Medical Biology - Theses

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Defining the intranodal spatial requirements for the formation and maintenance of long-lived T cell memory

Duckworth, Brigette Catherine ( 2023-10)

Immune memory is critical for providing superior protection against infection. Current vaccine strategies exploit immune memory with varying success, with most relying on humoral immunity while failing to elicit and maintain durable CD8+ T cell responses. As such, vaccine outcomes for pathogens which require a strong T cell response are poor. The next generation of vaccines against infectious disease and cancer require robust T cell memory. However, we lack the fundamental understanding of how the longevity of T cell memory is regulated so that we may optimise this strategy. During my PhD, I have made a series of discoveries that offer new insight into this problem. Firstly, I have discovered that memory cell differentiation is imprinted in the centre of draining lymph nodes (LNs). Secondly, I have identified a distinct intranodal location where memory T cells reside long-term. Lastly, I shed light on the changes to this positioning that occur as we age. Following infection, the differentiation of T cells is driven via dynamic interactions with multiple, distinct cellular subsets. I developed and employed a novel platform to quantify cell location in 3D to examine the spatial requirements that instruct T cell fate in intact LNs. Following viral infection, I established that CD8+ effector T cell fate correlates with positioning at the LN periphery, instructed by CXCR3 signalling. In the absence of CXCR3, T cells were retained in the LN paracortex and alternatively formed stem-like memory cell precursors. I also showed that CXCR3 ligands, CXCL9 and CXCL10 are expressed by spatially distinct dendritic and stromal cell subsets. Finally, I demonstrated that T cell location can be tuned, through deficiency in CXCL10 or type I IFN signalling, to promote effector or stem-like memory fates. Increasing evidence suggests that, in the steady-state, CD8+ central memory T cells (TCM) are positioned strategically in LNs; however, the mechanisms that regulate this location remain unknown. I used 3D light-sheet fluorescence microscopy of intact LNs to identify the location of TCM cells following the resolution of viral infection. In steady-state LNs, I showed that a higher density of TCM cells occupy the cortical ridge and interfollicular regions than naive T cells, which primarily reside in the T cell paracortex. This distinct TCM location was observed following various infection challenges and mRNA-LNP vaccination. Furthermore, in the LNs of aged mice, this TCM niche was disrupted and TCM cells relocated to the T cell paracortex. To explore cell-cell contacts regulating TCM location, I employed high-resolution confocal microscopy to identify specific dendritic and stromal cells which interact with TCM within this niche. Together, these findings suggest that TCM occupy a conserved and precise LN memory niche. This provides a platform for further spatial interrogation to determine how this niche promotes cell-specific interactions and sustains long-term TCM maintenance. In identifying the key mechanisms regulating the intranodal TCM niche, my work may contribute to improved vaccine strategies grounded in robust, long-lived T cell memory.
Characterisation of dual-specific Chimeric Antigen Receptor T cells against heterogeneous tumours

Hughes-Parry, Hannah Emily ( 2023-06)

