Biology subjects

Cook, G. P.

Publications and source records attributed to Cook, G. P..

16 recordsLinked to original sources

bioRxiv2026

Oncolytic virus-antibody combinations enhance immune-mediated killing of osteosarcoma

A major barrier to effective immunotherapy in osteosarcoma (OS) is the highly immunosuppressive tumour microenvironment (TME), which limits immune recognition and elimination of tumour cells. We evaluated a panel of oncolytic herpes simplex viruses (oHSVs) for their direct oncolytic activity, immune-modulatory properties and capacity to counteract OS-associated immunosuppression using immunologically relevant in vitro models. We demonstrate that established OS cell lines, primary cell cultures and dissociated OS cells from freshly resected tumour samples are susceptible to direct oncolysis by three oHSVs; HSV1716, HSV1716-GMCSF, and HSV47{Delta}, although susceptibility levels varied. Treatment of peripheral blood mononuclear cells from healthy donors and OS patients with oHSVs enhanced natural killer (NK) cell activation and promoted immune-mediated killing of OS cell lines and primary OS cell cultures. Among the three viruses, HSV1716-GMCSF exhibited the strongest immune-stimulatory effects and was uniquely capable of reducing the abundance of CD163+CD206+ immunosuppressive TAMs; use of this oHSV was therefore prioritised. We developed a multicellular spheroid model of OS, incorporating OS cells, mesenchymal stem cells and TAMs, which exhibits resistance to immune-mediated killing, better reflecting the immunosuppressive TME in patients. In this model, pairing HSV1716-GMCSF treatment with either anti-GD2 or anti-EGFR monoclonal antibodies (mAbs), selected according to OS tumour antigen expression, significantly increased immune-mediated tumour cell killing. These findings suggest that personalised combination strategies pairing oHSVs with appropriate mAbs provide a promising therapeutic approach for OS by integrating direct oncolysis, remodelling of the immunosuppressive TME and enhanced immune-mediated tumour destruction.

cancer biology↗

bioRxiv2026

Targeting of immune cells by human adenoviruses: CD46 receptor density influences entry of chimaeric Ad5F35 into natural killer cells

Most adenovirus (Ad) vectors are based on the genome of human Ad type 5 (Ad5), which targets their entry to cells that express the Coxsackie and Adenovirus Receptor (CAR or CXADR). However, certain human Ads do not use CAR, for example Ad35 interacts with cell-surface CD46 and Ad3 uses desmoglein 2 (DSG2) for cell entry. In this study, a comparison of different Ad receptors using transcriptomic and proteomic databases showed that CD46 is widely expressed across human cells and tissues whereas CAR and DSG2 are more restricted to epithelial cells. We have used a hybrid virus, Ad5F35, that comprises an Ad5 genome in which the Ad5 fibre was replaced with that of Ad35, thus retargeting the virus from CAR- to CD46-expressing cells and enabling transduction of primary human NK and T cells. However, lymphocytes required approximately 10 to 20-fold more Ad5F35 particles per cell (ppc) compared to A549 epithelial cells to achieve a similar level of transduction. Consistent with this, quantitation of the cell-surface density of CD46 molecules revealed approximately 100 CD46 molecules per {micro}m2 in primary NK cells compared with approximately 2000 CD46 per {micro}m2 in HeLa cells, a 20-fold difference. Cell-surface CD46 density was reduced by approximately 95% by RNA interference in HeLa cells to levels that approximate those found on NK cells. Lower CD46 density reduced transduction by Ad5F35 but this could be compensated for with increased MOI. Our results identify the density of cell surface CD46 as a critical determinant of Ad5F35 transduction and demonstrate that Ad5F35 is an efficient vector for gene delivery in primary human NK cells.

molecular biology↗

bioRxiv2025

Oncolytic Reovirus mediates innate-driven SARS-CoV-2 elimination in the absence of cell toxicity

