Microbiology & Immunology - Theses
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ItemGenerating an effective T cell-based influenza vaccine( 2023)Protective immunity against influenza virus is heavily dependent on humoral and cellular immune responses. Current influenza vaccines predominantly utilise antibody immunity, but as this is strain-specific, it leaves the population vulnerable to antigenic drift by influenza virus and importantly fails to protect against novel pandemic strains. CD8+ T cell immunity, on the other hand, due to its ability to recognise highly conserved antigenic determinants of influenza virus, enables the possibility of broadly protective universal immunity. In particular, CD8+ T cells that reside within the lung called tissue-resident memory T cells (TRM) are the responsible subset mediating cross-protection against influenza virus. However, current vaccines either do not or poorly generate CD8+ T cell responses. Therefore, this PhD thesis investigated the capacity of a novel T cell-based vaccine candidate to elicit lung CD8+ TRM and critical parameters required for the optimal induction of cross-protective influenza-specific lung CD8+ TRM. We investigated the cellular immune response evoked following a single-cycle replication-incompetent influenza vaccine candidate called S-FLU. Intranasal S-FLU immunisation generated lung CD8+ T cells and CD8+ TRM of reduced magnitude and functional avidity relative to natural influenza virus infection controls. Interestingly, the limited inflammatory profile of S-FLU immunisation conferred a clonally diverse CD8+ T cell and TRM profile in the lung. As a result, a greater propensity of these cells cross-reacted against a naturally occurring variant and prevented the development of T cell escape mutants. Our findings suggest the inflammatory milieu of a vaccine is an important consideration as this may influence the T cell receptor repertoire, resulting in downstream alterations in the cross-reactivity and capacity to subvert viral variants. Vaccine studies investigating protective efficacy must take into consideration pre- existing influenza-specific immunity generated by prior infections and the annual vaccination regime that occur over the course of an individual’s lifetime. Using a panel of live attenuated influenza virus vaccine candidates (cold-adapted and single-cycle), we next investigated the capacity of live attenuated influenza vaccines to elicit lung CD8+ TRM responses in the face of pre-existing immunity against the vaccine backbone. We determined that pre-existing antibodies specific for the vaccine backbone inhibited CD8+ T cell priming and therefore memory CD8+ T cell development and lung CD8+ TRM populations. Importantly, high dose vaccination could mitigate the impairment in CD8+ T cell priming, for which the resultant lung CD8+ TRM were protective against heterologous influenza virus challenge. Influenza infection can result in a transient depot of antigen long after viral clearance that influences influenza-specific CD8+ T cell responses, but it is unclear how this antigenic stimulation impacts the local cognate antigen-requisite lung CD8+ TRM compartment. Our studies suggest that residual antigen persistence is likely applicable to only certain epitopes. Furthermore, persistence of residual antigen activated naive CD8+ T cells that then formed CD8+ TRM populations in the lung, however these cells exhibited reduced polyfunctionality and longevity. Our results thus imply that lung CD8+ TRM generated from residual antigen following influenza viral clearance are unlikely to meaningfully participate in protection against re-infection with influenza virus. Overall, we show vaccines that evoke lung CD8+ T cells and CD8+ TRM of broad repertoire diversity are valuable against influenza virus variants and that this local immunity may be compromised in hosts with pre-existing humoral immunity against the vaccine backbone. As such, our work uncovered several insights in the optimal implementation of T cell-based vaccines aiming to induce universal protective immunity against influenza virus.
