Research Opportunities

Regulatory T cells (Tregs) have the ability to suppress allogeneic immune responses and have therefore become interesting as a possible cellular therapy.  Our group is leading a phase II randomised clinical trial utilising Tregs as a cellular therapy for kidney transplant recipients.

The proposed project aims to in depth investigate the local immune response within the transplanted allograft, specifically focusing on infiltrating regulatory T cells and their local interactions with other infiltrating immune cells, utilising renal biopsy samples from the cell therapy trial.  We hypothesise, that cellular therapy with regulatory T cells will result in trafficking of the Tregs cells to the allograft and creation of a local tolerogenic microenvironment.

We are proposing to utilise our unique collection of Treg treated patients’ biopsy samples employing the cutting-edge technology of spatial transcriptomics and single cell RNA sequencing to determine the cellular and molecular composition of leukocyte infiltrates in allograft biopsies, dissect cellular interactions in situ and determine Treg homing to the allograft.

The human microbiome is important for many aspects of our health and yet we currently lack the ability to modulate it for the vast majority of diseases. The key challenge is that it is a diverse ecological system, containing many strains and species of microbe, which needs new approaches and paradigms for the treatment of disease. This project will culture diverse anaerobic species of gut bacteria alongside bacterial pathogens to look for species combinations that can prevent or treat disease. The core methods will involve bacterial culture in the lab, but also germ-free mouse work to validate discoveries. A combination of ecological and evolutionary approaches will be applied to help rationally design multispecies communities that both treat disease but also avoid the evolution of resistance in pathogens. In this way, the goal is to help usher in a new set of treatments for microbiome-based diseases and reduce our reliance on antibiotics.

Most translational efforts for uncommon cancers stem from oncogenic mutations identified by sequencing. In contrast to genomic analyses, the tumor proteome has the potential to better approximate phenotype-inducing alterations, particularly in the absence of targetable driver mutations. However, tumor analyses using mass spectrometry can be challenging. Exosomes (small extracellular membrane-enclosed vesicles) may circumvent these issues and serve as a valuable, prioritized, “window” into the tumor cell proteome. Applying this reasoning to bile duct cancers (cholangiocarcinoma, CCA), we performed mass spectrometry on exosomes extracted from patient bile, revealing a 17-fold enhancement of the nuclear export protein XPO7. Immunohistochemistry analysis of XPO7 expression in 318 CCA patients unexpectedly demonstrated intense cytoplasmic staining. Within the cytosol we demonstrate that XPO7 exists in a molecular complex with the serine/threonine kinase SLK. shRNA-mediated knockdown of either XPO7 or SLK in CCA lines abrogated tumor organoid formation and reduced orthotopic tumor growth. To translate the target to patients, we identified tivozanib as a potent SLK inhibitor. Tivozanib treatment reduced tumor organoid formation in vitro and induced tumor regression in vivo in patient derived xenografts (n=2). Together, these findings reveal a novel cytosolic XPO7:SLK signaling axis that is targetable in CCA patients and we have already documented early responses with our accruing Phase I/II trial (NCT 04645160). It is however clear that single agents will not result in cures for patients with solid tumors, and a better understanding of the XPO7:SLK complex (including downstream oncogenic signaling axes) will be required to formulate and implement synergistic combination therapies.

Persistent inflammation is a pathogenic determinant in many common human diseases, including rheumatoid arthritis, Crohn’s disease, multiple sclerosis, and psoriasis, while playing a key role in the development of type-2 diabetes, obesity, certain forms of cancer, and neurodegenerative conditions such as Parkinson’s and Alzheimer’s diseases. The laboratory of Dr. Blackshear has identified tristetraprolin (TTP) as a protein that acts to restrict inflammation in mice by destabilising the mRNA of pro-inflammatory cytokines such as TNF, GM-CSF, CXCL1 and CXCL2. Mice where an instability-conferring element within TTP’s own mRNA was deleted using homologous recombination were remarkably resistant to inflammatory disease. Most importantly, these mice did not exhibit any pathology, presumably because of the relatively modest over-expression of TTP that was otherwise subject to normal regulation.  

However, studies of TTP’s biochemical and molecular activities have been hampered by the existence of three other members of the same protein family in the mouse.  Each of these proteins exhibits similar biochemical activities in cell transfection experiments and in cell-free assays, but knockout mice of each have led to dramatically different phenotypes, involving early development, hematopoiesis, and placental function rather than inflammation.  For this reason, we will use the fruit-fly Drosophila melanogaster, which expresses only a single TTP family member, known as TIS11. Using genetics, biochemistry, transcriptomics, and proteomics we will explore the role of TIS11 and extrapolate findings to experiments in mammalian systems at NIEHS.

