Publications by authors named "Joseph A Trapani"

174 Publications

Chimeric Antigen Receptor T cell Therapy and the Immunosuppressive Tumor Microenvironment in Pediatric Sarcoma.

Cancers (Basel) 2021 Sep 20;13(18). Epub 2021 Sep 20.

Children's Cancer Institute, Randwick 2031, Australia.

Sarcomas are a diverse group of bone and soft tissue tumors that account for over 10% of childhood cancers. Outcomes are particularly poor for children with refractory, relapsed, or metastatic disease. Chimeric antigen receptor T (CAR T) cells are an exciting form of adoptive cell therapy that potentially offers new hope for these children. In early trials, promising outcomes have been achieved in some pediatric patients with sarcoma. However, many children do not derive benefit despite significant expression of the targeted tumor antigen. The success of CAR T cell therapy in sarcomas and other solid tumors is limited by the immunosuppressive tumor microenvironment (TME). In this review, we provide an update of the CAR T cell therapies that are currently being tested in pediatric sarcoma clinical trials, including those targeting tumors that express HER2, NY-ESO, GD2, EGFR, GPC3, B7-H3, and MAGE-A4. We also outline promising new CAR T cells that are in pre-clinical development. Finally, we discuss strategies that are being used to overcome tumor-mediated immunosuppression in solid tumors; these strategies have the potential to improve clinical outcomes of CAR T cell therapy for children with sarcoma.
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http://dx.doi.org/10.3390/cancers13184704DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8465026PMC
September 2021

Lipid specificity of the immune effector perforin.

Faraday Discuss 2021 Sep 21. Epub 2021 Sep 21.

London Centre for Nanotechnology, University College London, 19 Gordon Street, London WC1H 0AH, UK.

Perforin is a pore forming protein used by cytotoxic T lymphocytes to remove cancerous or virus-infected cells during the immune response. During the response, the lymphocyte membrane becomes refractory to perforin function by accumulating densely ordered lipid rafts and externalizing negatively charged lipid species. The dense membrane packing lowers the capacity of perforin to bind, and the negatively charged lipids scavenge any residual protein before pore formation. Using atomic force microscopy on model membrane systems, we here provide insight into the molecular basis of perforin lipid specificity.
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http://dx.doi.org/10.1039/d0fd00043dDOI Listing
September 2021

Reprogrammed CRISPR-Cas13b suppresses SARS-CoV-2 replication and circumvents its mutational escape through mismatch tolerance.

Nat Commun 2021 07 13;12(1):4270. Epub 2021 Jul 13.

Cancer Immunology Program, Peter MacCallum Cancer Centre, Melbourne, Australia.

The recent dramatic appearance of variants of concern of SARS-coronavirus-2 (SARS-CoV-2) highlights the need for innovative approaches that simultaneously suppress viral replication and circumvent viral escape from host immunity and antiviral therapeutics. Here, we employ genome-wide computational prediction and single-nucleotide resolution screening to reprogram CRISPR-Cas13b against SARS-CoV-2 genomic and subgenomic RNAs. Reprogrammed Cas13b effectors targeting accessible regions of Spike and Nucleocapsid transcripts achieved >98% silencing efficiency in virus-free models. Further, optimized and multiplexed Cas13b CRISPR RNAs (crRNAs) suppress viral replication in mammalian cells infected with replication-competent SARS-CoV-2, including the recently emerging dominant variant of concern B.1.1.7. The comprehensive mutagenesis of guide-target interaction demonstrated that single-nucleotide mismatches does not impair the capacity of a potent single crRNA to simultaneously suppress ancestral and mutated SARS-CoV-2 strains in infected mammalian cells, including the Spike D614G mutant. The specificity, efficiency and rapid deployment properties of reprogrammed Cas13b described here provide a molecular blueprint for antiviral drug development to suppress and prevent a wide range of SARS-CoV-2 mutants, and is readily adaptable to other emerging pathogenic viruses.
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http://dx.doi.org/10.1038/s41467-021-24577-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8277810PMC
July 2021

Blockade of the co-inhibitory molecule PD-1 unleashes ILC2-dependent antitumor immunity in melanoma.

Nat Immunol 2021 07 7;22(7):851-864. Epub 2021 Jun 7.

Innate Pharma Research Labs, Marseille, France.

