2
Publisher: American Association for Cancer Research (AACR)
Date: 04-04-2023
DOI: 10.1158/2326-6066.22543848
Abstract: Supplementary Table S1
Publisher: The American Association of Immunologists
Date: 15-09-2017
Abstract: Mutations in the dedicator of cytokinesis 8 (DOCK8) gene cause an autosomal recessive form of hyper-IgE syndrome, characterized by chronic immunodeficiency with persistent microbial infection and increased incidence of malignancy. These manifestations suggest a defect in cytotoxic lymphocyte function and immune surveillance. However, how DOCK8 regulates NK cell–driven immune responses remains unclear. In this article, we demonstrate that DOCK8 regulates NK cell cytotoxicity and cytokine production in response to target cell engagement or receptor ligation. Genetic ablation of DOCK8 in human NK cells attenuated cytokine transcription and secretion through inhibition of Src family kinase activation, particularly Lck, downstream of target cell engagement or NKp30 ligation. PMA/Ionomycin treatment of DOCK8-deficient NK cells rescued cytokine production, indicating a defect proximal to receptor ligation. Importantly, NK cells from DOCK8-deficient patients had attenuated production of IFN-γ and TNF-α upon NKp30 stimulation. Taken together, we reveal a novel molecular mechanism by which DOCK8 regulates NK cell–driven immunity.
Publisher: American Association for Cancer Research (AACR)
Date: 2022
DOI: 10.1158/2326-6066.CIR-21-0224
Abstract: Targeting chromatin binding proteins and modifying enzymes can concomitantly affect tumor cell proliferation and survival, as well as enhance antitumor immunity and augment cancer immunotherapies. By screening a small-molecule library of epigenetics-based therapeutics, BET (bromo- and extra-terminal domain) inhibitors (BETi) were identified as agents that sensitize tumor cells to the antitumor activity of CD8+ T cells. BETi modulated tumor cells to be sensitized to the cytotoxic effects of the proinflammatory cytokine TNF. By preventing the recruitment of BRD4 to p65-bound cis-regulatory elements, BETi suppressed the induction of inflammatory gene expression, including the key NF-κB target genes BIRC2 (cIAP1) and BIRC3 (cIAP2). Disruption of prosurvival NF-κB signaling by BETi led to unrestrained TNF-mediated activation of the extrinsic apoptotic cascade and tumor cell death. Administration of BETi in combination with T-cell bispecific antibodies (TCB) or immune-checkpoint blockade increased bystander killing of tumor cells and enhanced tumor growth inhibition in vivo in a TNF-dependent manner. This novel epigenetic mechanism of immunomodulation may guide future use of BETi as adjuvants for immune-oncology agents.
Publisher: American Association for Cancer Research (AACR)
Date: 04-04-2023
DOI: 10.1158/2326-6066.C.6550757.V1
Abstract: Abstract Targeting chromatin binding proteins and modifying enzymes can concomitantly affect tumor cell proliferation and survival, as well as enhance antitumor immunity and augment cancer immunotherapies. By screening a small-molecule library of epigenetics-based therapeutics, BET (bromo- and extra-terminal domain) inhibitors (BETi) were identified as agents that sensitize tumor cells to the antitumor activity of CD8 sup + /sup T cells. BETi modulated tumor cells to be sensitized to the cytotoxic effects of the proinflammatory cytokine TNF. By preventing the recruitment of BRD4 to p65-bound i cis /i -regulatory elements, BETi suppressed the induction of inflammatory gene expression, including the key NF-κB target genes i BIRC2 /i (cIAP1) and i BIRC3 /i (cIAP2). Disruption of prosurvival NF-κB signaling by BETi led to unrestrained TNF-mediated activation of the extrinsic apoptotic cascade and tumor cell death. Administration of BETi in combination with T-cell bispecific antibodies (TCB) or immune-checkpoint blockade increased bystander killing of tumor cells and enhanced tumor growth inhibition i in vivo /i in a TNF-dependent manner. This novel epigenetic mechanism of immunomodulation may guide future use of BETi as adjuvants for immune-oncology agents. /