Sympathetic neurons have a wide range of physiological functions and their hypoactivity contributes to obesity and diabetes, among other syndromes. Sympathomimemic drugs rescue this deficiency but this drug class, mostly composed of brain-penetrant amphetamines and adrenergic agonist, is both cardiotoxic and highly controlled. Our recent publication puts forward new class of drugs named Sympathofacilitators that do not enter the brain and have an anti-obesity and cardio-neutral effect in vivo. The first in-class was published in Mahu I, Domingos, et al. Cell Metabolism 2020; Figure 3C of this paper demonstrated a neuro-facilitatory effect, rather than neuro-excitatory one.

This new class is in need of novel chemical entities which can be screened in vitro on cultured iPSC-derived sympathetic neurons. The screen would be based on fluorescent readouts of calcium activity reporter, screening for a facilitation of responses to acetylcholine (similar to Figure 3C of Mahu I, et al).

The prospect of identifying natural compounds that have a Sympathofacilitatory effect is tangible when performed in collaboration with the laboratory of Barry O'Keefe. The student will learn how to grow and scale-up iPSC-derived sympathetic neurons in the Domingos lab, and optimize an in vitro assay based on Figure 3C. The student will then transfer this knowledge to the lab of Barry O'Keefe where the screen will be performed using a fluorescent plate reader, robotic liquid handling, and a library of natural compounds.

We are interested in understanding the clonality of drug resistance in cancer (primarily in a haematological cancer called Multiple Myeloma (MM)). To achieve our goals, we have developed state of the art long-read single-cell sequencing approaches (termed scCOLOR-seq) that allow us to measure single clones within patient samples. This method allows for the simultaneous measurement of gene expression, exon mutations, exon SNPs and translocations. We apply this technology and develop cutting edge computational analysis solutions to understand the relationship between clonality and drug resistance in oncology. Furthermore, our lab is also working as part of the Human Cell Atlas (HCA) project and we have several international collaborations with immunologists, cancer biologists to support our work.

The aim of this project is to develop computational analysis strategies aimed at better defining specific MM clones within patients that are resistant to first line therapeutics. The work will involve combining long-read (Oxford Nanopore Technology) multi-modal datasets and performing machine learning approaches to better define high risk patients. This work is important to better understand drug resistance mechanisms in MM and identify patients that may respond less well to therapy.

Accumulation of misfolded proteins and aberrant protein aggregates are hallmarks of a wide range of pathologies such as neurodegenerative diseases and cancer. Under normal conditions, these potentially toxic protein species are kept at low levels due to a variety of quality control mechanisms that detect and selectively promote their degradation. Our lab investigates these protein quality control processes with a particular focus on ER-associated degradation (ERAD), that looks after membrane and secreted proteins. The ERAD pathway is evolutionarily conserved and in mammals, targets thousands of proteins influencing a wide range of cellular processes, from lipid homeostasis and stress responses to cell signaling and communication.

We investigate the mechanisms of ERAD using multidisciplinary approaches both in human and yeast cells. Using CRISPR-based genome-wide genetic screens and light microscopy experiments we identify and characterize molecular components involved in the degradation of disease-relevant toxic proteins. In parallel, we use biochemical tools to dissect mechanistically the various steps of the ERAD pathways. In this collaborative project with the Lea lab we will use structural approaches such as cryo-electron microscopy to gain insight into the molecular mechanisms of ERAD.

These studies, by providing mechanistic understanding of the ERAD process, may shed light on human diseases impacting ER function and may ultimately contribute to better therapeutics. 

The laboratory of Prof Bridge in Oxford focusses on understanding the pathways in the human visual system that can process residual vision after someone has had a stroke that affects the primary visual cortex.

The laboratory of Prof Leopold combines neuroimaging, behaviour and neurophysiology in a non-human primate model to better understand computation in the visual system, particularly relating to conscious perception.

The proposed PhD project would have 3 main objectives:

1. Quantitatively compare changes in retinotopic maps and population receptive fields in humans and non-human primates with damage to primary visual cortex.
2. Determine the visual pathways in the two species that are necessary and/or sufficient to provide residual vision within the blind region of the visual field.
3. Investigate the neural changes that occur as a result of visual training following the damage to the visual system in order to inform rehabilitation programmes for people who have suffered a stroke to the visual system.
   
   During the training programme, the student would have the opportunity to learn about multi-modal human neuroimaging approaches applied to both the healthy and the damaged visual system. This would be complemented by training in both neuroimaging and neurophysiology in the non-human primate.

