Title: Impact of obesity on phenotype and function of CD8+ tissue resident memory T cells in the liver
PhD student: Helena Horvatic
A fundamental advantage of immunological memory is the potential to react in a fast and vigorous manner upon secondary encounter with the same antigen. CD8+ TRM cells are one of the key players in the protection against reinfections due to their ability to develop rapid recall responses at the site of infection. Recent studies have shown that immunological memory loses its effectiveness in obese individuals that are of particular risk of re-infection. The enhanced incidence of reinfections in these patients was attributed to decreased CD8+ T cell responsiveness and impaired function. However, the exact underlying mechanisms are not well characterized. The aim of this project is to define how chronic inflammation of the liver during obesity might affect the differentiation, homeostasis, and function of hepatic CD8+ TRM cells. We exploit the combined expertise in the fields of chronic liver inflammation and viral infections in the Abdullah group in Bonn with the expertise in transcription regulators involved in T-cell biology and function in the Kallies group in Melbourne to study the development, homeostasis and function of the hepatic CD8+ TRM cells under obesity conditions. The delineated pathways could be targeted to improve the memory responses and to increase the effectiveness of vaccination in obese individuals.
Title: Identification of novel mediators of T cell function in chronic infection and cancer
PhD student: Leonie Heyden
Cytotoxic CD8+ T cells are one of the most important mediators of immunity against severe viral infections such as Covid-19 as well as cancer. However, when CD8+ T cells are persistently stimulated with high amounts of antigen as in severe or chronic infections or in tumors, they lose their functionality and become ‘exhausted’. Hallmarks of exhausted CD8+ T cells are high expression levels of inhibitory receptors such as PD-1 or CTLA-4 and compromised cytokine production, which in combination impairs control of viral infections and tumor growth. Although immunotherapies such as PD-1 checkpoint blockade have been shown to have the potential to successfully reboot T cell activity and result in improved survival of some cancer patients, other patients do not benefit from these therapies. Thus, novel therapeutic approaches are urgently needed. Long-term maintenance of T cell responses is mediated by a distinct population of T cells that displays characteristics of exhausted and memory T cells and acts as precursors of exhausted T cells. Notably, these cells are also responsible for the success of PD-1 checkpoint inhibition. The aim of this project is to identify and analyse molecular and metabolic pathways that lead to the development of exhausted T cells and their precursors by using different Lymphocytic Choriomeningitis Virus (LCMV) infection and tumor models.
The project will start in the Abdullah group in Bonn, Germany where different Cre mice and diverse infection and tumor models like the neo-LCMV infection or inducible Hepatocellular carcinoma HCC tumor model will be used to identify and characterize novel mediators of T cell function in chronic infection and cancer. With the expertise and extensive preliminary work by the Kallie’s group in Melbourne we will continue the work by using different mouse models like the Id3 reporter mouse, the Hobit reporter and different Cre mice.
Title: Molecular Definition of Melanoma Immune Equilibrium in Skin and Lymph Node Metastasis
PhD student: Freya Maria Kretzmer (completed)
Malignant melanoma is an aggressive type of skin cancer and a paradigm disease for our current understanding of tumour immune surveillance and the development of cancer immunotherapies. During tumour progression, both, immune and melanoma cells undergo an immunoediting process that is divided in three interconnected phases: the elimination, the equilibrium, and the immune escape. During tumour progression neoplasms may proceed sequentially through the different stages, but various factors within the tumour microenvironment can influence the process. In this context, recent evidence suggests that tissue-resident CD8+ memory T (TRM) cells play a critical role in melanoma surveillance by promoting the melanoma–immune equilibrium. The mechanisms how TRM cells limit tumour progression is yet unknown, but it has been found that TNF- deficient TRM cells in the skin had reduced capacity to achieve melanoma immune control. Therefore, we dissect the molecular pathways acting in melanoma cells during immune surveillance and tumour progression using tailored genomic and functional approaches. Based on our data, conditional control of cytokine signalling pathways in melanoma cells, in particular the TNF- signalling cascade, will help to analyse intrinsic and extrinsic mechanisms of melanoma cells. In order to achieve a comprehensive molecular characterisation, ribosomal proteins were fused with protein tags in melanoma cells using adapted CRISPR/Cas9 approaches in the Hölzel lab (Bonn, Germany). These modified melanoma cells will help to reveal intracellular mechanisms occurring upon immunotherapy in a subcutaneous melanoma model. Whereas in the Gebhardt lab (Melbourne), the genome engineered melanoma cells will be transferred into the epicutaneous melanoma model. This model will help to dissect the cellular crosstalk of melanoma cells with TRM cells and how this influences the intracellular pathways in melanoma cells.
