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ItemNo Preview AvailableA Quantitative Analysis of Natural Killer Cell Homeostasis, Competition, and Collaboration( 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.
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ItemUnderstanding how malaria-induced T-bet expression impacts the development of protective immunity to infection( 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.
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ItemGenerating CD8+ liver-resident memory T cell immunity against malaria( 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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ItemInterleukin-1 Is Unique in Its Ability to Modulate PD-L1 and PD-L2 Expression by Mo-DCs( 2022)Expression of PD-1 ligands PD-L1 and PD-L2 on the surface of tumour and immune cells has led to the widespread success of checkpoint blockade immunotherapy, yet despite decades of research, knowledge of the underlying mechanisms tumour cells implement to avoid recognition by the immune system is still evolving. Research from our laboratory has validated that human Mo-DCs can increase surface expression of PD-L1 and PD-L2 in the presence of inflammatory stimuli. PD-L1 on APCs has been implicated in the conversion of conventional T cells into Tregs, however the role that PD-L2 may play in this system has not been explored. Furthermore, the mechanism by which tumours can elicit expression of PD-1 ligands on the surface of APCs, and the impact that this may have on infiltrating T cell phenotype and function is incompletely characterised. In this study, human Mo-DCs were generated and assessed for their ability to simultaneously upregulate PD-L1 and PD-L2 in response to stimulation with proinflammatory cytokines. It was discovered that IL-1 could elicit upregulation of both PD-1 ligands more effectively than TNF, and IFN-gamma could induce low levels of PD-L1 but was unable to modulate PD-L2 expression. Other members of the IL-1 superfamily did not have the same ability as IL-1, and it appeared that the cellular response was limited to Mo-DCs as lymphocytes and macrophages did not respond similarly. While attempting to reproduce these results in a more biologically relevant system, it was discovered that A375 melanoma cells were able to lose their ability to modulate PD-L1 and PD-L2 expression, however modification of the culture conditions to mimic features of the tumour microenvironment partially restored this function. Further analysis of the supernatants of tumour cell-lines resulted in the identification of an inhibitory factor which antagonised the IL-1beta-mediated PD-L1 and PD-L2 upregulation by Mo-DCs, and the efficacy of this factor could be modulated by culture conditions. Finally, CD4 T cells cultured with cytokine-stimulated Mo-DCs expressing PD-L1 and PD-L2 showed increased proliferation and expression of FOXP3, however it was not possible to determine whether differentiation into functional Tregs had occurred. Overall, this study demonstrated that pro-inflammatory cytokines such as IL-1 can have dual functions that contribute to immunoregulation on specific cell types. Additionally, tumour cells were shown to have the capacity to produce factors which can positively or negatively modulate the immune response, and the secretion of these factors can be impacted by extracellular conditions. We were also able to demonstrate that co-culture of cytokine stimulated Mo-DCs with CD4 T cells promoted proliferation and expression of regulatory transcription factor FOXP3 by some T cells, suggesting that differentiation and function of these cells could be modulated by Mo-DCs. These findings have helped improve understanding of the mechanisms by which tumour cells resist the immune response or immunotherapy, and further identification of upstream modulators of PD-L1 and PD-L2 expression within the TME has the potential to uncover novel immunoregulatory factors which when targeted may provide a therapeutic advantage.