Our aim is to understand the sensory circuits that govern normal (protective) touch and pain, and how following injury or disease to the nervous system, pain can become chronic (neuropathic pain). Sensory neurons are heterogeneous neurons that innervate sensory targets (such as the skin), and extend central terminals which enter the dorsal horn of the spinal cord. We are interested in the role of molecularly defined subpopulations of sensory neurons, and how they contribute to the development and maintenance of neuropathic pain. We utilise chemogenetic and optogenetic strategies to selectively silence or activate sensory neuron function in mice. Combining this with behavioural paradigms to assess evoked and spontaneous pain, allows us to identify key populations of sensory neurons in pain processing, with the hope of identifying new druggable targets for future development. Further validation is undertaken in human iPSC derived sensory neurons. Students will learn and use a wide range of techniques including (but not limited to); Chemogenetics, optogenetics, viral vectors, mouse transgenics, animal behaviour, electrophysiology and calcium imaging.

The spinocerebellar ataxias (SCAs) are a complex group of neurodegenerative diseases that affect the cerebellum and result in the loss of motor coordination. No effective treatments exist for the SCAs, and there is a pressing need for better models in which to study the underlying disease-causing mechanisms and to identify potential therapies.

The aim of this project will be to develop novel stem cell-derived models to identify common pathological mechanisms in SCA that could be targeted therapeutically. The Becker group has identified several novel SCA mutations that highlight mGluR1-TRPC3-IP3R1 signaling as a key pathway affected in disease. Both research groups have developed complementary stem cell-derived and primary cerebellar models that provide unique systems to investigate the functional consequences of gene mutations affecting this pathway in the Purkinje cells, which are the neurons that are primarily affected in SCA.

The project will employ patient-derived induced pluripotent stem cells (iPSCs) as well as introduce gene mutations into iPSCs using CRISPR gene editing technology. Pluripotent stem cells will be differentiated into cerebellar neurons and three-dimensional organoids and deeply phenotyped using a combination of functional experiments including calcium imaging, super-resolution imaging, and morphological analyses. In addition, functional analyses will be carried out in primary Purkinje cells. Identified disease phenotypes will subsequently be screened for potential therapeutics.

Becker Group website: https://www.ndcn.ox.ac.uk/research/cerebellar-disease-group

Hammer Group website: https://irp.nih.gov/pi/john-hammer

How are different B cell populations developmentally linked in human health and disease?

We are investigating the generation, function and plasticity of B cell populations in human health. In particular, we are interested in how different lymphocyte subsets are developmentally linked and differences in function, and therefore providing a platform to understand how B cell fate may be different in human disease. We are defining how B cells select a particular developmental pathway, and will use this information to develop methods for modulating B cell function as potential therapeutic approaches.

How can B and T cells may be therapeutically modulated across cancers and autoimmune diseases?

There is accumulating evidence for the role of both T and B cells in modulating immune responses to both solid tumours and haematological malignancies. We are investigating the contributions, function and heterogeneity of B and T cells on the immune responses to tumours and their potential role in cancer detection and treatment. We are determining the nature of B and T cell immuno-surveillance, regulation and activation across cancers and autoimmune diseases, as well as the immunological features associated with better prognosis and immunomodulation. With this, we aim to highlight novel therapeutic avenues. Our lab is affiliated with the Oxford Cancer Centre (https://www.cancer.ox.ac.uk/research/research-themes/developments-in-immuno-oncology) and non-cancer clinicians, with strong clinical links to a wide range of hard-to-treat diseases.

What is the effect of genetic and environmental variation on B and T cell fate?

Immunological health relies on a balance between the ability to mount an immune response against potential pathogens and tolerance to self. B and T cells are key to the immune response by producing antibodies and cytotoxic T cells. B/T cell clones selectively expand following antigen recognition by B and T cell receptors (BCR and TCR) respectively. BCRs are the membrane-form of antibodies and are generated through DNA recombination resulting in the potential to recognise a vast array of pathogens. Defects in the ability to mount effective B cell or T cell responses have been implicated in infectious susceptibility, impaired surveillance of cancer and immunodeficiencies, whereas a breakdown of immunological tolerance has been attributed to autoimmune diseases such as through autoantibody production and reduced numbers of regulatory B/T cells. Through integrating genomics, bulk and single-cell transcriptomics, and metabolomics data, serological, B /T cell repertoire and viromics datasets we will investigate the effect of both genetic variation and environmental factors on B cell fate, regulation, and the relationship to disease susceptibility.