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ItemMucosal-associated invariant T (MAIT) cells and their function in bacterial infection( 2021)Mucosal-associated invariant T (MAIT) cells are a subset of innate-like alpha/beta T cells that recognize riboflavin metabolites presented by the monomorphic major histocompatibility complex (MHC) class I related protein-1 (MR1). The most potent antigen known to date is 5-(2-oxopropylidineamino)-6-D- ribitylaminouracil (5-OP-RU). MAIT cells are abundant in mucosal tissues and blood in humans. Upon bacterial infection, MAIT cells expand rapidly, with production of cytokines, including interferon-gamma (IFN gamma), tumor necrosis factor (TNF), granulocyte macrophage colony stimulating factor (GM-CSF) and interleukin-17 (IL-17), and cytotoxic granzymes. Their phenotype indicates that they play an important role in immunity. Previous studies showed a protective role in local infections, involving a single organ or tissue, but the mechanisms of MAIT cell-mediated protection in systemic infections are not fully understood. This study aimed to elucidate MAIT cell activation, protective role in primary infection and potential for a MAIT cell-based systemic vaccination to protect against Francisella tularensis live vaccine strain (LVS) and Legionella longbeachae. F. tularensis is a gram-negative intracellular bacterium which can cause systemic infection, in mice and humans. In this study, F. tularensis LVS was used to induce systemic infection in C57BL/6 (wild type) mice and in Mr1 -/- mice, which lack MAIT cells. A combination of CpGcombo (fused oligos for CpG-B and CpG-P) and synthetic 5-OP-RU antigen was used to vaccinate mice. Bacterial load, survival rate, and MAIT cell response kinetics and post-infection cytokine profiles were examined. MAIT cells expanded systemically and showed Th1-like cellular profile after infection with F. tularensis LVS. In several organs, C57BL/6 mice showed better control of bacterial burden compared with Mr1 -/- mice. Vaccination of MAIT cells with CpGcombo plus 5-OP-RU, but not CpGcombo alone, protected mice from infections by an otherwise lethal dose of F. tularensis LVS and L. longbeachae. Thus, MAIT cells displayed a protective role against systemic infections and potential to be boosted to protect against local and systemic infections. This study also showed that post infection, MAIT and non-MAIT alpha/beta-T cells manifest different contraction kinetics, indicating that these two groups could be regulated by different viability mechanisms. Specifically, the in vivo data illustrated that loss of receptor-interacting serine/threonine-protein kinase 3 (RIPK3, a key regulator of apoptosis and necroptosis), but not mixed-lineage kinase domain like pseudokinase (MLKL, a signaling molecule involved in necroptosis), preferentially increased MAIT cell abundance in a cell-intrinsic manner. In summary, the results in this thesis demonstrated that MAIT cells are critical in immune protection against systemic F. tularensis LVS infection, and are long lived with a differently regulated cell death and survival. These findings may inform the future development of vaccination strategies targeting MAIT cells.
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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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ItemNeuroimmune responses in viral infection( 2020)The regulation of the immune responses is important in maintaining good health. Interactions between the nervous and immune systems are increasingly studied and widely appreciated to be influential in orchestrating immune responses. T cells express adrenergic receptors (AR) that enable them to respond to neurotransmitters produced by the sympathetic nervous system (SNS), noradrenaline (NA) and adrenaline, inducing downstream signalling and modulating cell functions, although whether this is stimulatory or inhibitory in T cell antiviral responses is unclear. In this thesis, I examine the effects of SNS in various models of viral infection through chemical sympathectomy and treatment with AR agonists. Modulation of sympathetic signals in systemic infections with LCMV had minor influences on T cell responses but resulted in increased viral loads. Notably, the infection results in a loss of tyrosine hydroxylase (TH) positive sympathetic fibres in the spleen as early as day 3 post infection and is reflected by decreased NA splenic NA. The immune response may play a role with interferon g partially contributing to the depletion. Additionally, this thesis also investigates the capability of long-lasting resident memory T cell (TRM) responses in the highly innervated, immune-privileged cornea. Using a model of herpes infection of the cornea, I showed that T cells are effectively recruited to the cornea with a small heterogenous population able to persist following cessation of immune responses. These cells express CD69 and CD103, canonical markers of tissue residency, to varying degrees. Persistence, but not recruitment, of these cells is dependent on antigen availability at the cornea. These memory cells are capable of responding to secondary encounters with antigen. Moreover, circulating memory cells are also able to infiltrate the immune-privileged cornea more efficiently following infection. Together, these results highlight the important nuances in the regulation of immune responses by the nervous system.