Title: Dissecting mechanisms of immune-mediated control of melanoma
PhD student: Lewis Dylan Newland
The role of the host immune system in cancer is becoming increasingly apparent, demonstrated by the recent clinical success of cancer immunotherapies. Melanoma is an aggressive skin cancer which is ideal for investigating cancer-immune interactions due to its comparatively high immunogenicity. Disease recurrence is caused by melanoma cells that resist therapeutic eradication, particularly in terms of metastases. Therefore, it is of interest to define the immune mechanisms that can control these persisting melanoma cells so that such mechanisms can be harnessed to combat the danger of occult melanoma. Using a translatable epicutaneous melanoma model, work involving the Gebhardt group in Melbourne has demonstrated that tissue resident memory T cells (TRM cells) are key drivers of a melanoma-immune equilibrium in the skin. This is a situation in which the immune system prevents the outgrowth of the occult lesion without completely eradicating it. Using this pre-clinical model, we aim to extend on these findings by further elucidating the immune responses that control primary and metastatic disease. This will include high dimensional analysis of the tumour and immune contexture in controlled and progressing tumours to identify potential biomarkers associated with control. Additionally, melanoma cell lines that report for proliferation will be used to investigate the role of T cell-induced cell cycle arrest in controlled lesions. Using the expertise in molecular oncology of the Hölzel group in Bonn, we will generate new genetically modified melanoma cell lines for investigation of aspects of immune-mediated control of melanoma in the epicutaneous model.
Title: The influence of CCL17 and CCL22 on the adaptive immune response against Salmonella infections
PhD student: Manja Thiem (completed)
Dendritic cells (DCs) and macrophages express the chemokines CCL17 and CCL22. During the induction of adaptive immune responses, both chemokines promote interactions between T cells and DCs. CCL17 has been particularly associated with the pathogenesis of allergic and inflammatory diseases. Furthermore, the chemokine is involved in the migration and activation of both Th1 and Th2 cells. In comparison, the functions of CCL22 are mostly unknown although it has been associated with the recruitment of regulatory T cells (Tregs) and appears to play an immunosuppressive role. In this project we combine the expertise of the Strugnell lab in Salmonellae-driven infection models with that of the Villadangos lab in genetic engineering and biochemical approaches for identification of protein interactions (both in Melbourne) to complement the expertise of the Förster group in Bonn in analysing chemokine function and T cell migration. Using a mouse model for invasive Salmonellosis, we study the influence of CCL17 and CCL22 on the immune response against Salmonella enterica serovar Typhimurium after oral infection and during the vaccination. Specifically, we are interested in the differential susceptibility of the specific knockout mice for each or both chemokines and their associated receptor CCR4. We also want to analyse the influence of both chemokines on the development and recruitment of thymic and intestinal Tregs during infection and their importance regarding Salmonella-specific vaccination. Furthermore, we have evidence that CCL22 can elicit signaling responses in CCR4-deficient cells, giving rise to the hypothesis of another CCL22- (and CCL17-) specific receptor. In order to identify additional receptors for both chemokines, we want to utilize biochemical (e.g. Pull-Down Assay) and genetic approaches (e.g. CRISPR/Cas). For this purpose we have also established a chemokine receptor staining approach using biotinylated chemokines.
Title: IL-18R-dependent protection against bacterial pneumonia by epithelial cells
PhD student: Lara Oberkircher
Legionella spp are gram-negative bacteria that, when inhaled via infectious aerosols, may cause a severe type of bacterial pneumonia called Legionnaire's disease. Therefore, these pathogens pose a significant health risk. Unlike the majority of Legionella spp, Legionella longbeachae does not grow in aquatic habitats but in soil with potting mixes serving as the common source for infection. The collaborative project between the groups of Prof. van Driel, University of Melbourne, and Prof. Garbi, University of Bonn, aims to understand how immune responses against L. longbeachae are induced. The overarching aim is to identify critical pathways for protection against bacterial pneumonia that may be harnessed for the treatment of patients. Victoria Scheiding (first PhD student of the joint PhD program) uncovered a novel, noncanonical pathway of IL-18 mediated defence against L. longbeachae that does not involve IL-18R-stimulated IFN-γ release by lymphoid cells. The current student Lara Oberkircher will continue this work using an interdisciplinary approach of molecular, immunological, imaging and microbiological techniques to decipher the cellular and molecular mechanisms behind this novel pathway. Prof. Garbi will offer his expertise about innate and T cell responses and this will be combined with the expertise of Prof. van Driel in bacterial pneumonia and T cell responses to elucidate the detailed mechanisms governing the cross-talk between epithelial cells, myeloid cells and T cells during infection with L. longbeachae. We will also investigate whether tobacco smoke and influenza, two factors known to exacerbate bacterial pneumonia, hijack this pathway rendering the host more susceptible to L. longbeachae infection. These studies will highlight key mechanisms controlling Legionella replication and may contribute towards novel therapeutic strategies against bacterial pneumonia.
Title: Molecular and immunological mechanisms underlying inflammation during SARS-CoV-2 infection
PhD student: Alexandru Odainic
Since the SARS-CoV-2 outbreak in late 2019, the virus caused over 1.6 million deaths. Scientists worldwide are working hard on elucidating possible pathophysiological pathways, prognostic inflammatory markers as well as potential therapeutic targets.
Previous studies show that excessive immune responses played a major role in the pathophysiology of SARS, a coronavirus caused disease accompanied by severe lung injury and respiratory failure in patients. Based on current knowledge, the monocytes play a decisive role in the disease course.