Group 2 innate lymphoid cells (ILC2s) are essential to maintain tissue homeostasis. In cancer, ILC2s can harbor both pro-tumorigenic and anti-tumorigenic functions, but we know little about their underlying mechanisms or whether they could be clinically relevant or targeted to improve patient outcomes. Here, we found that high ILC2 infiltration in human melanoma was associated with a good clinical prognosis. ILC2s are critical producers of the cytokine granulocyte-macrophage colony-stimulating factor, which coordinates the recruitment and activation of eosinophils to enhance antitumor responses. Tumor-infiltrating ILC2s expressed programmed cell death protein-1, which limited their intratumoral accumulation, proliferation and antitumor effector functions. This inhibition could be overcome in vivo by combining interleukin-33-driven ILC2 activation with programmed cell death protein-1 blockade to significantly increase antitumor responses. Together, our results identified ILC2s as a critical immune cell type involved in melanoma immunity and revealed a potential synergistic approach to harness ILC2 function for antitumor immunotherapies.
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http://dx.doi.org/10.1038/s41590-021-00943-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7611091PMC
July 2021

Antigen-driven EGR2 expression is required for exhausted CD8 T cell stability and maintenance.

Nat Commun 2021 05 13;12(1):2782. Epub 2021 May 13.

John Curtin School of Medical Research, Australian National University, ACT, Australia.

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.
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http://dx.doi.org/10.1038/s41467-021-23044-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8119420PMC
May 2021

Tumor-specific cytolytic CD4 T cells mediate immunity against human cancer.

Sci Adv 2021 Feb 26;7(9). Epub 2021 Feb 26.

Department of Pathology and Immunology, Faculty of Medicine, University of Geneva, Geneva, CH-1211, Switzerland.

CD4 T cells have been implicated in cancer immunity for their helper functions. Moreover, their direct cytotoxic potential has been shown in some patients with cancer. Here, by mining single-cell RNA-seq datasets, we identified CD4 T cell clusters displaying cytotoxic phenotypes in different human cancers, resembling CD8 T cell profiles. Using the peptide-MHCII-multimer technology, we confirmed ex vivo the presence of cytolytic tumor-specific CD4 T cells. We performed an integrated phenotypic and functional characterization of these cells, down to the single-cell level, through a high-throughput nanobiochip consisting of massive arrays of picowells and machine learning. We demonstrated a direct, contact-, and granzyme-dependent cytotoxic activity against tumors, with delayed kinetics compared to classical cytotoxic lymphocytes. Last, we found that this cytotoxic activity was in part dependent on SLAMF7. Agonistic engagement of SLAMF7 enhanced cytotoxicity of tumor-specific CD4 T cells, suggesting that targeting these cells might prove synergistic with other cancer immunotherapies.
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http://dx.doi.org/10.1126/sciadv.abe3348DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7909889PMC
February 2021

SUGAR-seq enables simultaneous detection of glycans, epitopes, and the transcriptome in single cells.

Sci Adv 2021 Feb 19;7(8). Epub 2021 Feb 19.

Translational Haematology Program, Peter MacCallum Cancer Centre, Melbourne, Victoria 3000, Australia.

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.
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http://dx.doi.org/10.1126/sciadv.abe3610DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7895430PMC
February 2021

PVRIG is a novel NK cell immune checkpoint receptor in acute myeloid leukemia.

Haematologica 2020 11 5;Online ahead of print. Epub 2020 Nov 5.

Cancer Immunology Program, Peter MacCallum Cancer Centre, Melbourne, Australia; Sir Peter MacCallum Department of Oncology, The University of Melbourne.