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ItemNo Preview AvailableThe molecular and cellular basis of antigen recognition by CD1a-restricted T cells( 2022)In contrast to conventional T cells that recognise peptide antigens presented by MHC molecules, a group of “unconventional” T cells recognise lipid antigens presented by MHC-like CD1 family members, CD1a, CD1b, CD1c and CD1d. Studies suggest that CD1a-restricted T cells comprise a unique subset in human blood that recognise CD1a-lipid complexes and play a unique functional role in skin immunity. While they comprise a decent proportion of T cells compared to CD1d-restricted, natural killer T (NKT) cells, they remain relatively less well-understood. This thesis describes the phenotypic characterisation of CD1a-restricted T cells in human tissues directly ex vivo. Phenotypic analyses and single cell RNA-sequencing of CD1a-restricted T cells revealed that they are distinct from other CD1-restricted T cells. They did not express typical innate-like markers such as CD161, IL-18R, and PLZF, which are expressed by NKT cells, distinguishing them as a unique population of unconventional T cells. This thesis also elucidates how T cell receptors (TCRs) interact with CD1a-lipid complexes. Profiling the TCR repertoire of CD1a-restricted T cells, demonstrated that while diverse, there is a bias towards TCR variable genes that endow optimal TCR configurations to interact with CD1a and lipid antigens. Experiments with CD1a mutant cell lines revealed that individual TCRs bind at various sites across the entire binding cleft of CD1a, which likely increases the diversity of lipid antigens that can be recognised by CD1a-restricted T cells. Indeed, these T cells were observed to recognise numerous lipid antigens including self-lipids and dideoxymycobactin (DDM), a lipid antigen derived from Mycobacterium tuberculosis, with some CD1a-restricted TCRs even displaying cross-reactivity to lipids with distinct chemical structures. Reagents were developed as tools to study lipid-reactive T cells in macaques, especially for non-human primate models of disease. A suite of CD1 tetramers were generated to isolate CD1-restricted T cells in pig-tailed macaques and for preliminary enumeration and phenotypic analysis of CD1-restricted T cell subsets in macaque tissues. Lastly, tetramers were used to investigate CD1a-restricted T cells in human skin. Populations of lipid-reactive T cells and gd-T cells were isolated for phenotypic analysis and TCR sequencing, thus demonstrating that they may play a role in healthy skin. C12-15 alkyl-benzoate, a common oil in dermatological products, was identified as a novel CD1a antigen, suggesting a role for CD1a-restricted T cells in allergic dermatitis. These studies provide insight into the functional properties of CD1a-restricted T cells and their molecular interactions with CD1a-lipids. Collectively, they represent a step forward in characterising CD1a-restricted T cells and provide a greater understanding of their role in the immune system.
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ItemRole of IFN-y induced genes in cell autonomous defence against Legionellae( 2021)L. pneumophila and L. longbeachae are ubiquitous environmental bacteria that can cause a severe pneumonia, known as Legionnaires’ Disease, when contaminated aerosols are inhaled by susceptible humans. The co-evolution of the bacteria with their environmental hosts has equipped the bacteria with the ability to subvert the cell intrinsic host defence mechanisms in human cells, thereby allowing the pathogens to survive and replicate within the lung macrophages. During infection, Legionella establishes an intracellular niche, known as the Legionella containing vacuole (LCV). The biogenesis of the LCV is dependent on the defective organelle trafficking/intracellular multiplication (Dot/Icm) type IV secretion system, which translocates a large arsenal of bacterial effector proteins into the host cell. These effectors are known to modulate host metabolism and cell-autonomous defence, protect the integrity of the LCV and allowing the bacteria to acquire nutrients from the host to ensure intracellular survival and enable intracellular replication of the pathogen. It is known that during Legionella lung infection, the mammalian host mounts a robust inflammatory response, producing cytokines, such as TNF-alpha, IL-1-alpha, IL-6, IL-12 as well as type I interferons and IFN-gamma, which usually leads to the restriction of intracellular replication and culminates in the clearance of the infection. It was previously shown that IFN-gamma is crucial for host defence against Legionella in mice, since disruption of IFN-gamma signalling or IFN-gamma deficiency results in a high replication of Legionella in the lung as well as a failure to clear the infection from the host, despite the activity of other inflammatory cytokines. Exposure of cells to interferons (IFN), including IFN-gamma, results in the induction of a network of genes that combat infection, leading to so-called IFN-mediated cell-autonomous defence. This network is finely-tuned to balance efficient pathogen control while preventing collateral tissue damage. However, which interferon-induced genes and through what mechanism this strikingly potent restriction is mediated remains elusive for Legionella. In this study, we shed some light on the mode of action of the IFN-gamma induced host defence against Legionella. We identified a new mechanism of host defence mediated by interferon stimulated genes (ISGs), that results in the disruption of effector translocation into host cells by the Dot/Icm secretion system. We demonstrated that this mechanism is uniquely triggered by interferon signalling and is independent of well-known host defence mechanisms such as host cell death, direct bactericidal activities, inflammasome