NIHRDataInsights/HRCSData huggingface dataset:
Title: Epigenetic mechanisms that maintain immunological memory in CD4 T cells
Funder: Medical Research Council

Abstract: Immunological memory underpins adaptive immunity and is the basis for vaccination. WE aim to understand the epigenetic changes that occur during lymphocyte responses to infection which enable immunological memory. We recently revealed that in contrast to naïve T cells, memory T cells possess thousands of epigenetically primed DNase I Hypersensitive sites (pDHSs) in key inducible genes. We propose that pDHSs leave memory cells poised for rapid responses by maintaining key inducible genes in a more accessible and primed state. Because our previous studies were based mostly on in vitro findings, we now need to determine the importance of these epigenetic changes in endogenous antigen-specific memory CD4 T cell populations generated in vivo, and identify the mechanisms that maintain long term immunological memory. We will then determine how key signals known to support memory CD4 T cell responses impact on these regulatory elements. Collectively, these studies will reveal fundamental information on the mechanisms that establish and maintain immunological memory. This project brings together three local research groups to apply cutting edge epigenetic approaches to robust in vivo models of memory responses. Our specific goals are: 1. Use a transposase-mediated chromatin accessibility assay (ATAC) to demonstrate that pDHSs are maintained within endogenous antigen-specific memory CD4 T cell populations generated in vivo and dissect how the type of response affects these epigenetic changes. 2. Use gene knock-out models to determine the contributions of OX40 and IL-7 in the maintenance of pDHSS within memory populations in vivo. 3. Delete specific pDHSs from a human IL-3 gene BAC construct in a transgenic mouse model to establish that pDHSs are required for the inducible activation of human genes. 4. Use human T cells to study evolutionary conservation of pDHSs and show that they are equally relevant to human health and disease.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: INFLAMMATORY AND IMMUNE SYSTEM
Title: The integration of human T cell senescence and function at the molecular level
Funder: Medical Research Council

Abstract: We will use of freshly isolated T cell populations fromperipheral blood of young (<35 years) and older humans (>65 years). We will also isolate T cells from the site of recall antigen challenge in the skin of humans using suction blister technology that we developed. We have specific recruitment programmes for this and the ethical approval is in place for these studies. This project involves analysis of human leukocyte populations by multi-parameter flow cytometry. Using cell surface marker panels we will investigate T cells within mixed populations of leucocytes by flow cytometry or isolate these populations of cells by cell sorting. We will also separate these cells using magnetic bead technology. In addition, we utilize Western Blot to confirm flow cytometric data on expression of phosphorylated and non-phosphorylated signaling molecules. We will analyze the interaction between different molecules by co-immunoprecipitation followed by Western Blot analysis. All these techniques are currently in use in my laboratory. The proliferative assays of isolated T and NK cells include staining with the cell cycle marker Ki67, dye dilution or trittiated thymidine uptake assays. Cell death is measured by propidium iodide and annexin V staining. Cytokine production is measured by intracellular cytokine staining and telomerase activity using the telomere repeat amplification protocol (TRAP). We will measure cytotoxic activity by CD107a staining that indicates cytotoxic deganulation of these cells and also by a caspase 3 activation cytotoxic assay. We will transduce primary T cells with lentiviral vectors (pseudotyped with glycoprorein G of vesicular somatitis virus) containing short hairpin interfering RNA specific for AMPK, p38, JNK or ERK kinases to block the activity of these specific signaling components on functional activity.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: INFLAMMATORY AND IMMUNE SYSTEM
Title: Determining the potential universal applicability of a diurnal response to vaccination and the associated circadian immune processes
Funder: Medical Research Council