This study explored the novel immune checkpoint poliovirus receptor-related immunoglobulin domain-containing (PVRIG) in acute myeloid leukemia (AML). We showed that AML patient blasts consistently expressed the PVRIG ligand (poliovirus receptor-related 2, PVRL2). Furthermore, PVRIG blockade significantly enhanced NK cell killing of PVRL2+, poliovirus receptor (PVR)lo AML cell lines, and significantly increased NK cell activation and degranulation in the context of patient primary AML blasts. However, in AML patient bone marrow, NK cell PVRIG expression levels were not increased. To understand how PVRIG blockade might potentially be exploited therapeutically, we investigated the biology of PVRIG and revealed that NK cell activation resulted in reduced PVRIG expression on the cell surface. This occurred whether NK cells were activated by tumour cell recognition, cytokines (IL-2 and IL-12) or activating receptor stimulation (CD16 and NKp46). PVRIG was present at higher levels in the cytoplasm than on the cell surface, particularly on CD56bright NK cells, which further increased cytoplasmic PVRIG levels following IL-2 and IL-12 activation. PVRIG was continually transported to the cell surface via the endoplasmic reticulum (ER) and Golgi in both unstimulated and activated NK cells. Taken together, our findings suggest that anti- PVRIG blocking antibody functions by binding to surface-bound PVRIG, which undergoes rapid turnover in both unstimulated and activated NK cells. We conclude that the PVRIGPVRL2 immune checkpoint axis can feasibly be targeted with PVRIG blocking antibody for NK-mediated immunotherapy of PVRL2+ AML.
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http://dx.doi.org/10.3324/haematol.2020.258574DOI Listing
November 2020

Revisiting T Cell Tolerance as a Checkpoint Target for Cancer Immunotherapy.

Front Immunol 2020 23;11:589641. Epub 2020 Sep 23.

Peter MacCallum Cancer Centre, Melbourne, VIC, Australia.

Immunotherapy has revolutionized the treatment of cancer. Nevertheless, the majority of patients do not respond to therapy, meaning a deeper understanding of tumor immune evasion strategies is required to boost treatment efficacy. The vast majority of immunotherapy studies have focused on how treatment reinvigorates exhausted CD8 T cells within the tumor. In contrast, how therapies influence regulatory processes within the draining lymph node is less well studied. In particular, relatively little has been done to examine how tumors may exploit peripheral CD8 T cell tolerance, an under-studied immune checkpoint that under normal circumstances prevents detrimental autoimmune disease by blocking the initiation of T cell responses. Here we review the therapeutic potential of blocking peripheral CD8 T cell tolerance for the treatment of cancer. We first comprehensively review what has been learnt about the regulation of CD8 T cell peripheral tolerance from the non-tumor models in which peripheral tolerance was first defined. We next consider how the tolerant state differs from other states of negative regulation, such as T cell exhaustion and senescence. Finally, we describe how tumors hijack the peripheral tolerance immune checkpoint to prevent anti-tumor immune responses, and argue that disruption of peripheral tolerance may contribute to both the anti-cancer efficacy and autoimmune side-effects of immunotherapy. Overall, we propose that a deeper understanding of peripheral tolerance will ultimately enable the development of more targeted and refined cancer immunotherapy approaches.
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http://dx.doi.org/10.3389/fimmu.2020.589641DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7538772PMC
July 2021

Words of Advice: choosing the right lab for your post-doctoral fellowship.

FEBS J 2021 03 8;288(6):1734-1741. Epub 2020 Oct 8.

Rosie Lew Cancer Immunology Program, Peter MacCallum Cancer Centre, Melbourne, Vic, Australia.

For most researchers, the time they spend as a postdoc stands out as one of challenge, but also enormous personal and professional growth. This Words of Advice is intended to guide the choice of postdoctoral position to help make the venture a success and to launch the first chapter of a happy and fulfilling professional life.
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http://dx.doi.org/10.1111/febs.15565DOI Listing
March 2021

Challenges of PD-L1 testing in non-small cell lung cancer and beyond.

J Thorac Dis 2020 Aug;12(8):4541-4548

Cancer Immunology Program, Peter MacCallum Cancer Centre, Melbourne, VIC, Australia.

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http://dx.doi.org/10.21037/jtd-2019-itm-010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7475552PMC
August 2020

Intratumoral Copper Modulates PD-L1 Expression and Influences Tumor Immune Evasion.

Cancer Res 2020 10 18;80(19):4129-4144. Epub 2020 Aug 18.

Children's Cancer Institute, Lowy Cancer Research Centre, UNSW Sydney, Randwick, New South Wales, Australia.