Interplay between type I interferon (IFN) driven innate responses and viral antagonism strongly influences SARS-CoV-2 transmission and the COVID-19 disease course. Hence, variant adaptation includes diminished induction of IFN stimulated genes (ISG) and/or evasion of their effector functions. Exogenous IFN treatment "rewires" innate responses to drive virus elimination, yet therapeutic trials to date have been unremarkable. Resolving this paradox could translate to variant-agnostic innate immunotherapy. By contrast, oncolytic viruses (OV) exhibit profoundly attenuated innate antagonism, resulting in potent IFN responses despite the inherently immunosuppressive nature of tumour microenvironments. Moreover, OV only undergo lytic replication within innate-deficient malignant cells, and not in cells where sufficient innate responses exist. This, combined with previous studies showing that OV suppressed replication of underlying oncogenic viruses in tumours, we explored whether clinical grade oncolytic Orthoreovirus (Reo) superinfection could eliminate SARS-CoV-2 from immune-competent lung epithelial cell lines in the absence of toxicity. Reo exerted profound activation of innate responses, including when SARS-CoV-2 infection was already established, rewiring cells towards an antiviral state emulating that of Reo infection alone. Both intracellular and paracrine mechanisms induced ISG repertoires including multiple known anti-SARS-CoV-2 effectors, as well as others that remain unvalidated. Amongst these, we demonstrate the first direct evidence that MX2 and XAF1 restrict SARS-CoV-2 replication. Thus, with an excellent safety record, self-amplification, and respiratory tract tropism, we propose that Reo superinfection may provide a tractable alternative to recombinant cytokines for innate antiviral immunotherapy.

immunology↗

bioRxiv2025

Phospho-enol pyruvate carboxykinase inhibition limits inflammatory T cell activation

Following antigenic stimulation, T cells switch from a catabolic metabolic state maintained by low levels of nutrient uptake to an anabolic metabolism that sustains the biosynthetic and energetic demands of clonal expansion, differentiation and effector function. Much progress has been made in understanding the transcriptional and enzymatic regulation of activated T cell metabolism. However less is understood of the role for regulators of anaplerosis and cataplerosis such as phospho-enol pyruvate carboxykinases (PEPCK) in T cells. In the current work, we show that mitochondrial isoform PEPCK-M is upregulated following T cell activation whilst cytosolic PEPCK-C is not expressed. PEPCK inhibitors limited CD8+ T cell cytotoxic capacity and both CD4+ and CD8+ T cell inflammatory cytokine production. Suppression of T cell effector functions by PEPCK inhibitors was associated with decreased maximal mitochondrial respiration. These data suggest that PEPCK-M acts as a metabolic rheostat to enable optimal T cell activation.

immunology↗

bioRxiv2025

Oncolytic Maraba virus MG1 mediates direct and natural killer cell-dependent lysis of Ewing sarcoma

Ewing sarcoma (EWS) is a rare cancer of the bone and soft tissue, most prevalent in children and young adults. Treatment of EWS has progressed relatively little in over 30 years. Survival rates for patients, particularly those with metastatic and/or relapsed disease, remain poor, highlighting the urgent need for innovative treatment options. We have explored the therapeutic potential of the oncolytic Maraba virus strain MG1. We show that MG1 undergoes productive replication and exerts direct oncolysis of established EWS cell lines, doxorubicin-resistant EWS cell lines and patient-derived Ewing sarcoma (PDES) cell cultures more recently established from tumours. In contrast, primary mesenchymal stem cells (the likely cell of origin of EWS) were resistant to MG1, with IFN-I being a major determinant of tumour cell selectivity. MG1 treated PBMC produced IFN-I and killed EWS cells in vitro, in a natural killer (NK) cell-dependent manner. The ability of MG1 to kill EWS cells directly and to stimulate NK cell cytotoxicity against this tumour suggests that Maraba virus MG1 may provide therapeutic benefit for EWS patients where the efficacy of conventional treatments is currently limited.

cancer biology↗

bioRxiv2024

Enhancing oncolytic virotherapy by exosome-mediated microRNA reprograming of the tumour microenvironment.