Abstract: The project has three objectives: 1. To determine the impact of time of day on immune memory cell formation in humans. We will vaccinate subjects (aged 60-65) either in the morning (09.00) or afternoon (16.00), in line with routine GP surgery times, with the pneumonia vaccine Pneumovax 23 and the annual influenza vaccine. Subjects will provide blood samples at baseline, 4h, 24h, 7 days, 28 days and 1 year. Using CyTof methodology we will follow the ontogeny of the cellular immune response, assessing frequency of antigen specific plasmablasts and antigen specific IgM, IgA and IgG expressing B cells. We have CyTof reagents for 12 polysaccharide components of Pneumovax and influenza A and B strains. Antibody titres for these specific vaccine components will also be assessed to determine degree of class switching, magnitude and durability of the antibody response. 2. To determine differences in antigen presenting cell migration (skin DC and Langerhans cell) to lymph nodes with time of day. Skin painting with FITC in dibutylpthalate will be used and the migration of activated DCs and Langerhans cells from skin to draining lymph nodes will be monitored at different stages of the mouse circadian/activity cycle, equivalent to morning and evening in humans. 3. To determine the effect of time of day on germinal centre function and the role of clock genes in the function of germinal centre cells. Mice will be vaccinated with T dependent and T independent antigens at the extremes of the activity cycle and effects on initial BCR signalling, B cell translocation to the germinal centre T zone, and proliferation will be tested. To interrogate B cell intrinsic effects adoptive transfer will be used and germinal centre responses, affinity maturation, antibody titres and class switching monitored by immunostaining and RTPCR. Targeted deletion of circadian cycle genes, Bmal1 and RevErba, in B cells will allow us to determine their role in the circadian vaccination response.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS;3.4 VACCINES
Health categories: INFLAMMATORY AND IMMUNE SYSTEM
Title: Somatic mutation in Primary Sjögren's Syndrome
Funder: Medical Research Council

Abstract: Primary Sjögren's Syndrome (PSS) is a common autoimmune disease associated with significant morbidity due to sicca syndrome and a risk of death from systemic involvement or progression to lymphoma. Although a complex array of immune phenomenology has been documented, including multiple genetic risk loci and a suite of biomarkers associated with diagnosis and progression, the primary aetiology of PSS and of human autoimmune disease in general, remains obscure. Hypothesis: PSS is caused by somatic mutation of immune regulatory or checkpoint genes in lymphoid cells. Many lines of evidence are consistent with this hypothesis: a) immune tolerance is dependent upon multiple regulatory genes that when mutated in germline DNA cause autoimmunity; b) lymphocytes have a high intrinsic rate of somatic mutation and somatic mutation is a common age-dependent process in healthy human tissues; c) somatic mutation causes diverse inflammatory manifestations in histiocytosis and autoimmune syndromes in lymphoproliferative disorders. To test this hypothesis we bring together 1) the MRC-funded UK PSS Registry to provide tissue; 2) high resolution cell sorting of tissue lymphocytes; 3) state of the art sequencing and cancer genome biology. Aim 1 is to dissect the salivary glands of patients with PSS into multiple lymphoid populations. Aim 2 is use ultra-deep sequencing to identify rare somatic mutations and to map antigen receptor usage within specific lymphoid populations. Aim 3 is to link theses findings to existing models of PSS pathogenesis, to survey the range mutations causing PSS in a larger cohort and to investigate the presence of somatic mutation in peripheral blood. The demonstration of an acquired genetic aetiology of autoimmunity is anticipated to have a major impact on medicine, forming the basis of new diagnostic and prognostic tests and identifying the definitive molecular targets for therapy.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: INFLAMMATORY AND IMMUNE SYSTEM
Title: Human cell based assays to study the immunopathology of the inflammatory neuropathies
Funder: Medical Research Council

