Microbiology & Immunology - Theses
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ItemUnderstanding immune responses to severe influenza disease( 2021)Respiratory viral infections caused by Influenza A viruses (IAVs) are responsible for annual seasonal epidemics that cause mild, severe or fatal pulmonary disease and estimated to result in 243,000-640,000 deaths globally every year. Severe infections can result in lung tissue damage, acute respiratory distress syndrome (ARDS) as well as secondary bacterial pneumonia, all of which are detrimental to the host and can potentially lead to a fatal disease outcome. Many of those at high-risk of developing severe disease include children, the elderly and immunocompromised individuals. Although current influenza vaccines are the best way to combat the seasonal influenza epidemics, the vaccines need to be updated annually to match strains predicted to circulate in the upcoming influenza season. Frequent mutations in the virus can lead to strain mismatches, which can affect the protective efficacy of these vaccines and result in significant increases in infection rates, putting those who are most vulnerable at risk for developing severe disease. The aim of this PhD thesis was to investigate host factors and mechanisms associated with severe influenza disease to provide insights into identification and prediction of severe disease outcomes. Co-expression of surface CD38 and MHC-II on CD8+ T cells is recognized as a classical hallmark of activation in many acute and chronic viral infection settings. However, high and prolonged CD38+MHC-II+ expression can be also associated with severe and fatal disease outcomes. Our previous studies have found that the persistence of CD38+MHC-II+ CD8+ T cells is associated with impaired IFN-gamma production in H7N9-infected patients with fatal outcomes. In Chapter III, we sought to investigate the mechanisms underpinning the expression of CD38+MHC-II+ markers using wild type and transgenic mouse models of influenza infection. We provided evidence that CD38+MHC-II+ CD8+ T cells are recruited early to site of infection during severe influenza A virus infection and can be activated in an antigen-driven as well as bystander manner. Our adoptive transfer experiments also demonstrated that MHC-II molecules were not expressed intrinsically by CD38+CD8+ T cells, but acquired from antigen presenting cells by trogocytosis, and the CD38+MHC-II+ phenotype was crucial for optimizing T cell recall ability. In Chapter IV, we investigated disease signatures during the early phases of infection to identify features associated with clinical outcomes. While comparable viral titres were observed between H7N9 patients who survived or succumbed to infection, there was a trend of higher level of inflammation responses in those who died. Our transcriptome analyses of blood samples from fatal and recovered H7N9 patients found that the gene Olah, encoding for a fatty acid hydrolase, was expressed more than 80-fold higher in fatal H7N9 patients compared to those that recovered at early days after disease onset. We established an Olah-overexpressing A549 cell line, which allowed us to develop a qPCR platform to screen Olah expression in human tissues and cells. Our analyses of Olah distribution across different anatomical sites found Olah expression in human lung, spleen and PBMCs, with the most prominent expression detected in CD14+ cells within the human lungs. Subsequently in Chapter V, we generated and established OLAH knockout (OLAH KO) mice to further investigate the role of Olah during severe influenza A virus infection. Compared to wild-type mice, OLAH KO mice displayed milder disease symptoms, less body weight loss and were protected from severe and fatal disease outcomes following IAV infection. In addition, OLAH KO lungs exhibited lower viral titres, less tissue damage and had significantly reduced levels of inflammatory cytokines and infiltrating innate immune cells, indicating the importance of Olah in controlling early excessive host responses and disease severity. These effects were associated with reduced lipid droplets in virus-infected epithelial cells lining the bronchioles, thus defining a role for OLAH in driving lipid metabolism during IAV disease. Overall, the findings from this PhD thesis highlight the importance of CD38+MHC-II+ CD8+ T cells during severe influenza A virus infection and identified the mechanisms underlying severe IAV disease. We also identified Olah as a potential biomarker of severe disease and demonstrated its role in modulating early host immune responses via the formation of lipid droplets and defined its impact on disease outcomes. Overall, data generated in this PhD thesis advance our understanding of factors underlying fatal clinical outcomes and provide insights into the development of novel treatment strategies and biomarkers against severe influenza disease.