The Bonn based project at the Immunogenomics Unit lead by Susanne V. Schmidt (Institute of Innate Immunity, University of Bonn) aims to elucidate the molecular mechanisms of inflammatory programming of monocytes in different stages of COVID-19. Using RNAseq technologies, the transcriptomes of monocytes isolated from peripheral blood of asymptomatic, mild, and severe SARS-CoV-2 infected patients will be compared to healthy controls and recovered patients. The project will address the following questions: (1) which innate immune pathways are induced at different stages of SARS-CoV-2 infection; (2) are there specific chromatin-remodellers involved in inflammatory programming of monocytes in severe COVID-19 cases; (3) is there a central key player identified, which supports an overshooting and dysregulated inflammatory program in monocytes of severely infected patients; and (4) are there specific gene signatures in monocytes which allow predicting an asymptomatic, mild or severe course of COVID-19.
The exchange and collaboration with Sammy Bedoui (Peter Doherty Institute, University of Melbourne), whose expertise is the intercommunication of the innate and adaptive immunity during viral infections, will enable us to verify and investigate the discovered pathways and potential markers for different COVID-19 disease courses in mouse models.
Overall, this project focuses on revealing the immunological pathways, possible diagnostic markers as well as therapeutic targets for various COVID-19 courses, with a focus on the importance of the TAM receptor signaling by using precision immunology.
Title: Understanding and exploiting programmed cell death pathways during intracellular infections
PhD student: Sven Engel
Intracellular infections remain a major human health burden and the rise in antibiotic resistance threatens to bring us back to the pre-antibiotic era, where infectious diseases killed millions of people. We therefore require novel strategies that not only target the bacteria, but also improve host defence. Although programmed host cell death (i.e. pyroptosis, necroptosis, apoptosis) is thought to play an important role in controlling these pathogens, defects in individual pathways of programmed cell death often only cause minor defects in the in vivo control of such infections. This is likely related to redundancy that evolved in response to pathogen evasion strategies, which allows the host to compensate for evasion of one pathway through the activation of another. However, we know very little about the organisation, regulation and utilization of this system of redundancy in cell death pathways. New findings generated in a collaboration between the Bedoui group at the Doherty Institute and the Herold group at the Walter and Eliza Hall Institute suggest that a backup system allows the host to flexibly utilize different pathways of programmed cell death to eliminate infected cells. Building on these novel insights, we want to investigate how this backup system is organised and regulated, how the pathways are utilized for Salmonella control and if this system can be manipulated for therapeutic purposes. Furthermore, a global characterisation of bacterial and host factor requirements should be performed together with the expertise of Susanne Schmidt in Bonn. Here we want to analyse changes in the expression profile of infected cells as well as the infecting pathogen by performing RNA sequencing. Additionally, proteomic quantification of the secretome of infected cells will be conducted by a high-resolution mass spectrometric approach together with the expertise at Bonn University.
Title: Genetic analysis of leukocyte chemotaxis
PhD student: Nicole Dörffer
Regulated motility is a hallmark of immune cells. Yet, how these cells maintain continuous locomotion in chemokine gradients is unidentified. Especially how chemokine receptors are regulated during migration remains poorly understood. CC-chemokine receptor 7 (CCR7) is essential for immune cell distribution in mice and men. Together with its ligands, the chemoattractants CCL19 and CCL21, it regulates directed migration of dendritic cells (DCs) during immune responses and of homeostatic T-cell homing to lymphatic organs. CCR7 deficiency in DCs results in an impaired migration to lymphatic organs and T-cell priming. Loss of CCR7 was also observed after cholesterol ester synthesis was blocked. We therefore suspect that the lipid metabolism plays an important role in chemokine receptor presentation and cell migration. Furthermore, how chemokine presentation shapes directed migration of DCs in the interstitium, in perilymphatic spaces and lymphoid tissues is still under debate. Generally, chemokines may be freely diffusible, or immobilized to cellular or extracellular surfaces and are likely to form spatial concentration gradients which provide essential cues for chemo-/hapokinetic motility. Since the chemoattractant dynamics in the tissue is largely unknown, it remains elusive how DCs exactly integrate gradient information for directional migration within complex and dynamic environments. Within the underlying collaboration we will identify regulatory elements of CCR7 cell surface expression employing CRISPR/Cas9 technology and a flow-cytometry based genetic screen based on the expertise of the Mintern lab. Furthermore, we will test if potential target genes influence DC migration in a 2D-microfluidic device of the Kolanus lab by testing different chemokine gradients. Using a click-chemistry mass spectrometry reporter strategy, we will further unravel how lipids influence chemokine receptor expression and their impact on cell migration. Our aim is to understand the mechanism of CCR7 surface regulation which underlies its sustained signalling activity during directional DC migration.
