Microbiology & Immunology - Theses
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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.