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ItemUnderstanding human B cell and antibody responses against seasonal influenza viruses( 2020)Vaccination is the best available means to reduce the burden of seasonal influenza. However, current influenza vaccines need to be updated frequently to keep up with evolution among circulating viruses. Antigenic evolution, otherwise termed drift, is most rapid among A/H3N2 viruses, and the A/H3N2 component of vaccines is frequently updated. Despite this, influenza vaccine effectiveness against the A/H3N2 subtype has been poor in recent years, especially among previously vaccinated individuals. Protection induced by inactivated influenza vaccines is largely mediated by B cells and antibodies reactive against the head of the hemagglutinin (HA) protein, with help from T follicular helper cells. The cellular and molecular mechanisms that underlie the attenuating effects of prior vaccination and existing immunity are largely undefined. It has been suggested that existing antibodies clear or mask antigen, or that memory B cells induced by prior exposures competitively dominate responses so that B cells and antibodies become focused on epitopes that are shared between prior and prevailing vaccine strains. The aim of the work presented in this PhD thesis was to examine the impact of pre-existing immune responses induced by prior infection with different A/H3N2 strains on influenza vaccine immunogenicity. In depth antibody as well as B cell assessments were performed to understand the impact of existing antibodies and memory B cells following vaccination and provide insights into the design of new vaccine strategies. As a lead up to the ex vivo analysis of B cells from vaccinees, we first sought to understand how human naive versus memory B cells differentiate in vitro. Experiments were conducted in Chapter 3 to compare the stimuli required for their differentiation into plasmablasts, and subsequently understand how they change phenotypically once stimulated. Specifically, sorted human naive and memory B cells from healthy individuals were stimulated in vitro to induce differentiation into plasmablasts. Data obtained in this PhD thesis showed that stimulation with the Toll-like receptor (TLR) 7/8 agonist R848 in the presence of monocytes induced the highest activation of both naive and memory B cells. Conversely, stimulation with the TLR9 agonist CpG or with R848 in the absence of monocytes induced little to no differentiation of naive B cells but were able to stimulate memory B. cell differentiation. Despite robust differentiation into antibody secreting plasmablasts, naive-derived B cells remained phenotypically distinct from memory-derived B cells up to day 6 after in vitro activation, with differential expression of CD27, CD38 and CD20. This work resulted in a first-author publication in Clin Transl Immunol, 2019. The focus of Chapters 4 and 5 was to understand how prior influenza virus infection affects antibody and B cell responses to influenza vaccination. To address this question, vaccine responses were investigated in a unique influenza vaccine-naive cohort in Viet Nam, that had been monitored for both clinical and asymptomatic influenza virus infection for more than 9 years. In 2016, twenty-eight participants without documented A/H3N2 virus infection (since 2007) and 72 participants who had been infected with A/H3N2 viruses, belonging to a range of genetic clades, received an inactivated trivalent influenza vaccine containing an A/Hong Kong/4801/2014-like (H3N2) antigen. This work investigated whether influenza vaccination induced naive B cell responses specific for new epitopes or largely recalled B cells specific for conserved epitopes, common to the vaccine A/H3N2 component and prior infecting strains. Hemagglutination inhibition antibody titres were measured in pre- and serial post-vaccination sera against 40 A/H3N2 viruses spanning 1968-2018 to understand how the titre and cross-reactivity of antibodies against the HA head evolve. B cells were assessed by flow cytometry using a panel of phenotypic markers in addition to recombinant HA probes representing the vaccine and recently infecting strains (A/Perth/16/2009, A/Victoria/361/2011 and A/Switzerland/9715293/2013). Participants who had at least one pre-vaccination A/H3N2 virus infection had on average 2 to 3-fold higher vaccine-specific antibody titres, steeper titre rises in the weeks following vaccination (mean peak on day 14), and less titre decay by days 21 and 280 compared to participants without prior infection. Moreover, participants with prior infection exhibited greater and better-maintained titre rises against viruses that circulated a year after vaccination, indicating that prior infection extends the strain coverage of antibodies induced by vaccination. Notably, A/H3N2 viruses that circulated 275-340 days after vaccination caused illness in only 1.4% of participants with infection prior to vaccination and in 14% of participants without prior infection. This suggests that vaccine effectiveness can be enhanced by pre-existing immunity. However, it was also clear that the range of strains against which antibodies were induced was dictated by the strain with which participants were previously infected, indicating that vaccination may simply recall rather than update antibody-mediated immunity. HA-probe reactive B cell frequencies and activation status increased substantially after vaccination. The greatest increases in HA probe-reactive B cells were detected among participants who had recent prior infection, with the majority of B cells exhibiting cross-reactivity with prior strains. A modest but significant increase in the frequency of B cells that reacted with the HA of the vaccine strain, but not of past strains, could be detected in participants who lacked prior infection. The phenotype of vaccine HA single-positive B cells, including increased IgM expression, indicated that they may have been naive-derived B cells. Vaccination induced B cells that preferentially reacted with the HA of A/Perth/16/2009 and/or A/Victoria/361/2011 viruses, but not A/Switzerland/9715293/2013 viruses, among participants who had prior A/Perth/16/2009-like virus infection. However, B cells induced by vaccination in participants who had prior A/Switzerland/9715293/2013-like virus infection were equally cross-reactive with HA of all tested viruses. These results support the inference that immune responses to standard inactivated influenza vaccines are dominated and shaped by recalled memory B cells with limited activation of naive B cells to update immunity. Overall, this PhD thesis investigated how pre-existing immunity induced by documented influenza virus infection affected the humoral response to seasonal influenza vaccines in healthy adults. This work provides new insights into the capacity of influenza vaccines to stimulate naive B cells, which may be limited due to memory B cell dominance and to a lack of sufficient stimulation to activate naive B cells. This knowledge could be used to design new vaccine strategies and improve influenza vaccine-induced protection.