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ItemThe role of CD8+ tissue-resident memory T cells in melanoma immune surveillance.( 2018)In addition to its role in protecting the body from infection, the immune system can prevent the development of cancer in a process termed tumour immune surveillance. During this process, immune cells can either recognise and completely eliminate cancerous cells, or can suppress the outgrowth of malignant cells without completely eradicating them. This latter mode of control, designated ‘cancer-immune equilibrium’, can be sustained for extended periods of time in a manner dependent upon adaptive immune cells such as T cells. The vast majority of human cancers are spawned from epithelial tissues. However, long-lived CD8+ circulating memory T (TCIRC) cells such as effector memory T (TEM) cells and central memory T (TCM) cells are typically excluded from epithelial tissue compartments in the absence of robust inflammation. In contrast, CD8+ tissue-resident memory T (TRM) cells are a population of non-migratory immune cells that permanently occupy epithelial tissue sites without recirculating. CD8+ TRM cells provide efficacious protection against peripheral viral and bacterial infections and have recently been identified in a variety of human solid tumours, where they associate with improved disease outcome. However, a direct role for TRM cells in promoting natural immunity to cancer has yet to be demonstrated. In this thesis, we examined the contribution of CD8+ TRM cells to peripheral cancer immune surveillance and the mechanisms through which these cells protect against tumour progression. In order to study the peripherally localised anti-tumour immune response, we developed and characterised an orthotopic epicutaneous (e.c.) model of melanoma in mice that targets tumour growth to the outermost layers of skin. We found that a portion of mice receiving tumour cells e.c. remained free of macroscopic cancer long after inoculation, in a manner that depended upon immune cell mediated control. Spontaneous protection from progressive tumour development was associated with the formation of melanoma-specific CD69+CD103+ CD8+ skin TRM cells, whereas mice genetically deficient in TRM cell formation were highly susceptible to tumour growth. Importantly, tumour-specific skin TRM cells could protect against tumour development independently of TCIRC cells. Closer inspection of macroscopically tumour-free mice revealed that many harboured occult melanoma cells in their skin long after e.c. inoculation. These dormant melanoma cells were retained in the epidermis, where they were dynamically surveyed by tumour-primed CD8+ skin TRM cells. Ablation of skin TRM cells from macroscopically tumour-free mice that were initially protected from tumour development triggered late-stage tumour outgrowth, demonstrating that CD8+ TRM cells can suppress cancer progression by promoting a state of subclinical cancer-immune equilibrium. Further, our findings suggest that the cytokine tumour necrosis factor (TNF) may play a role in the induction and maintenance of this equilibrium state. Overall, we show that CD8+ TRM cells contribute to immune surveillance of peripherally localised cancers by upholding tumour-immune equilibrium. As such, our findings elucidate how cancers arising in epithelial compartments are subject to long-term and ongoing immune suppression. Collectively, our work provides critical insight and the impetus necessary to exploit CD8+ TRM cells as targets of cancer immunotherapies in order to improve solid cancer treatments in patients.
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ItemUsing dendritic cell receptors to enhance immunity( 2017)Dendritic cells (DCs) are the most potent initiators of immune responses, being highly specialised for the uptake and presentation of antigens (Ag) to activate T cells. Their priming potential can be harnessed to generate stronger immune responses by targeting Ag to DCs via monoclonal antibodies (mAbs) specific for DC-expressed surface receptors. This thesis builds upon the concept of targeting DCs in two main ways: firstly, by investigating a novel method of targeting adjuvant to DCs, and secondly, by investigating how DC-targeting constructs can be used to prime and boost responses. It was considered whether not only Ag, but also adjuvants could be targeted to DCs to improve their efficacy. A recent finding that the DC receptor DEC-205 can bind to and mediate the immunostimulatory effects of CpG oligonucleotide (ODN) adjuvants led to the hypothesis that CpG ODNs could be targeted to DCs via DEC-205 in order to enhance their potency. The interaction between DEC-205 and CpG ODNs was further characterised to determine the molecular properties of ODNs required for binding. This information was then used to enhance the DEC-205 binding capacity of a particular CpG ODN that normally only weakly