Therapeutic checkpoint antibodies blocking programmed death receptor 1/programmed death ligand 1 (PD-L1) signaling have radically improved clinical outcomes in cancer. However, the regulation of PD-L1 expression on tumor cells is still poorly understood. Here we show that intratumoral copper levels influence PD-L1 expression in cancer cells. Deep analysis of the The Cancer Genome Atlas database and tissue microarrays showed strong correlation between the major copper influx transporter copper transporter 1 (CTR-1) and PD-L1 expression across many cancers but not in corresponding normal tissues. Copper supplementation enhanced PD-L1 expression at mRNA and protein levels in cancer cells and RNA sequencing revealed that copper regulates key signaling pathways mediating PD-L1-driven cancer immune evasion. Conversely, copper chelators inhibited phosphorylation of STAT3 and EGFR and promoted ubiquitin-mediated degradation of PD-L1. Copper-chelating drugs also significantly increased the number of tumor-infiltrating CD8 T and natural killer cells, slowed tumor growth, and improved mouse survival. Overall, this study reveals an important role for copper in regulating PD-L1 and suggests that anticancer immunotherapy might be enhanced by pharmacologically reducing intratumor copper levels. SIGNIFICANCE: These findings characterize the role of copper in modulating PD-L1 expression and contributing to cancer immune evasion, highlighting the potential for repurposing copper chelators as enhancers of antitumor immunity. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/19/4129/F1.large.jpg.
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http://dx.doi.org/10.1158/0008-5472.CAN-20-0471DOI Listing
October 2020

Enhancing chimeric antigen receptor T-cell immunotherapy against cancer using a nanoemulsion-based vaccine targeting cross-presenting dendritic cells.

Clin Transl Immunology 2020 22;9(7):e1157. Epub 2020 Jul 22.

Cancer Immunology Program Peter MacCallum Cancer Center Melbourne VIC Australia.

Objectives: Adoptive transfer of chimeric antigen receptor (CAR)-modified T cells is a form of cancer immunotherapy that has achieved remarkable efficacy in patients with some haematological cancers. However, challenges remain in CAR T-cell treatment of solid tumours because of tumour-mediated immunosuppression.

Methods: We have demonstrated that CAR T-cell stimulation through T-cell receptors (TCRs) can generate durable responses against solid tumours in a variety of murine models. Since Clec9A-targeting tailored nanoemulsion (Clec9A-TNE) vaccine enhances antitumour immune responses through selective activation of Clec9A cross-presenting dendritic cells (DCs), we hypothesised that Clec9A-TNE could prime DCs for antigen presentation to CAR T cells through TCRs and thus improve CAR T-cell responses against solid tumours. To test this hypothesis, we used CAR T cells expressing transgenic TCRs specific for ovalbumin (OVA) peptides SIINFEKL (CAROTI) or OVA (CAROTII).

Results: We demonstrated that the Clec9A-TNEs encapsulating full-length recombinant OVA protein (OVA-Clec9A-TNE) improved CAROT T-cell proliferation and inflammatory cytokine secretion . Combined treatment using the OVA-Clec9A-TNE and CAROT cells resulted in durable responses and some rejections of tumours in immunocompetent mice. Tumour regression was accompanied by enhanced CAROT cell proliferation and infiltration into the tumours.

Conclusion: Our study presents Clec9A-TNE as a prospective avenue to enhance CAR T-cell efficacy for solid cancers.
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http://dx.doi.org/10.1002/cti2.1157DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7374388PMC
July 2020

Immune profiling of pediatric solid tumors.

J Clin Invest 2020 07;130(7):3391-3402

Cancer Immunology Program, Peter MacCallum Cancer Center, Melbourne, Victoria, Australia.

Pediatric cancers, particularly high-risk solid tumors, urgently need effective and specific therapies. Their outlook has not appreciably improved in decades. Immunotherapies such as immune checkpoint inhibitors offer much promise, but most are only approved for use in adults. Though several hundred clinical trials have tested immune-based approaches in childhood cancers, few have been guided by biomarkers or clinical-grade assays developed to predict patient response and, ultimately, to help select those most likely to benefit. There is extensive evidence in adults to show that immune profiling has substantial predictive value, but few studies focus on childhood tumors, because of the relatively small disease population and restricted use of immune-based therapies. For instance, only one published study has retrospectively examined the immune profiles of pediatric brain tumors after immunotherapy. Furthermore, application and integration of advanced multiplex techniques has been extremely limited. Here, we review the current status of immune profiling of pediatric solid tumors, with emphasis on tumor types that represent enormous unmet clinical need, primarily in the context of immune checkpoint inhibitor therapy. Translating optimized and informative immune profiling into standard practice and access to personalized combination therapy will be critical if childhood cancers are to be treated effectively and affordably.
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http://dx.doi.org/10.1172/JCI137181DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7324195PMC
July 2020

Differential cleavage of viral polypeptides by allotypic variants of granzyme B skews immunity to mouse cytomegalovirus.