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ItemMolecular mechanisms regulating CD8+ T cell differentiation following external cues( 2018)Upon activation, naive CD8 T cells undergo a program of proliferation and differentiation that results in the acquisition of effector functions. Optimal T cell activation requires the integration of multiple signals including cross-linking of the T cell receptor (signal 1), co-stimulation (signal 2) and soluble factors such as cytokines (signal 3). Once a CD8 T cell has received these three signals they differentiate into an effector cell, which are able to control infection by directly killing the infected cell. Once the infection is cleared, these effector cells contract by controlled cell death and a long-lived population of memory cells remain. These potent memory cells are the defining feature of adaptive immunity as they offer protection for the life of the host due to their unique capabilities to survive in the absence of antigen and respond rapidly to secondary challenge. Therefore, effective CD8 T cell memory is the goal of cell-mediated vaccination strategies. While it is well established that CD4 help is required for CD8 T cell memory formation, it is unclear when during CD8 differentiation this help is required. Further, the effect that CD4 help has on the transcriptional profiles of CD8 T cells and the molecular pathways they use during the generation and maintenance of memory CD8 T cells remains elusive. Using a mouse model of Influenza A virus infection, where priming occurs in the presence or absence of CD4 T cell help, we have pinpointed that help is required during the initial priming of CD8 T cells, and not during memory maintenance or recall. Genome wide RNA sequencing analysis of the transcriptional signatures between resting helped and unhelped memory CD8 T cells revealed surprisingly few differentially expressed genes. However, upon reactivation, helped memory CD8 T cells exhibited greater transcriptional up-regulation than their unhelped counterparts and utilization of alternate molecular pathways. Intriguing metabolism defects combined with similarities to an ‘exhausted phenotype’ suggest that help is required to defer a cell away from terminal differentiation, towards a memory cell. Further, our analysis revealed that CD4 T cell help during initial priming is essential for establishing a memory cell pool with enhanced transcriptional potential. Thus, CD4 T cell-dependent programming likely underpins rapid responsiveness, a key characteristic of memory CD8 T cells. Each stage of T cell differentiation; naive, effector and memory, are characterized by distinct transcriptional and functional profiles. However, the molecular mechanisms regulating the acquisition of these profiles remain poorly defined. Further, contention remains around the pathway of differentiation towards memory cell status. This thesis has compared the transcriptional profiles of each of these cell stages, aiming to identify any previously unappreciated genes, gene networks or TFs that may be vital during the differentiation of memory CD8 T cells. These transcriptional profiles were first compared globally, which highlighted the similarities between each of the cell stages. Based on transcriptional profiles of gene expression across each of the cell stages, two key genes were identified, Dmrta1 and Zbtb32. These were then validated and assessed to determine if they translate from mice models into human studies. The data shown in this thesis suggests each of these genes may be molecular signatures used to identify memory cells. Each gene should be further evaluated, but alone have validated the method of data mining and comparison to identify previously undescribed genes as having a role in cell differentiation. Finally, using mathematical modelling of in vitro activated cells combined with bioinformatic analysis of ATACSeq, this thesis also has explored the role of signal three on chromatin remodelling during CD8 T cell activation. Our analysis has identified that each cytokine has a slightly different impact on the degree of chromatin accessibility, and a combination of signal 3 cytokines resulting in the highest level of chromatin accessibility and subsequent activation, proliferation and downstream acquisition of effector function. We suggest signal 3 itself is an under-appreciated cascade of regulation during the activation of CD8 T cells by directly controlling chromatin structure. Taken together, this data suggests that each signal received by a CD8 T cell during activation is gearing it towards a particular fate. It can be speculated from this data that the default pathway after activation is towards terminal differentiation, that is, to become an effector cell, destined to die by controlled death after the infection is cleared. It is the cues from the environment that skew the cell away from this fate in a healthy environment, such as CD4 T cell help and signal three. Importantly, in chronic disease states such as HIV, or LCMV in mice, this balance is tilted and results in a mass of activated cells and exhaustion. Therefore, the work contained in this thesis demonstrates the importance of each factor during activation and their effect on not only the functional capacity of CD8 T cells, but also their fate.