The heterogeneity of solid tumours is a significant obstacle to the response and long-term remission of patient malignancies following Chimeric Antigen Receptor (CAR) T cell immunotherapy in the clinic. While long-term remission has been achieved against haematological cancers, relapses have frequently occurred several months post-treatment due to antigen escape, and solid tumour responses have been less effective. Therefore, to improve solid tumour elimination and prevent relapse, CAR T cell immunotherapy may be improved by the targeting of multiple tumour-associated antigens through dual-specific CAR T cells, in which T cells are engineered to express CARs against multiple antigens. Existing studies have observed significant improvements over single-specific CAR T cells; however, few studies have interrogated the underlying biology in immune competent systems. In this thesis, I explored whether dual-targeting CAR T cells targeting the HER2 and EGFRvIII tumour antigens were able to effectively clear heterogeneous tumours both in vitro and in vivo. I assessed their cytotoxic function and cytokine secretion against different heterogeneous tumour targets in vitro. I found that dual-specific CAR T cells exhibit enhanced killing of heterogeneous tumour cells, but not elevated levels of cytokine secretion or exhaustion markers compared to single-specific and pooled single-specific CAR T cells. This enhanced ability for multi-antigen targeting T cells to eliminate heterogeneous tumours allows for more complete clearance of the entire tumour cell population and may subsequently mitigate opportunities for antigen escape. To explore the utility of multitargeting tumour antigens in vivo, we used CRISPR technology to generate an immunocompetent mouse model (RHEO), tolerant to human HER2, EGFRvIII and OVA, to evaluate dual-targeted CAR T cell immunotherapy approaches. I demonstrate that administration of dual CAR T cells results in improved survival in vivo in RHEO mice using a heterogeneous intracranial tumour model, and a combination therapy, by combining dual CAR T cells with anti-CD137 agonist, results in complete intracranial tumour clearance in all mice. These results highlight the importance of evaluating CAR T cell efficacy in an immunocompetent mouse model. While targeting the entirety of a heterogeneous tumour with multi-targeting CAR T cells is critical for tumour elimination, combining CAR T cell immunotherapy with other immune modulatory agents may be necessary to achieve complete tumour clearance.
A Quantitative Analysis of Natural Killer Cell Homeostasis, Competition, and Collaboration

Hennessy, Robert John ( 2022-12)

Contemporary Immunology views Natural Killer (NK) cells as critical facilitators of immune protection in various pathological settings. Still, this has not always been the case; a somewhat challenging history of NK cell research has delayed full scientific appreciation of their importance and modus operandi, which rendered NK cells a mysterious and misunderstood immune cell subset for several decades. In more recent years, NK cells are receiving a resurgence in clinical attention owing to characterisation of their potent anti-tumour and immunomodulatory properties; however, as modern Immunology remains in the aftermath of an uncertain era for NK cells, harnessing this revolutionary therapeutic potential has proven difficult. NK cells are key inducers of early inflammation and systemic immune activation, as well as expert decision makers in the destruction of harmful cells versus protection of healthy tissue. As may be expected, catastrophic consequences can occur to a host if these processes are not properly regulated. There is growing appreciation in the research community regarding the sheer complexity and redundancy in regulatory processes that maintain NK cell homeostasis and functions, as well as the plethora of cytokines and cell-cell interactions that govern this regulated behaviour. As a means of dissecting these complex processes, we have applied a reductionist approach to study how various individual signals are integrated into the internal machinery of an NK cell to produce different outcomes. To this end, we applied quantitative methods previously established in adaptive T and B lymphocytes to delineate and quantify parameters relating to survival and proliferation. In this work, we uncovered that stimulatory proliferative signals from the cytokines IL-15, IL-18, and IL-12 are offset by enhanced propensity for NK cell death, which limits the overall efficiency of their expansion during stimulation. These responses were largely dependent on direct interactions between NK cells via Fas and FasL, which induce fratricidal killing of each other. These competitive relationships between fellow NK cells were heavily dependent on the type and dose of cytokine present. Further, our investigation of NK cell interactions led us to identify that NK cells also facilitate advantageous interactions with other NK cells in more homeostatic contexts, which were dependent on IL-15. We discovered that these homotypic collaborative interactions are the result of complex interactions and bidirectional signalling events between SLAM family receptors 2B4 and CD48, which together facilitate IL-15 responsiveness and education events, thereby enhancing NK cell fitness and function, respectively. This work offers valuable insights to improve in vitro culture protocols in the clinical cultivation of NK cells for immunotherapies, such as Adoptive Cell Therapy, as well as indicating broader and nuanced roles of immune and target cell interactions in the stimulation and regulation of NK cell fitness, function, and homeostasis.
Understanding how malaria-induced T-bet expression impacts the development of protective immunity to infection

Pietrzak, Halina Mary ( 2022)