Biochim Biophys Acta Proteins Proteom 2020 09 27;1868(9):140457. Epub 2020 May 27.

Rosie Lew Cancer Immunology Program, Peter MacCallum Cancer Centre, 305 Grattan Street Melbourne 3000, Australia; Sir Peter MacCallum Department of Oncology, The University of Melbourne 3050, Australia. Electronic address:

We investigated the molecular basis for the remarkably different survival outcomes of mice expressing different alloforms of the pro-apoptotic serine protease granzyme B to mouse cytomegalovirus infection. Whereas C57BL/6 mice homozygous for granzyme B (GzmB) raise cytotoxic T lymphocytes that efficiently kill infected cells, those of C57BL/6 mice congenic for the outbred allele (GzmB) fail to kill MCMV-infected cells and died from uncontrolled hepatocyte infection and acute liver failure. We identified subtle differences in how GzmB and GzmB activate cell death signalling - both alloforms predominantly activated pro-caspases directly, and cleaved pro-apoptotic Bid poorly. Consequently, neither alloform initiated mitochondrial outer membrane permeabilization, or was blocked by Bcl-2, Bcl-XL or co-expression of MCMV proteins M38.5/M41.1, which together stabilize mitochondria by sequestering Bak/Bax. Remarkably, mass spectrometric analysis of proteins from MCMV-infected primary mouse embryonic fibroblasts identified 13 cleavage sites in nine viral proteins (M18, M25, M28, M45, M80, M98, M102, M155, M164) that were cleaved >20-fold more efficiently by either GzmB or GzmB. Notably, M18, M28, M45, M80, M98, M102 and M164 were cleaved 20- >100-fold more efficiently by GzmB, and so, would persist in infected cells targeted by CTLs from GzmB mice. Conversely, M155 was cleaved >100-fold more efficiently by GzmB, and would persist in cells targeted by CTLs of GzmB mice. M25 was cleaved efficiently by both proteases, but at different sites. We conclude that different susceptibility to MCMV does not result from skewed endogenous cell death pathways, but rather, to as yet uncharacterised MCMV-intrinsic pathways that ultimately inhibit granzyme B-induced cell death.
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http://dx.doi.org/10.1016/j.bbapap.2020.140457DOI Listing
September 2020

ASC Modulates CTL Cytotoxicity and Transplant Outcome Independent of the Inflammasome.

Cancer Immunol Res 2020 08 22;8(8):1085-1098. Epub 2020 May 22.

QIMR Berghofer Medical Research Institute, Brisbane, Queensland, Australia.

The adaptor protein ASC (apoptosis-associated speck-like protein containing a CARD) is known to facilitate caspase-1 activation, which is essential for innate host immunity via the formation of the inflammasome complex, a multiprotein structure responsible for processing IL1β and IL18 into their active moieties. Here, we demonstrated that ASC-deficient CD8 T cells failed to induce severe graft-versus-host disease (GVHD) and had impaired capacity for graft rejection and graft-versus-leukemia (GVL) activity. These effects were inflammasome independent because GVHD lethality was not altered in recipients of caspase-1/11-deficient T cells. We also demonstrated that ASC deficiency resulted in a decrease in cytolytic function, with a reduction in granzyme B secretion and CD107a expression by CD8 T cells. Altogether, our findings highlight that ASC represents an attractive therapeutic target for improving outcomes of clinical transplantation.
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http://dx.doi.org/10.1158/2326-6066.CIR-19-0653DOI Listing
August 2020

Adoptive cellular therapy with T cells expressing the dendritic cell growth factor Flt3L drives epitope spreading and antitumor immunity.

Nat Immunol 2020 08 18;21(8):914-926. Epub 2020 May 18.

Cancer Immunology Program, Peter MacCallum Cancer Centre, Melbourne, Victoria, Australia.