Title: Systems analysis of paracrine effects after inflammasome activation
PhD student: Lena Standke
Myeloid cells like macrophages (MP) use several families of germline-encoded signalling receptors to recognize microbial pathogens and cell damage inflicted by pathogens. Among them the inflammasomes are unique as they do not trigger a transcriptional response, but a proteolytic cascade culminating in the activation of caspase-1 which cleaves precursor cytokines of the IL-1β family. However, inflammasome activation also induces the release of cellular proteins involved in inflammation and tissue repair. Additionally, inflammasome-activated MP undergo pyroptotic cell death and release their activated inflammasomes into the extracellular environment. While the activation mechanisms of inflammasomes and the activities of IL-1β family members have been extensively studied, little is known about how inflammasome-mediated unconventional release of proteins, pyroptotic cell death or the extracellularly released inflammasomes influences bystander cells, such as MP, endothelial cells or adaptive immune cells. Since inflammasome activation is important in the pathogenesis of multiple acute and chronic inflammatory disease, we aim to better understand cell-to-cell communication between inflammasome-activated cells and surrounding cells using a combination of systems biology, biochemical and cellular immunological approaches. The PhD student of the first IRTG2168 cohort, Christina Budden, discovered that the release of extracellular vesicles (EVs) is an important event that contributes to the paracrine effects of inflammasome activated cells. She isolated EVs secreted by a human macrophage cell line and investigated their effects on recipient macrophages. Building on this data, we will expand this knowledge on EV effects to further bystander cells using different in vivo and in vitro approaches. We anticipate that we will uncover and characterise novel functions of inflammasomes and provide a better understanding of how acute inflammatory reactions are turned off, how tissue repair can be induced by inflammatory triggers and how chronic inflammation may cause fibrotic tissue responses. The project will benefit from the complementary expertise in Melbourne (genomics, bioinformatics) and Bonn (imaging, inflammasomes).
Title: Unravelling the immune cell landscape in chronic CNS disorders
PhD student: Tarek Elmzzahi
Under steady-state conditions the central nervous system (CNS) houses a plethora of immune cells that serve vital homeostatic functions. In this project we investigate how the CNS immune cell composition varies over time in the context of three CNS disorders: neurodegeneration, autoimmunity, and chronic viral infection. In Bonn we will use a combination of high-content flow cytometry and single-cell RNA-sequencing (scRNA-seq) – areas of expertise in the Beyer lab – to map the transcriptomes and phenotypes of myeloid and non-myeloid cells in the aforementioned CNS disorders in comparison to age-matched wild-type animals. We will perform cross-disease comparison to determine disease-specific vs. overarching gene-expression patterns. Analysis of scRNA-seq data from brain-derived T cells will permit the reconstruction of T cell receptor (TCR) sequences, allowing for assessment of the diversity of the TCR repertoire as well as T-cell clonal expansion. An additional objective is the identification of the antigen-presenting cells that (re)activate T cells and shape the T cell repertoire in the diseased brain. In Melbourne we will focus on the molecular determinants of T cell migration to and residency in the CNS. The Kallies lab has shown that interleukin (IL)-33 regulates recruitment, expansion, and tissue-residency of regulatory T (Treg) cells in the periphery through binding to its receptor, ST2. The IL-33/ST2 axis also contributes to disease development in animal models for multiple sclerosis (MS) and Alzheimer’s disease (AD). Employing IL-33-deficient and ST2-deficient mice, we aim to define how IL-33 modulates T cell recruitment and CNS-tissue residency in the context of CNS disorders. Using IL-33 reporter mice, we will also analyze which cells are responsible for IL-33 production and Treg cell interactions in the brain. Moreover, we will utilize novel mouse models to examine the roles of molecular regulators of tissue T cell differentiation identified by the Kallies lab in the context of CNS diseases. As sex dimorphism is well-documented in immune responses, and CNS disorders exhibit a pronounced sex bias we will determine whether such differences in CNS disease susceptibility are dictated by distinct patterns of T cell recruitment or differentiation. Therefore, sex-specific patterns of immune cell activity in the CNS will be an important focus of the experimental work.
Title: Understanding regulatory T cell differentiation and function in chronic tissue inflammation and cancer
PhD student: Darya Malko
Regulatory T (Treg) cells are specialized immune cells of the T cell lineage that are indispensable for the maintenance of immune homeostasis and the prevention of immune mediated pathology. Therefore, Treg cells play central roles in a broad range of diseases including, cancer, autoimmunity as well as metabolic and inflammatory diseases. Treg cells undergo a distinct differentiation program that is essential to acquire a fully suppressive effector phenotype. During this process Treg cells undergo diversification and specialization and migrate to different non-lymphoid organs such as the adipose, gut or brain. Here they play critical roles in maintaining tissue integrity, repair and metabolism. Based on unpublished results of the Kallies laboratory (The Peter Doherty Institute for Infection and Immunity, Melbourne), effector Treg cell differentiation is regulated by a tightly controlled molecular network that is shared between inflamed and cancerous tissues. With the combined experience of the Beyer (German Center for Neurodegenerative Diseases, Bonn) and Kallies laboratories in this study we will examine the molecular control of effector Treg cell development, diversification, and function in non-lymphoid tissues with a focus on autoimmunity and cancer. Using the expertise of the Beyer laboratory on cutting-edge molecular techniques, including single cell RNA and chromatin accessibility (ATAC) sequencing, Treg cells in the context of the neurodegenerative autoimmune disease Multiple Sclerosis will be addressed using the in vivo mouse model of experimental autoimmune encephalomyelitis (EAE). Utilizing the in-depth knowledge on the molecular control of tissue-specific effector Treg cell adaptation of the Kallies laboratory, the development and function of tumor-associated Treg cells will be investigated using different knockout mouse models.
