Microbiology & Immunology - Theses
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ItemDissecting the role of gd T cells in T cell priming for liver stage immunity( 2023-11)Liver resident memory T cells (TRM) are poised for protection against repeat infection and rapidly form a robust defence against tissue-specific insults such as liver stage malaria. A direct correlation between liver stage immunity and gd T cells has been observed both in mice (Zaidi et al. 2017) and in humans (Seder et al. 2013; Ishizuka et al. 2016), but the precise molecular mechanisms by which these gd T cells exert their protective effect are yet to be defined. In mice, intravenous injection with radiation-attenuated sporozoites (RAS) confers sterile protection against challenge with live sporozoites. This protection is mediated by responding antigen-specific CD8+ and CD4+ T cells that migrate to the liver and form resident-memory T cells (TRM). In the absence of gd T cells, protective CD8+ liver TRM are not generated, leaving mice susceptible to reinfection. Using Plasmodium-specific T cells as a readout for effective immunity, we determined that IL-4 is important for the accumulation of CD8+ and CD4+ T cells. By utilising complex in vivo systems including mixed-bone marrow chimeras and adoptive transfer of gd T cells, we revealed that gd T cell-derived IL-4 is crucial for the expansion of antigen-specific CD8+ T cells. In addition, in vivo neutralisation of IL-12 or IFN-g confirmed a partial dependency for these cytokines, despite their traditionally opposing function to IL-4. Given IL-4, IFN-g and IL-12 all have a clear role in CD8+ T cell priming following RAS vaccination, we hypothesised that IL-4 and IFN-g synergise to enhance cDC1 activity. These findings led to our development of a novel model to reconstitute cDC1-deficient mice using CRISPR-edited primary dendritic cells. This model enabled the investigation of the mechanism by which gd T cell derived IL-4 leads to DC activation and therefore effective CD8+ T cell expansion for memory development. Collectively, this project has shown a significant role for IL-4 in the priming of malaria-specific CD8+ T cells and demonstrates a novel pathway for collaboration between gd T cells, cDC1s, and CD8+ T cells, revealing the potential for harnessing gd T cells in vaccination strategies against malaria.
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ItemFunctional antibody immunity in SARS-CoV-2 infection and vaccination( 2023-01)Since the emergence of SARS-CoV-2 in late-2019, the virus has caused substantial mortality in humans, with the confirmed death-toll exceeding 6.5 million lives and causing over-600 million confirmed cases. The introduction of COVID-19 vaccines has been instrumental in reducing the risk of hospitalisation and death globally. However, with the rise of novel variants, SARS-CoV-2 continues to cause high levels of infections, and thus the impact of the COVID-19 pandemic remains far from over. Antibodies play crucial roles in the protective immunological response to SARS-CoV-2. As such, this MPhil thesis explores the antibody response to SARS-CoV-2 in the context of both infection and vaccination using high-throughput multiplexing technologies. We began by investigating the systemic (plasma) and mucosal (saliva) antibody responses during acute (first-fourteen days) infection in adults and children, shown in Chapter 3 of this thesis. We used a cohort of individuals recruited from household clusters of COVID-19 infections during the first waves of ancestral-strain SARS-CoV-2 in Melbourne, Australia. Using systems serology analysis, we found that households with higher rates of secondary transmission to household contacts display greater levels of virus in the nasopharynx; and correspondingly display enhanced systemic antibody responses, suggesting a relationship between viral load and seroconversion. Additionally, we observed differential antibody response induced during acute infection between adults and children. Levels of IgA antibodies to SARS-CoV-2 spike (S)- and nucleocapsid-proteins were substantially lower, notably in the saliva, of children compared to adults. We speculate this in part may reflect that adults have greater prior exposure to endemic human coronaviruses. Given little information is known regarding the mucosal response in children, future studies are needed to profile the mucosal response more extensively in this population. The emergence of variants of SARS-CoV-2 are a challenge to public health. Many variants harbor mutations in the RBD of the SARS-CoV-2 S, a dominant target for neutralising and non-neutralising antibodies. In Chapter 4, we comprehensively characterised the effect of RBD mutations, including mutations present in the Omicron (BA.2) variant, on ACE2-binding affinity, as well as the functional antibody response induced in BNT162b2-vaccinated recipients and mild-convalescent SARS-CoV-2 infected individuals. The