Adoptive cell therapies using genetically engineered T cell receptor or chimeric antigen receptor T cells are emerging forms of immunotherapy that redirect T cells to specifically target cancer. However, tumor antigen heterogeneity remains a key challenge limiting their efficacy against solid cancers. Here, we engineered T cells to secrete the dendritic cell (DC) growth factor Fms-like tyrosine kinase 3 ligand (Flt3L). Flt3L-secreting T cells expanded intratumoral conventional type 1 DCs and substantially increased host DC and T cell activation when combined with immune agonists poly (I:C) and anti-4-1BB. Importantly, combination therapy led to enhanced inhibition of tumor growth and the induction of epitope spreading towards antigens beyond those recognized by adoptively transferred T cells in solid tumor models of T cell receptor and chimeric antigen receptor T cell therapy. Our data suggest that augmenting endogenous DCs is a promising strategy to overcome the clinical problem of antigen-negative tumor escape following adoptive cell therapy.
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http://dx.doi.org/10.1038/s41590-020-0676-7DOI Listing
August 2020

Cross-talk between tumors at anatomically distinct sites.

FEBS J 2021 01 21;288(1):81-90. Epub 2020 Apr 21.

Cancer Immunology Program, Peter MacCallum Cancer Center, Melbourne, Australia.

Cancer tissue is not homogenous, and individual metastases at different anatomical locations can differ from the primary tumor and from one another in both their morphology and cellular composition, even within an individual patient. Tumors are composed of cancer cells and a range of other cell types, which, together with a variety of secreted molecules, collectively comprise the tumor microenvironment (TME). Cells of the TME can communicate with each other and with distant tissues in a form of molecular cross-talk to influence their growth and function. Cross-talk between cancer cells and local immune cells is well described and can lead to the induction of local immunosuppression. Recently, it has become apparent that tumors located remotely from each other, can engage in cross-talk that can influence their responsiveness to various therapies, including immunotherapy. In this article, we review studies that describe how tumors systemically communicate with distant tissues through motile cells, extracellular vesicles, and secreted molecules that can affect their function. In addition, we summarize evidence from mouse studies and the clinic that indicate an ability of some tumors to influence the progression and therapeutic responses of other tumors in different anatomical locations.
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http://dx.doi.org/10.1111/febs.15316DOI Listing
January 2021

Efficient CRISPR/Cas9 Gene Editing in Uncultured Naive Mouse T Cells for In Vivo Studies.

J Immunol 2020 04 9;204(8):2308-2315. Epub 2020 Mar 9.

Peter MacCallum Cancer Centre, Melbourne, Victoria 3000, Australia; and

CRISPR/Cas9 technologies have revolutionized our understanding of gene function in complex biological settings, including T cell immunology. Current CRISPR-mediated gene editing strategies in T cells require in vitro stimulation or culture that can both preclude the study of unmanipulated naive T cells and alter subsequent differentiation. In this study, we demonstrate highly efficient gene editing within uncultured primary naive murine CD8 T cells by electroporation of recombinant Cas9/sgRNA ribonucleoprotein immediately prior to in vivo adoptive transfer. Using this approach, we generated single and double gene knockout cells within multiple mouse infection models. Strikingly, gene deletion occurred even when the transferred cells were left in a naive state, suggesting that gene deletion occurs independent of T cell activation. Finally, we demonstrate that targeted mutations can be introduced into naive CD8 T cells using CRISPR-based homology-directed repair. This protocol thus expands CRISPR-based gene editing approaches beyond models of robust T cell activation to encompass both naive T cell homeostasis and models of weak activation, such as tolerance and tumor models.
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http://dx.doi.org/10.4049/jimmunol.1901396DOI Listing
April 2020

Lipid order and charge protect killer T cells from accidental death.

Nat Commun 2019 11 27;10(1):5396. Epub 2019 Nov 27.

Killer Cell Biology Laboratory, Peter MacCallum Cancer Centre, 305 Grattan Street, Melbourne, VIC, 3000, Australia.