Title: Multi-layer single-cell omics applied to patients with infections
PhD student: Ioanna Gemünd
The sophisticated nature of cell-cell communications between different immune cell subsets majorly influences immune responses. Most studies that analyse human immune regulation focus on a cell type of interest or simplistic co-culture experiments. However, these approaches preclude the possibility to study cell interactions between all circulating immune cells. Previous analyses in our lab have provided insight into the transcriptional network of cell-to-cell communication using a whole blood culturing approach combined with single-cell RNA-sequencing. This approach was used to study cell-cell communications after the application of pro- and anti-inflammatory perturbations to whole blood of a healthy donor. In this project, we aim to analyse interactions of cells from both the innate and the adaptive immune system in a disease context, namely in viral infections such as influenza. Combining our experience in single cell genomics, especially with clinical samples, and bioinformatic analysis with Katherine Kedzierska’s complementary expertise in viral immunology in Melbourne, we will investigate interactions of cells of both, the innate and the adaptive immune system, in the context of infection and aging. To do this, we will use a single cell multi-omics approach that combines RNA-, TCR- and surfaceome-sequencing techniques. Heterogeneity of TCR repertoires will be determined after annual vaccination and after acute Influenza A infection within different age groups. Finally, we will link TCR repertoire to infection severity and define cell-cell communication loops in an influenza context with the prospect of building predictive models. The findings will have implications for the design of more effective vaccines, especially for groups of people with a compromised immune system, such as the elderly.
Title: Antiviral inflammasome activation in intestinal epithelial cells
PhD student: Florian Gohr
The innate immune system complements the adaptive immune system and serves as a first line of defence against infection. In this project we study how cells of the intestinal epithelium contribute to the detection and elimination of enteric viruses. To decipher the cell biology of inflammatory processes, we employ intestinal organoids as physiological models for the relevant tissues of the gastrointestinal tract. In particular, we aim to gain molecular and mechanistic understanding of virus-triggered inflammasomes, their cross-talk to other signalling pathways, and the resulting consequences for a complex tissue. Inflammasomes are macromolecular signalling complexes that integrate cytosolic information on cell damage or infection. They coordinate immune responses through the secretion of pro-inflammatory cytokines and initiate death of infected cells by pyroptosis. While the role and some mechanistic details of inflammasome assembly are well understood in myeloid cells, it is now clear that other cell types are also able to assemble inflammasomes. For many pathogens the intestine resembles an entry point into the body. The intestinal epithelial cells are the first to contact those pathogens. They have to discriminate pathogens from the innocuous gut microbiota and commence an appropriate response in order to restrict the infection and stimulate other parts of the immune system. In addition to pyroptosis and cytokine release, intestinal epithelial inflammasomes have been linked to cell expulsion, mucus secretion and eicosanoid release, which is critical in the defence against various enteric pathogens. Functional intestinal epithelium can be cultivated as 3-dimensional organoids. These maintain the relevant physiological context and immune functions, but allow manipulation with cell biological methods. To answer fundamental questions on inflammasomes and their role in responses to different enteric viruses we will introduce relevant genetic knock outs as well as novel biosensors into human organoids. We will further use nanobodies to specifically visualize or perturb proteins and their interactions. In a reciprocal approach we will introduce inflammasome reporters into recombinant viruses to deliver them into primary cells during infection experiments. This project will combine the cell biological tools and nanobodies available at the Schmidt laboratory at Bonn University with the expertise on RNA viruses and advanced microscopy in the Mackenzie laboratory in Melbourne to shed light on the molecular events that coordinate antiviral inflammatory responses in a complex tissue such as the intestinal epithelium. We will also benefit from the large collection of virus isolates from patients at VIDRL (Victorian Infectious Diseases Reference Laboratory).
Title: Lipid metabolism and immune regulation during flavivirus infection
PhD student: Alice Trenerry (completed)
Viruses are parasitic in nature and carry with them only a subset of fundamental genes. Their survival is dependent on the manipulation of host pathways and processes, and flaviviruses rely heavily on the subversion of host lipids for all stages of their life cycle. One enzyme in particular, fatty acid synthase, has been implicated as a pro-viral gene for several families of viruses including flaviviruses, and its perturbation often results in viral attenuation. FASN is a multifunctional, cytoplasmic enzyme, and in humans represents the only enzyme that can catalyse the de novo synthesis of fatty acids. In the context of immunology, fatty acid synthesis and oxidation are determinants of the metabolic state of a cell, and particular metabolic states can influence the function of immune cells. In the innate immune system, stimulation of FASN has been observed to be crucial for the differentiation and activation of inflammatory macrophages, as well as inflammasome activation and toll-like receptor signalling. It is also important for adaptive immunity, and can regulate dendritic cell function, as well as T and B cell responses. The Mackenzie lab at the University of Melbourne have been instrumental in understanding flavivirus replication processes and identifying host lipid factors necessary for replication. In this project we will be joining forces with the Schmidt lab in Bonn to take this one step further and elucidate whether the utilization of lipids by these viruses is contributing to the activation of inflammatory pathways, which may prove a viable therapeutic target to tackle inflammatory symptoms associated with severe flavivirus disease. We will take advantage of the specialized tools developed by the Schmidt lab to track and visualize activation of the NLRP3 inflammasome in response to infection in a diverse range of cell types, and determine the contribution of FASN to this pathway. We will also be investigating the impact of FASN inhibition on the polarization states of macrophages and the contribution of FASN and its products to TLR4 signalling in response to infection.