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ItemInvestigating the impact of influenza neuraminidase on viral replication( 2019)Infection with influenza A virus remains an important issue for healthcare systems worldwide due to the direct impact of seasonal epidemics on human morbidity and mortality and also the risk of emergent pandemics. Currently, preventive measures and treatments are not fully effective in controlling influenza virus infection. The mainstay of disease prevention is vaccination, with vaccines being updated annually and most commonly produced in embryonated chicken eggs. Influenza A viruses are classified by the surface glycoproteins haemagglutinin (HA) and neuraminidase (NA) that they express. The established role of influenza NA is the enzymatic cleavage of sialic acid from newly formed virions and from viral receptors on the surface of infected host cells at the site of virus budding. This is necessary to prevent virus aggregation and reattachment to the infected cell by the sialic acid-binding function of the HA, which would reduce the effectiveness of virus release. In this body of research, attention has been given to the potential effect of influenza NA on infectious virus yields in a variety of in vitro and in vivo systems. This project sought to determine whether an NA with intrinsically low enzyme activity for sialic acid would confer lower viral fitness and, if so, which stages of the infection cycle of influenza A virus are most affected. Specifically, this project has investigated the importance of sialidase activity of different H3N2 influenza viruses. Reverse engineered viruses differing only in the expression of different N2 NAs were constructed and tested for their yields in different host systems and for their ability to selectively remove sialic acids expressed in discrete linkage configurations (SA alpha2-3Gal and SA alpha2-6Gal) on host glycoproteins. The impact of altering the expression levels of sialic acid on host cell surfaces on the replication of viruses with strong and weak NA activity was also explored. It was shown that reverse engineered viruses expressing an NA with low cleavage activity for removal of sialic acid from a complex substrate yielded low infectious virus titres in embryonated chicken eggs and mouse lung, but not MDCK or A549 cells. This was despite the fact that wild-type virus from which the NA was selected did not show this deficit. In addition, viral fitness could not be restored in engineered viruses when NA was paired with the matched HA suggesting a gene other than HA was compensating for the weak NA activity in the wild-type virus. Mutation of the NA with lower activity to change amino acids near the active site to corresponding residues in a highly active NA was shown to increase the overall enzyme activity, but did not rescue high viral yields in chicken eggs. The reduced replication in eggs and mouse lung of viruses with weaker NA correlated with a pronounced decrease in the ability to cleave SA alpha2-3Gal. Importantly, low NA activity of reverse engineered viruses and associated reduction of viral yields in eggs was found to be due to a significant reduction in the amount of NA present on the virion. The low yield in eggs was also observed in viruses in which the weakly active NA and its matched HA were engineered onto the backbone of the virus strain used in reassortment to produce vaccine seeds for seasonal vaccine production. Acknowledging the present limitations of influenza disease prevention, this research indicates that NA may be inadequately represented in current seasonal vaccines, which may reduce their effectiveness. The study provides impetus to further understand mechanisms of virulence and optimise controls that limit influenza virus infection.
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ItemTranscriptional regulation and co-stimulatory signaling in antiviral T cell immunity( 2018)Special AT-rich binding protein-1 (SATB1) is a global chromatin organizer, promoting or repressing gene transcription in mice and human. In this PhD thesis, SATB1 expression was examined in humans across ages and tissues (Chapter 3). The molecular (Chapter 4) and functional (Chapter 5) role of SATB1 was investigated during anti-viral immunity in mice using an influenza (IAV) infection model. Additionally, the effect of CD27-mediated co-stimulation was studied in the context of HIV-1 infection (Chapter 6). SATB1 has pivotal roles during T cell development and maturation, with lineage fate decision in hematopoietic stem cells and gradual changes in SATB1 expression contributing to T cell development in the thymus in mice. In Chapter 3, SATB1 expression was analyzed across lymphocyte compartments from different human tissues and correlated with PD-1 expression in virus-specific CD8+ T cells. SATB1 expression in pediatric and adult donors showed that SATB1 expression was highest in the human thymus with differential expression levels from DN to DP thymocytes and down-regulation of SATB1 in peripheral T cells. Chapter 3 shows that SATB1 expression in the periphery is not static but follows fine-tuned