Research Opportunities

All viruses deliver or generate RNA in the cytosol. Cytosolic double-stranded RNA (dsRNA) is recognized by the innate immune sensors MDA5, LGP2 and RIG-I. Our group has shown that MDA5 forms filaments and cooperates with LGP2, TRIM14 and TRIM65 to recognize viral dsRNAs. Protein-RNA complexes containing both MDA5 and LGP2 activate the signaling hub protein MAVS to induce a potent antiviral interferon response. TRIM14 and TRIM65 are thought to promote crosslinking of filamentous MDA5/LGP2 complex. However, key aspects of how LGP2, TRIM14 and TRIM65 contribute to RNA sensing remain unclear. MDA5 and LGP2 are both known to bind dsRNA but whether the two proteins interact directly with each other to activate signaling is unknown. We used AlphaFold-Multimer to generate a molecular model of an MDA5-LGP2 heterodimer with high confidence metrics, but this model has not been tested experimentally. How TRIM-dependent filament crosslinking enhances signaling is also not understood.  The aims of this project are to use biochemical, structural, and cell-based approaches to obtain a detailed mechanistic understanding of the contributions of LGP2, TRIM14 and TRIM65 to viral RNA sensing by MDA5. The first aim of this project to test our AlphaFold model of the MDA5-LGP2 complex using structure-based mutations and functional assays. The second aim is to determine the structure of the MDA5 signaling holocomplex containing LGP2, TRIM14, and TRM65 using state-of-the-art cryo-EM image reconstruction approaches. These studies will provide important insights on the mechanism of RNA sensing by MDA5.  

References:

Rahul Singh, Yuan Wu, Alba Herrero del Valle, Kendra E. Leigh, Mark Cheng, Brian J. Ferguson & Yorgo Modis (2023) Contrasting functions of ATP hydrolysis by MDA5 and LGP2 in viral RNA sensing. DOI:10.1101/2023.05.25.542247  

Qin Yu, Alba Herrero del Valle, Rahul Singh & Yorgo Modis (2021) MDA5 autoimmune disease variant M854K prevents ATP-dependent structural discrimination of viral and cellular RNA. Nat. Commun., 12, 6668. DOI:10.1038/s41467-021-27062-5 

Tissue inflammation is a critical response for the front line of infections. However this same inflammatory response within the tissues is also potentially the most damaging – causing destruction to tissue cells and impeding the physiological functions of the tissue. The actions of immunosuppressive cell types, most potently regulatory T cells (Tregs) can counter this destructive inflammation, but the numbers of these cells within the tissues are limiting (Liston and Gray, Nature Reviews Immunology 2014). Through the use of gene therapy vectors we can deliver potent biologics that either enhance the number of immunosuppressive cell types, or replicate their function, within a tissue. We previously used this approach to create an anti-inflammatory pro-repair environment in the brain, protecting against brain inflammation (Yshii et al, Nature Immunology 2022) and aging (Lemaitre et al, EMBO Mol Med 2023). We have developed a novel system of gene delivery that allows an analogous correction of inflammation within the lung, with potential use in respiratory infections, and non-infectious inflammatory diseases. The proposed project will work on identifying the optimal biologics to deliver to the lung to drive repair during inflammation, will test the novel treatment in mouse models of respiratory disease, and will initiate testing in human lung tissue.

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.

Autoimmune central nervous system (CNS) demyelinating diseases, especially multiple sclerosis (MS) are a major public health burden and poorly controlled by current immunosuppressants. More precise immunotherapies with higher efficacy and fewer side effects are sought.

We are investigating genetic basis of autoimmunity genomic DNA sequencing of the largest cohort of MS patients and family members (14,000 specimens) in an effort to improve therapy. We are also evaluating the effectiveness and mechanism of an injectable myelin-based antigenic polyprotein MMPt to achieve antigen-specific treatment of MS. We find that it suppresses mouse experimental autoimmune encephalomyelitis (EAE) without major side effects. MMPt induces rapid apoptosis of the encephalitogenic T cells and suppresses inflammation in the affected CNS. Intravital microscopy shows that MMPt is taken up by perivascular F4/80+ cells but not conventional antigen-presenting dendritic cells, B cells, or microglia. MMPt-stimulated F4/80+ cells induce reactive T-cell immobilization and apoptosis in situ, resulting in reduced infiltration of inflammatory cells and chemokine production.

Our study will examine the anatomical and molecular aspects of new gene involvement and the effects of antigen-specific therapy to reveal new mechanisms that explain how cognate antigen suppresses CNS inflammation and may be applicable for effectively and safely treating demyelinating diseases.

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.