Title: Control of Barrier Immunology by Regular Exercise
PhD student: Marcel Michla
A major societal challenge over the last decades in the Western World and an important aspect of modern living is the reduced amount of physical movement combined with changes in dietary nutrition, leading to an increased incidence in chronic inflammation. Many inflammatory disorders manifest at barrier surfaces: allergies and asthma affect the lung, psoriasis affects the skin, while inflammatory bowel disease (IBD) results in chronic inflammation of the intestinal epithelium. Such inflammatory conditions are mainly driven by cytokine secretion of innate lymphoid cells (ILCs) and T helper cells. However, the etiology of these diseases is poorly understood, but is likely associated with changes in life-style coinciding with westernization, such as an overall increase in hygiene, abundant nutritional uptake, together with a reduced amount of physical activity. This project aims to unravel the effects of voluntary wheel running (VWR) in mice on the functionality of barrier immunology with particular focus on the regulation of metabolic pathways driving innate immune cell recruitment and T cell activation, differentiation and memory function. Based on exercise-induced changes in the host immune system we aim to elucidate to which extent a lack of exercise might on the one hand contribute to chronic inflammatory conditions (Crohn’s Disease, psoriasis or asthma), but also bacterial (Salmonella typhimurium) or viral (Influenza) infections. With our results we aim to assess whether exercise or an exercise-induced factor may be used as a potent therapeutic strategy in the future. The project will benefit from the complementary expertise of the MacKay lab in Melbourne (tissue resident T memory cells, bacterial and viral skin infection in in vivo models) and the Wilhelm lab in Bonn (dietary immunology, immunometabolism and barrier immunology).
Title: Molecular mechanisms governing TRM cell fate
PhD student: Andreas Obers
Tissue-resident memory T cells (TRM) are a distinct lymphocyte lineage that exist in peripheral tissues of the body. In contrast to circulating T cells, TRM cells are non-migratory and uniquely adapted to their tissue of residence for rapid recognition and response to invading pathogens. TRM cells can provide superior protection against local infection as compared to circulating T cells, as well as anti-tumor immune responses. This has led to much interest in developing therapeutic strategies to target this immune population. However, in some settings TRM cells may be detrimental, and aberrant TRM activation has been associated with autoimmune diseases including psoriasis or vitiligo. Thus, it is imperative to understand the molecular mechanisms governing TRM cell development in order to either promote its protective responses (infection/cancer) or dampen pathogenic responses (autoimmunity). Recent studies suggest that circulating memory T cells (TCIRC) and TRM might share a naïve common precursor cell. However, it is unclear at what point during an immune response these T cell populations diverge, and whether TRM cells indeed arise from a distinct committed precursor. Here, this project aims to elucidate the signals that regulate CD8+ T cell commitment to either the TCIRC or TRM lineage. This knowledge will refine our vaccine designs to selectively enhance protective TRM formation or attenuate pathogenic TRM formation in the context of autoimmune diseases. This project will be expanded in Christoph Wilhelm’s laboratory in Bonn, where we will interrogate how external environmental factors such as diet and exercise might influence CD8+ T cell differentiation and fate decisions during the course of infection.
Title: Structural basis of NLRP7 inflammasome formation
PhD student: Anja Kopp (completed)
As part of the innate immune system, inflammation plays a major role in defending the human body against pathogens. Cytosolic sensors known as NOD-like receptors (NLRs) recognize different stimuli caused by invading pathogens and can trigger an inflammatory response. Upon activation, these proteins assemble into oligomeric complexes and recruit the adapter protein ASC (apoptosis-associated speck-like protein containing a CARD). Multiple ASC molecules form speck-like filaments and can bind and activate pro-caspase-1. Active caspase-1 is responsible for the cleavage of pro-IL-1β and pro-IL-18, ultimately resulting in inflammation and a form of cell death called pyroptosis. There are four different subgroups within the family of NLRs, called NLRAs, NLRBs, NLRCs, and NLRPs. Human NLRP7 is expressed in a wide range of tissues and forms inflammasomes in response to bacterial acylated lipopeptides. Moreover, NLRP7 plays an important role in embryonic development. Nevertheless, the physiological functions NLRP7 exhibits in innate immunity and reproduction are diverse and not well understood. NLRP7 adopts the molecular PYD-NACHT-LRR domain architecture typical for the NLRP subgroup. The N-terminal PYD can form protein-protein interactions with the PYD of the adaptor molecule ASC. The central NACHT domain is responsible for NLR oligomerization and ATP hydrolysis, while the C-terminal LRRs are supposed to function in ligand sensing. The structure of full-length NLRP7 and its different activation states remain elusive until now. Using recombinant expression of full length human NLRP7 as well as separate domains in baculo virus infected Sf9 cells or bacterial E.coli cells, we aim to generate biochemical and structural data to understand the underlying mechanisms of NLRP7 activation and ASC and caspase-1 recruitment. The expertise of the Geyer laboratory at the University of Bonn on the biochemical and structural characterization of proteins will be combined with the expertise of the Masters laboratory at the University of Melbourne on the analysis of biological triggers that activate the inflammasome and induce pyroptosis. To this end, protein crystallization is applied and biochemical methods such as surface plasmon resonance (SPR) are used to study the interaction of NLRP7 with small molecules and other proteins. Moreover, NLRP7-specific nanobodies were generated as tools for structural and biochemical analyses, which will be carried out in the Geyer lab and the application in cellular assays which will be performed in the Masters lab.
