Minerva Elements Records

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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.
The role of lysine acetyl transferases and epigenetic regulators in T-cell mediated anti-tumour immune-responses

Pijpers, Lizzy Maria Gertruda ( 2023-09)

Adoptive cell therapy using chimeric antigen receptor (CAR) T cells has demonstrated remarkable success in treating haematological malignancies, resulting in the FDA approval of six CAR T cell products for the treatment of B cell Acute Lymphoblastic Leukaemia, multiple myeloma and non-Hodgkin lymphoma. However, CAR T cells have shown limited efficacy against solid tumours in the clinic, which is primarily attributed to the immunosuppressive nature of the tumour microenvironment and antigen heterogeneity of solid tumours. Hence, strategies to overcome these challenges are necessary for CAR T cells to be effective in solid malignancies. Improved clinical outcomes have been associated with CAR T cells exhibiting memory T cell characteristics. While the transcriptional control of memory differentiation is extensively studied, our understanding of the epigenetic mechanisms underlying these gene expression changes have yet to be fully understood. In this thesis, primary human and mouse (CAR) T cells were utilized to study the epigenetic landscape of CD8+ T cell activation and differentiation. A485, a novel histone acetyltransferase inhibitor of P300 and CBP was discovered in a small molecule compound screen to regulate memory T cell differentiation. Treatment of T cells with A485 resulted in memory phenotype differentiation with the corresponding transcriptional changes: upregulation of T cell memory associated genes and downregulation of T cell effector associated genes. In vivo data across various mice tumour models, collectively demonstrated the potent anti-tumour effect of pre-treating (CAR) T cells with A485 prior to adoptive transfer. Given that the P300 and CBP histone acetyltransferase inhibitor A485 was found to regulate memory T cell differentiation, the role of histone acetylation in T cell activation and differentiation was investigated. Histone acetylation was found to be rapidly enhanced upon T cell activation. In addition, P300 and CBP mediated histone acetylation was found to be critical for memory T cell differentiation in the time prior to first T cell division at 24 to 48 hours post activation. H3K27ac ChIP data suggest that P300 and CBP might preferentially acetylate effector-associated super enhancers at 2 hours post T cell activation, thus leading to memory T cell differentiation upon A485 inhibition. Although, P300 and CBP are often described as an entity, this thesis, proposes potential independent roles, with CBP involved in regulating memory T cell differentiation. Overall, the results presented in this thesis improve our understanding of the role of histone acetylation in T cell activation and differentiation.
Uncovering epigenetic regulators of bivalent MHC class I genes in cancer

Sparbier, Christina ( 2023-06)

Recent breakthroughs in cancer immunotherapy have transformed the management of many malignancies and renewed interest in the molecular understanding of tumour antigen presentation. However, despite the success of these therapies, resistance remains a significant challenge for many patients. One prominent resistance mechanism involves the disruption of major histocompatibility complex class I (MHC-I) antigen presentation, which has been demonstrated to occur through inactivating mutations or transcriptional silencing in the MHC-I antigen presentation pathway, with the latter presenting as a potentially reversible and, therefore, targetable mechanism of resistance. In this thesis, I investigate the epigenetic mechanisms underlying MHC-I resistance in cancer. Firstly, I elucidate the significance of the polycomb repressive complex 2 (PRC2) in maintaining transcriptional repression of MHC-I, which is conserved across different species, including humans, mice, and Tasmanian devils. The silencing of MHC-I by PRC2 in cancer cells facilitates the evasion of T-cell killing. However, I demonstrate that this can be overcome through genetic or pharmacological depletion of PRC2. By conducting ChIP-sequencing, I identify that silenced MHC-I genes in cancer cells exhibit bivalent modifications, specifically repressive H3K27me3 and activating H3K4me3 marks, which is a developmental process seen in embryonic stem cells and maintained during neural progenitor differentiation. Collectively, these findings reveal how cancer cells can co-opt an evolutionarily conserved, lineage-specific function of PRC2 to silence MHC-I antigen presentation and evade immune surveillance. Driven by the observation that bivalency is often dysregulated in cancer, I set out to identify the regulators of bivalent chromatin. Building upon the previous observation that MHC-I is bivalently modified, I leveraged this characteristic as a readout and conducted whole genome CRISPR/Cas9 screens to pinpoint key regulators involved. I uncover specific roles of the PRC2.1 and PRC1.1 sub-complexes in maintaining silencing of bivalent gene expression. Unexpectedly, I make the intriguing discovery that genetic depletion or pharmacological inhibition of Menin, traditionally known as a co-activator and a component of the KMT2A/B H3K4me3 methyltransferase complexes, phenocopies the effects of polycomb disruption. This results in the derepression of bivalent genes in cancer and human pluripotent and embryonic stem cells, findings which challenge the existing paradigm whereby disruption of the KMT2A/B and polycomb complexes is expected to have opposing effects on bivalent gene regulation. Furthermore, my research reveals an essential role of KMT2A/B in MHC-I gene expression following Menin inhibition and, therefore, highlights the existence of Menin-independent and Menin-dependent functions of KMT2A/B. Finally, I demonstrate that targeting the Menin-KMT2A interaction leads to the release and redistribution of KMT2A from active genes to bivalent genes, which creates a permissive chromatin environment that facilitates gene activation. My research has uncovered previously unknown roles for specific components of the KMT2A/B and polycomb complexes in regulating bivalency. Moreover, these findings have significant implications for cancer therapy. By identifying strategies to overcome transcriptional MHC-I repression, my work provides a compelling rationale for utilising inhibitors targeting PRC2 and Menin in the treatment of difficult-to-treat malignancies. Additionally, these insights offer potential avenues for developing novel therapeutic approaches to effectively treat cancers characterised by low MHC-I expression.
Deconstructing the brain tumour microenvironment using multimodal analysis