Abstract: The inflammatory neuropathies are significant causes of morbidity and mortality with sub-optimal diagnostic algorithms, biomarkers and treatments. Previous research has relied heavily on solid phase assays and animal models, with limited success. The proposed study aims to extend understanding of inflammatory neuropathy pathogenesis, improve diagnostic assays, and better guide future treatment decisions. This objective will be achieved by identifying pathogenic mechanisms and antibodies using human induced pluripotent stem cell (hiPSC) derived sensory neurons, spinal motor neurons, Schwann cells and myelinating co-cultures. The topographical pattern of antibody binding will then be assessed without a priori specification of putative molecular targets. Pathological processes and regeneration will be studied over longer time periods than is possible in other systems. Co-cultures will allow assessment of the deleterious effects of patient derived serum on axo-glial interactions and myelination. Factors modulating or protecting from injury, such as antibody internalisation, and complement activation and regulation, will be evaluated. Antigen targets will be identified by immunoprecipitation and mass spectrometry. Detected antibody specificities and neuropathological activity will then be linked back to key clinical features such as prognosis and the response to particular treatments. Specific hypotheses that will be addressed in the proposed research are; i) comparing sensory and motor neurons, and control and disease-associated cells, will identify reasons for the selective and regional sensitivity of specific peripheral nerve tissue to immune mediated injury, ii) cell based assays will offer improved diagnostic sensitivity and specificity, iii) patients whose serum show persistent neuropathic potential in culture are likely to continue to progress and/or recover more slowly, providing a biomarker to identify those most likely to benefit from additional treatment.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS;4.1 DISCOVERY AND PRECLINICAL TESTING OF MARKERS AND TECHNOLOGIES
Health categories: INFLAMMATORY AND IMMUNE SYSTEM
Title: Regulation of T cell development and function by the TNF induced signalling
Funder: Medical Research Council

Abstract: TNF is an inflammatory mediator implicated in the pathogenesis of a host autoimmune and inflammatory diseases in humans. In some of these conditions, blocking TNF activity has proved a clinically effective treatment. T cell immunity is implicated in many of the clinical settings in which TNF targeted intervention is being employed or considered, and yet our understanding of TNF signalling in T cells and how it may modulate their development and function is still rudimentary. Our recent work shows that TNF induced activation of NF-kB is critical for development of new T cells, and that making functionally mature T cells depends on these signals. Our hypothesis is that TNF and related family members deliver signals to developing thymocytes that regulate the functional maturation of new T cells, and may also mediate a similar regulatory role during T cell activation, affecting T cell survival and differentiation. By these two mechanisms, TNF signalling can influence both quality and quantity of T cell responses thereby influencing the outcome of adaptive immune responses. Our aims are therefore to : 1. To understand how TNF-family signalling controls functional maturation of new T cells in the thymus, 2. To determine the mechanisms by which TNF-family induced survival, death and differentiation signals regulate T cell activation, differentiation of effectors and generation of memory Our proposed studies will lead to fundamental advances in our understanding of how TNF signalling regulates both immune reconstitution and T cell activation and memory formation and promises to substantially improve our understanding of the full impact of TNF interventions on the T cell compartment as a whole. Improved understanding of TNF signalling in T cells will lead to a better understanding of how therapeutic intervention of TNF and its downstream signalling pathways can be best applied to modify T cell responses in a desirable manner and to a beneficial clinical outcome.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: INFLAMMATORY AND IMMUNE SYSTEM
Title: MICA: Development of PEGylated Domain I of beta-2-glycoprotein I as a new therapeutic agent for the antiphospholipid syndrome
Funder: Medical Research Council

Abstract: The antiphospholipid syndrome (APS) is an autoimmune disease in which autoantibodies interact with phospholipid-binding proteins, particularly beta-2-glycoprotein I (beta2GPI), leading to thrombosis, strokes and/or pregnancy morbidity. The only evidence-based treatment currently available is long-term non-specific anticoagulation with agents such as warfarin, carrying a significant risk of haemorrhage. No biologic targeted therapies exist. We, and others, have shown that the key autoantibody-antigen interaction in the pathogenesis of APS is with the N-terminal domain (Domain I or DI) of beta2GPI. Recombinant DI, expressed from bacteria, blocks binding of IgG purified from APS patients (APS-IgG) to beta2GPI in-vitro and inhibits thrombosis caused by these patient-derived APS-IgG in a mouse model. Our proposed therapeutic for APS is a PEGylated form of DI. PEGylation of small molecules improves half-life in-vivo, allowing feasible dosing regimens in patients, and two PEGylated agents are already in clinical use for other rheumatological diseases. We are collaborating with PolyTherics, a biopharmaceutical company with an innovative proprietary method of PEGylating proteins on disulphide bonds. A shared UCL/PolyTherics MRC CASE PhD student has established high-yield, high-purity expression of PEGylated DI, including three different PEG sizes, which retained the ability to inhibit the activity of IgG from patients with APS in assays of binding and clotting. In this project we will work with a CMO to optimise and scale-up the manufacture of PEG-DI and produce material to GLP standards. Pharmacokinetics and efficacy studies will show which PEG-DI variant has the optimal balance of half-life and retained activity. Toxicology studies in appropriately selected species will be carried out. The aim is to select a lead PEG-DI candidate for a first-in-man study and to determine from preclinical safety, efficacy and pharmacokinetic studies the dose range for such a study