Publisher: Oxford University Press (OUP)
Date: 24-01-2005
DOI: 10.1189/JLB.0704433
Abstract: Human Vγ9Vδ2 T cells pay a crucial role in early immune response to intracellular pathogens. In brucellosis infection, this population of cells is drastically increased in the peripheral blood of patients during the acute phase of infection. In vitro, Vγ9Vδ2 T cells exhibit strong cytolytic activity against Brucella-infected cells and are able to impair intracellular growth of Brucella suis in autologous macrophages. In this study, we have investigated the relative importance of contact-dependent mechanisms versus soluble factors in the intracellular growth and viability of B. suis. We show that Vγ9Vδ2 T cells use contact-dependent mechanisms, such as the release of lytic granules and Fas-mediated signals, to decrease intracellular B. suis through lysis of infected macrophages, but these mechanisms have little impact on Brucella survival. Moreover, we demonstrate that soluble factors secreted by Vγ9Vδ2 T cells can directly affect B. suis survival through their potent bactericidal effects. From these results, we conclude that Vγ9Vδ2 T cells are able to use a combination of mechanisms that reduce the total numbers of B. suis and thus, may benefit the host by limiting the spread of this intracellular pathogen.
Publisher: American Association for Cancer Research (AACR)
Date: 03-04-2023
DOI: 10.1158/2159-8290.22540163.V1
Abstract: Supplementary Figures 1-12
Publisher: American Association for Cancer Research (AACR)
Date: 04-04-2023
DOI: 10.1158/2326-6066.22543851
Abstract: Supplementary Figures S1-S5
Publisher: Springer Science and Business Media LLC
Date: 18-01-2008
Abstract: Human GraB (hGraB) preferentially induces apoptosis via Bcl-2-regulated mitochondrial damage but can also directly cleave caspases and caspase substrates in cell-free systems. How hGraB kills cells when it is delivered by cytotoxic lymphocytes (CL) and the contribution of hGraB to CL-induced death is still not clear. We show that primary human natural killer (hNK) cells, which specifically used hGraB to induce target cell death, were able to induce apoptosis of cells whose mitochondria were protected by Bcl-2. Purified hGraB also induced apoptosis of Bcl-2-overexpressing targets but only when delivered at 5- to 10-fold the concentration required to kill cells expressing endogenous Bcl-2. Caspases were critical in this process as inhibition of caspase activity permitted clonogenic survival of Bcl-2-overexpressing cells treated with hGraB or hNK cells but did not protect cells that only expressed endogenous Bcl-2. Our data therefore show that hGraB triggers caspase activation via mitochondria-dependent and mitochondria-independent mechanisms that are activated in a hierarchical manner, and that the combined effects of Bcl-2 and direct caspase inhibition can block cell death induced by hGraB and primary hNK cells.
Publisher: Wiley
Date: 19-10-2010
DOI: 10.1038/ICB.2010.122
Abstract: The movement of proteins within cells can provide dynamic indications of cell signaling and cell polarity, but methods are needed to track and quantify subcellular protein movement within tissue environments. Here we present a semiautomated approach to quantify subcellular protein location for hundreds of migrating cells within intact living tissue using retrovirally expressed fluorescent fusion proteins and time-lapse two-photon microscopy of intact thymic lobes. We have validated the method using GFP-PKCζ, a marker for cell polarity, and LAT-GFP, a marker for T-cell receptor signaling, and have related the asymmetric distribution of these proteins to the direction and speed of cell migration. These approaches could be readily adapted to other fluorescent fusion proteins, tissues and biological questions.