Malaria is a globally significant parasitic disease infecting millions of people annually. Clinical immunity to infection takes years of frequent exposure to develop and only partially protects the host against clinical symptoms, with individuals in endemic areas often developing chronic, asymptomatic infections. These observations suggest defects in the generation, maintenance, or effector capacity of immune memory induced in response to infection. Antibody responses are a critical component of clinical immunity to malaria. Recent work from our group demonstrated that inflammatory pathways contributing to the development of clinical malaria episodes play a negative role in the induction of humoral immunity. IFN-g produced in response to acute malaria infection was found to upregulate the expression of transcription factor T-bet in T follicular helper cells (Tfh), the key T cell subset required to provide help to B cells for the induction of protective antibody responses to infection. T-bet expression in Tfh cells impairs their normal differentiation and compromises downstream humoral responses to acute infection. The contribution of T-bet expression to the development of Tfh memory cells in malaria is unknown. To investigate this, the Tfh memory cell compartment was examined using PBMC samples from human P. vivax patients, and a murine model of severe malaria infection. Together, these analyses involving flow cytometry, adoptive transfer, and RNA-sequencing approaches revealed that the T-bet influences the composition and development of the Tfh memory cell compartment in malaria. Specifically, the main results from this investigation revealed that T-bet expression in CD4+ T cells impairs the development of Tfh central memory (TfhCM) cells which are an important compartment that support and bolster long-lived memory responses. This data provides evidence that malaria-induced inflammation negatively impacts the development of memory populations required for an efficient response to malaria, thus restraining a potent immune response to re-infection.
Manipulating cell death pathways to promote clearance of HIV-1

Garner, Sarah Elizabeth ( 2021)

HIV is a chronic retroviral infection first recognised in humans 40 years ago. Untreated, it leads to progressive CD4 T cell depletion and death approximately ten years post infection. Combination anti-retroviral therapy (cART) is very effective at controlling active HIV replication. However, it needs to be continued daily for the lifetime of the infected individual, leading to a large personal and societal cost. Although the lifespan of HIV infected individuals has approached that of the general population there continues to be excess morbidity and mortality from malignancies and cardiovascular disease. A cure for HIV has eluded the scientific community so far due to a latent reservoir of the virus existing in a small minority of memory CD4 T cells, which contain HIV DNA integrated into the cellular genome. The HIV DNA integrates can be replication competent or defective. The vast majority are defective but there exists a small pool of these cells that harbour replication competent virus. These latent cells containing integrated HIV DNA downregulate their cell differentiation markers compounding the search for these cells even further. Unfortunately, these cells are unaffected by cART and during cART interruption they can reactivate and infect naive CD4 T cells. Cell death and survival in HIV infection is balanced by host and viral factors. The most well characterised form of cell death in HIV infection is apoptosis, which can occur via both extrinsic and intrinsic pathways and can be triggered by multiple events. Actively infected cells die due to viral cytopathic effects and immune clearance, but central memory CD4 T cells infected with HIV appear to be more resistant to cell death via upregulation of important anti-apoptotic proteins that block the cell death pathways. However, this upregulation can be exploited to drive cells towards death by blocking their action. SMAC mimetics are compounds that drive cell death in extrinsic apoptosis by blocking the action of IAPs. This thesis explores the addition of SMAC mimetics to standard cART therapy with the hypothesis that by targeting these upregulated proteins this can deplete the latent reservoir of HIV infection. For the first time, I show that SMAC mimetics delay the time to viral rebound in HIS HIV mice. I also describe preliminary work targeting both the extrinsic and intrinsic apoptosis pathways.
The Purification, Identification, and Measurement Of RNA-Binding Proteins

Smith, Jeffrey Michael ( 2021)