Research activity codes: 5.1 PHARMACEUTICALS
Health categories: INFLAMMATORY AND IMMUNE SYSTEM
Title: Localised inhibition of thrombin at endothelial surfaces - a translational strategy to inhibit immune responses to localised antigen
Funder: Medical Research Council

Abstract: Monocytes are important in local inflammation and transplantation, where the number of infiltrating monocytes during rejection closely correlates with the severity of rejection, and the degree of functional impairment that results, indicating that strategies to limit monocyte infiltration might have significant impact on survival. The chemokines involved in monocyte recruitment are well-known. Although chemokine receptor antagonists have shown promise in models of inflammation, clinical development has been limited by toxicity. Underpinning this project is the discovery, by the supervisors, that hearts from transgenic mice expressing an endothelial cell (EC)-tethered direct thrombin inhibitor (hirudin) were completely resistant to antibody-mediated rejection, and were not infiltrated by monocytes (or NK cells), whereas in hearts in which thrombosis was inhibited but thrombin signalling (through protease activated receptor-1) was left intact, there was a very heavy monocyte infiltrate. From this they have invented Thrombalexin to use as a localized anticoagulant: my project will be to explore its capacity to inhibit localized inflammation via inhibition of chemokine expression. I will use well characterised models of mouse acute and chronic rejection to study the impact of Thrombalexin, localised in the transplant or injected intravenously, on chemokine gradient generation, monocyte recruitment and transplant survival. I will also investigate whether signalling through other members of the protease activated receptor family, can augment the protection offered by Thrombalexin; here I will also use a well characterised model of localised skin inflammation. Finally, I will study samples from the phase 1 trial of Thrombaelxin, to establish the link, in humans between thrombin signalling and chemokine gradient generation. This project will address fundamental aspects of the biology of inflammation and inform clinical development of a novel therapeutic.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS;5.1 PHARMACEUTICALS
Health categories: INFLAMMATORY AND IMMUNE SYSTEM
Title: Monocyte and endothelial cell interaction during sepsis
Funder: Medical Research Council

Abstract: Mortality from sepsis remains high, there is a need to improve understanding of the basic mechanisms and identify novel therapeutic targets. Recent murine data suggest monocytes patrol endothelium and may regulate the immune response. However little is known about how monocyte subsets interact with endothelium and how this regulates innate immunity. Monocytes are classified according to surface markers into classical (CD14++CD16-), non-classical (CD14+CD16++) and intermediate (CD14++CD16+). Colony-stimulating factor 1 (CSF1) may drive monocyte differentiation to the non-classical. These subsets seem to differ in function with non-classical involved in tissue repair and classical important for bacterial killing and the release of the anti-inflammatory cytokine interleukin (IL)-10. My preliminary data show that co-culture of human monocytes with lipopolysaccharide (LPS)-activated human microvascular endothelial cells leads to an increase in classical monocytes. I hypothesise an appropriate host response to endotoxaemia is for activated endothelium to enrich the classical subset, stimulating bacterial phagocytosis and transiently increasing anti-inflammatory cytokines to promote tissue repair. However, in sepsis these mechanisms are overwhelmed leading to a prolonged immunosuppressive state. Using my established co-culture system I shall determine if the increase in classical is contact-dependent, determine the role of CSF1 and assess the capacity of these monocytes to kill clinical strains of bacteria. I will assess monocyte function in vivo. Firstly, by studying monocytes before and after the administration of intravenous LPS to healthy volunteers under controlled conditions in an intensive care unit. Secondly, by isolating monocytes from patients with sepsis and then again in recovery. This will provide novel insights into the functional consequences of human monocyte-endothelial interactions and may suggest future therapeutic manipulations.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: INFLAMMATORY AND IMMUNE SYSTEM
Title: CHECKPOINT FOR B CELL SURVIVAL IN HUMAN GUT
Funder: Medical Research Council