Dinevska, Marija ( 2023-06)

Gliomas are a type of astrocytoma and are the most prevalent type of primary brain cancer, with the most aggressive form being glioblastoma (GBM), with a median survival of only 15 months. Rapid tumour cell invasion and progression is a significant challenge for patients and their oncologists and neurosurgeons, reducing treatment efficacy and inevitably leading to tumour recurrence. Cancer cells thrive by responding and adapting to cellular and non-cellular cues in the tumour microenvironment, including the extracellular matrix (ECM). However, little is known about ECM composition in brain tumours and how the ECM evolves during disease progression, and the impact of the ECM on immune cell localisation, cancer cell signalling and the functional activity of tumour cells. The PI3K and MAPK signalling pathways are typically dysregulated in GBM, and can activate the downstream transcription factor, CREB, which has been reported to regulate GBM malignancy. By integrating multiplex immunohistochemistry, histopathological staining, and spatial tissue analysis, as well as in vitro 3D GBM models, I investigated ECM composition in low- and high-grade glioma, and the spatial relationship between neoplastic cells, immune cells and the ECM in GBM tissue. My results demonstrated a grade-dependent increase in ECM deposition and an upregulation of type I and type IV collagen mRNA expression, which is associated with poor survival in patients with GBM. GBM cells and vascular cells were identified as key contributors of ECM protein deposition in GBM. Spatial analysis demonstrated that T-cells were predominantly located in perivascular niches in ECM-rich regions, while macrophages exhibited more efficient infiltration into tumour cell-rich regions. Extensive tissue remodelling contributes to cellular compartmentalisation in the tumour microenvironment and this compartmentalisation correlates with PI3K, MAPK and CREB activity, and histopathological hallmarks, including angiogenesis, tumour cell density and cell invasion. Inhibiting the PI3K and MAPK signalling pathways reduced 3D cell invasion and also facilitated a shift in the ECM composition, from a more fibrotic to a less fibrotic state. Taken together, the results suggest that the accumulation of ECM plays an important role in GBM progression, affecting both immune cell distribution and cancer cell signalling. These findings suggest that targeting the PI3K and MAPK pathways to ‘normalise’ the ECM could serve to enhance the efficacy of existing and novel therapies for GBM.
Novel Immunologic Assessment of Aplastic Anaemia and Post-Transplant Poor Graft Function for the Purposes of Therapeutic Intervention

Prabahran, Ashvind Anand ( 2023-03)

