Sir Peter MacCallum Department of Oncology - Research Publications
Permanent URI for this collection
Search Results
1 - 8 of 8
-
ItemSerglycin determines secretory granule repertoire and regulates natural killer cell and cytotoxic T lymphocyte cytotoxicity(WILEY, 2016-03)The anionic proteoglycan serglycin is a major constituent of secretory granules in cytotoxic T lymphocyte (CTL)/natural killer (NK) cells, and is proposed to promote the safe storage of the mostly cationic granule toxins, granzymes and perforin. Despite the extensive defects of mast cell function reported in serglycin gene-disrupted mice, no comprehensive study of physiologically relevant CTL/NK cell populations has been reported. We show that the cytotoxicity of serglycin-deficient CTL and NK cells is severely compromised but can be partly compensated in both cell types when they become activated. Reduced intracellular granzyme B levels were noted, particularly in CD27(+) CD11b(+) mature NK cells, whereas serglycin(-/-) TCR-transgenic (OTI) CD8 T cells also had reduced perforin stores. Culture supernatants from serglycin(-/-) OTI T cells and interleukin-2-activated NK contained increased granzyme B, linking reduced storage with heightened export. By contrast, granzyme A was not significantly reduced in cells lacking serglycin, indicating differentially regulated trafficking and/or storage for the two granzymes. A quantitative analysis of different granule classes by transmission electronmicroscopy showed a selective loss of dense-core granules in serglycin(-/-) CD8(+) CTLs, although other granule types were maintained quantitatively. The findings of the present study show that serglycin plays a critical role in the maturation of dense-core cytotoxic granules in cytotoxic lymphocytes and the trafficking and storage of perforin and granzyme B, whereas granzyme A is unaffected. The skewed retention of cytotoxic effector molecules markedly reduces CTL/NK cell cytotoxicity, although this is partly compensated for as a result of activating the cells by physiological means.
-
ItemAntigen-driven EGR2 expression is required for exhausted CD8+ T cell stability and maintenance(NATURE PORTFOLIO, 2021-05-13)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.
-
ItemSUGAR-seq enables simultaneous detection of glycans, epitopes, and the transcriptome in single cells(AMER ASSOC ADVANCEMENT SCIENCE, 2021-02)Multimodal single-cell RNA sequencing enables the precise mapping of transcriptional and phenotypic features of cellular differentiation states but does not allow for simultaneous integration of critical posttranslational modification data. Here, we describe SUrface-protein Glycan And RNA-seq (SUGAR-seq), a method that enables detection and analysis of N-linked glycosylation, extracellular epitopes, and the transcriptome at the single-cell level. Integrated SUGAR-seq and glycoproteome analysis identified tumor-infiltrating T cells with unique surface glycan properties that report their epigenetic and functional state.
-
ItemGranzyme B triggers a prolonged pressure to die in Bcl-2 overexpressing cells, defining a window of opportunity for effective treatment with ABT-737(NATURE PUBLISHING GROUP, 2012-07)Overexpression of Bcl-2 contributes to resistance of cancer cells to human cytotoxic lymphocytes (CL) by blocking granzyme B (GraB)-induced mitochondrial outer membrane permeabilization (MOMP). Drugs that neutralise Bcl-2 (e.g., ABT-737) may therefore be effective adjuvants for immunotherapeutic strategies that use CL to kill cancer cells. Consistent with this we found that ABT-737 effectively restored MOMP in Bcl-2 overexpressing cells treated with GraB or natural killer cells. This effect was observed even if ABT-737 was added up to 16 h after GraB, after which the cells reset their resistant phenotype. Sensitivity to ABT-737 required initial cleavage of Bid by GraB (gctBid) but did not require ongoing GraB activity once Bid had been cleaved. This gctBid remained detectable in cells that were sensitive to ABT-737, but Bax and Bak were only activated if ABT-737 was added to the cells. These studies demonstrate that GraB generates a prolonged pro-apoptotic signal that must remain active for ABT-737 to be effective. The duration of this signal is determined by the longevity of gctBid but not activation of Bax or Bak. This defines a therapeutic window in which ABT-737 and CL synergise to cause maximum death of cancer cells that are resistant to either treatment alone, which will be essential in defining optimum treatment regimens.
-
ItemFunctional Crosstalk between Type I and II Interferon through the Regulated Expression of STAT1(PUBLIC LIBRARY SCIENCE, 2010-04)Autocrine priming of cells by small quantities of constitutively produced type I interferon (IFN) is a well-known phenomenon. In the absence of type I IFN priming, cells display attenuated responses to other cytokines, such as anti-viral protection in response to IFNgamma. This phenomenon was proposed to be because IFNalpha/beta receptor1 (IFNAR1) is a component of the IFNgamma receptor (IFNGR), but our new data are more consistent with a previously proposed model indicating that regulated expression of STAT1 may also play a critical role in the priming process. Initially, we noticed that DNA binding activity of STAT1 was attenuated in c-Jun(-/-) fibroblasts because they expressed lower levels of STAT1 than wild-type cells. However, expression of STAT1 was rescued by culturing c-Jun(-/-) fibroblasts in media conditioned by wild-type fibroblasts suggesting they secreted a STAT1-inducing factor. The STAT1-inducing factor in fibroblast-conditioned media was IFNbeta, as it was inhibited by antibodies to IFNAR1, or when IFNbeta expression was knocked down in wild-type cells. IFNAR1(-/-) fibroblasts, which cannot respond to this priming, also expressed reduced levels of STAT1, which correlated with their poor responses to IFNgamma. The lack of priming in IFNAR1(-/-) fibroblasts was compensated by over-expression of STAT1, which rescued molecular responses to IFNgamma and restored the ability of IFNgamma to induce protective anti-viral immunity. This study provides a comprehensive description of the molecular events involved in priming by type I IFN. Adding to the previous working model that proposed an interaction between type I and II IFN receptors, our work and that of others demonstrates that type I IFN primes IFNgamma-mediated immune responses by regulating expression of STAT1. This may also explain how type I IFN can additionally prime cells to respond to a range of other cytokines that use STAT1 (e.g., IL-6, M-CSF, IL-10) and suggests a potential mechanism for the changing levels of STAT1 expression observed during viral infection.