BackgroundThere has been limited success of cancer immunotherapies in the treatment of ovarian cancer (OvCa) to date, largely due to the immunosuppressive tumour microenvironment (TME). Tumour-associated macrophages (TAMs) are a major component of both the primary tumour and malignant ascites, promoting tumour growth, angiogenesis, metastasis, chemotherapy resistance and immunosuppression. Differential microRNA (miRNA) profiles have been implicated in the plasticity of TAMs. Therefore, delivering miRNA to TAMs to promote an anti-tumour phenotype is a novel approach to reverse their pro-tumour activity and enhance the efficacy of cancer immunotherapies. Oncolytic viruses (OVs) preferentially replicate in tumour cells making them ideal vehicles to deliver miRNA mimetics to the TME. Importantly, miRNA expressed by OVs get packaged within tumour-derived extracellular vesicles (TDEVs), and release of TDEV is augmented by OV infection, thus enhancing the dissemination of miRNA throughout the TME. MethodSmall RNAseq was used to identify differentially expressed miRNA during TAM generation and following LPS/IFN{gamma} stimulation to induce an anti-tumour phenotype. Two differentially expressed miRNA identified, miR-155 and miR-19a, were cloned into oncolytic rhabdovirus (ORV), and anti-tumour efficacy was investigated using both in vitro and in vivo models of OvCa. ResultsThis study demonstrates that ORV infection enhances TDEV production in OvCa cell lines both in vitro and in vivo and that TDEV are preferentially taken up by myeloid cells, including TAMs. Small RNAseq identified 23 miRNAs that were significantly upregulated in anti-tumour TAMs, including miR-155-5p. While 101 miRNAs were downregulated during pro-tumour TAM differentiation, including miR-19a-3p. Culturing TDEV expressing miR-155 or miR-19a with TAMs reversed their immunosuppressive activity, as measured by T cell proliferation. While ORV-miR-155 enhanced the generation of anti-tumour T cells, only ORV-miR19a significantly improved survival of mice bearing ovarian tumours. ConclusionThis study demonstrates (i) that arming ORVs with immunomodulatory miRNA is an effective approach to deliver miRNA to myeloid cells within the TME and (ii) that miRNA have the capacity to reverse the tumour promoting properties of TAMs and improve the efficacy of cancer immunotherapies, such as OV.

cancer biology↗

bioRxiv2022

Pro- and anti-tumour activities of CD146/MCAM in breast cancer result from its heterogeneous expression and association with epithelial to mesenchymal transition

CD146, also known as melanoma cell adhesion molecule (MCAM), is expressed in numerous cancers and has been implicated in the regulation of metastasis. We show that CD146 negatively regulates transendothelial migration (TEM) in breast cancer. This tumour suppressor-like activity is supported by a reduction in MCAM gene expression and increased promoter methylation in tumour tissue compared to normal breast tissue. However, increased CD146/MCAM expression is associated with poor prognosis in breast cancer, a characteristic that is difficult to reconcile with inhibition of TEM by CD146 and its epigenetic silencing. Single cell transcriptome data revealed MCAM expression in multiple cell types, including the tumour vasculature and malignant epithelial cells. MCAM expressing tumour cells were in the minority and expression was associated with epithelial to mesenchymal transition (EMT). Furthermore, gene expression signatures defining invasiveness and a stem cell-like phenotype were most strongly associated with mesenchymal-like tumour cells with low levels of MCAM mRNA, likely to represent an intermediate or hybrid E/M state. Our results show that high levels of MCAM gene expression are associated with poor prognosis in breast cancer because they reflect tumour vascularisation and EMT. However, the inhibitory effects of CD146 on TEM are likely to be weakest in an intermediate state between the epithelial and mesenchymal phenotypes, consistent with highly tumourigenic nature of this population.

cancer biology↗

bioRxiv2022

Negative regulation of p53 by the poliovirus receptor PVR is a target of a human cytomegalovirus immune evasion molecule

Initially characterised for its role in maintaining genome integrity, p53 has emerged as a critical hub for coordinating cellular responses to diverse types of stress. Here we identify cell surface receptor loss as a signal for p53 induction. The poliovirus receptor (PVR) regulates angiogenesis, leucocyte adhesion and immune surveillance. We demonstrate that loss of PVR from endothelial cells also promotes cell cycle arrest through the induction of a p53 transcriptional programme. The p53 induction is post-translational and, despite remaining associated with MDM2, p53 exhibits reduced ubiquitination, aiding its stabilisation. Increased expression of PVR marks malignant or infected cells, and retention of PVR in the endoplasmic reticulum by human cytomegalovirus (HCMV) UL141 protein allows HCMV infected cells to evade immunity. We show that intracellular retention of PVR by UL141 prevents p53 induction, allowing HCMV to escape both the immune- and p53-mediated surveillance functions of PVR. These data reveal that p53 coordinates responses to changes in cell surface composition and that the cell intrinsic PVR-p53 pathway coupled with PVR-mediated immune surveillance functions provide a sensor mechanism to maintain expression of this multi-functional cell surface molecule.