RNA-binding proteins (RBPs) are classically regarded as facilitators of gene expression. In recent years, however, RNA-protein interactions have also emerged as a pervasive force in the regulation of homeostasis. The compendium of proteins with provable RNA-binding function has swelled from the hundreds to the thousands astride the partnership of MS-based proteomics and RNA Sequencing. At the foundation of these advances is the adaptation of RNA-centric capture methods that extract protein that has been crosslinked in its native environment. These methods reveal snapshots in time displaying an extensive network of regulation and a wealth of data that can be used for both the discovery of RNA-binding function and the molecular interfaces at which these interactions occur. This thesis describes the development of an extraction method that purifies RBP-RNA complexes. This method differentiates itself from other RBP-discovery protocols in that it 1) purifies these complexes so completely that RBP identification can be conducted qualitatively and without differential abundance analysis, 2) permits transcript-targeted capture with sequence-specific oligos, 3) permits global, sequence-agnostic capture 4) both RBP and its bound RNA are isolated intact and 5) can reliably interrogate RBPs at depths that exceed present methods without metabolic or molecular labelling. The performance of this method is first assessed with a census of proteins that directly interact with global, or targeted, RNA transcripts from model cell lines. These efforts are then extended to investigate how protein-RNA interactions change during transition from quiescence to proliferation and then contraction in primary murine CD8+ T cells. Finally, these studies demonstrate how cellular responses provoke different proteins to moonlight as RNA binders and sheds light on a network of complex, co-evolved molecular machines.
Investigating the Role of Oligomeric State in Chimeric Antigen Receptor Function Using de novo Designed Transmembrane Structures

Chandler, Nicholas John ( 2021)

Chimeric antigen receptor (CAR) T cell therapy has revolutionized the treatment of B cell malignancies by redirecting patient T cells to destroy cancer cells using engineered receptors. While CAR T cell therapies hold enormous potential as treatments in a wide range of tumour settings, treatments for non-B cell cancers have largely failed to significantly improve patient outcomes thus far. Furthermore, CAR therapies carry significant risk of inducing cytokine release syndrome (CRS), a potentially deadly toxicity caused by excessive release of inflammatory cytokines. The ability to minimize toxicity whilst maintaining adequate tumour cell-killing is therefore vital to the continued improvement of CAR therapies. We aimed to investigate the currently ill-defined relationship between CAR oligomeric state and potency using a novel protein engineering approach, with the aim of leveraging this knowledge to predictably modulate CAR activity. With de-novo protein design collaborators we identified synthetic transmembrane domain (TM) sequences that predictably formed defined homo-oligomeric structures. In addition to a previously validated trimeric TM sequence, I used X-ray protein crystallography to determine the structure of a dimeric TM peptide that agreed closely with its predicted structure. I inserted these novel oligomeric TM sequences into a well-established anti-HER2 CAR construct (comprising an anti-HER2 scFv attached via stalk/TM to costimulatory and stimulatory tail sequences) and validated their oligomeric state and signalling capacity in a mouse T cell line. When expressed in primary mouse T cells and incubated with HER2+ target cells, dimeric and trimeric CARs exhibited enhanced target cell killing compared to a reference anti-HER2 CAR. Using an in vivo mouse tumour model it was subsequently demonstrated that CAR oligomeric state correlates positively with CAR T cell anti-tumour efficacy. CARs encoding synthetic oligomeric TM’s also demonstrated a dramatic reduction in the release of inflammatory, CRS-associated cytokines within in vitro experiments. Using rational TM sequence mutations I identified lateral interactions between CARs and the endogenous T cell costimulatory molecule CD28 in primary mouse T cells as the key determinant of CAR cytokine release. These findings present an opportunity to improve efficacy and safety of CAR T cell therapies and warrant further validation in other clinically relevant CAR T cell disease models.
A quantitative framework for lymphocyte fate decisions

Horton, Miles Benjamin ( 2021)