The brain is a site of relative immune privilege, long considered isolated from the peripheral immune system. We recently identified a population of resident T cells in the healthy mouse and human brain, important for the maturation of microglia (Pasciuto et al, Cell 2020). By analysing the kinetics of migration between the blood and brain, we found that the key bottleneck controlling the number of anti-inflammatory regulatory T cells in the brain was the high rate of cell death the cells exhibit when housed within the brain. Through developing a unique tool, with potential therapeutic application, we were able to deliver a biologic directly to the brain and enhance the size of the regulatory T cell population. The approach protects mice from brain damage following traumatic brain injury, stroke and multiple sclerosis. In this project we wish to explore the immunological processes that drive damage during neuroinflammation, and to harness immune-modulating biologics to prevent damage to the brain. 

Immunity and immune-tolerance at mucosal and other barrier surfaces is vital for host survival and homeostasis with antibodies or immunoglobulins (Ig) playing a key role. However, the specific roles of B cell sub-types, particularly, the innate-like B-1 cell subset is poorly understood. The surgeon James Rutherford Morrison (1906) called the omentum in the peritoneal cavity the "abdominal policeman" and it promotes gut IgA production by peritoneal B-1 cells. IgA is the most abundantly produced antibody isotype and is known to be important for mucosal immunity. It is estimated that ~50% of IgA is derived from B-1 cells. In addition, B-1 cells are thought to be important because they make T cell-independent “natural” IgM circulating in our blood, and they can rapidly respond to mucosal perturbations such as an infection. By contrast, it will take conventional B-2 cells weeks to mount a germinal center (GC) reaction and generate antibodies that have undergone T cell-dependent affinity maturation and isotype switching. Textbooks currently do not entertain the possibility that B-1 cells can also participate in GC reactions. This project aims to challenge such an assumption. After all, B-1 cells in the gut mucosa and probably other mucosal tissues can undergo class switch recombination to IgA. However, the differentiation program that leads to this distinct pathway of IgA production is not well understood: for example, it is unknown if this process occurs outside or within GCs in mucosa-associated lymphoid tissues (MALT). Expertise on B-1 cells in the Muljo lab and the GC reaction in the Turner lab will be combined to explore this potentially paradigm-shifting research. Both wet-bench and bioinformatic research opportunities are available.

Students will learn about the fundamentals of transcriptional; epigenetic and post-transcriptional regulation; immunometabolism; in vivo CRISPR screening; CRISPR editing in primary B cells; and systems immunology. The combination of classical immunological techniques and cutting-edge, multi-disciplinary approaches will enable important discoveries to define the in vivo biology of B-1 cells. Ultimately, we seek novel insights that can be translated to inform vaccine design targeted to activate B-1 cells and/or therapeutics to inhibit their activity when necessary.
 

Recent publications:

Turner, D. J., Saveliev, A., Salerno, F., Matheson, L. S., Screen, M., Lawson, H., Wotherspoon, D., Kranc, K. R., and Turner, M. (2022). A functional screen of RNA binding proteins identifies genes that promote or limit the accumulation of CD138+ plasma cells. eLife, 11, e72313. PMID: 35451955; DOI: 10.7554/eLife.72313.

Osma-Garcia, I.C., Capitan-Sobrino, D., Mouysset, M., Bell, S.E., Lebeurrier, M., Turner, M. and Diaz-Muñoz, M.D. (2021). The RNA-binding protein HuR is required for maintenance of the germinal centre response. Nature Communications, 12(1):6556. PMID: 34772950; DOI: 10.1038/s41467-021-26908-2.

Wang, S., Chim, B., Su, Y., Khil, P., Wong, M., Wang, X., Foroushani, A., Smith, P. T., Liu, X., Li, R., Ganesan, S., Kanellopoulou, C., Hafner, M. and S. A. Muljo. (2109). Enhancement of LIN28B-induced hematopoietic reprogramming by IGF2BP3. Genes & Development, 33: 1048-1068. PMID: 31221665; DOI: 10.1101/gad.325100.119.

The Phosphoinositide 3-kinases (PI3Ks) are a family of lipid kinases that control diverse signalling pathways affecting gene-transcription, cellular adhesion and trafficking, autophagy and metabolism via the generation of PIP3. While some of these readouts are controlled by the evolutionarily conserved PI3K-AKT-FOXO, PI3K-AKT-mTOR axes, there is a diverse network of PI3K effectors that are less well studied, especially in lymphocytes, but which nonetheless can have profound effects on lymphocyte biology. We have recently used CRISPR/Cas9 to perform a targeted screen of PI3K effectors by generating a library that specifically targets PIP3-binding proteins. Screening for genes that affect T cell adhesion, we identified RASA3 as a key protein linking PI3K to the activation of the integrin LFA-1 and found that RASA3 is critical for T cell migration, homeostasis and responses to immunization. We have now generated extended CRISPR/Cas9 libraries that target the entire PI3K-ome (including the kinases, phosphatases and all known effector proteins). Potential projects include designing and implementing new screens for downstream readouts of PI function, including autophagy, endocytosis, regulation of humoral immunity in vivo or other readouts, and/or understanding how RASA3 regulates T cell function and the signaling pathways involving this key regulator of immune cell migration.