Killer T cells (cytotoxic T lymphocytes, CTLs) maintain immune homoeostasis by eliminating virus-infected and cancerous cells. CTLs achieve this by forming an immunological synapse with their targets and secreting a pore-forming protein (perforin) and pro-apoptotic serine proteases (granzymes) into the synaptic cleft. Although the CTL and the target cell are both exposed to perforin within the synapse, only the target cell membrane is disrupted, while the CTL is invariably spared. How CTLs escape unscathed remains a mystery. Here, we report that CTLs achieve this via two protective properties of their plasma membrane within the synapse: high lipid order repels perforin and, in addition, exposed phosphatidylserine sequesters and inactivates perforin. The resulting resistance of CTLs to perforin explains their ability to kill target cells in rapid succession and to survive these encounters. Furthermore, these mechanisms imply an unsuspected role for plasma membrane organization in protecting cells from immune attack.
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http://dx.doi.org/10.1038/s41467-019-13385-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6881447PMC
November 2019

Distinguishing perforin-mediated lysis and granzyme-dependent apoptosis.

Methods Enzymol 2019 21;629:291-306. Epub 2019 Aug 21.

Killer Cell Biology Laboratory, Peter MacCallum Cancer Centre, Melbourne, Australia; Sir Peter MacCallum Department of Oncology, University of Melbourne, Melbourne, Australia. Electronic address:

Perforin is an indispensable effector protein of primary cytotoxic lymphocytes (CTL or NK cells) that typically defend the host against virus infection, or gene-modified (chimeric antigen receptor-CAR) anticancer T cells. Perforin's pore-forming activity is necessary for the delivery of proapoptotic serine proteases, granzymes, into the cytosol of infected or cancerous target cells. The complete loss of perforin function is detrimental for the function of cytotoxic lymphocytes, and leads to fatal immune dysregulation in infants and predisposes the carriers of hypomorphic perforin mutations to various chronic inflammatory sequelae and blood cancers. Here, we describe several optimized and validated functional assays using purified effector proteins and cytotoxic lymphocytes that enable detailed analysis of perforin-mediated target cell death pathways.
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http://dx.doi.org/10.1016/bs.mie.2019.07.034DOI Listing
June 2020

Inhibition of the Cytolytic Protein Perforin Prevents Rejection of Transplanted Bone Marrow Stem Cells in Vivo.

J Med Chem 2020 03 26;63(5):2229-2239. Epub 2019 Sep 26.

QIMR Berghofer Medical Research Institute, 300 Herston Road, Herston, Queensland 4006, Australia.

Perforin is a key effector protein in the vertebrate immune system and is secreted by cytotoxic T lymphocytes and natural killer cells to help eliminate virus-infected and transformed target cells. The ability to modulate perforin activity in vivo could be extremely useful, especially in the context of bone marrow stem cell transplantation where early rejection of immunologically mismatched grafts is driven by the recipient's natural killer cells, which overwhelmingly use perforin to kill their targets. Bone marrow stem cell transplantation is a potentially curative treatment for both malignant and nonmalignant disorders, but when the body recognizes the graft as foreign, it is rejected by this process, often with fatal consequences. Here we report optimization of a previously identified series of benzenesulfonamide-based perforin inhibitors for their physicochemical and pharmacokinetic properties, resulting in the identification of , the first reported small molecule able to prevent rejection of transplanted bone marrow stem cells in vivo by blocking perforin function.
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http://dx.doi.org/10.1021/acs.jmedchem.9b00881DOI Listing
March 2020

Antigen-specific CD4 CD25 T cells induced by locally expressed ICOS-Ig: the role of Foxp3, Perforin, Granzyme B and IL-10 - an experimental study.

Transpl Int 2019 Nov 16;32(11):1203-1215. Epub 2019 Jul 16.

Department of Surgery, Austin Health, The University of Melbourne, Heidelberg, Vic., Australia.

We have previously reported that ICOS-Ig expressed locally by a PIEC xenograft induces a perigraft cellular accumulation of CD4 CD25 Foxp3 T cells and specific xenograft prolongation. In the present study we isolated and purified CD4 CD25 T cells from ICOS-Ig secreting PIEC grafts to examine their phenotype and mechanism of xenograft survival using knockout and mutant mice. CD4 CD25 T cells isolated from xenografts secreting ICOS-Ig were analysed by flow cytometry and gene expression by real-time PCR. Regulatory function was examined by suppression of xenogeneic or allogeneic primed CD4 T cells in vivo. Graft prolongation was shown to be dependent on a pre-existing Foxp3 Treg, IL-10, perforin and granzyme B. CD4 CD25 Foxp3 T cells isolated from xenografts secreting ICOS-Ig demonstrated a phenotype consistent with nTreg but with a higher expression of CD275 (ICOSL), expression of CD278 (ICOS) and MHC II and loss of CD73. Moreover, these cells were functional and specifically suppressed xenogeinic but not allogeneic primed T cells in vivo.
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http://dx.doi.org/10.1111/tri.13474DOI Listing
November 2019

Dual PD-1 and CTLA-4 Checkpoint Blockade Promotes Antitumor Immune Responses through CD4Foxp3 Cell-Mediated Modulation of CD103 Dendritic Cells.