T cells are a major component of adaptive immune response, continuously screening lymphoid tissues for antigen peptides presented by major histocompatibility complex (peptide/MHC or pMHC)3. These antigen peptides are recognized by T-cell receptors (TCRs). Thymocytes with a low-affinity TCRs mature into T cells and enter the lymphoid organs, where they are exposed to foreign antigen peptides by MHC molecules from antigen-presenting cells including macrophages, dendritic cells, and B-cells, during infection. When the T-cell receptors bind to antigenic peptide, T-cells are activated and undergo clonal expansion, resulting in immune response. CD8+ T cells play an important for immune response and viral clearing, but their role in protection and pathogenesis of SARS-CoV-2 remains poorly understood4. In addition to extensively studied spike protein, open reading frame 3a (ORF3a), a highly conserved protein within the Betacoronavirus subgenus, has been considered as a potential target for vaccines or therapeutics, with deletion of ORF3a resulting in decreased viral titer and morbidity. We have identified shared CD8+ T cell clonotypes responding to a ORF3a in COVID-19 infections.  Importantly, shared clonotypes in severe COVID-19 infections provides a target for development of novel antiviral immunotherapies. The aim of this project is to analyse shared TCR clonotypes in ORF3a recognition and provide structural basis for the recognition of ORF3a-pMHC complex by T-cell receptors.

Aggregations of the cytoskeletal protein Tau into neurofibrillary tangles and cleavage products of the Amyloid Precursor Protein (APP) into amyloid plaques are strongly associated with the progression of Alzheimer’s Disease (AD). However, it remains unclear whether these aggregates initiate and cause disease or are primarily by-products of more fundamental defects. Furthermore, the physiological and cellular roles of Tau and APP, which might be disrupted in AD, are not well understood.

We have developed a new cell model in the fruit fly, Drosophila melanogaster, to study the normal functions of fly APP and Tau. Remarkably, we find that they are both involved in the biogenesis and secretion of two multimolecular signalling complexes, which are formed in endosomes: exosomes, which are small secreted vesicles, and supermeres, newly identified aggregates of protein and RNA. Their secretion is disrupted when pathological versions of Tau or cleaved APP are expressed in these cells. Using the fly model, we have already identified multiple additional regulators of these APP- and Tau-dependent processes, some of which are implicated in other neurodegenerative diseases. This project will employ neuronal and cancer cell lines to investigate which of these mechanisms are conserved in human cells and how they affect both exosome and supermere signalling. Again, informed by genetic screens in flies, we will then test whether Tau- and APP-induced defects in secretion are suppressed by genetic manipulations of other genes, which might provide new therapeutic targets going forward.

More than 90% of the drugs that enter clinical trials fail because of lack of efficacy or unexpected toxicity. This high failure rate has been partly attributed to the use of in vitro cellular assays and animal models that do not reproduce human physiology and pathology during preclinical drug development. This gap in clinical predictability in drug development is especially severe for CNS disorders, in which many therapeutics must cross the blood-brain barrier (BBB) to be effective. There has been a steady development and evaluation of drug delivery mechanisms that bypass the BBB to reach the target site in the CNS, and ultrasound (US)-mediated therapeutics extravasation has been one of such tools. However, while there have been several in vivo animal and clinical studies done on the focused US-guided BBB disruption, there have not yet been an established in vitro model in which US has been utilized to understand the BBB tight junction disruption and penetration. This project seeks to develop a microfluidics-based BBB model in order to: (1) investigate brain endothelial cell behaviour in microenvironment when stimulated by US and microbubbles; (2) establish whether US provides a means to enhance drug delivery through the BBB using the in vitro microfluidics-based assay platform; (3) investigate the stimulation of any inflammatory responses arising from ultrasound/microbubble exposure (in collaboration with NCI, Frank Lab). The work will contribute to greater understanding of interactions between US-mediated microbubble disruption of the BBB, especially benefitting the physicians working to utilize the technique in clinical settings and the researchers developing microbubbles to establish and optimize BBB drug delivery during the drug R&D pipeline.

Antibiotics help clear an ongoing infection, but they can also cause significant collateral damage: they select for drug-resistant strains and cause dysbiosis to the commensal gut microbiota. This antibiotic-induced selection for resistance within the microbiome can facilitate the spread of resistant pathogens to extra-intestinal infections and to other patients (see our recent work: Stracy et al. Science. 2022, 375 (6583), 889-894).

This project will aim to understanding how antibiotics cause collateral damage to the microbiota leading to subsequent resistant infections. This will involve experiments with synthetic and human-derived microbial communities as well as developing microscopy methods to understand the effect of antibiotics on the micro-scale biogeography of the microbiota. The aim is to characterise the key factors that determine how antibiotics affect a patient’s resident microbial population and cause resistant opportunistic pathogens to spread. This will be used to help develop new ways to minimize the spread of antibiotic resistance both within and between patients. The project would suit applicants with a strong background in microbiology and keen interest in developing new ways to combat antibiotic resistance.