We have several lines of research that accommodate excellent PhD candidates. These revolve around the theme of RNA viral pathogens, antibodies/B-cell responses and immunodeficiencies.

The first involves understanding Immune Correlates of protective immunity, specifically which types of B-cell response and their fine specificities are important for protection against specific RNA viral pathogens (RNA viruses from HIV, HCV to Ebola) how B-cell responses to correlate with protection by vaccines to specific pathogens. The 2nd project involves using broadly neutralizing monoclonal antibodies to develop improved and novel vaccines against notoriously variable viruses. The 3rd project involves understanding how the resident virome in primary, acquired or induced immunodeficiencies leads to chronic immune activation and poor prognosis, with an emphasis on mucosal immunity.

We have the largest world-wide collection of patients suffering from rare-inherited immunodeficiency that have been whole-genome sequenced (1500+ cases). Using established analytical expertise the candidate will use novel methods to interrogate and filter potential genetic mutations, we will identify novel candidate genetic loci in patients grouped by disease phenotype or familial relationship. Candidate genetic loci will be investigated using CRISPR-editing of patient derived material (lymphoblastoid, fibroblast and iPS cell lines). Confirmatory studies at mRNA, protein and functional level will be carried out to validate the link between variant and disease.

In this project, we will investigate how different stimuli including IgG-immune complexes and TLR ligands affect the ability of DCs to influence T cell autocrine complement regulation. This is of relevance to our understanding of how inflammation is propagated in autoimmunity and for vaccination boost strategies.

The inflammasome consists of a cytosolic NOD-like receptor, an adaptor molecule (ASC) and an effector molecules caspase 1.  Once activated the inflammasome processes inflammatory cytokines such as interleukin 1 beta (IL1B) and IL18 as well as driving an aggressive form of cell death (pyroptosis).  Inflammasomme protein complexes are central to sustaining inflammation in acute diseases (like COVID-19 associated ARDS) or chronic conditions (such as Alzheimer’s Disease, Parkinson’s, diabetes, arthritis).  Patients with rare autoactivating mutations in the NLR proteins have basally active inflammasomes leading to severe autoinflammatory syndromes. How inflammasome complexes form within the cell, particularly in patients with autoactivating mutations in NLRs are poorly understood.  

The aims of this project are as follows:
1.      Identify the molecular mechanisms by which the gain of function mutations causes constitutive activation of the NLRs
2.      Determine why gain of function mutations in different NLRs (NLRP3 and NLRC4) result in differences in inflammasome cytokine production with NLRP3 biased towards IL1B and NLRC4 towards IL18
3.      Visualise how gain of function mutations alter inflammasome formation by visualising the protein complexes at super resolution and atomic resolution

This project will study how the inflammasome forms using state of the art microscopy techniques including live super resolution imaging and cyroelectron microscopy tomography.  The consequences of the gain of function mutations on inflammasome formation will be studied using these techniques in cell lines where the key proteins are tagged and the gain of function mutations introduced by CRISPR/Cas9 (many of which are already available within the laboratory).  This work will be extended to consider cells from patients with these diseases to map back the biology and the imaging onto the cell line models.   

Asthma is the world’s commonest chronic lung disease, affecting 350 million people worldwide. The advent of novel ‘biologic’ therapies targeting specific phenotypes of asthma is currently revolutionizing the treatment of patients with type 2 inflammation. However, there are no specific treatments available for the 50% of patients with type 2 low disease. The Levine group has identified a novel pathobiologic mechanism involving dysregulation of apoplipoproteins, which may play an important role in this phenotype by regulating the recruitment and function of innate and adaptive immune cells, which may have relevance for resistance to corticosteroids. Peptide mimetics of these molecules have potential as novel therapies for asthma, especially for patients with type 2 low neutrophilic inflammation. Dr Hinks group uses in vitro, murine and ex vivo human studies on highly phenotyped asthmatics to explore the biology of the inflamed airway mucosa, particularly innate and adaptive immune cells. Through this collaboration the student would use a range of techniques and a mix of wet lab science and human experimental medicine to understand the translational potential of apolipoprotein biology in human asthma.