cell biology↗

bioRxiv2022

Molecular dynamics simulations reveal membrane lipid interactions of the full-length lymphocyte specific kinase Lck

The membrane-bound lymphocyte-specific protein-tyrosine kinase (Lck) triggers T cell antigen receptor signalling to initiate adaptive immune responses. Despite many structure-function studies, the mode of action of Lck and the potential role of plasma membrane lipids in regulating Lcks activity remains elusive. Advances in molecular dynamics simulations of membrane proteins in complex lipid bilayers have opened a new perspective in gathering such information. Here, we have modelled the full-length Lck open and closed conformations available from crystallographic studies and simulated its interaction with the inner leaflet of the T cell plasma membrane. In both conformations, we found that the unstructured unique domain and the structured domains including the kinase interacted with the membrane with a preference for PIP lipids. Interestingly, our simulations suggest that the Lck-SH2 domain interacts with lipids differently in the open and closed Lck conformations, demonstrating that lipid interaction can potentially regulate Lcks conformation and in turn modulate T cell signalling. Additionally, the Lck-SH2 and kinase domain residues that significantly contacted PIP lipids are found to be conserved among the Src family of kinases, thereby potentially representing similar PIP interactions within the family.

biophysics↗

bioRxiv2022

TGFβ limits Myc-dependent TCR-induced metabolic reprogramming in CD8+ T cells

T cell activation is dependent upon the integration of antigenic, costimulatory and cytokine-derived signals and the availability and acquisition of nutrients from the environment. Furthermore, T cell activation is accompanied by reprogramming of cellular metabolism to provide the energy and building blocks for proliferation, differentiation and effector function. Transforming growth factor {beta} (TGF{beta}) has pleiotropic effects on T cell populations, having both an essential role in the maintenance of immune tolerance but also context-dependent pro-inflammatory functions. We set out to define the mechanisms underpinning the suppressive effects of TGF{beta} on mouse CD8+ T cell activation. RNA-sequencing analysis of TCR-stimulated T cells determined that Myc-regulated genes were highly enriched within gene sets downregulated by TGF{beta}. Functional analysis demonstrated that TGF{beta} impeded TCR-induced upregulation of amino acid transporter expression, amino acid uptake and protein synthesis. Furthermore, TCR-induced upregulation of Myc-dependent glycolytic metabolism was substantially inhibited by TGF{beta} treatment with minimal effects on mitochondrial respiration or mTOR activation. Thus, our data suggest that inhibition of Myc-dependent metabolic reprogramming represents a major mechanism underpinning the suppressive effects of TGF{beta} on CD8+ T cell activation.

immunology↗

bioRxiv2021

The CXCR6/CXCL16 axis links inflamm-aging to disease severity in COVID-19 patients

Advancing age and chronic health conditions, significant risk factors for severe COVID-19, are associated with a pro-inflammatory state, termed inflamm-aging. CXCR6+ T cells are known to traffic to the lung and have been reported to increase with age. The ligand of CXCR6, CXCL16, is constitutively expressed in the lung and upregulated during inflammatory responses and the CXCR6/CXCL16 axis is associated with severe lung disease and pneumonia. Genome-wide association studies have also recently identified 3p21.31, encompassing the CXCR6 gene, as a susceptibility locus for severe COVID-19. We assessed numbers T cells expressing the chemokine receptor CXCR6 and plasma levels of CXCL16, in control and COVID-19 patients. Results demonstrated that circulating CD8+CXCR6+ T cells were significantly elevated with advancing age, yet virtually absent in patients with severe COVID-19. Peripheral levels of CXCL16 were significantly upregulated in severe COVID-19 patients compared to either mild COVID-19 patients or SARS-CoV-2 negative controls. This study supports a significant role of the CXCR6/CXCL16 axis in the immunopathogenesis of severe COVID-19.