Publisher: Springer Science and Business Media LLC
Date: 18-08-2006
Publisher: Elsevier BV
Date: 04-2011
Publisher: Rockefeller University Press
Date: 17-10-2011
DOI: 10.1084/JEM.20110345
Abstract: In humans, DOCK8 immunodeficiency syndrome is characterized by severe cutaneous viral infections. Thus, CD8 T cell function may be compromised in the absence of DOCK8. In this study, by analyzing mutant mice and humans, we demonstrate a critical, intrinsic role for DOCK8 in peripheral CD8 T cell survival and function. DOCK8 mutation selectively diminished the abundance of circulating naive CD8 T cells in both species, and in DOCK8-deficient humans, most CD8 T cells displayed an exhausted CD45RA+CCR7− phenotype. Analyses in mice revealed the CD8 T cell abnormalities to be cell autonomous and primarily postthymic. DOCK8 mutant naive CD8 T cells had a shorter lifespan and, upon encounter with antigen on dendritic cells, exhibited poor LFA-1 synaptic polarization and a delay in the first cell ision. Although DOCK8 mutant T cells underwent near-normal primary clonal expansion after primary infection with recombinant influenza virus in vivo, they showed greatly reduced memory cell persistence and recall. These findings highlight a key role for DOCK8 in the survival and function of human and mouse CD8 T cells.
Publisher: American Association for Cancer Research (AACR)
Date: 04-04-2023
DOI: 10.1158/2326-6066.22544822
Abstract: Supp Video 1
Publisher: American Association for Cancer Research (AACR)
Date: 03-04-2023
DOI: 10.1158/2159-8290.C.6549299.V1
Abstract: Abstract Pharmacologic inhibitors of cyclin-dependent kinases 4 and 6 (CDK4/6) are an approved treatment for hormone receptor–positive breast cancer and are currently under evaluation across hundreds of clinical trials for other cancer types. The clinical success of these inhibitors is largely attributed to well-defined tumor-intrinsic cytostatic mechanisms, whereas their emerging role as immunomodulatory agents is less understood. Using integrated epigenomic, transcriptomic, and proteomic analyses, we demonstrated a novel action of CDK4/6 inhibitors in promoting the phenotypic and functional acquisition of immunologic T-cell memory. Short-term priming with a CDK4/6 inhibitor promoted long-term endogenous antitumor T-cell immunity in mice, enhanced the persistence and therapeutic efficacy of chimeric antigen receptor T cells, and induced a retinoblastoma-dependent T-cell phenotype supportive of favorable responses to immune checkpoint blockade in patients with melanoma. Together, these mechanistic insights significantly broaden the prospective utility of CDK4/6 inhibitors as clinical tools to boost antitumor T-cell immunity. Significance: Immunologic memory is critical for sustained antitumor immunity. Our discovery that CDK4/6 inhibitors drive T-cell memory fate commitment sheds new light on their clinical activity, which is essential for the design of clinical trial protocols incorporating these agents, particularly in combination with immunotherapy, for the treatment of cancer. i This article is highlighted in the In This Issue feature, p. 2355 /i /
Publisher: American Association for Cancer Research (AACR)
Date: 04-04-2023
DOI: 10.1158/2326-6066.22544822.V1
Abstract: Supp Video 1
Publisher: American Association for Cancer Research (AACR)
Date: 04-04-2023
DOI: 10.1158/2326-6066.22544825
Abstract: Supplementary Video Legends and Supplementary Figures 1-10
Publisher: Wiley
Date: 02-2006
DOI: 10.1111/J.1440-1711.2005.01415.X
Abstract: Lymphocyte function is regulated by complex signalling responses to erse extracellular inputs, and a cell will often receive multiple, conflicting signals at one time. The mechanisms by which a lymphocyte integrates these signals into a single cellular response are not well understood. An important factor in the integration of signals likely involves the regulation of access of signalling molecules to cell surface receptors and of receptor signals to morphological determinants within the cell. Recent studies have led to important advances in our understanding of both the mechanisms by which signals are compartmentalized in T cells and the physiological role played by such compartmentalization. We review progress in the field, with a particular focus on membrane microdomains or lipid rafts and on cell polarity.