expression dynamics with downregulation from naïve to antigen-specific CD8+ T cells, likely to be antigen- and tissue-dependent. These data led to the hypothesis that fine-tuned SATB1 expression is necessary for maintaining fate-potential in developing and mature, peripheral T cells. Several molecular mechanisms have been identified for gene regulation by SATB1 with wide-range impacts on the overall chromatin landscape. Previous studies in our laboratory showed that SATB1 mRNA levels are high in naïve, but low in effector CD8+ T cells. The impact of SATB1 in repressing transcriptional programs in naïve CD8+ T cells, prior to its downregulation in effector T cells, was addressed in Chapter 4 of this study. ChIP-Sequencing analysis was performed to decipher genomic binding sites of SATB1 in naïve and effector CD8+ T cells. SATB1 ChIP-Seq data demonstrated that SATB1 binding sites were predominately distal to transcriptional start sites, likely to harbor transcriptional enhancer sites, with reduced SATB1 binding sites in effector over naïve CD8+ T cells. To understand the effects of SATB1 on the transcriptional regulation in naïve and IAV-specific CD8+ T cells, SATB1 imposter mice (SATB1imp/imp) were used in this PhD study. In these mice, Satb1 contains a point mutation in the DNA-binding domain encoding position. SATB1 protein expression in SATB1imp/imp mice persists but is dysfunctional with reduced DNA-binding capability. CD8+ T cells from SATB1imp/imp mice showed up-regulation of certain gene profiles, especially at the naïve stage, such as Pdcd1, Ctla4 and Ccl5, characteristic of activated or exhausted T cells. In Chapter 5, an IAV infection model was used, to examine the effects of dysfunctional SATB1 in IAV-specific CD8+ T cell response generation. CD8+ T cell numbers were consistently reduced in SATB1imp/imp mice with significantly reduced IAV-specific CD8+ T cell numbers in lungs on d10 post-infection. SATB1imp/imp CD8+ T cells exhibited an early overexpression of PD-1 from the naïve stage and reduced polyfunctionality within IAV-specific SATB1imp/imp CD8+ T cells. Using a bone marrow chimera approach, in which mice were reconstituted with a mixture of wildtype and SATB1imp/imp-derived lymphocytes, data showed that reduced T cell numbers and PD-1 overexpression are T cell intrinsic in SATB1imp/imp mice. Immunotherapies, including anti-PD-1, anti-CD27 and histone deacetylase inhibitors, are often used in clinical trials to manipulate activation of T cells. In Chapter 6, we used CD27-mediated stimulation to understand the effect on CD4+ T cells with and without HIV-1 infection. CD27 is a co-stimulatory receptor of the TNF-family, expressed on naïve and central memory T cells. Non-permanent stimulation via CD27 leads to increased primary and memory antiviral CD8+ T cell responses in mice. Here, in humans, CD27-mediated stimulation of CD4+ T cells via its ligand CD70 exhibited profound activation potential in vitro, with high CD4+ T cell proliferation and GzmB production. To examine whether this high activation potential could trigger re-activation of viral transcription in latently infected CD4+ T cells, we re-stimulated CD4+ T cells with conventional α-CD28 or CD27-mediated co-stimulation in an in vitro latency model. Unexpectedly, re-stimulation via CD27 of CD4+ T cells led to reduced viral reactivation compared to α-CD28 stimulation of CD4+ T cells. However, similar transcriptional reactivation levels were obtained when CD4+ T cells isolated from HIV+ individuals on ART were re-stimulated with the two protocols. Strikingly, pre-stimulation of CD4+ T cells prior to in vitro HIV-1 infection showed a trend towards reduced HIV-DNA integration and overall infection. This suggests that CD27-mediated stimulation could lead to activation of antiviral mechanisms that reduces CD4+ T cells HIV-1 infection. Overall, this PhD study provides an in-depth understanding of the transcriptional and co-stimulatory regulations of T cell differentiation in response to viral infections. SATB1’s ability to regulate immune checkpoint molecules, such as PD-1 by its DNA-binding capability in antiviral immunity highlights its significance in future PD-1-related cancer and HIV-1 immunotherapy trials used to reverse T cell exhaustion.
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ItemNo Preview AvailableTracking human CD8+ and γδ T cell receptor repertoire dynamics to understand the impact on immune responses towards influenza viruses( 2018)Seasonal IAV epidemics cause severe morbidity and mortality, resulting in up to 250,000 -600,000 deaths worldwide annually, especially in the young, elderly, immunocompromised, pregnant and those with co-morbidities. Given the segmented nature of the viral genome and a rapid mutational rate, newly-emerging influenza viruses have the potential to cause pandemics. Current seasonal vaccination regimens elicit neutralizing antibodies (nAbs), which require yearly updates to account for the antigenic evolution of influenza viruses. Alternative strategies such as the development of a universal vaccine that can provide broad protection by eliciting immune responses across different