immunology↗

bioRxiv2021

Multi-scale simulations of the T cell receptor reveal its lipid interactions, dynamics and the arrangement of its cytoplasmic region

The T cell antigen receptor (TCR-CD3) complex initiates T cell activation following recognition of peptides presented by Major Histocompatibility Complex (pMHC)-encoded proteins. The ligation of pMHC to TCR{beta} induces Src family kinases activity via the cytoplasmic tails of the CD3{delta}{varepsilon}, CD3{gamma}{varepsilon} and {zeta}{zeta} dimers. The TCR-CD3 topology is well understood, but little is known about its conformational dynamics and arrangement of its cytoplasmic tails, limiting our grasp of the signalling mechanism. Here, we investigated the entire TCR-CD3 embedded in an asymmetric lipid bilayer using molecular modelling and multi-scale molecular dynamics simulations. Our study demonstrates conformational changes in the extracellular and transmembrane domains, and the arrangement of the TCR-CD3 cytoplasmic tails. The TCR{beta} variable regions were the most flexible in the extracellular domain. The cytoplasmic tails formed highly interlaced structures while some tyrosine sidechains within the immunoreceptor tyrosine-based activation motifs (ITAMs) of the CD3{varepsilon} and {zeta} subunits dynamically penetrated the hydrophobic core of the bilayer. Ionic interactions between the cytoplasmic tails and phosphatidylinositol phosphates (PIP2 and PIP3) in the inner leaflet of the lipid bilayer led to the formation of a distinct annular lipid fingerprint around the TCR-CD3 complex. These results combined with available experiential data increase our understanding of the TCR-CD3 activation mechanism and highlight the importance of membrane lipids in regulating T cell activation. Significance statementThe T cell receptor (TCR-CD3) detects antigenic peptides displayed by major histocompatibility complexes (pMHC) to instigate activation of T cell adaptive immunity. Despite significant structural and functional knowledge of TCR-CD3 topology, the membrane interactions and dynamics of its cytoplasmic moieties remain elusive. Interactions of TCR-CD3 cytoplasmic tails with membrane lipids may regulate their phosphorylation by Src-family kinases, the first intracellular event required for T cell activation. Using the static 3D structure of TCR-CD3 resolved by cryo-electron microscopy, we provide novel insights into the protein-lipid interactions of the complete TCR-CD3 embedded in a bilayer closely mimicking its native membrane environment. Our study sheds light on the dynamics of the TCR-CD3 at near-atomic resolution and further aids in deciphering its activation mechanism.

biophysics↗

bioRxiv2020

Oncolytic virus treatment differentially affects the CD56dim and CD56bright NK cell subsets in vivo and regulates a spectrum of human NK cell activity

Natural killer (NK) cells protect against intracellular infection and cancer. These properties are exploited in oncolytic virus (OV) therapy, where anti-viral responses enhance anti-tumour immunity. We have analysed the mechanism by which reovirus, an oncolytic dsRNA virus, modulates human NK cell activity. Reovirus activates NK cells in a type I interferon (IFN-I) dependent manner, resulting in STAT1 and STAT4 signalling in both CD56dim and CD56bright NK cell subsets. Gene expression profiling revealed the dominance of IFN-I responses and identified induction of genes associated with NK cell cytotoxicity and cell cycle progression, with distinct responses in the CD56dim and CD56bright subsets. However, reovirus treatment, acting via IFN-I, inhibited NK cell proliferative responses to IL-15 and was associated with reduced AKT signalling. In vivo, human CD56dim and CD56bright NK cells responded with similar kinetics to reovirus treatment, but CD56bright NK cells were transiently lost from the peripheral circulation at the peak of the IFN-I response, suggestive of their redistribution to secondary lymphoid tissue. These results show that reovirus modulates a spectrum of NK cell activity in vivo, encompassing direct action on tumour cells and the regulation of adaptive immunity. Such activity is likely to mirror NK cell responses to natural viral infection.

immunology↗

bioRxiv2019

Expression profiling of single cells and patient cohorts identifies multiple immunosuppressive pathways and an altered natural killer cell phenotype in glioblastoma