Publisher: American Society for Microbiology
Date: 04-2004
DOI: 10.1128/IAI.72.4.2303-2311.2004
Abstract: Brucella spp. are facultative intracellular bacteria that can establish themselves and cause chronic disease in humans and animals. NK cells play a key role in host defense. They are implicated in an early immune response to a variety of pathogens. However, it was shown that they do not control Brucella infection in mice. On the other hand, NK cell activity is impaired in patients with acute brucellosis, and recently it was demonstrated that human NK cells mediate the killing of intramacrophagic Mycobacterium tuberculosis in in vitro infection. Therefore, we have analyzed the behavior of Brucella suis infecting isolated human macrophages in the presence of syngeneic NK cells. We show that (i) NK cells impair the intramacrophagic development of B. suis , a phenomenon enhanced by NK cell activators, such as interleukin-2 (ii) NK cells cultured in the presence of infected macrophages are highly activated and secrete gamma interferon and tumor necrosis factor alpha (iii) impairment of bacterial multiplication inside infected cells is marginally associated with the cytokines produced during the early phase of macrophage-NK cell cocultures (iv) direct cell-to-cell contact is required for NK cells to mediate the inhibition of B. suis development and (v) inhibition of B. suis development results from an induction of NK cell cytotoxicity against infected macrophages. Altogether, these findings show that NK cells could participate early in controlling the intramacrophagic development of B. suis in humans. It seems thus reasonable to hypothesize a role for NK cells in the control of human brucellosis. However, by impairing the activity of these cells in the acute phase of the illness, the pathogen should avoid this control.
Publisher: Springer Science and Business Media LLC
Date: 03-04-2017
DOI: 10.1038/CMI.2017.9
Publisher: Wiley
Date: 20-02-2019
Abstract: Mutation of Dedicator of cytokinesis 8 (DOCK8) has previously been reported to provide resistance to the Th17 cell dependent EAE in mice. Contrary to expectation, we observed an elevation of Th17 cells in two different DOCK8 mutant mouse strains in the steady state. This was specific for Th17 cells with no change in Th1 or Th2 cell populations. In vitro Th cell differentiation assays revealed that the elevated Th17 cell population was not due to a T cell intrinsic differentiation bias. Challenging these mutant mice in the EAE model, we confirmed a resistance to this autoimmune disease with Th17 cells remaining elevated systemically while cellular infiltration in the CNS was reduced. Infiltrating T cells lost the bias toward Th17 cells indicating a relative reduction of Th17 cells in the CNS and a Th17 cell specific migration disadvantage. Adoptive transfers of Th1 and Th17 cells in EAE-affected mice further supported the Th17 cell-specific migration defect, however, DOCK8-deficient Th17 cells expressed normal Th17 cell-specific CCR6 levels and migrated toward chemokine gradients in transwell assays. This study shows that resistance to EAE in DOCK8 mutant mice is achieved despite a systemic Th17 bias.