Cancer Immunol Res 2018 09 17;6(9):1069-1081. Epub 2018 Jul 17.

Cancer Immunology Program, Peter MacCallum Cancer Centre, Melbourne, Victoria, Australia.

Immunotherapy is widely accepted as a powerful new treatment modality for the treatment of cancer. The most successful form of immunotherapy to date has been the blockade of the immune checkpoints PD-1 and CTLA-4. Combining inhibitors of both PD-1 and CTLA-4 increases the proportion of patients who respond to immunotherapy. However, most patients still do not respond to checkpoint inhibitors, and prognostic biomarkers are currently lacking. Therefore, a better understanding of the mechanism by which these checkpoint inhibitors enhance antitumor immune responses is required to more accurately predict which patients are likely to respond and further enhance this treatment modality. Our current study of two mouse tumor models revealed that CD4Foxp3 cells activated by dual PD-1/CTLA-4 blockade modulated the myeloid compartment, including activation of conventional CD103 dendritic cells (DC) and expansion of a myeloid subset that produces TNFα and iNOS (TIP-DCs). CD4Foxp3 T cell-mediated activation of CD103 DCs resulted in enhanced IL12 production by these cells and IL12 enhanced the therapeutic effect of dual PD-1/CTLA-4 blockade. Given the importance of these myeloid subsets in the antitumor immune response, our data point to a previously underappreciated role of CD4Foxp3 cells in modulating this arm of the antitumor immune response. .
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http://dx.doi.org/10.1158/2326-6066.CIR-18-0291DOI Listing
September 2018

Tumor immune evasion arises through loss of TNF sensitivity.

Sci Immunol 2018 05;3(23)

Immune Defence Laboratory, Cancer Immunology Program, Peter MacCallum Cancer Centre, Melbourne, Victoria 3000, Australia.

Immunotherapy has revolutionized outcomes for cancer patients, but the mechanisms of resistance remain poorly defined. We used a series of whole-genome clustered regularly interspaced short palindromic repeat (CRISPR)-based screens performed in vitro and in vivo to identify mechanisms of tumor immune evasion from cytotoxic lymphocytes [CD8 T cells and natural killer (NK) cells]. Deletion of key genes within the tumor necrosis factor (TNF) signaling, interferon-γ (IFN-γ) signaling, and antigen presentation pathways provided protection of tumor cells from CD8 T cell-mediated killing and blunted antitumor immune responses in vivo. Deletion of a number of genes in the TNF pathway also emerged as the key mechanism of immune evasion from primary NK cells. Our screens also identified that the metabolic protein 2-aminoethanethiol dioxygenase (Ado) modulates sensitivity to TNF-mediated killing by cytotoxic lymphocytes and is required for optimal control of tumors in vivo. Remarkably, we found that tumors delete the same genes when exposed to perforin-deficient CD8 T cells, demonstrating that the dominant immune evasion strategy used by tumor cells is acquired resistance to T cell-derived cytokine-mediated antitumor effects. We demonstrate that TNF-mediated bystander killing is a potent T cell effector mechanism capable of killing antigen-negative tumor cells. In addition to highlighting the importance of TNF in CD8 T cell- and NK cell-mediated killing of tumor cells, our study also provides a comprehensive picture of the roles of the TNF, IFN, and antigen presentation pathways in immune-mediated tumor surveillance.
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http://dx.doi.org/10.1126/sciimmunol.aar3451DOI Listing
May 2018

Down-regulation of a pro-apoptotic pathway regulated by PCAF/ADA3 in early stage gastric cancer.

Cell Death Dis 2018 05 1;9(5):442. Epub 2018 May 1.

Sir Peter MacCallum Department of Oncology, The University of Melbourne, Parkville, VIC, Australia.

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.
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http://dx.doi.org/10.1038/s41419-018-0470-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5906598PMC
May 2018
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