-
ItemInitiation of apoptosis by granzyme B requires direct cleavage of Bid, but not direct granzyme B-mediated caspase activation(ROCKEFELLER UNIV PRESS, 2000-11-20)The essential upstream steps in granzyme B-mediated apoptosis remain undefined. Herein, we show that granzyme B triggers the mitochondrial apoptotic pathway through direct cleavage of Bid; however, cleavage of procaspases was stalled when mitochondrial disruption was blocked by Bcl-2. The sensitivity of granzyme B-resistant Bcl-2-overexpressing FDC-P1 cells was restored by coexpression of wild-type Bid, or Bid with a mutation of its caspase-8 cleavage site, and both types of Bid were cleaved. However, Bid with a mutated granzyme B cleavage site remained intact and did not restore apoptosis. Bid with a mutation preventing its interaction with Bcl-2 was cleaved but also failed to restore apoptosis. Rapid Bid cleavage by granzyme B (<2 min) was not delayed by Bcl-2 overexpression. These results clearly placed Bid cleavage upstream of mitochondrial Bcl-2. In granzyme B-treated Jurkat cells, endogenous Bid cleavage and loss of mitochondrial membrane depolarization occurred despite caspase inactivation with z-Val-Ala-Asp-fluoromethylketone or Asp-Glu-Val-Asp-fluoromethylketone. Initial partial processing of procaspase-3 and -8 was observed irrespective of Bcl-2 overexpression; however, later processing was completely abolished by Bcl-2. Overall, our results indicate that mitochondrial perturbation by Bid is necessary to achieve a lethal threshold of caspase activity and cell death due to granzyme B.
-
ItemDown-regulation of a pro-apoptotic pathway regulated by PCAF/ADA3 in early stage gastric cancer(NATURE PUBLISHING GROUP, 2018-04-18)The loss of p300/CBP-associated protein (PCAF) expression is associated with poor clinical outcome in gastric cancer, and a potential bio-marker for invasive and aggressive tumors. However, the mechanism linking loss of PCAF to the onset of gastric cancer has not been identified. Given that PCAF and its binding partner transcriptional adaptor protein 3 (ADA3) were recently shown to regulate the intrinsic (mitochondrial) pathway to apoptosis via epigenetic regulation of phosphofurin acidic cluster sorting proteins 1 and 2 (PACS1, PACS2), we analyzed PCAF, ADA3, and PACS1/2 expression in 99 patient-matched surgical samples ranging from normal gastric mucosa, through pre-malignant chronic gastritis and intestinal metaplasia to stage I-III invasive cancers. PCAF mRNA levels were not reduced in either pre-malignant state but were significantly down-regulated in all stages of gastric cancer, commencing at AJCC stage I (p < 0.05), thus linking reduced PCAF expression with early malignant change. Furthermore, patients with combined reduction of PCAF and PACS1 had significantly poorer overall survival (p = 0.0257), confirmed in an independent dataset of 359 patients (p = 5.8 × 10e-6). At the protein level, PCAF, ADA3, and PACS1 expression were all significantly down-regulated in intestinal-type gastric cancer, and correlated with reduced progression free survival. We conclude that a pro-apoptotic mechanism centered on the intrinsic (mitochondrial) pathway and regulated by PCAF/ADA3 can influence the progression from premalignant to malignant change, and thus act as a tumor suppression mechanism in gastric cancer.
-
ItemA role for multiple chimeric antigen receptor-expressing leukocytes in antigen-specific responses to cancer(IMPACT JOURNALS LLC, 2016-06-07)While adoptive immunotherapy using chimeric antigen receptor (CAR)-modified T cells can induce remission of some tumors, the role of other CAR-modified leukocytes is not well characterized. In this study, we characterize the function of leukocytes including natural killer (NK) cells, macrophages and CAR T cells from transgenic mice expressing a CAR under the control of the pan-hematopoietic promoter, vav, and determine the ability of these mice to respond to ERB expressing tumors. We demonstrate the anti-tumor functions of leukocytes, including antigen specific cytotoxicity and cytokine secretion. The adoptive transfer of CAR T cells provided a greater survival advantage in the E0771ERB tumor model than their wildtype (WT) counterparts. In addition, CAR NK cells and CAR T cells also mediated increased survival in the RMAERB tumor model. When challenged with Her2 expressing tumors, F38 mice were shown to mount an effective immune response, resulting in tumor rejection and long-term survival. This was shown to be predominantly dependent on both CD8+ T cells and NK cells. However, macrophages and CD4+ T cells were also shown to contribute to this response. Overall, this study highlights the use of the vav-CAR mouse model as a unique tool to determine the anti-tumor function of various immune subsets, either alone or when acting alongside CAR T cells in adoptive immunotherapy.