strains of influenza viruses are ugently needed. CD8+ T cells recognize internal conserved segments of the viral protein and thus have the potential to provide cross-strain immunity. The efficient immune response of a CD8+ T cell is dictated by the recognition of peptide-MHC complex by its T cell receptor (TCR). However, the response magnitude varies with age and immunogenetics. Similarly, innate γδ T cells are potently activated by stress-induced ligands, independently of classical antigen-presenting molecules and could provide immediate effector function for novel influenza immunotherapies and vaccines. However, much of human γδ T cell biology remains understudied. Therefore, this PhD aimed to determine TCR dynamics of both γδ T cells and influenza-specific CD8+ T cells across different age groups, anatomical locations and influenza infection. Aim 1 explored the diversity of the human γδ T cell TCR repertoire and how γδ T cells are actived by influenza viruses. We implemented a single-cell RT-PCR for paired analysis of the TCRγ and TCRδ chains and developed an in vitro infection model of influenza-infected lung epithelial cells co-cultured with peripheral blood mononuclear cells (PBMCs). We performed a thorough repertoire analysis of ex vivo γδ T cells from cord blood, young adult, elderly adult and human tissues (spleen, lung and lymph node). Our analyses found diverse and private γδ T cells in cord blood and spleen, while those in young adults and lungs were highly focused towards invariant γ9δ2 TCRs. Elderly adult γδ T cells displayed expansion of private or the invariant γ9δ2 clonotypes. Using the in vitro infection model of influenza, we next investigated γδ TCRs which produced IFNγ during an in vitro influenza infection and PBMC co-culture. Our results demonstrated that the majority of responding γδ T cells harbored γ9δ2 TCRs. We observed heterogeneity in the influenza response between cord blood, young adult and elderly adults. γδ T cells within cord blood and the elderly adults had minimal IFN- production in the absence of γ9δ2 TCRs. Thus, this study provided an understanding on how γδ T cells contribute to immune protection during influenza infection and which TCRs are important to elicit across all age groups vulnerable to influenza virus infection. In Aim 2 and 3, we tracked the repertoire dynamics of influenza specific CD8+ T cells across age groups and tissue locations. Since HLA-A*02:01-restricted M158-66 viral peptide has high sequence conservation and elicits immunodominant CD8+ responses, we focused our analyses on HLA-A*02:01-M158+CD8+ TCRs. A robust response elicited by HLA-A*02:01-M158+CD8+ TCRs is governed by the presence of the public TCR signature, TRBV19/TRAV27 (CDR3 motif “GGSQGNL”/“SIRSYEQ”). Our study demonstrated the loss of this public TCR and presence of large private clonotypes in the HLA-A*02:01-M158+CD8+ TCRs isolated from the elderly donors, as compared to young adults who maintained high frequencies of public TCRs. Our study showed, for the first time, HLA-A*02:01-M158+CD8+ T cells were present in human tissues (spleen, lung and lymph nodes) obtained from young adults. Furthermore, lung tissue-resident HLA-A*02:01-M158+CD8+ T cells and those isolated from spleen and lymph nodes displayed a prominent presence of public TCRs. Overall, we showed a loss of public TCRs with aging and we speculate that this is a mechanism underlying reduced immune responsiveness during influenza infection with aging. Moreover, the presence of public TCRs in distal tissues could provide a reservoir to replenish “optimum” TCRs at the site of infection. The magnitude of antigen-specific CD8+ T cells can be influenced by the different Human Leukocyte Antigens (HLAs) expressed by an individual, thus contributing to the phenomenon of CD8+ T cell immunodominance hierarchy. Using the known highly conserved immunodominant epitopes restricted by 6 HLAs that have broad coverage towards influenza viruses across different ethnicities, we compared the response magnitude of these epitope-specific CD8+ T cells to that of HLA-A*02:01-M158+CD8+ T cells. We showed that individuals co-expressing HLA-B*27:05 and HLA-A*02:01 had higher magnitude of B*27:05-NP383+CD8+ T cell responses compared to that of A*02:01-M158+CD8+ T cells. Our findings showed that B27/NP383+CD8+ T cells had higher functional capacity as compared to A02/M158+CD8+ T cells. Moreover, TCRs of A02/M158+CD8+ T cells from heterozygous donors showed a reduction or complete loss of the public TCR present in A*02:01+B*27:05- individuals. This suggested that the reduction in the observed magnitude of response was partly attributed to changes within the A02/M158 TCR repertiore. Overall, this PhD contributes to our understanding of innate and adaptive T cell compartments during influenza virus infection. It provides evidence that influenza-specifc γδ and CD8+ T cell immune responses are affected by age, HLA genotype and alterations in the TCR repertoire. These findings form an important foundation for future research developing universal vaccines against influenza viruses and immunotherapies against viral infections or cancer.
