Medical Biology - Theses
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ItemMeasuring B-lymphocyte responses in human health and primary immunodeficiency( 2019)Production of high-quality, specific antibody is essential for lifelong protection against pathogens. Generation of antibodies is the end result of a complex and highly regulated process of B cell development and signalling. Defects anywhere in this process can lead to immune dysregulation, resulting in primary immunodeficiency and/or autoimmunity. This thesis explores a new hypothesis – that the majority of cases of primary immunodeficiency, and likely other complex immune disorders, are caused by the combinatorial effect of multiple small defects in B cell function or ‘health’ that sum to cause disease in patients. Further, that we can measure and model these defects quantitatively in vitro. To test this hypothesis, we focused on Common Variable Immunodeficiency (CVID), a clinically heterogenous primary immunodeficiency, united by antibody deficiency, where most cases are sporadic and, presumably, polygenic. In this thesis, we outline the development of a novel in vitro pipeline and accompanying parametric mathematical modelling tools to identify and measure the functional cause of immunodeficiency in individual patients. These in vitro assays were first developed and calibrated on healthy donors, to measure healthy human B cell responses to T-dependent and T-independent stimulation. These assays measure cell division, death, differentiation, isotype switching and antibody production, to reveal the innate programming of B lymphocytes in response to T-independent and T-dependent stimuli. Here, we observed, for the first time, autonomous programming of the division-burst size (division destiny) in human B cells, a phenomena previously demonstrated in murine lymphocytes. We applied cyton modelling to human lymphocytes for the first time, to explain the parameters underlying the synergy between two signals. Additionally, by testing a number of unrelated healthy donors, we established a healthy donor ‘range’ of B cell responses for comparison to patients. We subsequently applied these assays to a cohort of CVID patients and identified a number of quantitative differences. These included: 1. A striking, severe early survival defect of patient naive B cells; 2. A defect in the ability of patient naive B cells to differentiate to antibody secreting cells and produce switched antibody, and; 3. Reduced proliferation of patient naive B cells in response to T-independent stimulation. We utilised mathematical modelling tools to demonstrate that this reduced proliferation is explained by a combination of multiple small defects in the parameters that control the B cell response (times to divide, times to die etc) that sum together in a non-linear manner to magnify their impact on immune responses. Furthermore, the relative contribution of each parameter to the final immune deficiency was individually determined and found to be unique for each patient. This work significantly improves our understanding of the functional causes of immunodeficiency and offers a clear path toward improved clinical diagnosis and targeted treatment strategies for individual patients.
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ItemThe Transcription Factor T-bet in the Control of Germinal Centre Dynamics in Malaria( 2019)With reductions in the global malaria burden stalled, this preventable and curable infectious disease caused by the Plasmodium parasites, remains a public health challenge that affects the world’s most vulnerable populations. Naturally acquired immunity plays an important role in protection from disease; however, there is long-standing evidence that it requires years of repeated infections to develop. The reasons for this are largely elusive, but immuno-epidemiological studies support that protective antibodies and memory B cells are short-lived and inefficiently generated to infection. Moreover, recurrent infections are associated with an expansion of atypical memory B cells that may have impaired function. Histological analyses revealed significant disorganisation of the spleen in severe malaria patients, which led to the concept that acute infection may undermine the acquisition of B cell memory. T helper 1 pro-inflammatory responses induced by blood-stage infection were subsequently shown to compromise the induction of humoral immunity by inhibiting effective T follicular helper (Tfh) cell differentiation and germinal centre (GC) reactions. The relative contribution of the T helper 1 lineage-defining transcription factor, T-bet, in CD4+ T cells and B cells to GC development in malaria, was investigated using the P. berghei ANKA blood-stage infection model. T-bet expression in CD4+ T cells limited the differentiation of Tfh cells that supported GC development in the spleen. This led to an impaired generation of antibody-secreting cells and memory B cells following infection. In addition to its impact on CD4+ T cells, T-bet was highly up-regulated in GC B cells elicited by infection, and limited the magnitude of the GC response in a B cell-intrinsic manner. Strikingly, T-bet expression in the B cell compartment modulated the transcriptional landscape of GC B cells to promote the GC dark zone program but constrained light zone development. In particular, T-bet suppressed expression of the regulator of G-protein signaling 13, which down-regulates the responsiveness of B cells to migrate towards the chemokine CXCL12, for effective dark and light zone transition within the GC. T-bet-driven dark zone skewing of the GC reaction following malaria infection associated with enhanced somatic hypermutation of GC B cells, and improved the avidity of antibodies against the parasite. Therefore, this thesis supports a model in which malaria-elicited inflammation mediated by T-bet, exquisitely modulates the dynamics of the GC reaction, promoting GC B cell dark zone polarization that promotes the generation of B cells with increased affinity for antigen, consequently enhancing affinity maturation. This provides novel insight into the cellular mechanisms that underlie the development of humoral immune responses in malaria, and has implications for other chronic infections and autoimmune disease that are characterised by a similarly potent inflammatory milieu.