activation as well as proteasome and autophagy-mediated degradation. By utilising mRNA sequencing of IFN-gamma and type I interferon-stimulated macrophages, we identified possible factors that mediate this inhibition: ISG15 and PARPs. These proteins have not previously been implicated in Legionella host defence and represent a unique opportunity to increase our knowledge of interferon mediated cell-autonomous host defence. Currently, more than 65 Legionella species are known and roughly half of them have been clinically associated with infection, frequently in immune compromised patients. After L. pneumophila, L. longbeachae is the second most common causative agent of Legionnaire’s Disease worldwide and is the leading causative agent in Australia and south-east Asia. Despite this, knowledge about the pathogenesis of L. longbeachae is minimal. Therefore, during this study, we also aimed to provide new insights into the pathogenesis of L. longbeachae infection and characterise the impact of IFN-gamma on immune control. We observed unique features of L. longbeachae infection in comparison to L. pneumophila, such as the ability to survive within a wider range of lung phagocytes, dampening of the cytokine response of the host and translocation of effectors into all lung phagocytes tested. These unique features may enable L. longbeachae to subvert the host defence more efficiently than L. pneumophila and thus replicate to higher numbers. Furthermore, we were able to show that IFN-gamma is crucial for host defence against L. longbeachae in vivo, with neutrophils and monocyte derived cells dependent on IFN-gamma signalling to mediate their bactericidal properties. In addition, we were able to demonstrate that IFN-gamma stimulation restricts L. longbeachae Dot/Icm secretion system effector translocation into host cells. Overall, this study substantiates the importance of IFN-gamma in host defence against Legionella and supports the need to broaden research efforts to non-L. pneumophila species. Investigation and deeper understanding of critical host defence mechanisms can be used as a starting point to develop anti-infective agents against pathogens targeting the process of effector translocation or effector mediated manipulation of host function and cell-autonomous defence.
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ItemThe viral glycoproteins of the Hepaciviruses. Structural and functional studies to inform vaccine design( 2022)Hepatitis C virus (HCV) infection is a major global health burden, with an estimated 71 million people infected worldwide. Vaccine design for HCV is challenging for multiple reasons, including high sequence variability of the glycoprotein E2, as well as the lack of a small animal challenge model in which to test vaccine candidates. This thesis addresses aspects of both challenges. Glycoprotein E2 is present on the virion surface and is a major target of neutralizing antibodies which can prevent infection. The N-terminal hypervariable region 1, HVR1 (residues 384-411), of E2 is an immunodominant region within E2 and elicits neutralizing antibodies that are usually isolate specific. We previously identified a novel murine monoclonal antibody, MAb33, which binds to an unusual epitope bridging HVR1 and the adjacent target of broadly neutralizing antibodies referred to as epitope I (residues 412-423). MAb33 potently neutralizes genotype 1a viruses and can cross-neutralize 3 different HCV genotypes. This study defined the epitope of MAb33 to include residues within the E2 region 401-415 and resolved its structure in complex with its epitope. The epitope adopts an alpha-helical conformation with residues G406, A407 and N410 involved in direct polar interaction with the antibody. The helical structure of the epitope differs from the extended conformation of other E2 crystal structures that include this region, suggesting that it could be conformationally flexible. Sero-surveys of HCV positive individuals have identified significant reactivity to a peptide encompassing the MAb33 epitope, indicating a role for MAb33-like antibodies in natural infection. To address the need for an immune competent model for HCV vaccine development, a Rodent hepacivirus (RHV) was investigated as it shows close evolutionary relatedness and virological similarities with HCV and represents an important potential model of HCV pathogenesis and host responses. While the T cell response to RHV has been characterized, detailed studies characterizing the RHV E2 glycoprotein, the development of the humoral immune response after infection and in vaccination studies are lacking. This thesis characterized the antigenic and immunogenic properties of RHV E2. A minimal ectodomain expressed as a soluble protein was defined (residues 418-603), which has 4 sites for N-linked glycans and 12 cysteine residues. The development of the anti-E2 antibody response in outbred rats infected with RHV was analysed and the appearance of anti-E2 antibodies observed at 28 days post-infection. RHV E2 was assessed as a potential vaccine antigen in immunisation/challenge studies in rats. Rats received a E2 protein prime followed by a protein boost, or a combined vaccination of E2 protein and a simian adenovirus (ChAdOx1) encoding the non-structural proteins NS3-NS5B from RHV. Both groups failed to generate anti-E2 antibody prior to challenge at 6 weeks. Following challenge with RHV, anti-E2 antibodies appeared 14 days later, with most animals seroconverting by 28 days post challenge. These studies are the first of their kind to define a soluble ectodomain of the RHV E2 protein and explore the development of anti-E2 antibodies in infection.
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ItemManipulating cell death pathways to promote clearance of HIV-1( 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.