During an adaptive immune response activated B and T lymphocytes undergo rapid clonal expansion and generate extensive cellular heterogeneity. How lymphocytes guarantee the emergence of functional diversity amongst responding cells is not fully understood. In this thesis, the strategies utilised by the adaptive immune system for the diversification of B and T cells is investigated at the cellular, molecular and clonal levels in a quantitative manner. Activated B cell heterogeneity is predominantly driven by two critical programs. Firstly, the differentiation of antibody-secreting cells (ASCs) and secondly, the diversification of antibody isotype by class switch recombination (CSR). The regulation of these two processes was investigated through combined clonal and molecular analysis using a high-throughput proliferative lineage tracing approach to study ASC differentiation and CSR across thousands of clones. Two distinct fate programs emerged. Firstly, the timing of ASC differentiation within clones was strongly correlated. Diversity in commitment to the ASC lineage is established early and could be traced to the naive founder cell, from where it is transmitted to all progeny during clonal expansion. In striking contrast, isotype switching was highly variable across related cells irrespective of common ancestry, revealing a highly stochastic, cell-autonomous process regulated late within activated single cells. Further analysis demonstrated that single cells faced with a choice of two heavy chain isotypes solve the conflict using stochastic selection that is independent of their clonal lineage. As the principle molecular drivers of CSR are well known, their variation amongst single cell within clonal families was measured. Extensive variation was demonstrated in the expression of both activation-induced cytidine deaminase (AID) and the transcription of the germline noncoding RNAs. Furthermore, there was no correlation between AID expression and germline transcription, nor was the expression of distinct germline transcripts correlated. Thus, the net effect of stochastic influences over these two components can account for the single cell autonomy governing CSR. This stochastic molecular mechanism of CSR was developed into a quantitative model that accurately described and predicted B cell fate decisions across cell division and under varying experimental conditions. Quantitative analysis was applied to multi-parameter data of CD8 T cell heterogeneity, generated in response to diverse external stimulation. Using a combination of novel and established analytical techniques, the influence of time and division progression on T cell diversification, and their control by external signals, was accurately measured. The results of this investigation was subsequently used to construct a kinetic model of time- and division-dependent expression patterning for the molecule CD69 under varying external conditions. This model accurately described the expression dynamics of CD69 over time and division and highlighted the utility of a quantitative modelling approach to understanding CD8 T cell heterogeneity. Collectively, the work presented in this thesis represents a set of quantitative principles that describe lymphocyte fate decisions.
Identification of novel regulators of B lymphocyte biology

Trezise, Stephanie Elise ( 2020)

The differentiation of B cells into antibody secreting cells (ASCs) and the production of protective antibodies is a critical part of the adaptive immune response to infection. ASCs are also important for the formation of immunological memory which provides protection against reinfection and the generation of ASCs is the goal for almost all current vaccination strategies. Despite the importance of these cells, we still lack a complete understanding of the factors that control B cell differentiation into ASCs, ASC survival and antibody secretion, all of which must be tightly regulated to ensure an optimal immune response. Here, I have developed a CRISPR/Cas9 mediated arrayed screening approach for the identification of novel positive and negative regulators of primary mouse B cell proliferation, survival, differentiation into ASCs and antibody secretion. By interrogating multiple gene sets I have identified all elements within the ASC gene signature that are essential for the in vitro generation of ASCs. I have also identified several novel negative regulators of the B cell differentiation process (AB124611, Arhgef18, A430078G23Rik, Fam43a, Pold1, Ripk3, Rnf130 and Rps6ka5). This work has also uncovered a novel role for 6 genes, (Cdv3, Hspa5, Sec61a1, Selk, Sumo2, Vcp) in driving the proliferation of B cells. One of these genes, Cdv3, has no previous association with proliferation in any cell type and presents an exciting new candidate for further investigation. I have demonstrated that within the ASC gene signature there are 35 genes which are essential for efficient antibody secretion. Interestingly, many of these genes are components of the ER protein processing pathway, however, not all elements of this pathway appear to be essential for antibody secretion. These results raise the possibility of there being a specific pathway for antibody secretion, or that the genes identified in this thesis may represent weak links in the ER protein processing pathway which could potentially be exploited therapeutically to inhibit antibody secretion in disease settings. Finally, I have used an Irf4 deficient mouse model to uncover a novel role for Irf4 in the development of the peritoneal B-1a population. I have shown that Irf4-/- mice lack peritoneal B-1a cells and by examining multiple stages of B-1a cell development I have demonstrated that in the absence of Irf4, B-1a cell development is blocked at the transitional B-1a stage. By employing RNA sequencing to analyse the transcriptional profiles of the remaining Irf4-/- B-1 cells and analysis of previously published ChIP sequencing data, I have revealed a potential role for Irf4 in directly activating the expression of Bhlhe41, a transcription factor that is required for B-1a cell development and homeostasis. Together, the results from this thesis build upon decades of previous work on the genetic regulation of B cell biology. Integrating the novel regulators of B cell proliferation and differentiation that I have identified in this thesis into the current model of ASC generation will improve our understanding of how the decision between undergoing differentiation or maintaining the B cell fate is made. A detailed understanding of how this fate decision is made has far reaching implications for human health and disease as this information can be used to inform vaccine design, reveal the causes of immunodeficiencies or highlight novel avenues for targeting pathogenic ASCs in autoimmunity and cancer.
The ontogeny of effector regulatory T cells