Publisher: American Association for Cancer Research (AACR)
Date: 04-04-2023
DOI: 10.1158/2326-6066.C.6550431
Abstract: Abstract Combined inhibition of BRAF, MEK, and CDK4/6 is currently under evaluation in clinical trials for patients with melanoma harboring a i BRAF sup V600 /sup /i mutation. While this triple therapy has potent tumor-intrinsic effects, the impact of this combination on antitumor immunity remains unexplored. Here, using a syngeneic i Braf sup V600E /sup Cdkn2a sup −/− /sup Pten sup −/− /sup /i melanoma model, we demonstrated that triple therapy promoted durable tumor control through tumor-intrinsic mechanisms and promoted immunogenic cell death and T-cell infiltration. Despite this, tumors treated with triple therapy were unresponsive to immune checkpoint blockade (ICB). Flow cytometric and single-cell RNA sequencing analyses of tumor-infiltrating immune populations revealed that triple therapy markedly depleted proinflammatory macrophages and cross-priming CD103 sup + /sup dendritic cells, the absence of which correlated with poor overall survival and clinical responses to ICB in patients with melanoma. Indeed, immune populations isolated from tumors of mice treated with triple therapy failed to stimulate T-cell responses i ex vivo /i . While combined BRAF, MEK, and CDK4/6 inhibition demonstrates favorable tumor-intrinsic activity, these data suggest that collateral effects on tumor-infiltrating myeloid populations may impact antitumor immunity. These findings have important implications for the design of combination strategies and clinical trials that incorporate BRAF, MEK, and CDK4/6 inhibition with immunotherapy for the treatment of patients with melanoma. /
Publisher: American Association for Cancer Research (AACR)
Date: 04-04-2023
DOI: 10.1158/2326-6066.22544816.V1
Abstract: Supp Video 2
Publisher: American Association for Cancer Research (AACR)
Date: 04-04-2023
DOI: 10.1158/2326-6066.22544828
Abstract: Cell lines used in this study.
Publisher: American Association for Cancer Research (AACR)
Date: 04-04-2023
DOI: 10.1158/2326-6066.C.6550757
Abstract: Abstract Targeting chromatin binding proteins and modifying enzymes can concomitantly affect tumor cell proliferation and survival, as well as enhance antitumor immunity and augment cancer immunotherapies. By screening a small-molecule library of epigenetics-based therapeutics, BET (bromo- and extra-terminal domain) inhibitors (BETi) were identified as agents that sensitize tumor cells to the antitumor activity of CD8 sup + /sup T cells. BETi modulated tumor cells to be sensitized to the cytotoxic effects of the proinflammatory cytokine TNF. By preventing the recruitment of BRD4 to p65-bound i cis /i -regulatory elements, BETi suppressed the induction of inflammatory gene expression, including the key NF-κB target genes i BIRC2 /i (cIAP1) and i BIRC3 /i (cIAP2). Disruption of prosurvival NF-κB signaling by BETi led to unrestrained TNF-mediated activation of the extrinsic apoptotic cascade and tumor cell death. Administration of BETi in combination with T-cell bispecific antibodies (TCB) or immune-checkpoint blockade increased bystander killing of tumor cells and enhanced tumor growth inhibition i in vivo /i in a TNF-dependent manner. This novel epigenetic mechanism of immunomodulation may guide future use of BETi as adjuvants for immune-oncology agents. /
Publisher: Elsevier BV
Date: 12-2021
Publisher: Elsevier BV
Date: 02-2005
Publisher: Begell House
Date: 2015
DOI: 10.1615/CRITREVIMMUNOL.2015014417
Abstract: A synapse is a specialized structure that forms when the plasma membrane of two cells come into close contact to facilitate communication and signaling. Cells of the immune system form 'immunological' synapses that have an ordered structure and are essential for immune cell activation, function and homeostasis. Optimal synapse formation is not only critical for the generation of effective immunity against pathogens but is also essential for immune surveillance against cancer and for the prevention of immune disorders. Not surprisingly, defective synapse formation can therefore have severe consequences for human health, culminating in poor immune function leading to immunodeficiency disease or failure to detect and control infected or cancerous cells. Here, we discuss the immunological synapse formed by cytotoxic lymphocytes in both immunodeficiency diseases and anticancer immunity and touch on novel therapies that may alter or enhance synapse formation.