Abstract: Peripheral B cell development and selection occurs after B cells exit the bone marrow and enter the blood as transitional B cells and before maturation. Having observed that transitional type 2 (T2) B cells selectively enter the gut and become activated, we proposed that human gut hosts a selective stage in B cell development. We reasoned that a checkpoint would be evidenced by divergence in cell behaviour in a population: cells would have alternative fates (eg life or death) dependent on a selective process. However whilst funded by MR/L009382/1 we did not observe this in T2 cells in GALT. We therefore analysed a subsequent stage in T2 B cell development implicated by mouse models: marginal zone (MZ) B cells. We identified CD27+IgD+ cells in GALT that are clonally separate from classical memory cells. They are members of the same clones as CD27+IgD+ cells that circulate in the blood and they also have clone members in GALT GCs. CD27+IgD+ cells from GALT are relatively non-viable in culture and express very high levels of negative B cell regulators TACI and FCRL4. In contrast, classical memory B cells from GALT and CD27+IgD+ cells in blood do not. We propose a model for MZ B cell development and selection in human gut and wish to interrogate and build the model further by asking the following questions: What are the key differences between CD27+IgD+ B cells in the gut compared to the blood? What induces the CD27+IgD+TACIhiFCRL4hi state? What does survival of CD27+IgD+TACIhiFCRL4hi cells depend on? The gut is involved in B cell development in many species. We propose that understanding the role played by GALT and the microbiota in human MZ B cell development as planned here will result in a paradigm shift in B cell biology that will support investigation of B cell associated pathologies and underpin novel approaches to establishing human B cell tolerance.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: INFLAMMATORY AND IMMUNE SYSTEM
Title: Regulation of IL-1 production by an E2 ubiquitin conjugase
Funder: Medical Research Council

Abstract: IL-1 (IL-1alpha and IL-1beta) are related inflammatory cytokines that regulate homeostasis, antimicrobial immunity and inflammation. IL-1b is produced upon caspase-1 activation, and deletion of genes encoding IL-1b, its receptor or caspase-1 protects mice against autoinflammation. Importantly, blocking IL-1 signalling in humans improves clinical symptoms in chronic conditions, including gouty arthritis and familial fever syndromes. IL-1 cytokines are transcriptionally induced by NF-kB in phagocytic cells. However, how IL-1 proteins are turned over remains unclear. We recently described the E2 ubiquitin conjugating enzyme UBE2L3 as a post-translational regulator of IL-1 protein levels in human and mouse macrophages. We found that UBE2L3 silencing increased the cellular pool of pro-IL-1b protein available for processing by caspase-1. On the other hand, UBE2L3 over-expression enhanced K48-ubiquitylation and proteasomal degradation of pro-IL-1b. UBE2L3 is associated with autoinflammatory diseases such as lupus and Crohn's colitis, and is an essential gene. However, its roles in the immune system remain unclear. In this proposal we aim to study the molecular mechanisms of UBE2L3 action in vitro and in vivo. We will investigate site-specific ubiquitylation of IL-1 and the involvement of UBE2L3 associated E3 ligases. How UBE2L3 activity is regulated in cells by its own ubiquitylation will also be determined. IL-1-driven inflammation in vivo will be studied using a newly derived myeloid-specific conditional Ube2l3-deficient mouse. Models of IL-1-linked inflammation, such as response to lipopolysaccharide and monosodium uric acid crystal-induced peritonitis, will shed light on the systemic roles of UBE2L3. Together, these studies will clarify mechanisms of UBE2L3-dependent down-regulation of IL-1. Our studies on UBE2L3 should have wide implications, and could be exploited to design new strategies to dampen IL-1 for clinical benefits.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: INFLAMMATORY AND IMMUNE SYSTEM