binds DEC-205. Enhanced DEC-205 binding was found to significantly improve the stimulatory capacity of this ODN, demonstrating that targeting adjuvant to DCs could be a viable method to improve adjuvant potency. Another receptor, CD14, has also been reported to bind CpG ODNs, so the potential for CD14 to act in synergy with DEC-205 was investigated. However, CD14 was not observed to mediate the uptake or stimulatory effects of CpG ODNs. The identification of natural ligands of DEC-205 is critical for understanding its physiological function. Although ODNs are synthetic molecules, their binding to DEC-205 may signify that DEC-205 is capable of binding other types of DNA that structurally resemble ODNs. A panel of biological DNA samples was screened for DEC-205 binding. While none of the DNA samples were observed to bind DEC-205, some DNA samples were found to bind another receptor, RAGE, suggesting a role for RAGE as a detector of both pathogenic and self-DNA. Most vaccines must be administered more than once, or “boosted”, to achieve optimal efficacy, and DC-targeted vaccines should be no exception. However, our data suggested that simply administering the same DC-targeting construct twice does not effectively boost the response. This was due to interference from the primary antibody response, which can cross-react with and neutralise a subsequently administered boosting construct. To overcome this issue, the efficacy of various heterologous prime-boost strategies designed to reduce the reactivity of the primary response against the boosting construct was assessed. Ultimately, a combination of anti-Clec9A and anti-XCR1 targeting constructs was found to induce the least cross-reactivity and strongest response after boosting. These findings contribute to the development of better adjuvants and immunisation strategies that optimise the efficacy of DC-targeted vaccines. More broadly, they also highlight the value of understanding the underlying biological mechanisms that drive immune responses, which can then be applied to the rational design of more effective vaccines.
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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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ItemCharacterization of T cell populations in human skin( 2016)Human skin serves as a primary barrier to pathogenic and environmental assault. As part of the frontlines of the skin immune system, normal skin contains a vast number of T cells. With advances in immunofluorescent staining, confocal microscopy and fluorescence activated cell sorting, enumeration and characterization of these cutaneous T cells can now be performed with improved accuracy. Furthermore, several subsets of T cells, including mucosal-associated invariant T (MAIT) cells and resident memory T (TRM) cells, have only recently been described and their exact distribution in normal human skin remains to be elucidated. The relationship of T cells to hair follicles, ubiquitous appendages in human skin, also requires further investigation. This dissertation presents a comprehensive assessment of the distribution and composition of major T cell subsets in human skin at steady state. The gene expression profiles of skin and blood T cells were compared to determine a transcriptional signature for cutaneous T cells. We also conducted a preliminary investigation of the novel T cell subsets MAIT cells and TRM, exploring their presence in normal and diseased skin. Finally, we used a targeted laser capture microdissection approach to study chemokine expression in healthy hair follicles and in the autoimmune hair loss condition alopecia areata. Our findings reconfirm the strikingly anisotropic arrangement of T cells in normal human skin, with preferences for superficial perivascular and perifollicular localization. A resident cutaneous population of MAIT cells was found, with increased frequency in the blistering skin condition dermatitis herpetiformis. Surprisingly, a strong disparity was observed between the transcriptional profiles of skin-tropic T cells isolated from normal blood and skin. As the skin-derived T cells showed significant enrichment for genes from the TRM transcriptional core signature, we hypothesized that the skin may contain additional as-yet undefined TRM or TRM-like populations. Analysis of alopecia areata hair follicles revealed the presence of perifollicular T cells of a TRM phenotype that persisted long after active hair loss had ceased. Gene expression studies of laser microdissected follicles further highlighted transcriptional abnormalities in alopecia areata follicles that spanned the natural history of the disease. The results from this dissertation form a foundation for further study of conventional and emerging T cell subsets in skin and hold implications for the pathogenesis of the dermatological diseases dermatitis herpetiformis and alopecia areata.