Title: Nucleic acid sensing by CD8 T cells and NK cells during viral infections
PhD student: Adham Mohamed
Immune cells possess receptors capable of sensing pathogen molecules and structures such as the highly conserved microbe-associated molecular patterns (MAMPs) like sugars, lipids, or proteins. Viruses are commonly recognized in humans by either the localization (e.g. DNA in cytosol), structures (e.g. dsRNA) or modification to their nucleic acid (e.g. 3p-dsRNA) that distinguish them from their host-derived counterparts. As such, mammalian cells sense these different structures using different cytosolic nucleic acid receptors such as retinoic acid-inducible gene I (RIG-I), melanoma differentiation-associated protein 5 (MDA5), cyclic GMP-AMP synthase (cGAS) and stimulator of interferon genes (STING). Recognition of foreign nucleic acid can trigger a type-I interferon (IFN-I) dominated anti-viral immune response, including cell-autonomous anti-viral defence mechanisms, which include the induction of apoptosis, as well as the production of soluble mediators such as chemokines and cytokines (e.g. IFN-I), which in turn can both enhance anti-viral response in neighbouring cells and attract immune cells. Although it is well understood how activated antigen presenting cells like monocytes, macrophages or dendritic cells activate lymphocytes, how direct activation of innate immune receptors in lymphocytes modulates their function has been poorly understood. Interestingly, many of these receptors that recognize viral infection appear constitutively expressed in NK and T cells. The aim of this project is to analyse the impact of nucleic acid receptor activation on both CD8 T cell and NK cell effector function. We will be utilising the tools and systems present within the Brooks lab in Melbourne, including multiple models of viral infection, the capacity of virus-infected primary NK cells and T cells to respond to transformed cells or cognate antigen respectively. Specifically, the effects of activation of the cytosolic nucleic acid receptor RIG-I, MDA5, CGAS and STING will be assessed. The complementary expertise of the Schlee lab in Bonn will allow us to analyse the mRNA expression pattern in NK cells and T cells, followed by the activation of signalling pathways downstream of these nucleic acid receptors in combination with lymphocyte receptor stimulation. Taken together, the project will provide insights as to both, the extent to which cytolytic lymphocytes can exert effector functions despite sensing viral infection by either intrinsic or extrinsic means, as well as the impact of viral infection on the breadth of effector functions exhibited by these cells.
Title: Tolerance induction in Hemophilia A using FVIII fusion proteins
PhD student: Andrea Maione
Hemophilia A is a congenital bleeding disorder caused by the deficiency in functional coagulation factor VIII (FVIII), which leads to prolonged bleeding times. To prevent bleedings, patients with severe Hemophilia A are prophylactically treated with recombinant FVIII protein (rFVIII). However, up to 30% of these individuals develop anti-FVIII antibodies (also known as "inhibitors") that neutralize the function of the administered rFVIII. To eradicate these inhibitory antibodies, an immune tolerance induction (ITI) regime was developed for inhibitor-bearing Hemophilia A patients, characterized by repetitive high dose infusions of rFVIII, based on the potential immunological response of the high zone tolerance. The ITI strategy results in stable, detectable serum FVIII levels in up to 70% of all cases. Although this protocol is the clinical gold standard, nothing is known about the molecular mechanism underlying this process. Therefore, we will establish an in vivo mouse model to mimic the ITI in Hemophilia A mice to then determine which cell population is responsible for B cell suppression. On a cellular level, regulatory T cells as well as MDSCs will be analyzed for their potential suppressive capacity. Furthermore, the involvement of inhibitory surface molecules as well as cytokines will be evaluated. Subsequently we aim to enhance the efficacy of the Immune Tolerance Induction protocol by taking advantage of FVIII fusion proteins, for example, FVIII coupled to albumin. The latter is already reported to prolong the half-life of Factor IX (FIX) as a fusion protein, which could improve the tolerance regimen. FVIII fusion proteins may be processed and/or presented differently than FVIII alone. Therefore, DCs and FVIII-specific B cells will be analyzed in regard to protein routing and its dependency on the neonatal Fc receptor. We will therefore combine the expertise of Dr Janine Becker-Gotot at Bonn University on suppressive cell subsets and the expertise of Prof Paul A Gleeson in Melbourne on protein processing and presentation to study the effect of FVIII-fusion proteins on tolerance induction. Finally, the success of tolerance induction by ITI is depending on the health status of the patients during the ITI regime, since infections lead to tolerance breakdown. Hence, we aim to elucidate if tolerance achieved by fusion proteins is robust to infections in contrast to tolerance induced by FVIII alone.