RBD of variants of concern (VOCs) displayed greater affinity to ACE2 than ancestral SARS-CoV-2 RBD. Furthermore, using a surrogate neutralisation assay that examined 39-naturally occurring RBD mutations, we showed reduced capacity of plasma antibodies to block ACE2-binding to VOCs. Additionally, we show a reduced capacity by RBD-binding plasma antibodies to engage Fc-receptors (FcRs), suggesting the potential for RBD-binding antibodies to recruit Fc effector functions are compromised by mutations in the RBD. These findings highlight the capacity of viral variants with RBD mutations to subvert host antibody responses. Lastly, with the observation that S-binding antibodies are attenuated by mutations found in variants, it is thought enhancing the magnitude of the antibody response may be useful in offering protection against SARS-CoV-2. As such, in Chapter 5 we investigated the effect of administration of a novel priming vaccine strategy designed to augment antibody responses – Bacillus Calmette-Guerin (BCG) immunisation – prior to immunisation with two homologous doses of either COVID-19 vaccine BNT162b2 or ChadOx1 on the antibody responses to the SARS-CoV-2 S. No differences in the SARS-CoV-2 antibody levels between BCG- and non-BCG-vaccinated individuals was observed, suggesting this strategy may not substantially improve the efficacy of current COVID-19 vaccines. Further studies are needed to determine whether this reflects the time window (>1 year) between BCG-vaccination and COVID-19 vaccines, or whether BCG- priming is not a suitable option for augmenting COVID-19 antibody responses. In summary, our work provides greater insights into the antibody response to SARS-CoV-2, of which can be further explored in prospective work.
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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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ItemCharacterising novel cytotoxic T lymphocyte dysfunction by spatiotemporal analysis, and optimizing adoptive T cell therapy against cancer while preventing autoimmune side effect.( 2022)Adoptive cell therapy (ACT) is an emerging strategy for cancer treatment. However, ACT is not always successful because of various factors. In ACT using cytotoxic T lymphocytes (CTLs), T cell dysfunction is one of the most significant causes of failure. Our laboratory has previously discovered a novel type of T cell dysfunction, which we named “stunning”. We used a mouse model of ACT in the context of B-cell lymphoma. Here, mice were injected with Eu-myc lymphoma cells expressing ovalbumin (OVA) followed by injection of OVA-specific CTLs (OT-I CTL). In this model, we found that the presence of a large tumour burden led to rapid deletion and inactivation of OT-I CTLs in an antigen-specific manner. We hypothesised that at early stage after ACT, one CTL encounters a large number of antigen-presenting tumour cells within a short period of time, and this multiple interaction leads to stunning. Therefore, we aimed to investigate (1) How tumour cells and CTLs localise in organs, (2) Exact timing when stunning occurs, (3) How frequently CTLs interact with tumour cells, and (4) How CTLs behave after encountering tumour. In order to obtain these questions, we utilise both static (confocal) and dynamic (intravital) imaging techniques in this research. Using confocal fluorescence imaging, we found that in the spleen and lymph nodes, the proportion of antigen-specific CTLs that localised in T cell zones was decreased under a higher tumour burden. We next investigated the motility of CTLs in the lymph nodes by intravital imaging and found that motility was decreased in the context of high tumour burden. Accordingly, we propose the like between spatiotemporal characteristics of T cells and their dysfunction, which might be a potential therapeutic target. In addition to T cell dysfunction, another shortcoming of ACT is the potential for autoimmunity. If adoptively transferred CTL has specificity to an antigen that is shared with self cells, self-tissues are also susceptible to the attack by CTLs. Several clinical trials of ACT resulted in severe self-tissue destruction. Thus, balancing anti-cancer CTL responses with prevention of autoimmunity is paramount to the success of ACT. Typically, tumour-associated antigens are highly expressed in tumour cells compared to normal untransformed cells. Therefore, we hypothesised T cells with a lower affinity for antigen may be better candidates for use in ACT expecting that the T cell would only recognise the antigens on the tumour but not the ones on healthy tissues. We utilised OT-3 CTLs which express a lower affinity TCR than that of the OT-I cells. After optimising the concentration of IL-2 and cellular density for in vitro T cell priming, we found that OT-3 CTLs were still able to kill targets as effectively as OT-I CTLs. This provides evidence that low-affinity T cells may act as candidates for ACT.