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ItemIdentification and characterization of proteins and mechanisms involved in the uptake and traffic of vitamin B related antigens( 2021)Major histocompatibility complex, class I-related (MR1) presents Vitamin B-related antigens (VitBAg) at the cell surface to activate mucosal associated invariant T (MAIT) cells, directing homeostasis and immune responses. Although previous work has suggested endocytosis as a participant in MR1 presentation, how these antigens are captured by the cell is currently unknown. It is likely that MR1 ligands are uptake as metabolites for they have several structural similarities with molecules known to be transported through solute carrier (SLC) transporters. Here, we shown that flavins are pathway-specific inhibitors of MR1-5-OP-RU, and do not inhibit MR1-Ac-6-FP upregulation. We revealed that 5-OP-RU, ribityl lumazine (RL) and bacterial VitBAg, but not folate derived ligands, enter the cell through SLC52A family of riboflavin transporters, as their expression increases MR1 presentation and MAIT cell activation in a riboflavin modulated manner. In contrast, knock-outs models SLC52A family drastically reduce the incorporation of RLs but do not abolish the capacity to present 5-OP-RU through MR1. In fact, pathway specific inhibitors of MR1-5-OP-RU and MR1-Ac-6-FP extend to nucleosides, nucleobases and other drugs, arguing for the contribution of more SLC transporters in their uptake. Likewise, MR1 presentation during infection is increased by ligand-producing bacteria located in the cytosol, stating a cytosolic step to reach empty MR1 molecules. Finally, we showed that 5-OP-RU alters the metabolome of cells like LPS, leading to changes in their transcriptome profile. Our results unveil a new route for 5-OP-RU, RL and bacterial VitBAg uptake through SLC52A transporters, contributing to their capture and modulating MR1 presentation, together with a new potential role of 5-OP-RU as a pathogen-associated molecular patterns (PAMPs) molecule.
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ItemT cell response to an MHC-II restricted epitope of rodent malaria( 2021)Malaria is caused by different Plasmodium species that can infect a variety of animals including humans and rodents. The life cycle of these parasites is complex and includes a liver stage followed by a blood-stage in their vertebrate hosts. While the host’s immune response against each of these stages is incompletely understood, CD4 T cells are known to play an important role in immunity to Plasmodium infection during both stages. This project aims to examine the specific CD4 T cell response to a novel MHC II-restricted epitope in Plasmodium infection in C57BL/6 mice, and to characterise the protective capacity of these T cells. To this end, we made use of a recently generated TCR transgenic mouse line, termed PbT-II, which responds to a so far unknown Plasmodium derived epitope. In this project, the PbT-II epitope was identified as derived from heat shock protein 90, residues 484 to 496 (Hsp90484-496 or abbreviated DIY). Different priming methods, such as injection of an anti-Clec9A antibody attached to the Hsp90 epitope (aClec9A-DIY), infection with P. berghei ANKA (PbA) infected red blood cells (iRBCs) or immunisation with radiation attenuated PbA sporozoites (RAS), were used to characterise PbT-II memory cell formation. Results revealed the formation of memory PbT-II cells expressing surface markers associated with central memory T cells (TCM), effector memory T cells (TEM) and tissue resident memory T cells (TRM). Given the importance of tissue-resident memory T cells in peripheral immunity, mainly studied in CD8 T cells, we focused our study on the formation and function of CD4 TRM cells in the liver. Parabiosis studies using RAS vaccinated mice confirmed the liver residency of a CD69+ PbT-II cell population. Gene expression profile analysis revealed that these CD4 T cells expressed a core gene signature similar to that of CD8 resident memory T cells. Furthermore, differences in the gene expression profile of PbTII TRM cells generated via different protocols, suggested lineage specific effector mechanisms, such as IL-4 production or perforin expression, for subsets of CD4 TRM cells in the liver. As CD4 T cells can potentially act against both the liver and blood-stage of Plasmodium infection, we sought to investigate the protective potential of PbTII effector and memory cells for both of these stages. While none of the PbT-II priming methods resulted in a reduction of liver parasite burden upon sporozoite infection, mice injected with large numbers of in vitro polarized PbT-II Th1 or Th2 cells showed reduced parasitemia after PbA blood-stage infection. Surprisingly, most of these mice were protected from experimental cerebral malaria (ECM), although they were not able to clear PbA blood-stage infection.