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ItemElucidating the role of T-box protein family members in influenza-specific T cell immunity( 2017)CD8+ T lymphocytes are specialised cells responsible for immunity to intracellular pathogens and cancer. Upon exposure to its cognate antigen, a naïve CD8+ T cell undergoes a program of differentiation and proliferation into a pool of effector CD8+ T cells. Following clearance of infection, this pool contracts and a pool of long lived memory CD8+ T cells remain. Transcription factors (TFs) from the T-box group of proteins are known to play a key role in differentiation but their molecular mechanism remains unclear. This thesis aims to understand the molecular mechanism, that T-box members T-bet and Eomes play in CD8+ T cell differentiation and also the role that the epigenetic modifier KDM6b plays. Utilising a model of influenza A virus (IAV) infection and adoptive transfer of Tbx21-deficient (-/-) cells, we show that while T-bet is dispensable for early CD8+ T cell participation and cellular division, it is essential for full expansion of a virus-specific effector CD8+ T cell response. While Tbx21-/- IAV-specific CD8+ T cells are able to divide early after infection, Tbx21-/- CD8+ T cells are unable to sustain CD8+ T cell lineage specific cytokine expression. Whole transcriptome analysis showed a global dysregulation in early programming events where IAV-specific Tbx21-/- CD8+ T cells expressed genes associated with various CD4+ T cell lineages. Finally, analysis of histone signatures within the Ifng locus demonstrated that T-bet deficient CD8+ T cells were unable to activate “poised” enhancer elements compared to wild-type (WT) CD8+ T cells, correlating with diminished Ifng transcription. In all, these data support a model whereby CD8+ T cell activation induces rapid induction of T-bet. This ultimately leads to correct CD8+ T cell programming and differentiation via T- bet interactions with regulatory genetic elements that promote appropriate chromatin remodelling events at key gene loci. KDM6b is a lysine demethylase that has specific activity against histone 3, lysine 27 di-, and tri-methylation (H3K27me2/3). This histone modification is associated with a repressive transcriptional state, or, when found co-located with the active H3K4me3 mark, marks a bivalent epigenetic state. Resolution of bivalency at fundamental TF loci has been shown to be a key mechanism for the initiation of CD8+ T cell differentiation. This thesis aimed to understand if KDM6b was required for resolution of bivalency at the loci of key TFs involved in CD8+ T cell differentiation. To address this question, we developed a conditional short hairpin RNA (shRNA) mouse model targeting KDM6b. In this model, the Kdm6b shRNA will target the Kdm6b locus and result in degradation of Kdm6b mRNA and subsequently decrease KDM6b activity. Preliminary data using this mouse model showed that the endogenous CD8+ T cell response to IAV was impeded with KDM6b knockdown. Moreover, the Tbx21 promoter remains bivalent in these mice, compared to luciferase hairpin controls. Surprisingly, this is not a general mechanism as other TF promoters were resolved, suggesting there is specific targeting of KDM6b to the T-bet locus in IAV-specific CD8+ T cells. Eomesodermin (Eomes) has previously been reported to play a role in development of the memory CD8+ T cell pool, specifically central memory (TCM) CD8+ T cells. Analysis of endogenous IAV-specific Eomes-/- CD8+ T cell responses demonstrated that this TF is essential for the development of the memory CD8+ T cell pool, particularly TCM. Interestingly, secondary challenge showed that Eomes is dispensable for a secondary IAV-specific effector CD8+ T cell response but essential for maintenance of the secondary memory CD8+ T cell pool. Furthermore, transcriptome analysis of primary memory CD8+ T cells showed surprisingly few differentially expressed genes in the absence of this TF. Taken together, this data suggests a role for Eomes in the development and long term maintenance of memory CD8+ T cells, but not the programming and function of these cells. These data show that the T-box proteins play specific roles in different stages of T cell differentiation. T-bet plays a role rapidly following initiation of differentiation, likely by interacting with regulatory genetic elements at key CD8+ T cell signature genes to maintain CD8+ T cell identity and fate. Eomes is involved later during the immune response to shape the memory CD8+ T cell pool and maintain these populations. Finally, KDM6b appears to be involved in the resolution of bivalency at fundamental TF loci. This information can be used to inform future vaccine or drug target strategies that enhance or modulate the CD8+ T cell response.
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ItemIdentification and characterization of C-type lectin receptors for respiratory viruses( 2015)Studies described in this thesis have characterized interactions between respiratory viruses and different C-type lectin receptors. A transfection-based approach was used to express mannose- and galactose-specific C-type lectin receptors and to examine their ability to promote attachment, entry and infection of target cells by influenza A viruses. In addition, we investigated the ability of C-type lectin receptors to promote infection by other respiratory viruses, including mumps virus, Newcastle disease virus, respiratory syncytial virus and parainfluenza virus type-3.