Teh, Peggy Pek Gee ( 2020)

Regulatory T (Treg) cells are critical for the maintenance of immune homeostasis and peripheral tolerance. Different subsets of Treg cells have recently been described with many studies showing the importance of context-specific differentiation of Treg cells, in particular within non-lymphoid organs. These non-lymphoid organ Treg cells have a fully suppressive Treg cell phenotype with an effector function and are termed effector (e)Treg cells. However, the ontogeny of eTreg cells have not yet been fully described. Additionally, molecular determinants of the eTreg cell program remain incompletely understood. My thesis examines the transcriptional events that regulate the generation of eTreg cells during their thymic development, their homeostasis and response to infection. Using different gene targeted mouse models at steady state and in viral infection models, I studied the intrinsic molecular mechanisms that contribute to eTreg cell differentiation. In particular, I focused on follicular Treg (TFR) cells, which constitute the eTreg cell subset of the germinal centre. The molecular control of eTreg cell fate and function converges on the transcription factors IRF4 and Blimp-1. IRF4 is induced by antigen receptor signals and cooperates with AP-1 factors, BATF and JUN, to regulate transcriptional networks involved in lymphocyte differentiation, function and metabolism. For example, these factors regulate genes important for antibody class switch recombination in B cells and functional differentiation of distinct CD4 T helper (Th) subsets, including Th2, Th9, Th17 and T follicular helper cells. IRF4 expression in Treg cells is critical for effector differentiation, yet the precise mechanisms of how IRF4 regulates the transcriptional program of TFR cells remains unknown. Using a novel transgenic IRF4 reporter mouse we found that IRF4 is highly expressed in TFR cells. Using IRF4 knockout mouse models, we demonstrate that IRF4 is necessary for TFR cell generation in a Treg cell-intrinsic manner. IRF4 controls important aspects of the transcriptional program that drives TFR cell differentiation, including genes essential for Treg cell migration. Furthermore, I identified the transcription factor c-Maf to be essential for TFR cell generation and demonstrated its central role in maintaining a follicular program in Treg cells. Subsequently, using ribonucleic acid (RNA)-sequencing, we generated a “follicular signature” of gene expression from the combined analysis of TFH and TFR cells. Integrated transcriptional analyses showed that in the absence of either IRF4 or c-Maf, the majority of the follicular signature genes were downregulated, indicating that these two transcriptional regulators, aside from Bcl6 are indispensable for follicular TFR cell development. Finally, analyses of IRF4 and c-Maf DNA binding sites, identified by chromatin induced precipitation (ChIP)-sequencing, in combination with open chromatin regions in follicular T cell specific loci, we showed that the precise orchestration of distinct sets of genes is required to promote conserved aspects of the follicular T cell fate. In conclusion, my thesis describes how a key transcriptional network orchestrates fundamental steps in TFR cell differentiation and function, which contributes to the understanding of eTreg cell biology.

Medical Biology - Theses

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