Engraftment post allogeneic stem cell transplantation (alloSCT) is defined by two intersecting domains; 1) restoration of normal haematopoiesis, 2) confirmation that this haematopoiesis is derived from a donor source. Poor Graft Function (PGF) is defined by the presence of multilineage cytopenias in the setting of complete donor chimerism and is a significant complication of alloSCT as it causes morbidity and mortality due to effects of bone marrow failure (BMF). Aplastic anaemia (AA) is an acquired, BMF syndrome resulting in the autoimmune destruction of haematopoietic tissue and subsequent pancytopenia. PGF is a controversial syndrome due to the numerous ways it has been defined in prior literature. In this thesis I investigate the underlying immunologic mechanisms as well as novel treatments for both PGF and AA. I will define the risk factors and outcomes for PGF in a large retrospective analysis of institutional databases. To assess the underlying immunology of PGF and AA I will apply multiple analytic techniques to patient samples. Finally, I describe a clinical trial framework that forms the basis for a currently operating Phase II study evaluating the efficacy and safety of atorvastatin and n-acetyl cysteine (NAC) in the treatment of PGF and relapsed refractory AA. The framework for this study can also be utilised to test novel therapies discovered as part of this thesis.
Harnessing unconventional T-cells for vaccines and immunotherapies in pre-clinical animal models

Barber-Axthelm, Isaac ( 2023-03)

Unconventional T-cells represent a heterogenous population of CD3+ T-cells that recognise protein and non-protein antigens through MHC-unrestricted mechanisms. Unconventional T-cells also undergo cytokine- or surface receptor-mediated activation independent of T-cell receptor ligation, a characteristic that is commonly associated with innate immune cells and permits rapid responses to stimuli. These cells have diverse effector responses following activation, including direct cytolytic activity against target cells, proinflammatory cytokine production to mediate other immune responses, and antigen presentation to conventional CD4+ and CD8+ T-cells. Several unconventional T-cell subsets have also been explored as immunotherapeutics, in part due to our ability to readily expand them pharmacologically, with some expressing highly conserved public T-cell receptors. Current knowledge gaps with unconventional T-cell immunotherapies includes our understanding of the frequency and phenotype of therapeutic cells in different tissue compartments, and how this is impacted by changes in pharmacological expansion protocols. Additionally, several unconventional T-cell subsets can augment conventional T- and B-cell responses associated with humoral immunity. However, the contribution of these unconventional T-cell populations to conventional adaptive immune responses against protein vaccines or viral infection is not well understood. The overarching aim of this thesis is to characterize unconventional T-cell in the context of immunotherapeutics and during vaccine-elicited immune responses, in pre-clinical animal models. Vgamma9Vdelta2 T-cells are a subset of unconventional T-cells that recognises endogenous and exogenous phosphoantigens and have garnered significant interest for immunotherapies to treat cancer and infectious diseases. While studies in pre-clinical animal models have shown promise, the clinical efficacy with Vgamma9Vdelta2 T-cell therapy has been limited. In Chapter 2, we characterised the Vgamma9Vdelta2 T-cell population at steady-state and following in vivo pharmacological expansion in pigtail macaques. We found the tissue distribution of pharmacologically expanded Vgamma9Vdelta2 T-cells changed based on the antigen administration route. Additionally, our pharmacological expansion protocol drove marked CCR6 downregulation and granzyme B upregulation in expanded Vgamma9Vdelta2 T-cells. Our results highlight how changes to pharmacological expansion protocols can alter the phenotype and tissue distribution of the expanded cell population, which is important to consider as this will likely impact therapeutic efficacy. Lymph nodes are a critical site of adaptive immune responses and the generation of antigen specific Tfh and BGC cells following vaccination. However, vaccine draining lymph node identification to study these responses is hindered by anatomical variations in lymphatic drainage between individuals, and lymph nodes being arranged in clusters with only a subset draining the vaccine site. To improve the identification of vaccine draining lymph nodes in preclinical animal models, we developed a vaccine strategy to label draining lymph nodes with tracking dyes (Chapter 3). We show that protein vaccines co-formulated with tattoo ink accurately labels vaccine draining lymph nodes in both mice and nonhuman primates. Ink-containing lymph nodes had higher frequencies of antigen specific BGC and Tfh cells compared to lymph nodes without ink. Furthermore, the ink coformulation was compatible with flow cytometry-based assays and did not alter the vaccine immune response serologically or at the B- and T-cell level. Unconventional T-cells are capable of humoral immune responses through multiple mechanisms including conventional antigen presentation, co-stimulatory signalling to Tfh cells, and providing both cognate and non-cognate B-cell support. Whether different unconventional T-cell subpopulations significantly contribute to the humoral immune response following vaccination or viral infection has not been well established. In Chapter, 4, we evaluated the contribution of unconventional T-cells to conventional adaptive immune responses elicited by vaccines or influenza infection, using transgenic mice that individually lack gamma delta T-cells, MAIT cells, and NKT cells. We found transgenic animals had comparable serological, Tfh, and BGC responses following immunisation with clinically-relevant vaccine formulation or subclinical influenza infection. Our findings indicate these unconventional T-cell subpopulations are not individually essential for mounting a robust humoral immune response to protein vaccines or viral infection. These results also raise questions about compensation between these unconventional T-cell populations, or a potential lack of unconventional T-cell recruitment to vaccine- or viral-mediated immune responses. Collectively, we evaluated unconventional T-cells in the context of immunotherapies and conventional humoral immune responses, in preclinical animal models. Better animal model characterisation will likely improve clinical translatability of candidate vaccines and therapeutics. Future utilisation of and improvements to the labelling techniques described here will help interrogate vaccine responses in regional draining lymph nodes in preclinical animal models. Our findings also raise important questions about modifying in vivo Vgamma9Vdelta2 T-cells treatment protocols to improve therapeutic cell delivery to target tissues, and modifying vaccine formulations to better recruit different unconventional T-cell populations as part of the humoral immune response.
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.
Generating CD8+ liver-resident memory T cell immunity against malaria