Title: Immune escape mechanisms of advanced ovarian cancer
PhD student: Helena Boll
Ovarian cancer is one of the leading causes of cancer-related death in women and is frequently diagnosed at later stages due to the lack of early clinical symptoms. Debulking surgery, cisplatin-based chemotherapy, anti-angiogenic drugs and PARP inhibitors form the basis of an effective first line therapy. Nevertheless, long-term outcomes are still unsatisfactory and acquired cisplatin resistance is a key challenge underscoring the need for alternative strategies such as immunotherapy. Clinical trials investigated the efficacy of immune checkpoint blockade (ICB), but response rates were significantly lower than in other cancer types. The CRISPR/Cas9 genome editing technology will be utilised to engineer murine ovarian cancer cells to recapitulate human pathophysiological mutations. By using syngeneic and orthotopic mouse models, this project aims to identify the underlying mechanisms of how ovarian cancer cells escape T cell-based immunotherapies. In Bonn, the molecular expertise and in vivo mouse models will be used to delineate novel strategies to improve current efforts of establishing immunotherapy. In Melbourne, the unique opportunity to work with large cohorts of patient-derived materials in the Peter MacCallum Cancer Centre will enable us to generate organoids of metastatic ovarian cancer which mimic tumour heterogeneity, thereby allowing us to test therapeutic approaches in a more human relevant context.
Title: Cellular and molecular dynamics of splenic myeloid cells during blood-stage malaria
PhD student: Katharina Mauel
Malaria is one of the most widespread infectious diseases, causing alarming numbers of deaths annually due to the lack of efficient treatment or vaccination. Mosquitos transmit the Plasmodium parasites to the human host, where upon differentiation during the asymptomatic liver-stage, Merozoites are released that invade red blood cells (RBC) and initiate the symptomatic blood-stage. The spleen is the major organ to clear infected RBC, a process that is mainly executed by macrophages. Splenic macrophages occupy distinct anatomical niches with specific homeostatic and immunological functions. Yet, the individual impact of these macrophage subpopulations, their developmental origin and role in the cross-talk with the adaptive immune system during malaria pathophysiology is poorly understood. Here, we will use genetic mouse models to characterize the origin and function of splenic macrophage subsets during acute malaria and after resolution. The spatiotemporal dynamics of resident and recruited macrophages will be addressed by multi-colour flow cytometry as well as highly multiplexed single-cell spatial analysis of the spleen using co-detection by indexing (CODEX). We will also investigate the stem cell compartment in the bone marrow as a potential niche for the parasite to persist. With advanced transcriptomic approaches, we will identify differentially regulated pathways and signaling molecules in splenic immune cells. Conditional genetic depletion models will inform on the most relevant macrophage subpopulations controlling the infection. The involvement of splenic macrophages in the generation of parasite-specific immunity will be evaluated with a Plasmodium-specific T cell model using intravital microscopy. With the combined experience in the ontogeny and function of tissue resident macrophages of the Mass group (LIMES Bonn) and in malaria and tissue-resident memory T cells of the Heath group (PDI Melbourne) this study will dissect the role of distinct myeloid cell populations in infectious diseases and identify inter-cellular networks and intra-cellular signalling events to uncover new checkpoints that could be targeted for preventing systemic inflammation leading to malaria-induced lethality.
Title: Role of G4-quadruplex in myeloid dendritic cells
PhD student: Rebecca Linke
The detection of checkpoint molecules such as programmed death 1 (PD-1), programmed death ligand 1 (PD-L1) and cytotoxic T lymphocyte antigen 4 (CTLA-4) have changed the understanding and the treatment approaches of many malignant diseases. These checkpoint molecules can be expressed on T cells (such as PD-1), on tumor cells (PD-L1), but also on antigen-presenting cells (e.g. PD-L1, CTLA-4) and are known to regulate T cell activation, proliferation and exhaustion. G-quadruplexes (G4) are an alternative DNA structure which can form in living cells, where they could lead to gene expression changes, replication stalling and increased genome instability. In so far all tested cells, G4 formation correlates with altered cellular function. To date, G4 formation within dendritic cells (DC) has not been explored yet. We speculate that G4 stabilization within the PD-L1 gene, leads to a significant down regulation of its expression which should alter DC phenotype, function and its ability to induce T cell responses. In the proposed research plan, we will combine the synergistic expertise of Katrin Paeschke (molecular and biochemical assay on G4 DNA and RNA molecules), Annkristin Heine (medical and immunotherapeutic approaches) at the University of Bonn with the expertise of Nicholas Williamson (mass spectrometry and proteomic approaches) at Melbourne University to encipher the molecular function and relevance of G4 formation for PD-L1 function. The obtained data will be set into context of immune-therapeutic aspects that could lead to new ideas how G4 formation could be used to support current immunotherapeutic approaches.