Publisher: American Association for Cancer Research (AACR)
Date: 04-08-2019
DOI: 10.1158/2326-6066.CIR-18-0428
Abstract: Chimeric antigen receptor (CAR) T-cell therapy has proven successful in the treatment of hematological malignancies, notably acute lymphoblastic leukemia and B-cell lymphoma. However, the efficacy of CAR T cells against solid tumors is poor, likely due to tumor-associated immunosuppression. Here, we demonstrated that antagonizing the “inhibitor of apoptosis proteins” with the clinical smac-mimetic, birinapant, significantly enhanced the antitumor activity of CAR T cells in a tumor necrosis factor (TNF)-dependent manner. Enhanced tumor cell death occurred independently of the perforin-mediated granule exocytosis pathway, underscoring the cytotoxic potential of CAR T-cell–derived TNF. Combining CAR T-cell therapy with birinapant significantly reduced established tumor growth in vivo, where either therapy alone was relatively ineffective. Using patient biopsy-derived tumoroids, we demonstrated the synergistic potential of combining CAR T-cell therapy with smac-mimetics. Taken together, we identified CAR T-cell–derived TNF as a potent antitumor effector, which can be further harnessed by smac-mimetics.
Publisher: American Association for Cancer Research (AACR)
Date: 03-04-2023
DOI: 10.1158/2159-8290.22540160.V1
Abstract: Supplementary Table S1
Publisher: American Association for Cancer Research (AACR)
Date: 04-04-2023
DOI: 10.1158/2326-6066.C.6550431.V1
Abstract: Abstract Combined inhibition of BRAF, MEK, and CDK4/6 is currently under evaluation in clinical trials for patients with melanoma harboring a i BRAF sup V600 /sup /i mutation. While this triple therapy has potent tumor-intrinsic effects, the impact of this combination on antitumor immunity remains unexplored. Here, using a syngeneic i Braf sup V600E /sup Cdkn2a sup −/− /sup Pten sup −/− /sup /i melanoma model, we demonstrated that triple therapy promoted durable tumor control through tumor-intrinsic mechanisms and promoted immunogenic cell death and T-cell infiltration. Despite this, tumors treated with triple therapy were unresponsive to immune checkpoint blockade (ICB). Flow cytometric and single-cell RNA sequencing analyses of tumor-infiltrating immune populations revealed that triple therapy markedly depleted proinflammatory macrophages and cross-priming CD103 sup + /sup dendritic cells, the absence of which correlated with poor overall survival and clinical responses to ICB in patients with melanoma. Indeed, immune populations isolated from tumors of mice treated with triple therapy failed to stimulate T-cell responses i ex vivo /i . While combined BRAF, MEK, and CDK4/6 inhibition demonstrates favorable tumor-intrinsic activity, these data suggest that collateral effects on tumor-infiltrating myeloid populations may impact antitumor immunity. These findings have important implications for the design of combination strategies and clinical trials that incorporate BRAF, MEK, and CDK4/6 inhibition with immunotherapy for the treatment of patients with melanoma. /
Publisher: Springer Science and Business Media LLC
Date: 04-12-2020
Publisher: Microbiology Society
Date: 07-2000
DOI: 10.1099/0022-1317-49-7-643
Abstract: The use of alkaline phosphatase fusion methodology to identify Helicobacter pylori exported proteins enabled the identification of an open reading frame (ORF) encoding a highly immunogenic, previously uncharacterised exported protein. The predicted aminoacid sequence displays a typical N-terminal signal peptide and contains regions of C-terminal hydrophobicity consistent with a membrane-associated protein. Southern blot analysis revealed that the gene encoding the protein was absent in several Helicobacter spp. and a combination of PCR and sequence analysis of the lified gene showed that it is highly conserved amongst isolates of H. pylori. To obtain pure recombinant protein, the gene encoding the protein was cloned and expressed as a beta-galactosidase (beta-gal) fusion in Escherichia coli and the protein was purified by affinity chromatography and proteolytic cleavage of the beta-gal portion. The purified protein, which has an apparent mol. wt of 18 kDa, was recognised by antibody present in 71% of sera from patients infected with H. pylori, but in only 16% of sera from patients with unrelated or no gastrointestinal disease, by Western blot assays. These results indicate that the 18-kDa protein from H. pylori is immunogenic and is expressed in vivo.