Chua, Yu Cheng ( 2022)

Liver resident memory CD8+ T (Trm) cells are attractive vaccine targets for malaria (Plasmodium) liver-stage immunity and can be effectively generated by glycolipid-peptide (GLP) vaccines. To gain insight into underlying mechanisms, we examined the requirements for priming, differentiation, long-term maintenance, and secondary boosting of liver Trm cells. We found that type I conventional dendritic cells (cDC1) were essential for priming CD8+ T cell responses, during which exposure to IL-4, most likely provided by activated type I natural killer T (NKT) cells, enhanced liver Trm cell formation. In addition, optimal generation of liver Trm cells required exposure to a combination of vaccine-derived inflammatory and antigenic signals post-priming, with antigen recognition being associated with enhanced Trm cell longevity. After primary immunisation with GLP vaccines, boosting of liver Trm cells could be achieved with the same GLP vaccine but a substantial delay was required for optimal boosting. This appeared to be due to NKT cell anergy post-priming as NKT cell-independent heterologous boosting could be achieved much earlier. Overall, our study revealed that the generation of liver Trm cells by GLP vaccination is IL-4 and cDC1 dependent, with longevity increased by post-priming antigenic signals and homologous boosting influenced by NKT cell recovery. Like many other malaria subunit vaccines, however, the utility of GLP vaccines is somewhat limited by the scarcity of protective CD8+ T cell epitopes. This issue is particularly prominent in the context of rodent P. berghei ANKA (PbA) infection of B6 mice, an extensively studied model of malaria. Using a combination of mass-spectrometry and in-silico approaches, we generated a library of 400 PbA-derived MHC I-restricted epitopes, from which we identified 4 immunogenic candidates that each reproducibly stimulated CD8+ T cells after pre-erythrocytic and blood-stage infections of B6 mice. Further characterisation of one of these peptide candidates, Db163, revealed cross-reactivity with a known immunogenic, but non-protective peptide PbA GAP5040-48. Targeting two additional epitopes, Db100 and Db177, by GLP vaccines induced substantial CD8+ liver Trm cells but these responses lacked protective efficacy against sporozoite challenge. The fourth epitope is derived from the PbA X, a predominantly late liver-stage antigen. Promisingly, this epitope could be targeted by a GLP vaccine to evoke liver Trm cell-mediated immunity against malaria in B6 mice. This protective immunity was remarkably long-lived with liver Trm cells persisting for at least 210 days. Furthermore, we demonstrated that X-specific liver Trm cells could execute a protective immune response cooperatively with those specific for PbA TRAP130-138, leading to improved sterile immunity even against high-dose sporozoite challenges. Lastly, the discovery of two novel HLA-A 02:01-restricted epitopes within the P. falciparum X proteins provides a future opportunity to dissect their usefulness as human vaccine candidates. Overall, this thesis provides novel mechanistic insights to maximise liver Trm cell formation and longevity after vaccination. Additionally, this thesis identifies novel antigenic targets of liver Trm cells that could be exploited for vaccination to induce immunity against malaria.

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