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ItemUnderstanding CD8+ T cell responses to seasonal and pandemic influenza viruses( 2015)In the absence of neutralizing antibodies, CD8+ T cells can provide protection and ameliorate disease severity during influenza A virus (IAV) infection. Thus, there is great interest in the development of a CD8+ T cell-mediated IAV vaccine directed towards the relatively conserved internal proteins of IAV. The rational design of a CD8+ T cell mediated vaccine requires a greater understanding of factors, which govern the development of an optimal CD8+ T cell response. However, despite the breadth and depth of murine studies, little is known about human immune responses towards IAV. This PhD thesis focuses on the identification and characterisation of IAV-specific CD8+ T cell responses towards IAV to determine key factors required for the development of an optimal immune response. In particular, this PhD thesis examines the importance of the CD8+ T cell receptor (TCR) in inter-species but not intra-species cross-reactivity. A systematic screening approach was utilised for the unbiased detection of immunodominant IAV-specific CD8+ T cell responses regardless of donor HLA profile. Nucleoprotein (NP) was the most immunogenic protein of IAV, in which seven novel and highly conserved CD8+ T cell epitopes restricted by a range of HLA alleles were identified. Interestingly, these were clustered within the carboxyl terminal 2/3 of NP, suggesting that the epitope-rich regions within NP present a promising target for a CD8+ T cell mediated cross-strain protective influenza vaccine. IAV replicates using an RNA-dependant RNA polymerase, therefore rapidly evolves, and it is unknown how human CD8+ T cells respond to distinct IAV strains. The potential for inter-strain cross-reactivity was assessed using the HLA-B*37:01-restricted NP338 peptide in which two major variants bearing mutations at positions 6 and 7 were identified. In vitro assays revealed that CD8+ T cells stimulated with one NP338 peptide could recognise and respond to the NP338 variants. Human single-cell multiplex RT-PCR for detection of TCRαβ repertoire revealed that this strain cross-protection was attributable to diverse and cross-reactive TCRαβ repertoires, which can recognise the subtle differences within the distinct NP338-pHLA complexes. This suggests that diverse and cross-reactive TCRαβ repertoires are important in the recognition of variant IAV peptides and may provide protection in the face of novel IAV strains emerging in the human population. Having identified inter-species cross-reactivity, intra-species cross-reactivity (heterologous cross-reactivity) was investigated between two sets of HLA-A*02:01 restricted peptides, IAV-M158 and EBV-BMLF-1280 or IAV-NA231 and HCV-NS31073. Despite the sequence homology of the peptide pairs, no cross-reactivity was observed either ex vivo or after in vitro expansion in healthy or HCV-infected donors. Structural analysis revealed that each pHLA complex displays a distinct conformation and TCRαβ analysis showed that the TCRαβ repertoire used for the recognition of the different pHLA complexes was unique, demonstrating that CD8+ T cells are highly specific due to their TCRαβ and although they display some inter-species cross-reactivity, it is unlikely that intra-species cross-reactivity is common. CD27 is constitutively expressed on the majority of memory human T cells at any given stage of life, however, little is known about the effect of CD27 co-stimulation in human lymphocytes. CD27-negative cells were superior in function and cytotoxic potential directly ex vivo, suggesting the importance of CD27 co-stimulation on CD27-expressing lymphocytes. Using a plate bound CD70 (pCD70) monoclonal antibody (mAb) the role of CD27 co-stimulation was investigated. Co-stimulation with pCD70 was required for naïve CD27hiCD45RA+CD8+ T cell proliferation, accelerated Granzyme B acquisition and enhanced the phenotypic plasticity of CD27-expressing CD8+ T cells. This suggests that CD27 co-stimulation is vital during CD8+ T cell priming and enhances recall, similar to murine data and thus might be manipulated for future vaccines or immunotherapies. Overall, this PhD thesis has expanded our understanding of human CD8+ T cell responses towards IAV, in particular the importance of a cross-reactive and diverse TCR repertoire for protection against distinct IAV strains. Furthermore, my PhD work discusses characteristics that govern an “optimal” CD8+ T cell responses, thus providing important foundations for future research into universal immunity against influenza viruses.
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ItemAn investigation into how the TLR-2 agonist Pam2Cys stops influenza and its sequelae( 2015)Influenza continues to be a major public health concern with up to 1 billion people being infected annually. Mitigation of the disease and its symptoms are effected by the use of seasonal influenza vaccines i.e. vaccines against the currently circulating strains of influenza virus and a small number of antiviral drugs which are often used in concert. The development of antiviral resistance to the antiviral drugs and the need for vaccines to accommodate pathogen evolution, limit the effectiveness of these treatments and as a consequence focus has been directed on developing alternate treatments to combat influenza. Immunostimulants are a new class of antiviral agents that are currently being examined and function by activating the host’s own innate immune system. Because these agents target the host and not the pathogen, they are unlikely to be plagued with issues of resistant strains and can also be used to combat a broad range of pathogens including influenza strains yet to be encountered. When administered intranasally, the Toll-like receptor-2 (TLR-2) ligand S-[2,3-bis(palmitoyloxy)propyl]cysteine (Pam2Cys) has been shown to alter the pulmonary immune environment in a matter of hours in such a way that the host is protected from infection with influenza virus. This study has identified those components of the innate immune system which are elicited and/or activated by Pam2Cys and are responsible for the immediate protective effect. In the absence of an effective influenza vaccine, treatment with Pam2Cys also allows the induction of influenza-specific immunity on exposure to the virus providing the opportunity for population immunity to be achieved naturally. Furthermore, treatment with Pam2Cys reduces the morbidity and mortality associated with secondary bacterial infections which are a major contributor to the mortality associated with influenza. The work described here provides an opportunity for clinical development of Pam2Cys to combat the influenza virus and other respiratory pathogens