Publisher: Elsevier BV
Date: 06-2005
DOI: 10.1016/J.IMMUNI.2005.04.009
Abstract: T cell shape is dictated by the selective recruitment of molecules to different regions of the cell (polarity) and is integral to every aspect of T cell function, from migration to cytotoxicity. This study describes a mechanism for the regulation of T cell polarity. We show that T cells contain a network of asymmetrically distributed proteins with the capacity to dictate the subcellular localization of both cell surface receptors and morphological determinants in T cells. Proteins from the Scribble, Crumbs3, and Par3 complexes, previously shown to regulate epithelial polarity, were polarized in T cells containing either uropods or immunological synapses. Reduction in Scribble expression prevented the polarization of cell surface receptors and prevented morphological changes associated with uropod formation, migration, and antigen presentation. By dynamically coordinating molecular distribution throughout the T cell, this network provides a mechanism by which T cell function and polarity are linked.
Publisher: Proceedings of the National Academy of Sciences
Date: 05-12-2006
Abstract: Lymphocyte function in vivo is dictated by multiple external cues, but the integration of different signals is not well understood. Here, we show that competition for the axis of polarization dictates functional outcomes. We investigated the effect of ligation of the immunoregulatory cell surface receptor, CD46, on lymphocyte polarity during antigen presentation and cytotoxic effector function. Ligation of CD46 on human T cells prevented recruitment of the microtubule organizing center, CD3, and perforin to the interface with the antigen-presenting cell and caused a reduction in IFN-γ production. In human NK cells, similar changes in polarity induced by CD46 ligation inhibited the recruitment of the microtubule organizing center and perforin to the interface with target cells and correlated with reduced killing. These data indicate that external signals can alter lymphocyte polarization toward antigen-presenting cells or target cells, inhibiting lymphocyte function.
Publisher: Springer Science and Business Media LLC
Date: 13-05-2021
DOI: 10.1038/S41467-021-23044-9
Abstract: Chronic stimulation of CD8 + T cells triggers exhaustion, a distinct differentiation state with diminished effector function. Exhausted cells exist in multiple differentiation states, from stem-like progenitors that are the key mediators of the response to checkpoint blockade, through to terminally exhausted cells. Due to its clinical relevance, there is substantial interest in defining the pathways that control differentiation and maintenance of these subsets. Here, we show that chronic antigen induces the anergy-associated transcription factor EGR2 selectively within progenitor exhausted cells in both chronic LCMV and tumours. EGR2 enables terminal exhaustion and stabilizes the exhausted transcriptional state by both direct EGR2-dependent control of key exhaustion-associated genes, and indirect maintenance of the exhausted epigenetic state. We show that EGR2 is a regulator of exhaustion that epigenetically and transcriptionally maintains the differentiation competency of progenitor exhausted cells.
Publisher: American Association for Cancer Research (AACR)
Date: 03-04-2023
DOI: 10.1158/2159-8290.22540160
Abstract: Supplementary Table S1
Publisher: American Association for the Advancement of Science (AAAS)
Date: 23-03-2007
Abstract: A hallmark of mammalian immunity is the heterogeneity of cell fate that exists among pathogen-experienced lymphocytes. We show that a iding T lymphocyte initially responding to a microbe exhibits unequal partitioning of proteins that mediate signaling, cell fate specification, and asymmetric cell ision. Asymmetric segregation of determinants appears to be coordinated by prolonged interaction between the T cell and its antigen-presenting cell before ision. Additionally, the first two daughter T cells displayed phenotypic and functional indicators of being differentially fated toward effector and memory lineages. These results suggest a mechanism by which a single lymphocyte can apportion erse cell fates necessary for adaptive immunity.