Glioblastoma (GBM) is an aggressive cancer with a very poor prognosis. Generally viewed as weakly immunogenic, GBM responds poorly to current immunotherapies. To better understand this problem we used a combination of NK cell functional assays together with gene and protein expression profiling to define the NK cell response to GBM and explore immunosuppression in the GBM microenvironment. In addition, we used transcriptome data from patient cohorts to classify GBM according to immunological profiles. We show that glioma stem-like cells, a source of post-treatment tumour recurrence, express multiple immunomodulatory cell surface molecules and are targeted in preference to normal neural progenitor cells by natural killer (NK) cells ex vivo. In contrast, GBM-infiltrating NK cells express reduced levels of activation receptors within the tumour microenvironment, with hallmarks of TGF-{beta} mediated inhibition. This NK cell inhibition is accompanied by expression of mutiple immune checkpoint molecules on T cells. Single cell transcriptomics demonstrated that both tumour and haematopoietic-derived cells in GBM express multiple, diverse mediators of immune evasion. Despite this, immunome analysis across a patient cohort identifies a spectrum of immunological activity in GBM, with active immunity marked by co-expression of immune effector molecules and feedback inhibitory mechanisms. Our data show that GBM is recognised by the immune system but that anti-tumour immunity is restrained by multiple immunosuppressive pathways, some of which operate in the healthy brain. The presence of immune activity in a subset of patients suggests that these patients will more likely benefit from combination immunotherapies directed against multiple immunosuppressive pathways.

immunology↗

bioRxiv2019

CD99 regulates cancer cell transendothelial migration and endothelial cell function via CDC42 and actin remodelling

Metastasis requires tumour cells to cross endothelial cell (EC) barriers and this occurs using mechanisms similar to those used by extravasating leucocytes during inflammation. The cell surface receptor CD99 is expressed by leucocytes and EC and participates in inflammatory transendothelial migration (TEM). CD99 is also expressed by tumour cells and we have analysed its role in tumour progression and cancer cell TEM. In a xenograft model, CD99 expression inhibited the metastatic progression of human breast cancer. In vitro, tumour cell CD99 was required for adhesion to ECs. However, tumour cell CD99 inhibited the invasion of the endothelial barrier by breast and prostate cancer cells and TEM itself. Furthermore, tumour cell CD99 depletion was associated with cytoskeletal remodelling. Loss of EC CD99 enhanced endothelial barrier function and reduced tumour cell TEM. Mechanistically, CD99 loss enhanced the expression and activity of CDC42, a known cytoskeletal organiser. CDC42 positively regulates EC angiogenic activity and the enhanced CDC42 activity resulting from loss of EC CD99 increased angiogenesis. As a signal transduction hub, CDC42 activity impacts upon many of the hallmarks of cancer. The functional link between CD99 and CDC42 identified here implicates CD99 in regulating these diverse pathways by modulation of CDC42 activity.

cell biology↗

bioRxiv2019

Site-directed M2 proton channel inhibitors enable synergistic combination therapy for rimantadine-resistant pandemic influenza

Pandemic influenza A virus (IAV) remains a significant threat to global health. Preparedness relies primarily upon a single class of neuraminidase (NA) targeted antivirals, against which resistance is steadily growing. The M2 proton channel is an alternative clinically proven antiviral target, yet a near-ubiquitous S31N polymorphism in M2 evokes resistance to licensed adamantane drugs. Hence, inhibitors capable of targeting N31 containing M2 (M2-N31) are highly desirable.\n\nRational in silico design and in vitro screens delineated compounds favouring either lumenal or peripheral M2 binding, yielding effective M2-N31 inhibitors in both cases. Hits included adamantanes as well as novel compounds, with some showing low micromolar potency versus pandemic \"swine\" H1N1 influenza (Eng195) in culture. Interestingly, a published adamantane-based M2-N31 inhibitor rapidly selected a resistant V27A polymorphism (M2-A27/N31), whereas this was not the case for non-adamantane compounds. Nevertheless, combinations of adamantanes and novel compounds achieved synergistic antiviral effects, and the latter synergised with the neuraminidase inhibitor (NAi), Zanamivir. Thus, site-directed drug combinations show potential to rejuvenate M2 as an antiviral target whilst reducing the risk of drug resistance.

microbiology↗