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ItemMemory CD4 T cells in skin at steady-state and following infection( 2015)Peripheral tissues such as the skin, gut and lungs are exposed to the environment, making efficient immunosurveillance of these organs critical. This in part relies on T cells that gain access to these barrier sites. There has been an increasing interest in recent years on memory T cells permanently lodged in peripheral tissues, termed resident memory T cells or TRM. The CD8+ subset of TRM are relatively well characterized. However, there is much less information available on CD4+ T cells in peripheral compartments, especially to what extent these cells are retained in the tissue or recirculate. In this thesis, we examine CD4+ T cells in skin, with a focus on their migration patterns in the steady state as well as following the resolution of inflammation. These studies show that in mouse flank skin at steady state, the vast majority of CD4+ T cells are in equilibrium with the circulation, indicating that they constitutively migrate between the skin and blood. Viral infection or contact sensitization results in a sustained, localized increase in number of memory CD4+ T cells in skin, which is due to the generation of a long term-retained or resident population, as well as enhanced recruitment of circulating cells from the bloodstream. The hair follicle is key in mediating this numerical increase, with memory CD4+ T cells persisting in clusters surrounding these structures in conjunction with a high density of antigen presenting cells. T cells in such clusters are highly dynamic and rely on factors produced by other cells within the cluster, namely dendritic cell- and CD8+ T cell-derived CCL5, but do not appear to require persisting viral antigen. Furthermore, the majority of memory CD4+ T cells that produce the anti-viral cytokine IFNγ upon secondary infection are in close association with hair follicles. Thus, the follicle may provide an environment in which efficient T cell activation can occur in the skin, allowing for the initiation of rapid immune responses. These results highlight the complex nature of CD4+ memory T cells that are found within the skin and reveals the importance of tissue-specific factors in determining the localization of these cells within the tissue.
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ItemUnconventional T cells: from basic biology to multiple myeloma( 2016)Most studies of human T cells have focused on peptide-MHC-restricted CD8+ and CD4+ T cells. There are many T cell subsets however that do not conform to the same paradigms as these cells, but rather exhibit non-MHC-restriction, recognising for example lipid Ag presented by the CD1 family, or vitamin-B derivative Ags presented by the MHC-related protein 1 (MR1). Moreover, many of these ‘unconventional T cells’, including natural killer T (NKT) cells, mucosal-associated invariant T (MAIT) cells and γδ T cells circulate at high frequency in the steady state, pre-primed to elicit a rapid effector response. As such, in comparison to their conventional T cell counterparts, unconventional T cells likely play differing roles in health and disease. Studying the biology of these cells, from their fundamental biology to their roles in disease settings, is critical not only to gaining a comprehensive understanding of human immunity, but may provide new avenues for immune manipulation in therapeutic settings. This thesis explores the fundamental biology of MR1-restricted T cells. While previous studies suggest that the MAIT TCR-α chain is fixed, (using TRAV1-2 paired with TRAJ33), this thesis shows that MAIT TCRs can also pair TRAV1-2 with TRAJ12 and TRA20. Alternate TRAJ usage was shown to be permitted by a conserved germline encoded tyrosine residue (Tyr95α) within the TRAJ genes, which allowed the non-canonical MAIT TCRs to recognise MR1-Ag complex within a consensus PRR-like mode of Ag-recognition. Moreover, it is shown that a subpopulation of MAIT cells can not only recognise riboflavin-derivative Ags but are also capable of detecting folate-derivative Ags presented by MR1. A molecular basis for this observation is provided, showing that Ag-discrimination by the MAIT TCR is mediated via CDR3β hypervariability. Finally, diverse populations of atypical MR1- restricted T cells from both the αβ and γδ T cell lineage are identified and characterised at a cellular and structural level. These findings provide a basis for a role for MR1-mediated T cell immunity beyond that of the MAIT-MR1-riboflavin axis, including in diseases of non-microbial aetiology such as cancer. Multiple myeloma is a haematological malignancy that, despite considerable improvements in patient outcomes with modern therapies, remains incurable. The conventional T cell compartment in myeloma patients is highly dysfunction, yet little is known about the role of unconventional T cells. This thesis furthermore characterises the unconventional T cell compartment in multiple myeloma, with a focus of MAIT cells and γδ T cells. Both MAIT cells and a subset of γδ T cells known as Vγ9Vδ2 γδ T cells are shown to be highly reduced in the circulation and marrow of myeloma patients and furthermore these cells have perturbed function. Of importance, these T cell subsets have strong antimicrobial activity, and a loss of these cells may contribute to the high mortality associated with infectious disease exhibited by myeloma patients. Furthermore, MAIT cells were shown to have the capacity to directly lyse myeloma cell lines when pulsed with microbial antigen, opening the possibility that these cells may be harnessed in a therapeutic setting during the treatment of myeloma or other malignancies.