Publisher: Hindawi Limited
Date: 2011
DOI: 10.1155/2011/521863
Abstract: Many infectious agents utilize CD46 for infection of human cells, and therapeutic applications of CD46-binding viruses are now being explored. Besides mediating internalization to enable infection, binding to CD46 can directly alter immune function. In particular, ligation of CD46 by antibodies or by measles virus can prevent activation of T cells by altering T-cell polarity and consequently preventing the formation of an immunological synapse. Here, we define a mechanism by which CD46 reorients T-cell polarity to prevent T-cell receptor signaling in response to antigen presentation. We show that CD46 associates with lipid rafts upon ligation, and that this reduces recruitment of both lipid rafts and the microtubule organizing centre to the site of receptor cross-linking. These data combined indicate that polarization of T cells towards the site of CD46 ligation prevents formation of an immunological synapse, and this is associated with the ability of CD46 to recruit lipid rafts away from the site of TCR ligation.
Publisher: American Association for the Advancement of Science (AAAS)
Date: 04-05-2018
DOI: 10.1126/SCIIMMUNOL.AAR3451
Abstract: Whole-genome CRISPR screens identify resistance to TNF-mediated killing by T and NK cells as a tumor immune evasion mechanism.
Publisher: Springer Science and Business Media LLC
Date: 06-08-2021
DOI: 10.1038/S41467-021-25009-4
Abstract: The function of MR1-restricted mucosal-associated invariant T (MAIT) cells in tumor immunity is unclear. Here we show that MAIT cell-deficient mice have enhanced NK cell-dependent control of metastatic B16F10 tumor growth relative to control mice. Analyses of this interplay in human tumor s les reveal that high expression of a MAIT cell gene signature negatively impacts the prognostic significance of NK cells. Paradoxically, pre-pulsing tumors with MAIT cell antigens, or activating MAIT cells in vivo, enhances anti-tumor immunity in B16F10 and E0771 mouse tumor models, including in the context of established metastasis. These effects are associated with enhanced NK cell responses and increased expression of both IFN-γ-dependent and inflammatory genes in NK cells. Importantly, activated human MAIT cells also promote the function of NK cells isolated from patient tumor s les. Our results thus describe an activation-dependent, MAIT cell-mediated regulation of NK cells, and suggest a potential therapeutic avenue for cancer treatment.
Publisher: EMBO
Date: 09-2021
Publisher: American Association for Cancer Research (AACR)
Date: 04-04-2023
DOI: 10.1158/2326-6066.22544813.V1
Abstract: Supp Video 3
Publisher: American Association for Cancer Research (AACR)
Date: 04-04-2023
DOI: 10.1158/2326-6066.22544807.V1
Abstract: Supp Video 4
Publisher: American Association for Cancer Research (AACR)
Date: 03-04-2023
DOI: 10.1158/2159-8290.22540163
Abstract: Supplementary Figures 1-12
Publisher: Springer Science and Business Media LLC
Date: 18-05-2015
DOI: 10.1038/ONC.2015.167
Abstract: Scribble complex proteins maintain apicobasal polarity, regulate cell fate determination and function as tumour suppressors in epithelial tissue. Despite evidence that the function of Scribble is maintained in the lymphocyte lineage, we still understand little about its role as a tumour suppressor in haematological malignancies. Using the Eμ-myc model of Burkitt's lymphoma we investigated the role of Scribble in lymphomagenesis. We found that contrary to its well-documented tumour suppressor role in epithelial tissue, loss of Scribble expression delayed the expansion of peripheral B cells and delayed the onset of Eμ-myc-driven lymphoma. This was despite upregulated ERK phosphorylation levels in Scribble-deficient tumours, which are associated with loss of Scribble expression and the development of more aggressive Burkitt's lymphoma. Interestingly, the developmental stage of lymphoma was unaffected by Scribble expression challenging any role for Scribble in fate determination in the haematopoetic lineage. These data provide evidence for oncogenic properties of Scribble in Myc-driven B-cell lymphomagenesis, reinforcing recent findings that overexpression of a mutant form of Scribble can act as an oncogene in epithelial cells. Our results support the growing appreciation that the tumour regulatory functions of Scribble, and other polarity protein family members, are context dependent.
Publisher: Elsevier BV
Date: 2007
No related grants have been discovered for Jane Oliaro.